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TISSN: 0971-7196Journal ofVolume 30 Number 1 June 2006(Released August 2006)ParasiticDiseasesThe Official Organ of the Indian Society for ParasitologyHFOR PARASITOLOSOCIETY INDIANEGYPublished byThe Indian Society for ParasitologyHkkjrh;ij1973thohfolKkuka?Electronic version available on ISP Websitewww.parasitologyindia.org

JOURNAL OF PARASITIC DISEASES(ISSN: 0971-7196)Editor-in-ChiefProfessor Prati Pal SinghNational Institute of PharmaceuticalEducation and ResearchSector-67, Phase-XS. A. S. Nagar-160 062, IndiaManaging EditorDr. Varsha GuptaDeptt. of MicrobiologyGovt. Medical College & HospitalChandigarh-160 032Assistant Managing EditorDr. Savita SinghNational Institute of PharmaceuticalEducation and ResearchS. A. S. Nagar-160 062Advisory BoardDr. A. B. Chaudhary, KolkataProf. R. C. Mahajan, ChandigarhDr. G. P. Dutta, LucknowDr. V. P. Sharma, New DelhiProf. N. K. Ganguly, New DelhiEditorial BoardProf. M. C. Agrawal, JabalpurProf. Irfan Ahmed, AligarhDr. H. K. Bajaj, HissarProf. H. S. Banyal, ShimlaProf. Neelima Gupta, BareilleyProf. B. C. Harinath, SevagramDr. D. C. Kaushal, LucknowDr. S. L. Hoti, PondicherryProf. R. Madhubala, New DelhiProf. Sandeep Malhotra, AllahabadProf. Nancy Malla, ChandigarhDr. J. R. Rao, IzatnagarProf. M. L. Sood, LudhianaDr. B. K. Tyagi, MduraiJournal of Parasitic Diseases is published biannually by the Indian Society for Parasitology in June and Decemberin each calender year. The subscription price for libraries and other multi-reader organizations for each number isRs. 400 in India and US $ 100 elsewhere. Subscription by Demand Draft in favour of 'The Indian Society forParasitology' should be sent to Dr. J. K. Saxena, Secretary, The Indian Society for Parasitology, Division ofBiochemistry, Central Drug Research Institute, Chattar Manzil, Lucknow - 226 001, India.

JOURNAL OF PARASITIC DISEASESVolume 30, Number 1, June 2006The official organ ofTHE INDIAN SOCIETY FOR PARASITOLOGYCentral Drug Research Institute, Lucknow-226 001, IndiaEditorial Office: National Institute of Pharmaceutical Education and ResearchSector-67, Phase-X, S. A. S. Nagar-160 062, IndiaPhone: 0172-2214682-87; Fax: 0172-2214692; E-mail: drppsinghjpd@yahoo.co.in

Journal of Parasitic DiseasesCopyright © 2006 The Indian Society for ParasitologyAll Rights ReservedNo part of this publication may be reproduced or utilized in any form or by any means, electronic ormechanical including photocopying, recording or any other information storage and retrieval systemwithout the permission in writing from copyright owner.

JOURNAL OF PARASITIC DISEASESVolume 30 Number 1 June 2006CONTENTSA Note from the New Editor-in-Chief1-3ReviewsHistochemical, biochemical and immunological studies in Haemonchus contortus(Nematoda: Trichostrongyloidea) - an Indian perspective. M. L. SoodImmunological perspectives and malaria vaccine. H. S. Banyal and N. Elangbam4-1516-29Original papersIsolation and characterization of the paraflagellar rod proteins of Leishmaniadonovani. A. Lahiri and A. BhattacharyaOnchocercosis in Benue state, Nigeria: comparative epidemiological studiesamongst the Etulo and Idoma ethnic groups. E. A. Omudu and B. O. AtuDistribution of iron in plasma, erythrocytes and tissues of calves with theprogression of Theileria annulata infection. N. Sangwan and A. K. SangwanMosquito breeding in riceland agro-ecosystem near Chennai, Tamil Nadu, India. J.Ravindran and J. WilliamsRandom amplified polymorphic DNA of Trichomonas vaginalis isolates fromTarbiz, Iran. R. Jamali, B. Zareikar, A. Kazemi, M. Asgharzadeh, S. Yousefee, R.Estakhri, S. Montazer and A. GhazanchaeiTwo new species of Trypanosoma from freshwater fish (Heteropneustes fossilis andChanna punctatus) from Bareilley, India. D. K. Gupta, N. Gupta and R. GangwarImpact of anthelmintic therapy on live weight gain in gastrointestinal nematodeinfectedgoats. A. K. Jayraw and Y. V. Raote30-3637-4041-4445-5253-5758-6364-67

Ultrastructure, differential density and distribution pattern of polymorphicmicrotriches in tegument of Stilesia globipunctata infecting Ovis aries (sheep). C.Venkatesh, K. Ramalingam and V. VijayalakshmiThe protozoan fauna living in the digestive system of Periplaneta americana inKolkata, West Bengal, India. J. Ghosh and A. GayenChelatrema neilgherriensis n. sp. (Trematoda: Gorgoderidae) infecting thefreshwater fishes from Noolpuzha river in Wynad district, Kerala, India. K. T.Manjula and K. P. Janardanan68-7576-8081-84Short communicationsHaemato-biochemical studies on fowl coccidiosis in layer birds. N. D. Hirani, J. J.Hasnani, R. S. Joshi and K. S. PrajapatiRe-redescription of Dissurus farrukhabadi Verma, 1936 (Digenea -Echinostomatidae) with a discussion of the genus Dissurus Verma, 1936. P. C.Gupta and R. B. SinghA case of vaginal bleeding due to leech bite. R. P. Ganguly, M. S. Mukhopadhyayand K. K. PatraField evaluation of a rapid immunochromatographic test kit for the diagnosis ofPlasmodium falciparum and non-falciparum malaria parasites from Sonpurdistrict, Assam. C. Rajendran and S. N. Dube85-8889-9192-9394-97

Journal of Parasitic Diseases: Vol. 30, No. 1, June 2006, 1-3J P DA Note from the New Editor-in-Chief“What the Author Expects from the Editors”. This has The Members of Editorial Board, who must be expertsbeen expressed by Dr. Earl H. Wood of Mayo Clinic as in their respective fields, in turn, have a very important“I expect the editor to accept all my papers, accept responsibility of sending the manuscripts to suitablethem as they are submitted, and publish them Reviewers/Referees/Consultants, determine thepromptly. I also expect him or her to scrutinize all quality of these manuscripts and then send theirother papers with utmost care, especially those of my recommendations to the Editor-in-Chief. Theircompetitors”.recommendations regarding the suitability of themanuscripts for publication, revision or rejection areMy association with Journal of Parasitic Diseasescrucial for the Editor-in-Chief in making a final(started as Indian Journal of Parasitology) is bothdecision, and thus, in maintaining the qualitypersonal and somewhat historical, and goes nearly 30standards of the journal. Therefore, the Editor-inyearsback when in 1976 the first issue of the journalChief must decide upon an appropriate size of thewas released in the main conference hall of the CentralEditorial Board taking into consideration the expertiseDrug Research Institute, Lucknow. Certainly, it mustof its Members which should be in consonance withhave been born out of the hard work andboth the dimensions of the scope of the journal and theuncompromised dedication of several Members of thenumber of manuscripts to be handled.Society, and the vision of its Founder Editor-in-Chief,late Dr. B. N. Singh. And with this issue, in a new style The Reviewers/Referees/Consultants read, evaluateand format, my tenure as Editor-in-Chief has just and return the manuscripts along with their commentsbegun.prepared in accordance with the instructions provided.These comments must be specific and not abstract,The Editor-in-Chief of a journal is ultimatelypoint errors related both to fact and interpretations,responsible for its quality standards and acceptabilityindicate inaccuracies and ambiguities, and also mustby the audience it addresses. His or her singular goalclearly suggest as to how to condense, enlarge andshould be to deliver the readers a package of goodimprove upon the style of writing. In biologicalscience in a good and simple language. Therefore, thesciences, hardly 5% manuscripts are accepted withoutfinal decision to accept or not to accept a manuscriptany revision. “The Guidelines for Reviewers,for publication or to send it back for modification mustAmerican Society for Microbiology”, formulatedbe the responsibility of the Editor-in-Chief only. Forbased on the policies recommended by a committee ofarriving at such a decision, he or she should carefullythe Council of Biology Editors is usually very helpfulgo through the manuscript and take into considerationfor the Reviewers/Referees/Consultants. The helpthe recommendations, and the reports and commentsprovided by them is generally acknowledged in theof the Editorial Board Members and Reviewers/form of listing their names in the last issue of the year.Referees/Consultants, respectively. However, for theBecause a manuscript is the intellectual property of itsresolution of complicated and controversial matters,authors, it must be treated as a privileged confidentialthe Editor-in-Chief should also be willing to take thecommunication throughout, till published.help of Advisory Board Members, should such asituation arise.The Managing Editor of a journal is usually not

2 Prati Pal Singhinvolved in decisions related to the acceptance or non- parasitology. This certainly does not mean that theacceptance of a manuscript. Rather, he or she is newer cellular and molecular, and other important andsupposed to provide the Editor-in-Chief various emerging dimensions of parasitology shall besupport services during the review process. However, neglected. Second, the journal should orient itself inthe main function of a Managing Editor starts only such a way, that it must make an impact on the conductafter a manuscript has been accepted for publication, of the theory and practice (including clinical) ofand he or she takes the responsibility to convert it into a parasiotology in our country. This may not turn out toprinted product i. e. a published paper. The Copy be an easy task. The third, I feel is that there are severalEditors and Production Editors, usually associated areas in parasitology which continue to remainwith big journals are responsible for the final product controversial, conflicting, confusing andas related to grammar, spellings, syntax, style conjecturing. Therefore, Journal of Parasiticpolishing etc., and the quality of page layout and Diseases must specifically publish comprehensiveimage resolution, respectively. Therefore, in my and accessible reviews which should try to focus onopinion, the entire Editorial Board must meet at least these areas of parasitology. Towards this end, I intendonce during the year, and our annual meetings can be to invite manuscripts from experts who would bethe most appropriate occasions for such meetings. willing to specifically contribute such reviews.However, this notwithstanding, the journal intends toIt is aptly said that the reputation of a journal primarilypublish at least two invited reviews on variousdepends upon the quality of research papers itimportant aspects of parasitology in its each issue.publishes. I shall, therefore, with the help of Advisors,Members of the Editorial Board and Reviewers/ Parasitology research is a very big field spanningReferees/Consultants, strive hard to take the standards mainly into areas like agricultural, veterinary, medicalof our journal to new heights. Towards this end, I shall and general parasitology. These areas, in turn, haveexpand the current Editorial Board, both in terms of their own sub-areas and allied areas. Put together, theythe number of its Members and the diversity of their all constitute a big world of parasitology. In ourfields of specialization to encompass various aspects country Journal of Parasitic Diseases is probably theof parasitology. The speed of the editorial processing only journal that is specifically devoted to parasitesof the manuscripts submitted for publication leading and the diseases caused by them. Therefore, it shouldto its final acceptance or non-acceptance is also be the aim of the journal to adequately address all suchimportant. I shall try to strictly follow the 15-day areas and sub-areas effectively. Additionally, Journalduration for the Reviewers/ Referees/Consultants to of Parasitic Diseases should also try to address tosubmit their reports. To the extent possible, I shall areas related to parasitology and community/society,encourage the use of electronic mail for sending the parasitology and economics, and parasitology andmanuscripts for review and for receiving the review nation development.reports. This can be expected to reduce both time andcost.One of the most important policies that I have alreadyintroduced is the “Tutorial Editorial Policy”. ThisThe job of an Editor-in-Chief is a very responsible one. policy aims towards working of the Editorial BoardI would, therefore, be a remiss if I did not acknowledge with the authors, to improve the quality of theirthe attendant challenges that I expect to face. First, I submitted manuscripts so as to make thembelieve, is to implement again the very vision with publishable. Experience has shown that a greater andwhich the journal was conceived and started. There is intense interaction between the authors and Editorialno doubt that the primary constituency of Journal of Board has always been beneficial in improving bothParasitic Diseases is basic parasitology. During my the quality of science and of writing. Scientificinformal discussions with various members of the writings are different from any other type of writingsSociety, I gathered the impression that a large number and at times not easy, and thus require considerableof them feel that the journal has seriously departed practice. Unfortunately, in our country, unlike infrom its primary constituency. Certainly, such wheels several other countries, most of the universities do notmust have turned slowly over a period of time. I would, have a formal course related specifically to scientifictherefore, with the help of our authors/contributors writings. It is now well known that in many reputedand Editorial Board Members, like to improve upon journals, mostly the manuscripts are not turned downthis state and try to move the journal back to basic because of bad science but bad writing. The effects of

Editor's Note3this policy may not become visible very soon, but sets of problems, and may not be able to make printcertainly in the long run it is going to be beneficial, journals obsolete so soon.both to the authors and the journal. I would like to takethis opportunity to assure our authors that the entireIn closing, I would like to sincerely thank Dr. V. P.Editorial Board is on their side, to help them inSharma (Chairman) and Members of the committee topublishing good science. Here, I must bring to yourconstitute new Editorial Board Prof. M. S. Jairajpurikind attention that our journal has a big problem of not(former President of the Society), Prof. Veena Tandonhaving a suitable number of quality manuscripts(President of the Society), Prof. Nancy Malla (formeravailable for publication. And, this often results in theEditor-in-Chief), Dr. J. K. Saxena (Secretary of thedelayed release of a particular issue of the journal.Society), Dr. L. M. Tripathi (Treasurer of the Society),Dr. S. Dutta and Dr. S. L. Hoti, and to the Society atIn an editorial titled “On the Future of Scholarly large, for the honour of offering me this importantJournals”, published in Science (17 April 1998, 279, responsibility and for reposing confidence in me. I359), the author Alen M. Edelson has very should like to take this opportunity to especially thankappropriately emphasized the potential impact of Prof. R. C. Mahajan and Dr. G. P. Dutta for theirdigital/electronic publishing on print publishing. valuable suggestions. I should also like to thank thePresently, most of our print journals are suffering from authors of this issue and to the future authors. I deem itfinancial crunch because of declining library budgets, my greatest pleasure to thank Prof. P. Ramarao,escalating costs, reduction/cancellation in Director, National Institute of Pharmaceuticalsubscriptions and, of course, increasing manuscript Education and Research, for his help andpressure. Digital/electronic publishing, on the other encouragement without which it would not have beenhand, can be expected to be cost-effective and would possible for me to undertake this responsibility.allow greater and relatively more convenient Finally, I thank our printer Mr. Lalit Azad, for his keenaccessibility to scientific information. Therefore, I interest and excellent work in a timely manner. I ambelieve in the need that Journal of Parasitic Diseases confident that with our joint efforts, Journal ofalso must run with the times, and should evolve Parasitic Diseases will continue to maintain its highstrategies to digitalize it in a bigger way, including on- standards and surge to new heights. I shall continue toline submission and processing of manuscripts. interact with you all from time to time.Nevertheless, it is becoming increasingly clear thatelectronic journals will also be fraught with their ownPrati Pal Singh, Ph. D., F. N. A. Sc.Editor-in-Chief

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 4–15ReviewJ P DHistochemical, biochemical and immunological studiesin Haemonchus contortus (Nematoda: Trichostrongyloidea)- an Indian perspectiveM. L. SoodDepartment of Zoology, Punjab Agricultural University, Ludhiana.ABSTRACT. Haemonchus contortus is the most pathogenic nematode parasite of sheep/goats intropics/subtropics. Presently, the different aspects of H. contortus research in histochemistry[absorptive surfaces-structure and composition (the body wall, the gut), anthelmintic effects;spicules and gubernaculum], biochemistry [inorganic elements; carbohydrates (glucose andglycogen utilization, glycolysis, TCA cycle, pentose phosphate pathway]; amino acids(composition, metabolism, anthelmintic effects); lipids (composition, metabolism, enzymes,anthelmintic effects), proteins; nucleic acids; nutrition (digestive enzymes), biochemistry intaxonomy; miscellaneous] and immunology have been reviewed in Indian context.Key words: biochemistry, Haemonchus contortus, histochemistry, immunology, IndiaINTRODUCTIONHeamonchus contortus (Rud.,1803) commonlyknown as the twisted stomach worm, is a bloodsuckingnematode parasite, primarily occurring in theabomasum (Fig.1) of small ruminants, notably sheepand goats. It has been ranked as the most importantparasite of small ruminants in all regions across thetropics/subtropics (Anonymous, 1992).Haemonchosis, the disease caused by this nematode isresponsible for considerable economic lossesworldwide. In hyperacute disease, death of the hostmay occur within one week without significant signs(Fraser, 1991).Over the years, attempts have been made to studyvarious aspects of Haemonchus spp. from differentparts of the world (see Sood and Kapur, 1982a).Earlier, Haemonchus research in India was reviewedCorresponding author: Prof. M. L. Sood, 500/4, Model Town(Club lane), Ludhiana - 141 002, Punjab, India.E-mail : mlsood@rediffmail.comFig. 1. A part of goat abomasum cut open to show Haemonchuscontortus, in situ. Inset, adult female and male (From Sood andKapur, 1982; with permission from Shidha Publications,Ludhiana, India).(Sood, 1981) under the titles : morphology anddistribution, biochemistry, immunology,development and cytology, life-history, pathogenesis,

Haemonchus histochemistry, biochemistry and immunology5clinical symptoms, diagnosis, epidemiology, the cuticle (Sood and Kaur, 1977), has been welltreatment and prophylaxis. Subsequently, in a review documented (Sood and Kaur, 1976).on haemonchosis in India, Sood (2003) coveredepidemiology, pathology, diagnosis and control, The histochemical studies on the body wall of H.including resistant strains. Presently, the upcoming contortus (Sood and Kalra, 1977) have revealed thatareas of Haemonchus research, and the ones not fully the cuticle is mainly proteinaceous in nature. Thecovered in the above reviews have been taken up. lipids and PAS-positive materials are only present inHopefully, these three reviews, covering separately cortical layers. In addition, haemoglobin and acidthe various aspects, would provide an up-dated phosphatase (AcPase) are also present. Glycogen,consolidated account of 'Haemonchus to lipids, RNA, AcPase and alkaline phosphatasehaemonchosis' in India.(AkPase) have been reported in the hypodermis. Theoval dense body is composed of keratinous andHISTOCHEMISTRYcollagenous proteins associated with acidmucopolysaccharides. Muscles carry a greaterHistochemical studies have been made on the concentration of glycogen granules andabsorptive surfaces (including in vitro anthelmintic phospholipids. The functional significance of theseeffects) and of the spicules and gubernaculum.components has been fully discussed.Absorptive surfacesThe gut: Singh and Johl (2001) studied the structureParasitic nematodes have two absorptive surfaces- the of the fore-gut (stomodaeum). Histomorphology ofexternal surface or the body wall and the gut, though the intestine has also been studied (Johl, 2003). Soodlatter is the main functional unit from nutrition point of and Sehajpal (1978) made morphological,view. The body wall in nematodes is composed of the histochemical and biochemical studies. The intestinalcuticle, an underlying cell layer (hypodermis or epithelium (IE) is provided with a well-developedepidermis), and the longitudinally orientated somatic brush border (BB) which contains periodic acid-musculature (Wright, 1987). Nematode cuticle is one Schiff- positive mucoproteins. The IE stores glycogenof the most complex acellular structures synthesized and lipids. It stains diffusely for phospholipids andby a living organism. In order to understand the general proteins and also for terminal-NH 2 group. The2+ 3+transport of solutes in the body wall, experimental presence of Fe and Fe containing pigments anddetermination of diffusion coefficients of NaCl and activities of AcPase and AkPase, glucose-6-KCl in adult H. contortus at 298. 16 k has been made phosphatase (G-6-Pase) and 5'-nucleotidase have(Sood et al., 1999).been abserved in IE. Biochemically, pH optimum forintestinal AcPase has been found to be 4.8. BB showsThe nematode gut consists of three parts, a muscular, positive reactions for Acpase and G-6-Pase, andcuticular lined pharynx (oesophagus), a relatively negative for AkPase and 5'-nucleotidase. Presence ofstraight intestine, whose wall is one cell thick and a enzymes in the BB is related to extracellular digestionrectum or cloaca, which like pharynx is lined by and absorption of nutrients.cuticle (Bird, 1971; Chitwood and Chitwood, 1974).Nematodes do not have a continuous muscle sheet Anthelmintic effectsaround the intestine and the pseudocoelomic fluid is inStudy of alterations induced in the absorptive surfacesdirect contact with the basement membrane. The(and neuromuscular system) of the parasites by the innematode intestine is, therefore, an ideal cellvitro incubations with the anthelmintic drugs is ofmonolayer to study the transport mechanisms, theprime importance, particularly in view of theonly diffusion barrier being the basement membranerepeatedly reported resistant strains of H. contortus.(Barrett, 1981).The mechanisms by which the drugs act on theseAbsorptive surfaces in H. contortus have been absorptive surfaces are quite obscure. During theirreviewed (Sood, 1999), under the titles: structure and absorption, anthelmintics are expected to induce somecomposition, immunology and anthelmintic effects. cellular and chemical changes in the absorptivesurfaces. Anthelmintics during their absorptionStructure and compositionaffect/and/or modify the enzyme activity and may alsoalter the normal metabolism of the absorptiveThe body wall: The morphology of the vulvarsurfaces.configurations in the female H. contortus, formed by

6 SoodAnthelmintic effects (and other aspects) on theabsorptive surfaces have been reviewed (Kaur andSood, 1986).The morphological and histochemical effects of invitro incubations with thiabendazole, moranteltartrate, tetramisole hydrochloride and piperazinehexahydrate on the body wall and intestine have beeninvestigated (Sood and Kaur, 1982). Similar effects ofdl-tetramisole (TMS) and rafoxanide (RFX) havebeen observed for AkPase, AcPase adenosinetriphosphatase (ATPase) and G-6-Pase (Kaur andSood, 1982 d). Also, histological study on the effectsof these two anthelmintics has been made (Kaur andSood, 1983a). In vitro alterations induced by Nilzan(NLZ) and albendazole (ABZ) have also been studiedin AcPase, AkPase, ATPase, G-6-Pase, cytochromeoxidase, monoamine oxidase, non-specific esterases,acetylcholine esterase (AChE), and in succinic, lactic,glutamate and glucose-6-phosphate dehydrogenases,reduced nicotinamide, adenine dinucleotidediaphorase and reduced nicotinamide adeninedinucleotide diaphorase (NADH-D) and reducednicotinamide adenine dinucleotide phosphatediaphorase (Kaur and Sood, 1990). Also, the effects ofNilverm (NLV) and Nilverm forte (NLF) (Kaur andSood, 1992a) and thiophenate (TP) and fenbendazole(FBZ) (Kaur and Sood, 1992b) have been abserved forthese enzymes except NADH-D. Also, themorphological and histochemical effects of ALB,FBZ, TP, TMS, dl-tetramisole and oxyclozanide (TO),and levamisole HCl (LMS) have been studied on theabsorptive surfaces (and neuromuscular system) ofadult H. contortus. ABZ, FBZ, TP, TO, HCl and LMSreduced the quantity of neutral mucopolysaccharidesin the intestine. TO and ABZ caused the loss of acidmucopolysaccharides in the microvilli of intestine.Loss of lipids from the intestine was evident after TO,ABZ, FBZ and TP treatments. LMS causedaccumulation of very large lipid droplets in theintestine (Kaur and Sood, 1996).In general, morphological changes are most evident inthe intestine and muscles. Enzymatic intensities andalterations induced by the in vitro incubations of thedrugs in H. contortus are stronger in the intestine thanin any structure, indicating it to be the main structureinvolved in the absorption and also in the action ofvarious anthelmintics. Different structures of theparasite respond differently to a particular drug,indicating the response and function correlation. Thealterations induced by the drugs in the intestine (aswell as neuromuscular system) and hypodermisemphasize that both absorption and motility of theparasite are effected.Spicules and gubernaculumMorphological and histochemical studies on thespicules and gubernaculum (Sood and Kaur, 1983),have revealed that these are mainly proteinaceous. Thesclerotized part consists mainly of keratin andcollagen. The presence of traces of AcPase in thespicules may represent some metabolic activity. Theprotoplasmic part is composed of proteins,carbohydrates and lipids.BIOCHEMISTRYMuch of the interest in parasite biochemistry comesfrom the ways in which the metabolic pathways havebeen modified to suit the highly specialized parasiticmode of life. In addition to intrinsic interest, parasitebiochemistry has great practical importance throughchemotherapy and vaccine production, and inunderstanding of the complex association involved inthe host-parasite relationship. However, informationin parasite biochemistry is patchy, and mainly thelarge-sized worms have been exploited (Barrett,1981).Of the various biochemical parameters, in H.contortus, carbohydrates and lipids have been studiedmore extensively compared to others. Further, most ofthese studies are restricted to adult form, though in L 3carbohydrate catabolism has been studied in detail.Moreover, variation in metabolic pathways due tostrains poses a great problem. Biochemistry ofHaemonchus has been reviewed (Kapur and Sood,1987a) under the titles: chemical composition(carbohydrates, lipids, proteins, amino acids, nucleicacids, hormones, inorganic elements, pigments),absorption/transport, biosynthesis (carbohydrates,lipids, proteins, amino acids, nucleic acids),catabolism/utilization (carbohydrates-glycolysis,TCA cycle), carbon dioxide fixation, electrontransport system, pentose phosphate pathway,glyoxylate cycle, strain variations in energymetabolism; lipids; proteins; biochemistry intaxonomy and miscellaneous.Inorganic elementsThese play a significant role in the physiology ofparasites. Their impotance is also demonstrated by theinfluence of mineral deficiencies in host's diet.

Haemonchus histochemistry, biochemistry and immunology7Various elements detected in H. contortus (Sood and of glycogen utilization is similar both in male andKapur, 1980) include phosphorous (P), zinc (Zn), female (Premvati and Chopra, 1979). Under in vitrocalcium (Ca), iron (Fe), magnesium (Mg), copper conditions, the worms readily utilize glucose from the(Cu), manganese (Mn), boron (B) and potassium (K), medium (Chopra and Premvati, 1977; Kaur and Sood,in decreasing concentrations in female. In the male 1982a). At the same time, glucose is excreted and thisalso, same elements are present, with the exception of excretion is possibly at the expense of endogenousB. The relative order of occurrence in the male is glycogen reserves, which falls rapidly with time. AnP>Ca>Zn>Fe>Mn>Mg>Cu>K. Presence of more Ca, outstanding feature of carbohydrate catabolism inP and Fe in male has been discussed.nematodes is the production of reduced organic endCarbohydratesproducts, even under aerobic conditions.The in vitro effects of TMS and RFX on theseCarbohydrates form the chief energy source inconstituents have also been studied (Kaur and Sood,parasitic nematodes. In view of the importance of1982a).carbohydrates in helminths, any difference in theircarbohydrate metabolism and that of their hosts might The pathway of carbohydrate breakdown in H.be usefully exploited in helminth control. The contortus has been worked out as follows:outstanding feature of carbohydrate breakdown innematodes is the production of reduced organic end Glycolysis: Adult worm utilizes glucose bothproducts and this occurs even under aerobic aerobically and anaerobically. Under both aerobic andconditions.anaerobic conditions, end products of glucosemetabolism include CO 2, propanol, acetate, and n-Carbohydrate metabolism in H. contortus has beenpropionate as the major and ethonol, lactate andreviewed (Kaur and Sood, 1983b) under the titles :succinate as the minor components. It is unique amongglucose and glycogen utilization, carbohydratenematodes that propanol is a major excretory product.metabolism, including the glycolytic pathway, theThis is possibly produced by reduction of propionatepathway of CO 2 fixation, the TCA cycle and theand hence glucose catabolism in H. contortus does notpentose phosphate pathway.indicate a major departure from the known pathwaysGlucose and glycogen utilization: Glucose in very of anaerobic glucose utilization. Production of lactateimportant energy source for many helminths from glucose in H. contortus has been demonstratedinhabiting the gut of vertebrates. It is generally by Chopra and Premvati (1977). In female 90-100% ofbelieved that helminths absorb glucose against a glucose and in male 80-90% is catabolized to lactate.concentration gradient and use their endogenous Both glucose consumption and lactate productioncarbohydrates as an energy source only when it is decrease with the progression of incubation.unobtainable from outside. Similarly, glycogen inmost of the nematodes provides a significant reserveEnzymes of glycolysis have been detected in adult H.store of energy, particularly in forms which arecontortus (Kaur and Sood, 1982b). These includeparasitic in animals and which exist in environmentshexokinase, phosphoglucomutase, phospho-of low oxygen tension.glucoisomerase, aldolase, glycerladehyde-3-phosphate dehydrogenase, phosphoglycerate kinase,Amount of glucose and glycogen contents in H. phosphoglyceromutase-enolase-pyruvate kinase,contortus has been determined. It contains pyruvate kinase (PK), and lactate dehydrogenaseconsiderable amount(8-12% fresh tissue of glycogen; (LDH). Low PK and LDH activities suggested anChopra and Premvati, 1977; Premvati and Chopra, alternate pathway from phosphoenolpyruvate. LDH1979; Kaur and Sood, 1982a). Famale has more exhibits optimum activity (about 180 nmoles/min/mgglycogen than male (Premvati and Chopra, 1979). protein at a pH of 6.6 and a temperature of 37° CThis is possibly related to the reproductive role of (Kapur et al., 1985). Thermostability of LDH has alsofemale rather than with general metabolism. Adult been studied (Harpreet et al., 1991).worm utilizes glycogen reserves rapidly. In male, itdecreases less rapidly with time, while lin female, itVarying degrees of inhibition of glycolytic enzymesdecreases exponentially with time. Thus, in female, have been observed with 50 µg/ml of TMS and RFXrate of glycogen utilization at any time is directly treatments, former being more effective. These effectsproportional to glycogen content. However, initial rate may block the glycolytic pathway and deprive the

8 Soodparasite of its ATP production (Kaur and Sood, 6-phosphate dehydrogenase (GDPH) and 6-phosphogluconate dehydrogenase, as also the in vitroeffects of TMS and RFX on these enzymes have beenTCA cycle: Studies on aerobic catabolism ofdemonstrated (Kaur and Sood, 1985). GDPH exhibitscarbohydrates in helminthology is an important areaactivity (4 nmoles/min/mg protein) at pH 7.4 and 37º Cof research. In small nematodes, the TCA cycle plays(Kapur et al., 1984). Thermostability of GPDH,an important but not exclusive part in their energyseparately for male and female worms has beeneconomy (Ward, 1982). The occurrence of TCA cyclestudied (Harpreet et al., 1991).in parasites has not been demonstrated with the sameprecision as in vertebrate tissue or bacteria. A Synthesis of nucleic acids in adult H. contortus, is alsofunctional TCA cycle may exist in the developing eggs indicative of the operation of pentose phosphateand larvae of H. contortus. The presence of aerobic pathway (Kapur and Sood, 1986c).enzyme systems in the larvae may be a preparation forthe next stage of development, since development ofAmino acidslarvae to adults requires oxygen. Various enzymes of Amino acids commonly found in proteins, also occurthe TCA cycle viz. aconitase (ACO), isocitrate as free acids. There are number of amino acids whichdehydrogenase (ICDH), succinate dehydrogenase are never found as constituents of proteins, but play(SDH), fumerate reductase (FR), fumarase and malate important metabolic roles. Therefore, it is essential todehydrogenase (MDH) have been detected in adult H. study both free and bound amino acids.contortus (Kaur and Sood, 1983c). Low activities ofACO and ICDH suggest that TCA cycle has a minor Composition: In adult H. contortus, both free andfunction and the pathway of CO fixation is the major bound acids are present (Kapur and Sood, 1984b). The2pathway in the energy metabolism of the parasite. level of free amino acids is 191 mg in male and 523 mgIncorporation of carbon into proteins and in female per 100 g on fresh weight basis. Various14carbohydrates (Kapur and Sood, 1986c). and of C acids in free form include aspartic acid (Asp),into various amino acids (Kapur and Sood, 1984b), glutamic acid (Glu), leucine + isoleucine (Leu+Ile),also give evidence for the TCA cycle operation in H. tryosine (Try), lysine+histidine (Lys+His),contortus.glycine+serine (Gly+Ser), arginine (Arg),valine+methionine (Val+Met), cystine+cysteineIn a study on the effects of pH and temperature, Kapur (Cys-Cys+Cys), β-alanine (Ala) and α−Ala inet al. (1984), found that SDH exhibits maximum decreasing order of concentration in female, and inactivity (about 120 nmoles/ min/mg protein) at anmale, the order of occurrence is: Asp>Glu>Tyr>βoptimumpH of 8.2 and temperature 32° C. pHAla>Lys+His>Gly+Ser>Leu+Ile>Val+Met>Agr.optimum for ICDH (about140nmoles/min/mgThus, in male Cys-Cys+Cys and α−Ala are missing. Inprotein) and MDH (about 30 nmoles/min/mg protein)female, concentration of Leu, Ile and Asp isis 9.0 and 7.8 respectively; optimum temperaturesignificantly higher than in male, and in male it is ofbeing 32º C for both the enzymes (Kapur et al., 1985).Tyr, β−Ala, Lys+His and Glu. However, concentrationThe in vitro effects of TMS and RFX on various of Val, Met, Gly, Ser and Arg is similar in both theenzymes at 50 µg/ml, have shown varying degrees of sexes. Asp is present in the highest concentration ininhibition of SDH and FR. At the same concentration, both the sexes and the acid, in least concentration isthe activities of other enzymes remained unaltered Arg in male and α-Ala in female. Like other(Kaur and Sood, 1983c). Also, the in vitro effects of invertebrates, in H. contortus too, free amino acidNLZ, ABZ, NLV, NLF, TP and FBZ have been studied pools are dominated by one or two amino acids, i.e.,on LDH (Kaur and Sood, 1993).Asp in female and Asp, Glu in male. Proline (Pro) notreported has been earlier demonstrated by NigamPentose phosphate pathway: This is an alternative (1979). Higher amount of Met in female than in malepathway of glucose catabolism. There is, however, no indicates the extent of transaminase reactions. Moredefinite evidence that this pathway is involved in of Lys in male in indicative of its role in maintainingenergy metabolism in parasitic nematodes. Its main sperm viability (having a histone-like function) as infunction may be to provide NADPH and C 5 and C 7 humans. As in birds, more of Glu in male may play asugars for synthetic reactions (Barrett, 1976). In H. role in the maintenance of osmolarity and pH ofcontortus, key enzymes of this pathway, viz. glucose- seminal plasma.

12 Soodconcentration (Kocher et al., 2002a). The female of phosphoenolpyruvate and ATP is generated, whenfractions had a particularly high activity of protease (s) the latter is reduced to pyruvate. Activity of ME hasin comparision with the male fractions, especially of been studied in adult H. contortus (Kaur and Sood,membrane-bound enzymes in the anterior half. 1982b). The in vitro treatments with TMS had noInhibition/activation studies revealed the presence of significant effect. However, after RFX treatment, MEfour kinds of protease (s) in the cystosolic and activity was reduced to 38%.membrane-bound fractions. Protease (s) in differentfractions are purified to a greater extent by higherKaur and Sood (1982a) studied the effect of TMS andconcentrations of saturated ammonium sulphateRFX on total volatile fatty acids under in vitrosolutions, i.e., ranging from 50 to 65%. Theconditions.purification study revealed the presence of multipleforms of protease (s) in cystosolic and membrane-IMMUNOLOGYbound extracts of H. contortus.Chemotherapy is an inadequate means of controllinghaemonchosis because of the development ofStudies have also been made to elucidate the inhibition resistance to anthelmintics. In view of the severeof protease (and lipase activity) from excretory- pathogenicity and economic losses due tosecretory products, cystosolic and membrane bound haemonchosis, various other strategies, includingfractions of male and female H. contortus by breed resistance, biological control andimmunoglobulins raised in rabbits against cytosolic immunological control through vaccine are beingfraction (Kocher et al.,1996).studied. Immunology is an exciting area forBiochemistry in taxonomyformulating new products for therapeutic anddiagnostic purposes. In immunological controlPossibility of the use of electrophoresis in methods, with vaccines as targets, specificpolyacrylamide gel for taxonomy of helminths is well immunodominant antigens play a significant role.established. Electrophoretic analysis of proteins ofImmunoparasitology of Haemonchus infestation hasknobbed and linguiform morphs of female H.been reviewed (Kapur et al., 2001). The topicscontortus revealed marked differences (Sood anddiscussed are: vaccination, genetic control, immuneKapur, 1982a). Proteins of knobbed form have 13responese, transfer of immunity, immunodiagnosticsfractions, and those of linguiform 14. Further, inand host effects.knobbed form, there are 3 bands at cathode and 2 atanode, while in linguiform morph, at cathode, there The various aspects of H. contortus immunology,are 5 bands and at anode only one. This observation reported from India are as follows:(along with other evidences) tends to support the viewof subspeciation in H. contortus.Kaur et al. (2002a) used Dot-ELISA to study kineticsof hyperimmune sera of rabbits immunized with adultMiscellaneousH. contortus. A high titre sera were obtained inimmunized rabbits and observed to be maintained forGlutamate dehydrogenase (GDH) plays a central role180 days, in response to adult H. contortus solublein amino acid deamination and in the formation of α-extract which can be of significant value in the identiaminonitrogen groups form ammonia. Its optimum fication of immunodominant antigens and theiractivity (162 nmoles/min/mg protein) is at pH 7.0 and further characterization using natural host sera.temperature of 27° C (Kapur et al., 1984). Earlier studies of Sood et al. (1996) have shown thatThermostability of GDH has also been studied competitive-inhibition Dot-ELISA using adult H.(Harpreet et al., 1991. The in vitro effects of NLZ, contortus antigen is able to detect as low as 10 pg of theABZ, NLV, NLF, TP and FBZ have been studied in antigen and can be suitably applied at the field leval forAkPase and AcPase. All the six drugs caused decrease mass screening of infected animals. To enhance thisin the enzyme levels of AkPase, the maximum effect sensitivity Kapur-Ghai et al. (2004) have developedbeing exerted by NLZ. In AcPase, marked decrease in avidin-biotin ELISA for detection of H. contortusthe level was observed, maximum effect being caused antigens.by NLF (Kaur and Sood, 1993).Immunodominant antigens of adult H. contortus thatMalic enzyme (ME) is one of the enzymes involved in can evoke a protective immune response in the hostCO 2 fixation and its presence suggests an alternate fate have been identified (Kaur et al., 2002b). Since the

Haemonchus histochemistry, biochemistry and immunology13antigenic component with MW 91.2 KDa was and smooth gave three precipitin lines with rabbitrecognized in the immune sera of all the rabbits and antisera. Also, lines unite in a manner indicatingalso in sera collected at intervals, it is suggested as the marked antigenic differences between the three types.immunodominant component of adult H. contortus. Since serological techniques are considered to besensitive tools in taxonomy, the present work hasTwo low molecular weight proteins of 15 and 22 KDa indicated that taxonomic importance should bewere isolated from the extract of adult H. contortus by attached to the vulvar configurations in female H.gel filtration conA-sepharose and affinity contortus.chromatography on antibody-sepharose. Theseproteins were also indentified in the excretorysecretoryREFERENCESproducts of adult parasites. Antibodies Anonymous. 1992. Distribution and impact of helminthagainst these proteins were identified in the sera of diseases of livestock in developing countries. FAO animalanimals infected with H. contortus. Upon production and health paper 96. Food and Agricultureimmunization, these proteins protected animals Organization of the United Nations, Rome.against challenge H. contortus infection, as eggBarrett B. 1976. Bioenergetics in helminths. In: Biochemistryshedding and worm burden significantly reduced. The of Parasites and Host-Parasite Relationship. H van Denimportance of these antigens in the host-parasite Bossche (Edt.). North Holland Biomedical Press,relationship has been discussed (Joshi and Singh, Amsterdam.1999).Barrett J. 1981. Biochemistry of Parasitic Helminths.Preliminary studies carried out by Kocher et al. MacMillan Publisher, Ltd., London and Basingstoke.(2002b) to determine the relationship between level of Bird AF. 1971. The Structure of Nematodes. Academic Press,circulating immune complex (CIC) and the possibility London.of ascerting mortality caused by H. contortusChitwood BG and Chitwood MB. 1974. Introduction toinfestation in sheep and goat, have revealed thatNematology, University Press, Baltimore, Maryland.determination of CIC level may prove to be importantparameter for early diagnosis of the state of the host Chopra AK and Premvati G. 1977. Glucose metabolism andimmunized with H. contortus.lactic acid production in sheep nematodes. Indian J Parasita1:93-96.To detect anthelmintic resistance using thiabendazole,Fraser CM. 1991. The Merck Veterinary Manual. A handbooka larval development assay (LDA) has been of diagnosis, therapy and disease prevention and control forstandardized. A linear dose-response relationship was the veterinarians. Merck and Co. Inc. Rahway, USA,observed between the probit of larval development of pp.205-215.the logarithms of anthelmintic concentration of bothHarpreet, Sood ML and Soni GL. 1991. Thermostability ofegg hatch assay and LDA (Singh et al., 2003).some enzymes of Haemonchus contortus and TrichurisSood and Kapur (1981) observed immunologicaltrichura. Indian J Parasit 15: 171-173.changes in the spleen of rabbits in response to antigens Johl M. 2003. Histomorphological variations in someof H. contortus female. The study of sections showed gastrointestinal nematodes of domestic ruminants. In:hyperplasia of reticuloendothelial cells and Helminthology in India. ML Sood (Edt.). Internationalduplication of red pulp. Haemosidrin pigment was Book Distributors, Dehra Dun , India, pp.453-466.also observed. A few eosinophils and neutrophils were Joshi P and Singh BP. 1999. Isolation and characterization ofalso seen at the cortical region. Antibodies being two low molecular weight protective antigens ofproteins, their synthesis is similar to that of other Haemonchus contortus. Indian J Anim Sci 69: 284-288.proteins and hyperplasia of reticuloendothelial cellsJoshi P and Singh BP. 2000. Purification and characterization ofindicates increased protein synthetic activity.cholinesterase from Haemonchus contortus. Indian JIn support of the biochemical techniques in taxonomyBiochem Biophys 37: 192-197.in H. contortus (Sood and Kapur, 1982b), Kapur J, Parmar A and Sood ML. 2001. Immunoparasitology ofimmunodiffusion patterns of antigens from Haemonchus infestation. Prof. VN Capoor Comm.Vol.phenotypically different females have been studied Sandeep Kumar Malhotra (Edt.). Ankit Publication,Allahabad, pp. 26-34.(Sood and Kapur, 1982c). The studies indicate that thethree phenotypes---smooth, linguiform, and knobbed Kapur J and Sood ML. 1984a. Haemonchus contortus: Lipiddiffer from each other serologically. Linguiform14biosynthesis from C -labelled acetate and glucose. Zbl Vetantigens gave five precipitin lines, knobbed four lines Med B 31:225-230.

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J Kapur and ML Sood (Edts.).Narendra Publishing House, Delhi, India, pp. 97-154.Kapur J and Sood ML and Soni Gl. 1984. Effect of pH andtemperature on four dehydrogenase enzymes ofHaemonchus contortus. Acta Vet Hung 32:165-170.Kapur J, Sood ML and Soni Gl. 1985. Haemonchus contortus:Effect of pH and temperature on some dehydrogenases. ActaVet Hung 33:19-23.Kapur-Ghai J, Thapar V, Kaur K and Sood ML. 2004.Kaur R and Sood ML. 1982c. In vitro effect of anthelmintics onmalic enzyme and cholinesterase of Haemonchus contortus(Nematoda : Trichostrongylidae). Indian J Parasit 6:267-268.Kaur R and Sood ML. 1982d. In vitro effect of anthelmintics onthe phosphatases of Haemonchus contortus (Rud., 1803).Indian J Parasit 6: 279-274.Kaur R and Sood ML. 1983a. Effects of anthelmintics on theabsorptive surf aces of adult Haemonchus contortus in vitro:a histoligical study. Folia Parasit 30:146.Kaur R and Sood ML. 1983b. Review article. 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Haemonchus histochemistry, biochemistry and immunology15pentose phosphate pathway enzymes of Haemonchus contortus Sood ML and Kapur J. 1980. Research note. Inorganic elements(Nematoda :Trichostronogylidae). Indian J Parasit 9:159- in the adults of Haemonchus contortus (Nematoda :160. Trichostrongylidae).J Helminth 54:253-254.Kaur R and Sood ML. 1986. Effects of anthelmintics onHaemonchus contortus (Nematoda : Trichostrongylidae).Vet Res Comm 10:21-36.Kocher DK, Ahuja SP and Sood ML. 1996. Proteases andlipases as host protective antigens in Haemonchuscontortus. In:Parasitic Diseases-new horizons. Proc EighthNat Cong Vet Parasit Nat Symp Mol Parasit. DP Banerjee,JD Ghosh and SK Gupta (Edts.). Department of VeterinaryParasitology, CCS Haryana Agricultural University, Hisarpp. 82-86.Kocher DK, Ahuja SP and Sood ML. 2000. Absorption and14incorporation of C-labelled amino acids in Haemonchuscontortus. J Vet. Parasit 14 : 129-131.Kocher DK, Ahuja SP and Sood ML. 2002a. Charaterizationand purification of cystosolic and membrane-boundprotease (s) in adult s of Haemonchus contortus. J Vet Med B49:71-75.Kocher DK, Ahuja SP and Sood ML. 2002b. Relationshipbetween circulating immune complex (CIC) level andmortality of host immunized with Haemonchus contortus. JParasit Dis 26:46-47.Lee DL. 1965. The Physiology of Nematodes. Oliver and Boyd,Edinburgh and London.Nigam SC. 1979. Amino acid composition of nematodeparasites. Indian J Helminth 31:69-71.Premvati G and Chopra AK. 1979. In vitro variation ofglycogen content in three sheep nematodes. Parasitology78:355-359.Rodwell VW. 1990. Harper's Biochemistry. RK Mdrray, DKRanner, PA Mayes and YW Rodwell (Edts.). 22nd ed.,Prentice Hall International Inc. Connecticut.Singh D, Swarnkar CP, Khan FA and Bhagwan PSK. 2003.Standardization of larval development assay for detection ofanthelmintic resistance in Haemonchus contortus. J VetParasit 17:21-25.Singh J and Johl M. 2001. Observations on the foregut(stomodaeum) of Haemonchus contortus Rud., 1803. UttarPradesh J Zool 21:139-145.Sood ML and Kapur J. 1981. Immunologic changes in thespleen of rabbits in response to antigens of Haemonchuscontortus female (Nematoda : Trichostrongylidae). FoliaParasit 28:272.Sood ML and Kapur J. 1982a. Haemonchus of ruminants- abibliography of titles prepared from HelminthologicalAbstracts covering the period 1932-1980. ShikhaPublications, Ludhiana, India.Sood ML and Kapur J. 1982b. Electrophoretic analysis ofproteins of knobbed and linguiform morphs of femaleHaemonchus contortus (Nematoda : Trichostrongylidae).Helminthologia 19:273-278.Sood ML and Kapur J. 1982c. Haemonchus contortus:Immunodiffusion patterns of antigens from phenotypicallydifferent females. Expl Parasit 53:164-169.Sood ML and Kapur J. 1989. Review article. Lipidcomposition and metabolism of Haemonchus contortus(Nematoda :Trichostrongylidae). Indian J Parasit 13:367-371.Sood ML and Kaur C. 1976. Studies on vulvar configurations inHaemonchus contortus (Rud., 1803) from goats atLudhiana, India. Riv di Parassit 37:13-33.Sood ML and Kaur C. 1977. Morphological and histologicalstudies on the vulvar configurations in Haemonchuscontortus (Rud., 1803). Folia Parasit 24:111-115.Sood ML, Kaur G, Parmar A and Kapur J. 1996. Developmentof Dot-ELISA for detection of Haemonchus contortusantigen. Helminthologia 33:73-75.Sood ML and Kaur M. 1983. Morphology and histochemistryof the spicules and gubernaculum of Haemonchus contortus(Nematoda : Trichostrongylidae). Folia Parasit 30:249-255.Sood ML and Kaur R. 1982. The in vitro effects of some drugson the morphology and histochemistry of adultHaemonchus contortus (Nematoda : Trichostrongylidae).Helmin-thologia 19:61-70.Sood ML and Sehajpal K. 1978. Morphological, histochemicaland biochemical studies on the gut of Haemonclus contortus(Rud., 1803). Z Parasitenkd 56 : 267-273.Sood ML. 1981. Haemonchus in India. Parasitology 83: 639- Sood ML, Sood M and Sood ML. 1999. Experimental650.determination of diffusion coefficients of NaCl and KCl inHaemonchus contortus at 298.16K. J Indian Chem SocSood ML. 1999. Absorptive surfaces of Haemonchus contortus 76:410-411.(Nematoda : Trichostrongyloidea). JPAS 1 : 225-227.Ward PFV. 1982. Aspects of helminth carbohydrateSood ML. 2003. Haemonchosis in India. In: Helminthology in metabolism. Parasitology 84:177-194.India. ML Sood (Edt.). International Book Distributors,Dehra Dun, India, pp. 575-617.Wright KA. 1987. The nematodes' cuticle---its function,homology, analogy-a current consensus. J Parasit 73:1077-Sood ML and Kalra S. 1977. Histochemical studies on the body 1083.wall of nematodes : Haemonchus contortus (Rud., 1803)and Xiphinema insigne Loos, 1949. Z Parasitenk51 : 265-273.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 16–29ReviewImmunological perspectives and malaria vaccineJ P DH. S. Banyal and N. ElangbamDepartment of Biosciences, Himachal Pradesh University, Shimla.ABSTRACT. Even after more than a century of efforts to either eradicate or control malaria, it stillremains a major vector borne parasitic disease that affects nearly a third of world's population,mainly in poor developing countries. Increasing incidences of parasite resistance to available antimalarialdrugs and of mosquitoes to commonly used insecticides necessitates an alternative combatstrategy for the control of malaria. The development of an effective malaria vaccine is one suchstrategy. Herein, we focus on various immunological aspects of malaria including target antigensthat can serve as potential candidate vaccines, and on the status and prospects of the development ofa malaria vaccine.Keywords: immune response, malaria, Plasmodium, target antigens, vaccineINTRODUCTIONMalaria, derived from the Italian word for bad air wasattributed to fevers in populations living in the vicinityof marshes. Edwin Smith Surgical Papyrus 1600 B. C.,indicated about the disease while the Greek physician,Hippocrates gave the first accurate clinical descriptionof malarial fever in 400 B. C. (Boyd, 1949). Malarialfevers were known in ancient China and Arabiancountries, and have also been mentioned in ancientIndian literature like 'Charaka Samhita'.Meckel in 1847 was the first person to observe blackgranules embedded in protoplasmic masses in theblood of a severely ill malarious patient. In 1880, aFrench army physician, Charles-Louis-AlphonseLaveran observed exflagellation of a parasite anddescribed it as Laverania falcipara in 1884. Mansonsuggested mosquitoes to be the host for malariaparasite's extrinsic development which encouragedSir Ronald Ross to investigate the fate of malariaparasite in various mosquito species in India. In 1898,Corresponding author: Prof. H. S. Banyal, Laboratory ofParasitology and Immunology, Department of Biosciences,Himachal Pradesh University, Shimla-171 005, H. P., India.E-mail: hsbanyal@yahoo.co.inRoss succeeded in completely elucidating thesporogony of Plasmodium relictum in Culex pipensfatigans. Meanwhile, Italian worker Bignami in 1899succeeded in infecting a healthy volunteer withPlasmodium falciparum through the bites ofmosquito. At the same time, sporogony in P.falciparum and P. vivax was elucidated in anophelinemosquitoes (Bastianelli and Bignami, 1899; Grassi etal., 1899a), and also the development of sporogonicstages of P. malariae in Anopheles claviger (Grassi etal., 1899b). Differential descriptions of P. vivax and P.malariae were given by Grassi and Feletti (1892) andfor P. falciparum by Welch (1897). The fourth humanmalaria parasite species, P. ovale was described in1922 (Stephens, 1922).Even after more than a century of efforts to eithereradicate or control malaria, it still remains a majorglobal health hazard and one of the most importantvector borne human diseases prevalent in morethan107 countries that affects nearly a third of theworld's population. Malaria is prevalent mainly inpoor developing countries and causes more deathsthan any other parasitic disease with sub-SaharanAfrica accounting for nearly 90% of the cases. Otherplaces like South-East Asia, Oceania, Middle East and

Malaria vaccine development17cellular and humoral arms of immunity are tightlybound through cytokines which control the immuneresponse with both antibody and cellular immunityplaying critical roles in protective immunity. Themechanisms of non-specific innate defense are poorlydefined. Neutrophils, mononuclear phagocytes andnatural killer (NK) cells appear to play an importantrole(s) in innate immunity to malaria infections.Humoral immunity: B-cells are primarily concernedwith production of antibodies, which form humoralimmune response. In malaria endemic areas, inductionof strong humoral immune responses involvingpredominantly IgM and IgG have been reported(Chelimo et al., 2005; Couper et al., 2005). Antibodiesact against different stages of parasites, and arepredominantly effective against erythrocytic stages.IgG 1 and IgG 3, the two cytophilic isotypes inhumans, predominate in malaria protected individualsand IgG2a in P. yoelii has been reported to protectrodents (Druilhe et al., 2005).The possible mechanisms of action of antibodiesinclude inhibition of Mz invasion of erythrocytes andinhibition of intraerythrocytic development ofparasites or both. Antibodies can also causeneutralization and agglutination of Mzs. Protectiveantibodies have very limited direct effect on parasitegrowth and invasion but act in co-operation with bloodmonocytes, known as antibody-dependent cellularinhibition (ADCI). In ADCI, soluble mediators arereleased at the time of schizont rupture triggered by thecontact between Mz surface components andcytophilic antibodies bound to monocytes whichdiffuse in the serum and block multiplication ofsurrounding parasites at the uninucleate stage(Bouharoun-Tayoun et al., 1995). Tumor necrosisfactor-α (TNF-α) has been implicated in ADCI and isthought to inhibit the ring stages of parasitedevelopment.Cell-mediated immunity: The importance of cell-mediated response in parasitaemia suppression inanimal models was suggested by the resolution ofacute malaria infection by several Plasmodium sp. inB-cell deficient but not athymic mice (Grun andWeidanz, 1981; Cavacini et al., 1990). Acquisitionand maintenance of protective immunity to malaria isT-cell dependent as T-cells are essential both inregulating antibody formation and in inducingantibody independent immunity (Webster et al.,2005). T-cells act as helpers for antibody response butalso as effector cells as they can inhibit parasite growthLatin America also face serious malaria problems. InAfrica, every 30 seconds, a child dies of malaria.Various strategies have been adopted to control thespread of malaria through vector elimination andchemotherapy. Taking into account the increasingresistance of Plasmodium to chemotherapeuticagents, and of Anopheles to conventional insecticides,there is a critical need for an effective malaria vaccineto combat malaria. Great advances have been made inunderstanding the immunological perspectives ofmalaria which should help in the fight against thedisease.LIFE CYCLE OF MALARIA PARASITEMalaria is caused by a protozoan parasite,Plasmodium, four species of which infect humans:Plasmodium falciparum, P. vivax, P. ovale and P.malariae. Mortality and morbidity are mainly due to P.falciparum, although P. vivax is more widespreadgeographically. Malaria parasite has a complex lifecycle alternating between a vertebrate, ranging from areptiles to mammals, and an arthropod host femaleanopheline mosquitoe. Each bite of an infected femaleAnopheles inoculates 5-20 sporozoites which, within30 min, find their way into hepatocytes. Eachsporozoite multiplies and differentiates intracellularlyinto a liver stage trophozoite and ultimately a schizont.Rupture of hepatocytes releases merozoites (Mzs) intocirculation, which continue a cycle of red cell invasionand multiplication causing clinical manifestations ofthe disease. Within erythrocyte, each Mz develops intoa trophozoite that matures and divides, generating aschizont that gives rise to up to 32 Mzs within 48 or 72h depending upon the species. The Mzs upon invadingnew erythrocytes either maintain the bloodschizogony or some of them differentiate into male orfemale gametocytes which are ingested by the bloodfeeding mosquito. In the mosquito gut, thegametocytes emerge as gametes and fertilize toproduce motile ookinetes which burrow into the gutwall of mosquito to form oocysts. Finally, sporozoitesreleased into the body cavity ultimately find their wayto the salivary glands, and are injected to a new hostduring the next blood-meal.IMMUNOLOGY OF MALARIAMalaria infection gives rise to immune responses bythe host which are regulated both by the innate andadaptive immune systems. Immunity to malariainvolves both cell-mediated and humoral immunemechanisms through both T-cells and B-cells. The

18 Banyal and Elangbamin vitro. The cell-mediated immune effector immunity against blood-stage malaria parasites.mechanisms include macrophage activation by CD4+ T-cells are essential for immune protectioninterferon-γ (IFN-γ ) derived from γ T cells, NK cells against asexual blood stages in both murine andor T helper1 (Th 1) and inhibition of parasite growth human malaria infections. Adoptive transfer of CD4+and development inside hepatocytes mediated by T-cells from immune donor mice to nude miceCD8+ cytotoxic and IFN-γ producing T cells (Tsuji suppresses acute infection. The antibody-independentand Zavala, 2003). Cell-mediated immune responses mechanisms of action of CD4+ T-cells have beenmay protect against both pre-erythrocytic and suggested to involve cytokines and NO and T-cellserythrocytic stage parasites.(Seixas et al., 2002). In P. chabaudi infection T-cellsare stimulated during primary infection and theirImmunity against pre-erythrocytic stages: In pre- expansion is observed during recovery from acuteerythrocytic stage immunity, infected hepatocytes infection. CD4+ T-cells act through the release ofexpressing major histocompatibility complex (MHC) cytokines that may exert parasiticidal or parasitostaticmolecules are the primary target of cell-mediated effect, activate macrophages and provide help forimmune responses. Both CD 4+ T-cells and CD8+ T- antibody production by B-cells. Activated T-cells butcells recognize parasite derived peptides presented by not T-cells from malaria naïve donors inhibit parasiteclass I and II MHC molecules, respectively, present on replication in erythrocytes in vitro, suggesting theirthe surface of infected hepatocytes. CD8+ T-cells are protective function (Perlmann and Troye-Blomberg,thought to be the primary mediators against pre- 2002).erythrocytic stages (Meraldi et al., 2005). It has beensuggested that CD 8+ T-cells interact with MHCantibodyIn humans, T-cells act not only as helpers for anpeptide complex on the surface of infectedresponse but also act as effector cells as theyhepatocytes and secrete IFN-γ, which in turn induces can inhibit parasite growth in vitro. As humanthe infected hepatocytes to produce nitric oxide (NO) erythrocytes do not express MHC antigens, lysis ofthat renders the parasite non-infectious. CD8+ T-cells infected-erythrocytes by CD8+ cytotoxic Trather than CD8+ cytotoxic T lymphocytes (CTLs) per lymphocytes (CTLs) plays no role in protectionse, are the critical effector cells of pre-erythrocytic against blood-stage parasites. Sterile immunitystage immunity.induced by repeated low grade infection of red cellswithout any detectable antibodies suggests that T-cellImmunity against erythrocytic stages: Erythrocytic mediated protection operates in humans (Pombo et al.,stage immunity is thought to involve both antibodies 2002).and T-cell. Antibodies are presumed to work againstparasite proteins on the surface of erythrocytes and Role(s) of cytokines: In malaria, parasite killingprevent the sequestration of parasites in requires the production of inflammatory cytokinesmicrocirculation resulting in their destruction by like IFN-γ, interleukin (IL)-1 and IL-6 and others thatspleen. Anti-Mz antibodies may also act in other can have deleterious systemic effects and have beenprotective mechanisms like complement-mediated correlated with malaria pathology. The cytokineslysis and act through co-operation with Fc bearing TNF-α, IL-1 and IL-6 have been connected withcells. Parasite proteins present intracellularly or severe malaria (Artavanis-Tsakonas et al., 2003). Inexpressed on the surface of Mzs are also target of murine malaria infection, TNF-α and IFN-γ activateantibodies that prevent invasion of erythrocytes by macrophages to phagocytose parasitized-erythrocytesparasites (Banyal and Inselburg, 1985). In humans, and release NO that causes destruction of parasitecytophilic antibodies IgG1 and IgG3 may bind to Mzs which results in protection from severe disease (Goodand thus facilitate their uptake by phagocytes or and Doolan, 1999). In humans, TNF-α, IFN-γ and NOmediate antibody-dependent cellular cytotoxicity are associated with the resolution of fever and parasite(ADCC) or ADCI (Tebo et al., 2001). Pouvelle et al. clearance (Kremsner et al., 1996). However, high(1991) reported that antibodies can penetrate the levels of circulating TNF-α are associated with severeinfected RBC through the parasitophorous ducts and P. falciparum malaria and high levels of IFN-γ withbind to the intracellular parasite.fever. IL-12, produced by mononuclear phagocytes isStudies in animal models have implicated T-cell involved in protection against pre-erythrocytic andmediated, antibody-independent mechanism(s) in blood-stage infection by a Th1 anti-malaria response(Doolan and Hoffman, 1999). In contrast, anti-

Malaria vaccine development19inflammatory cytokines like IL-10 and transforming expressed in the hepatocytes are considered primarygrowth factor-β (TGF-β) counteract the production effectors with IFN-γ playing a major role (Plebanskiand cytopathic effects of the pro-inflammatory and Hill, 2000).cytokines (Omer et al., 2000). Lower than normalCircumsporozoite proteins: Protective antibodieslevels of circulating TGF-β are seen in patients withagainst sporozoites are mainly directed againstsymptomatic P. falciparum infection. Also risk ofcircumsporozoite proteins (CSP). A 44K CSPfebrile illness is associated with high ratios of IFN-γ,exhibited protection against P. berghei (Potocnjack etTNF-α or IL-12 to TGF-β (Dodoo et al., 2002) which al., 1980). Passive protection by polyclonal antibodiesemphasize the importance of the balance between pro- against central repeat has also been reported for P.and anti-inflammatory cytokines. TGF-β plays an berghei and P. yoelii (Egan et al., 1987; Wang et al.,essential role in down regulating the production of 1995). P. vivax CS-derived synthetic peptides havepotentially pathogenic pro-inflammatory cytokines shown good antigenicity and immunogenicityand may be a novel mechanism of pathogen- mediated eliciting both humoral and cellular responses (HerreraTGF-β activation. TNF plays a central role both in et al., 2004; 2005).protection and pathogenesis of malaria, whereaselevated levels of serum IFN-γ enhance production ofLiver stage antigens (LSA): Immunity during thisTNF, NK and T-cells and have been identified asstage is mostly mediated by cellular-dependentmechanisms involving CD8+ T-cells, CD4+ T-cells,potential sources of IFN-γ in malaria infection.NK cells and T-cells. Studies in mice indicate thatNO can modulate anti-microbial activity, smooth IFN-γ produced by activated CD8+ T-cells inducemuscle contraction, cytokine production and has been infected- hepatocytes to synthesize NO which hascorrelated with malaria immunity as well as potent anti-parasitic activity (Doolan and Hoffman,pathogenesis (MacMicking et al., 1997). NO has also 2000). Some of the identified liver stage antigensbeen implied in protection from blood-stage malarial include LSA-1, LSA-2 and LSA-3. LSA-1 isparasites in humans as plasma NO levels increased in expressed specifically in liver stage parasites and nopatients with P. falciparum and P. vivax.homologue has been identified in mouse or nonhumanprimate malarias. It is a 200 kDa protein withTarget antigens: Problems of the resistance toconserved sequence across strains and involved inchemotherapeutic agents and insectides emphasizenaturally acquired responses in human protection.the urgency of a suitable malaria vaccine. TheLSA-1 specific protective immune responses includescomplex life cycle of malaria parasite with distinctCD8+ T-cells, IFN-γ, IL-10 and antibodies in naturaldevelopmental stages expressing multiple antigenstransmission. Daubersies et al. (2000) reportedcould provide targets of immune responses. There areprotection of chimpanzees against P. falciparum byessentially six targets for a malaria vaccine in theimmunization with LSA-3, 200 kDa protein expressedwhole life cycle of the parasite namely, sporozoites,both in sporozoites and liver stages and is highlyliver stages, Mzs, infected-erythrocytes, parasiteconserved. Pf LSA-3 DNA immunization inducedtoxins and sexual stages.potent Th 1 response with protective properties andSporozoite: In 1967, Nussenzweig and co-workers conferred protection against P. yoelii challenge inshowed that mice immunized with radiation mice.attenuated sporozoites of P. berghei were protectedSporozoite surface protein 2 (SSP-2): SSP-2 is alsoagainst challenge with infectious sporozoites. Thisknown as thrombospondin-related anonymous proteinimmunization confers sterile protective immunity(TRAP) involved in sporozoite invasion and is carriedwhich is species-specific but not strain specificinto hepatocytes along with CSP ( Muller et al., 1993).(Nussenzweig et al., 1967). Immunization with heatSSP-2 contains a sulphated glycoconjugated bindingkilled, formalin treated or lysed sporozoites could notpeptide sequence needed for parasite invasion.induce protection emphasizing the requirement forAntibodies to SSP-2 prevent sporozoites fromlive sporozoites in the hepatocytes for protectiveinvading hepatocytes in vitro.immunity. Intrahepatocytic parasites are a majortarget of protective immunity induced by Merozoiteimmunization with irradiated sporozoites and CD8+T-cells specific for epitopes of parasite proteins The Mz is the only extracellular stage of the malaria

20 Banyal and Elangbamparasite in the human host other than the sporozoitemaking it a visible target for antibodies. Antibodiestargeting Mz proteins, mainly its surface proteins,interfere in the invasion process through agglutinationfollowed by phagocytosis or blocking of Mzerythrocyteinteraction. Several Mz surface proteinshave been identified and some are targets for leadingmalaria candidate vaccines.Mz surface proteins (MSP): MSP-1 is the mostabundant protein on the surface of Mzs (Polley et al.,2003). It is a 190230 kDa protein on the surface of Mzthat is processed into smaller fragments at the time ofinvasion of erythrocytes and exists as a non-covalentlylinked complex of four fragments (83, 28, 34 and 42kDa). The C-terminal 42 kDa fragment (MSP-142)undergoes further processing to form MSP-1 33, whichis shed, and MSP-1 19, which remains on the Mz surfaceand is taken into the newly invaded erythrocytes.Monoclonal antibodies to MSP-1 19 inhibit Mzinvasion in vitro, and sera from P. falciparum immuneMSP-2 is encoded by a single gene and is a 4552 kDaintegral membrane glycoprotein anchored on thesurface of Mz by a glycosylphosphatidyl inositol(GPI) moeity (Weisman et al., 2001) and a target ofhost protective immune responses as monoclonalantibodies specific to MSP-2 have inhibited parasitesgrowth in vitro. Mice immunized with conservedregions of P. falciparum MSP-2 have been protectedagainst challenge with P. chabaudi. Antibodies toMSP-2, mainly IgG 3, have been detected in sera ofpeople living in endemic areas. Human trials of multisubunit vaccines containing MSP-2 have beenundertaken both in non-exposed and malaria-exposedindividuals (Genton et al., 2000).MSP-3 is a secreted polymorphic antigen associatedwith erythrocytic schizonts and Mzs. P. falciparumMSP-3 has been shown to range from 40-76 kDadepending on the isolate and has been implicated ininduction of ADCI (Audran et al., 2005; Druilhe et al.,2005). P. vivax MSP-3 is associated with but notadult humans and P. chabaudi immune mice revealed aanchored in the Mz membrane and is structurallymajor role for MSP-1 specific antibodies inrelated to P. falciparum MSP-3 and 140 kDa MSP of P.19knowlesi (Galinski et al., 1999).mediating the invasion-inhibition (O'Donnell et al.,2001). The 19 kDa fragment is reported to be highly P. falciparum MSP-4 is a 40 kDa protein containing aconserved in P. falciparum and contains a series of single epidermal growth factor (EGF)-like domain atcysteine residues that are conserved among species ofplasmodia infecting humans, primates and rodents(Daly et al., 1992).Different effector mechanisms of antibodies againstMSP-1 are being suggested which primarily involveinhibition of erythrocyte invasion by Mzs (Tolle et al.,1993; Locher et al., 1996; O'Donnell et al., 2001;Vukovic et al., 2003), blocking the processing oflarger mature MSP-1 protein on the Mz surface(Blackman et al., 1994) and through macrophage Fcreceptors to induce ADCC (Bouharoun-Tayoun et al.,1995; Ravetch and Clynes, 1998). MSP-119-specificimmunoglobulin IgG 3 monoclonal antibody canpassively transfer protection to mice deficient in Fc-ãRI receptors whose macrophages cannot bind IgG 3(Vukovic et al., 2000). Studies in P. yoelii model byWipasa et al. (2002) show that antibodies specific toMSP-1 19 alone can induce protective immunity and Proteolytic processing of MSP-1 precursor producesthat effector T-cells specific to MSP-1 19 play no role in two components p 36 (MSP-636) and p 22 (MSP-722)immunity. However, such antibodies must be which are associated with the shed MSP-1 complex.the C-terminus that is synthesized at the late ring stageand transported to the parasite surface, anchored to theMz membrane by a GPI moiety. Studies analyzed aregion of chromosome 2 in P. falciparum andidentified 3 clustered genes that encode GPI-anchoredMz surface proteins in tandem MSP-2, MSP-5 andMSP-4 and MSP-5 encoding a 40 kDa protein locatedon the Mz surface (Marshall et al., 1998). Ahomologue of P. falciparum MSP-4 and MSP-5 in P.chabaudi designated Pc MSP 4/5 encoding a protein ofapparently 36 kDa contains a single EGF-like domainnear the C-terminus. Murine homologue of MSP-4induces protective immunity in mice against lethalchallenge with P. yoelii. Anti-MSP-4 antibodies arehighly prevalent and present at high level inindividuals in malaria endemic area and are mainlyIgG 1 and IgG 3.produced during challenge. Specific cellular immune The 36 kDa protein is derived from a larger precursorresponses induced by MSP-1 can be protective against MSP-6 and so designated as MPS-6 36. Antibodiesexoerythrocytic forms of P. yoelii (Kawabata et al., against recombinant protein containing the C-terminal2002). of MSP-6 36 bound to parasite surface or theparasitophorous vacuole within schizonts (Trucco et

Malaria vaccine development21al., 2001). MSP-6 reactive antibodies are generated in erythrocyte subpopulation for Mz invasion have beena natural human infection and antibodies on an MSP-3 intensely studied as a possible means of blocking Mzpeptide cross reacted with MSP-6 which suggest it to attachment to erythrocytes. In P. falciparum,be a target of ADCI. The 22 kDa protein designated glycophorin A is the erythrocyte receptor for MzsMSP-7 22 is the result of protease cleavage of precursor whose major ligand is the erythrocyte-bindingMSP-7 expressed in mature schizonts (Pachebat et al., antigen-175 (EBA-175) located in the microneme.2001). Another asexual stage parasite protein of P. Blocking an EBA-175 binding site inhibits parasitefalciparum containing two EGF-like domains near the multiplication in vitro and immunization of AotusC-termini is designated as MSP-8 (Black et al. 2001). monkeys with recombinant EBA-175 region IIinduced anti-parasite effect (Jones et al., 2001). In P.Apical membrane antigen (AMA)-1: Apical vivax, Duffy antigen receptor for chemokines (DARC)membrane antigen is a trans-membrane protein is the receptor in erythrocyte for Mz which expressespresent on the surface of Mzs and involved in the Duffy-binding protein (DBP; Yazdani et al., 2005). P.parasite invasion of erythrocytes. AMA-1 has been vivax and P. knowlesi DBPs and their orthologue EBAidentifiedin all Plasmodia as a relatively conserved 175 bind well-defined glycoprotein motifs onsequence and synthesized de novo as a 66 kDa protein erythrocyte membrane. P. vivax reticulocyte bindingexcept in P. falciparum and P. reichenowi. In P. proteins (PvRBP-1 and PvRBP-2) attach tofalciparum and P. reichenowi, AMA-1 is a 83 kDa reticulocyte enriched erythrocytes. PvRBP-1 andprotein processed by proteolytic cleavage between PvRBP-2 have molecular masses of 325 kDa and 330different domains into a 66 kDa form (Pf AMA-1 66)kDa, respectively, and share similar structureswhich is further proteolytically cleaved and shed as (Galinski et al., 2000). PvRBP-2 appears to besoluble ectodomain (Howell et al., 2001). P. distantly related to a 235 kDa rhoptry protein of P.falciparum Pf 83/AMA-1 is the analogue of 66 kDa P. yoelii. P. falciparum genes homologous to P. vivaxknowlesi protective Mz protein, Pk 66/AMA-1 that is RBP-1 and -2 encoding high molecular mass proteinsexpressed in late schizonts and localized in the Mz of > 300 kDa are expressed in late schizonts. Twoapex. The full length 83-kDa remains apically PvRBP-2 orthologues of P. falciparum termed P.restricted while the processed 66-kDa becomes falciparum RBP-2 homologues a and b (Pf RBP 2-Hacircumferentially associated with the Mz surface. An and Pf RBP 2-Hb) along with RBP-2 of P. vivax and P.Escherichia coli expressed recombinant P. falciparum yoelii 235 kDa protein, constitute an importantAMA-1 induced in vitro growth inhibitory anti-AMA- Plasmodium family important for Mz invasion.1 antibodies which recognize both strain specific andconserved epitopes and show AMA-1 to be a natural Parasitized erythrocytestarget of protective antibody responses (Hodder et al.,Parasite antigens expressed on the infected-2001). Recombinant AMA-1 proteins either alone orerythrocytes are targets for antibodies to act on, andin combination with MSP-1 have also been evaluatedseveral such molecules have been identified. Ringforits efficacy against blood-stage malaria in animalinfected erythrocyte surface antigen (RESA) is one ofmodels (Burns et al., 2003).the most studied with molecular weights 155 and 130Several of the potential malaria blood-stage vaccine kDa, and anti-RESA antibodies raised in mice inhibitcandidate antigens are expressed on the rhoptries, parasite growth in vitro (Chopra et al., 2000). RESAapical organelles involved in erythrocyte invasion. has also been used as a component in multiantigenRhopty Asociated Protein (RAP-1) is synthesized as a blood-stage vaccine together with MSP-1 and-2 in86 kDa precursor N-terminally cleaved to generate 82 Phase I clinical trials. The serine-rich proteinkDa molecule, P 82, that is further processed to give a (SERP/SERA) is a 120 kDa soluble protein expressed68 kDa molecule, P 68 (Howard et al., 1998). Purified in the schizont stage, whereas glutamate rich proteincomplexes of RAP-1 and RAP-2 used in experimental (GLURP) is expressed during all stages of the parasiteimmunization of Saimiri monkeys have shown partial development in human host as a 220 kDa protein.protection against P. falciparum infection, whereas Several immuno-epidemiological studies haveanti-RAP monoclonal antibodies have shown identified high anti-Ro-GLURP IgG levels asinhibition of parasite replication in vitro (Ridley et al., predictors of protection against high parasitaemia and1990).febrile malaria episodes (Soe et al., 2004). There is anassociation between protection against febrile malariaProteins involved in the targeting of particular

22 Banyal and Elangbamand presence of anti Ro-GLURP antibodies in intense characteristic arrangement of cysteine containingmalaria transmission areas, and further that increasing domains within the proteins. Monoclonal antibodieslevels of IgG 1 and IgG 3 antibodies are associated against these proteins have been effective in blockingwith reducing P. falciparum parasite densities the infectivity of the parasites to mosquitoes(Lusingu et al., 2005). Erythrocyte membrane (Templeton and Kaslow, 1999). (ii) Ps 25 andproteins (EMP) e. g. EMP 1, 2 and 3 are also located on Ps 28 antigens. These are surface proteins expressederythrocyte membrane; however, Pf EMP-3 is on the zygotes and mature ookinetes of malariaexpressed not only on erythrocyte surface but also by parasite. The ookinete surface proteins of P.liver stage parasites and sporozoites (Gruner et al., falciparum, Pfs 25 and Pfs 28, are target antigens for a2001). possible vaccine and their homologous proteins haveParasite toxinsbeen cloned from P. vivax and other species. P.falciparum Pfs 25 has been tested in Phase I clinicalThe toxic basis of malarial pathogenesis was first trials in human volunteers (Kaslow and Shiloach,conjectured by Camillo Golgi in 1886 (Golgi, 1886). 1994). Yeast produced recombinant Pvs 25 and Pvs 28Malaria GPI is considered a candidate toxin as it are highly immunogenic and antisera recognizedinduces cytokine like TNF-α and adhesion expressioncorresponding molecules expressed by field-isolatedin macrophages and vascular endothelium which areparasites in Thailand (Sattabongkot et al., 2003).associated with malaria pathogenesis. Antibodies to CURRENT STATUS AND PROSPECTS OFGPI lipid domains have been associated with MALARIA VACCINESprotection against malaria. GPI is a highly conservedpro-inflammatory endotoxin of parasite origin and The development of a malaria vaccine remains ansynthetic anti-GPI can be used as a plausible anti-toxic urgent need for most people living in malaria endemicvaccine (Schofield et al. 2002).regions. Two lines of evidence suggest the feasibilitySexual stage antigensof a malaria vaccine: firstly, immunity can be acquiredas a result of natural exposure to infection, andVarious studies have demonstrated that antibodiessecondly, numerous experimental malaria infectionsdirected against the sexual-stage antigens can preventin animal models and human volunteers can befertilization in the mosquito thereby interrupting theprotected through various immunization strategies.transmission of malaria. Transmission blockingThe distinct developmental stages of malaria parasitevaccines (TBVs) would reduce or interrupt malariaprovide numerous targets for vaccine development.transmission in human and mosquito populationsBecause the production of live, attenuated or killed-within a community as a whole but confer noinactivated malaria vaccine is not practical, the aimprotection to an individual recipient. Such ahas been to develop sub-unit vaccines. In this, part ortransmission blocking vaccine when given incomplete antigens are identified from the pathogen'scombination with a pre-erythrocytic or blood-stageproteomic complement, which can induce protectivevaccine would prevent or reduce the spread ofimmunity to the whole pathogen on vaccination. Theparasites which become resistant to such vaccines andgeneral target for a subunit vaccine has been towould thus prolong the effective life of other malariaidentify critical target antigens at each stage of the life-vaccines. Antigens expressed on the surface of sexualcycle of malaria parasite. Another strategy is tostages i.e. gametocyte, gamete, zygote and ookinete ofassemble peptide sequences from a range of parasitemalaria parasite are being considered as promisingantigens into different combinations that are thentargets for developing a transmission blockingtested for immunogenicity in animal models andvaccine. These vaccines induce antibodies in humanhuman volunteers. In the new generation of DNA-host that inhibit parasite development withinbased subunit vaccines, DNA sequences from P.mosquito vector, thus, blocking parasite transmission.falciparum parasites have been inserted into plasmidCloning of genes and subsequent recombinantDNA molecules (DNA vaccines) or variousproteins have shown to induce transmission-blockingrecombinant attenuated DNA viruses (recombinantantibodies in animal models. Two groups of antigensviral vaccines) to generate candidate vaccines (Wanghave been explored to block the propagation of sexualet al., 1998). DNA vaccines are taken up by host cells,parasites. (i) Pfs 48/45 and Pfs 230 antigens. Theseprotein expressed and T-cells are primed to formproteins belong to a family unique to Plasmodium withmemory T-cell populations while in recombinant viral

Malaria vaccine development23vaccines, cells are actively infected and express the CS-based particle vaccine that uses the highlyrecombinant malaria proteins. Both vaccines induce immunogenic hepatitis B core antigen (HBcAg) as ahigh levels of effector T-cell immune responses which delivery platform. It includes T-cell epitopes and B-in combination with antibody responses are both cell epitopes and is engineered so that the B-cellcritical in protection against malaria. Three stages in epitopes are exposed on the surface of virus-likethe life-cycle of malaria parasite are targeted for particles (VLPs), whereas the T-cell epitopes arevaccine development (Table I).within the interior. Three Phase I clinical trials inhealthy, malaria-naïve adults have been undertaken.Pre-erythrocytic vaccines: These would prevent thePhase I trials in UK and Germany evaluated aluminumdisease by targeting sporozoites or schizont infectedhydroxide and Montanide ISA 720 formulations safeliver cells and by preventing the release of primaryand well tolerated (Walther et al., 2005).Mzs from infected hepatocytes. Such a vaccine thatsuccessfully interferes with pre-erythrocytic Multi-epitope TRAP: This vaccine constructdevelopment would terminate infections before they consists of plasmid DNA or recombinant attenuatedhad any chance of causing clinical illness and would live viral vector like adenovirus, fowl pox andbe beneficial to travellers and residents of malaria modified vaccinia Ankara (MVA) in a prime-boostendemic countries. Numerous vaccine constructs strategy, the most advanced of which is a multihavebeen developed with CS protein being the prime epitope (ME) string fused to TRAP. The ME portiontarget.contains two B-cell, 14 CD8+ and three CD4+epitopes from six sporozoite and/or liver stageRTS, S/ASO 2A: This vaccine candidate is the mostantigens, including CS, LSA-1 and LSA-3. Severaladvanced in clinical development and is based on thephase I and Phase IIb sporozoite challenge studiesCS protein of P. falciparum comprising of twohave been conducted in malaria-naïve adults in UKpolypeptides RTS and S expressed in yeast. RTS is awhich is reported to induce high T-cell responsessingle polypeptide chain comprising of CS protein(McConkey et al., 2003). Phase I safety trials werefused to hepatitis B surface antigen (HBsAg), while Sinvestigated in adults and children in The Gambia alsois a polypeptide of HBsAg. The two polypeptides(Moorthy et al., 2003) and a further Phase IIb proof ofassemble to form composite particulate structureconcept trial is being conducted in The Gambia.(RTS, S) that constitute the vaccine antigen. Thisvaccine when administrated with the adjuvant Plasmid DNA vaccines: Intense efforts have beenASO2A, an oil in water emulsion consisting of the made to develop effective DNA-based vaccines to theimmunostimulants monophosphoryl lipid A and liver and blood stages with combinations of plasmidsaponin derivative QS 21, has shown good DNA vaccines targeting one or more P. falciparumimmunogenicity and efficacy in clinical trials. Phase antigens. Several single and combination vaccines,II safety and immunogenicity trials in malaria naïve including CS alone and CS with SSP2/TRAP, LSA-1,and malaria-immune subjects have shown it to be safe EXP-1 and LSA-3 (MuStDO-5) have undergoneand immunogenic and conferred 50% sterile Phase I and IIa clinical trials (Doolan and Hoffman,immunity in volunteers but immunity waned with time 2001). DNA vaccines require viral boosting to inducelasting only up to six months in a few cases. Phase I strong T-cell immunogenicity.field evaluation in malaria experienced adults wasdone in Gambia (Doherty et al., 1999). Later aAsexual blood-stage vaccines: A second strategy forrandomized, double-blind controlled Phase IIbvaccine development is to target immune responsesefficacy study was carried out in malaria-experiencedagainst the asexual blood stages with the aim toadult Gambian men that gave overall efficacy of 34%prevent or contain the disease process by suppressing(Bojang et al., 2001). Efficacy trial in children inparasite replication either by preventing Mz invasionMozambique and Phase I trials in children in Theof erythrocytes or by attacking parasite inside hostGambia gave encouraging results. A Phase IIb proof oferythrocytes. A blood stage vaccine would be effectiveconcept efficacy study was done in children inin reducing mortality in endemic countries. TheSouthern Mozambique which showed that the vaccineprincipal target of asexual stage vaccine is the Mz withwas highly immunogenic and had an efficacy of 58%the major target proteins like MSP-1, 2, 3 and AMA-1.against severe malaria (Alonso et al., 2004).A vaccine based on the C-terminal of P. falciparumMSP-1 consisting of a 42 kDa protein produced as aICC/-1132 CS/hepatitis B core particle: This is a lyophilized recombinant antigen expressed in E. coli

24 Banyal and ElangbamTable I: Malaria vaccines in clinical trialsStages Vaccine/antigen Research group Trial phasePre-erythrocytic RTS,S: Hybrid P. falciparum GSK / WRAIR/MVI Phase IIbStageCSPHBsAg particles + AS02adjuvantCSP C-ter peptide + Montanide Dictagen / Lausanne Univ Phase IbISA 720ICC-1132: Hybrid CSP Apovia/MVI Phase IImultiepitope-HBc VLPsDNA vaccines (including US Navy/ Vical Phase IMuStDO-5: CSP/LSA-1/LSA-3/EXP1/TRAP)Live recombinant FPV- or Oxford Univ/ Oxxon/MVI Phase IbMVA-CSP + LSA-1 epitopeLive recombinant MVA- Oxford Univ/Oxxon Phase Ibmultiepitope string + TRAPLive recombinant Ad-CSP Crucell/GSK/WRAIR/NIAID Phase IaOther live recombinant vaccines Oxford Univ; NYU Preclinical(MVA, cold-adapted influenzavirus)-CSPLSA-3 (long peptides; Pasteur Institute/WRAIR/ GSK Phase Ialipopeptide;recombinant)LSA-1, SALSA, other Hawaii Biotech; Epimmune Preclinicalliver-stage antigensAsexual MSP-1 42 kD + AS02 GSK/WRAIR/MVI Phase Ib/ IIerythrocytic AMA-1 MVDU; NIAID Phase Ibstage PfCP 2.9: MSP-1-AMA-1 fusion Sec Military Med Univ Shanghai/protein (yeast) + Montanide Wanxing harmaceuticals/WHO Phase IISA 720Other MSP-1 derivatives NIAID; Hawaii Biotech; AECOM; Preclinical to(peptides, Salmonella or Univ Maryland Phase IBCG recombinants)MSP-3 long peptides Pasteur Institute/ AMANET/EMVI Phase IbGLURP long peptide EMVI/SSI Phase IMSP-3-GLURP hybrid long EMVI/SSI Phase Ipeptide + Montanide ISA 720MSP-4, -5 Monash PreclinicalCombination B: MSP-1, -2, Queensland Med Res Institute/ Phase IIRESA + MontanideWEHRISE36 Osaka Univ/Biken Phase IOther blood-stage antigens Various groups Preclinical(EBA-175,EBP2, MAEBL,RAP-2, EMP-1, DBL-á..)Sexual stage PfS25 (yeast) NIH Phase IPvS25 and other sexual-stage NIH PreclinicalantigensSource: www.who.int/vaccine_research/documents/en/status_table.pdf.

Malaria vaccine development25has shown great promise (Angov et al., 2003). Thisvaccine reconstituted with ASO2A, falciparum Mzprotein 1 (FMP-1)/ASO2A was found to be safe andimmunogenic in mice, rabbits and rhesus macaques.The safety and immunogenicity trial has beenconducted in malaria-naïve individuals. A Phase Isafety study in malaria-experienced Kenyan adultshas been completed and is scheduled to begin a Phase Istudy in children in malaria-endemic region ofwestern Kenya.A chimeric fusion of domain III of AMA-1 and the 19-kDa portion of MSP-1 called P. falciparum chimericprotein 2 (Pf CP-2.9) containing conserved portions ofboth proteins is also being developed (Genton andCorradin, 2002). Pf CP-2.9 expressed in Pichiapastoris has demonstrated good immunogenicity toboth portions of antigen in mice, rabbits and non-human primates. A preclinical trial showed itseffectiveness, and a Phase I trial in malaria-naïvehealthy adults with Montanide ISA720 formulation isunder way that began in February 2006.polymorphism and polyclonal B-cell activation.Protection in endemic areas takes years to develop andthere is no clearly defined immune response that caneffectively protect against the disease. There is alsothe need to develop novel adjuvants that can enhancethe cell-mediated immunity, and also for thedevelopment of novel vectors and vaccineformulations that will produce optimal protectiveimmune responses. Some of the recent vaccine trialslike RTS, S which showed 58% efficacy against severedisease have given encouraging results. Malariavaccine development is at an exciting stage with anoptimistic outlook. Recent advances in understandingthe immune mechanisms and the volume ofinformation generated from malaria parasite genomewould help in better understanding and identificationof critical antigens for vaccine development. Perhaps,given the complexity of the parasite, vaccine based onall or many antigens would be more effective than theone based on a single antigen.ACKNOWLEDGMENTSThe idea of a multi-component vaccine against blood- Ms. N. Elangbam is grateful to University Grantsstage parasites was approached by Patarroyo and Commission, New Delhi, for financial assistance incolleagues with a synthetic peptide vaccine SPf66, the form of a Junior Research Fellowship (Nationalcontaining sequence from MSP-1 and two other Eligibility Test).blood-stage antigens combined with CS protein(Patarroyo et al., 1987). The vaccine underwent fourREFERENCESPhase III clinical trials in different locations. Studies Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Milman J,in Colombian adults and children (Valero et al., 1993) Mandomando I, Spiessens B, Guinovart C, Espasa M,Bassat Q, Aide P, Ofori-Anyinam O, Navia MM, Corachanand in Tanzanian children (Alonso et al., 1994) gaveS, Ceuppens M, Dubois MC, Demoite MA, Dubovsky F,encouraging results but no significant protection was Menendez C, Tornieporth N, Ballou WR, Thompson R andobserved in studies in Gambian infants (D'Alessandro Cohen J. 2004. Efficacy of the RTS, S/AS02A vaccineet al., 1995) and in Thailand (Nosten et al., 1996).against Plasmodium falciparum infection and disease inyoung African children: randomized controlled trial. LancetSexual-stage vaccines: The third vaccine strategy is 364: 1411-1420.the development of a sexual-stage vaccine. Antigens Alonso PL, Smith T, Schellanberg JR, Masanja H, Mwankuyseof gametocyte 48/45 kDa and 230 KDa and the 25 kDa S, Urassa H, de Azevedo IB, Chongela J, Kobero S,and 28 kDa post-fertilization antigens are leading Menendez C, Hurt N, Thomas MC, Lyimo E, Weiss NA,candidates for transmission blocking vaccine (TBV). Hayes R. Kitva AY, Lopez MC, Kilama WL, Teuscher T andTanner M. 1994. Randomised trial of efficacy of SPf66Pvs 25 adjuvanted with aluminum hydroxide isvaccine against Plasmodium falciparum malaria in childrencurrently in Phase I clinical trial. Pvs 25 adjuvanted in Southern Tanzania. Lancet 344: 1175-1181.with cholera toxin (CT) when used to immunize intranasallyinduced significant Th-2 type immune Angov E, Aufiero BM, Turgeon AM, Van Handenhove M,Ockenhouse CF, Kester KE, Walsh DS, McBride JS, Duboisresponse in mice and the antisera completely blockedMC, Cohen J, Haynes JD, Eckels KH, Heppner DG, Ballouparasite transmission (Arakawa et al., 2003).WR, Diggs CL and Lyon JA. 2003. Development and preclinicalCONCLUSIONS analysis of a Plasmodium falciparum Mz surfaceprotein-1 (42) malaria vaccine. Mol Biochem Parasitol 128:Immunity to malaria is complex and not clearly195-204.defined with the parasite exhibiting various Arakawa T, Tsuboi T, Kishimoto A, Sahabongkot J, Suwanabunimmunoevasive mechanisms like antigenic variation,N, Rungruang T, Matsumoto Y, Tsuji N, Hisaeda H, StowersA, Shimabukuro I, Sato Y and Torii M. 2003. Serum

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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 30–36J P DIsolation and characterization of the paraflagellar rodproteins of Leishmania donovaniA. Lahiri and A. BhattacharyaImmunoparasitology Research Unit, Department of Zoology, University of Calcutta, Kolkata.ABSTRACT. The paraflagellar rod (PFR) is a unique cytoskeletal structure present in thekinetoplastid protozoans but absent from their mammalian hosts. It is a massive network of wovencytoskeletal filaments attached to one face of the common 9+2 axoneme of the flagellum. The PFR isnecessary for proper parasite motility, viability and successful infection. Biochemical studiescarried out on the PFR have revealed that it is composed of both major and minor proteincomponents. In this paper, purification of the PFR proteins of Leishmania donovani has beendescribed, which involves a combination of flagella isolation, non-ionic detergent treatment andrestricted proteolysis. Scanning electron microscopy was done to monitor the process of isolation offlagella from intact cell bodies. The major PFR proteins have been identified as two distinct bandsof mol wt 76 kDa and 68 kDa, by using sodium dodecyl sulphate polyacrylamide gel electrophoresisanalysis. The ultrastructure of the flagellum was studied by using a transmission electronmicroscope.Keywords: axoneme, flagellum, kinetoplastid, paraflagellar rodINTRODUCTIONLeishmania donovani, an intracellular protozoanparasite, causes Kala-azar (visceral leishmaniasis) inhumans. The parasite is transmitted by various speciesof female sandflies (Phlebotomus argentipes). Itexists in two morphological forms: the promastigote,residing in the gut of female sandflies, and theamastigote, living in the reticuloendothelial system ofthe mammalian hosts. The promastigotes possess afull-length, free-flagellum whereas it is rudimentaryin amastigotes.The flagellum of promastigotes helps in motility and isinvolved in hemidesmosomal attachment to thechitinous itima, and maintenance of the parasitewithin the sandfly gut (Killick-Kendrick, 1979;Killick-Kendrick et al., 1974; Walters et al., 1987).Corresponding author: Dr. A. Bhattacharya, Immuno-parasitologyResearch Unit, Department of Zoology, University of Calcutta, 35Ballygunge Circular Road, Kolkata-700 019, W.B., India.The flagellar modes of attachment have been observedwith other kinetoplastids also and appear to beessential for their survival within the insect vector(Killick-Kendrick, 1979; Rowton et al., 1981;Vickerman, 1973; Vickerman and Preston, 1976). Theflagellum of Leishmania is also involved inchemotactic responses (Bray, 1983). Structurally, theflagellum of Leishmania has a typical 9+2microtubular axoneme and also possesses afilamentous, lattice-like structure called theparaflagellar rod (PFR) or paraxial rod. Themicrotubules of the axoneme are arranged in a precisepattern of nine outer double microtubules and twoseparate central ones.The PFR is a unique cytoskeletal structure found inkinetoplastids, euglenoids and some dinoflagellates(Bastin et al., 1996; Hyams, 1982; Cachon et al.,1988). The paraxial rod of kinetoplastida is similar tothat of euglenoids, although apparently, they appear tobe morphologically distinct from each other (Farina et

The PFR proteins of Leishmania donovani31al., 1980; Fuge, 1969; Gallo and Schrevel, 1985). The significant homology has been reported betweenPFR is a massive network of woven cytoskeletal major PFR proteins and other known proteinsfilaments, running alongside the typical eukaryotic (Imbodem et al., 1995).9+2 axoneme of the flagellum (Russell et al., 1983).Recent molecular studies have demonstrated that theThe ultrastructural study of the flagellum includingPFR is necessary for proper promastigote motility andthe PFR and the purification of the PFR proteinsviability (Santrich et al., 1997; Bastin et al., 1998).provides an important guideline to the researchers forAblation of a specific molecule of the PFR resulted inunderstanding the complete biology of this structure.mutant cells that were paralyzed, indicating theThe parasite L. donovani represents a standard modelessential role of PFR in cell motility (Deflorin et al.,for studying unique structures such as the PFR1994). Also, ATPase activity was detected in the PFRbecause it is one of the most common humanof euglenoids (Moreira-Leite et al., 1999). Thepathogens in tropical countries such as India.ultrastructure of kinetoplastid PFR appears largelysimilar in all species of the group (De Souza andMATERIALS AND METHODSSouto-Padron, 1980; Beard et al., 1992). The structure Parasite culture: The promastigotes were cultured athas been divided in three distinct zones in relation to 22-25° C in liquid M-199 medium supplemented withthe axoneme namely, proximal, intermediate and 10% heat-inactivated fetal bovine serum (FBS, Sigmadistal (Freymüller and Camargo, 1981). The proximal Aldrich, St. Louis, MO, USA; Morgan et al., 1950).and distal regions each contain filaments of 7-10 nm The culture medium was filtered under sterilethat intersect at an angle of 100°. The intermediate conditions in a culture room with laminar flow. Theregion contains thin (5 nm) filaments that intersect at medium was kept for 48 h at room temperature toan angle of 45° and connect the proximal and distal check for any contamination, and then stored at -20° C.regions (Maga and LeBowitz, 1999). Attachment Aliquots of the above culture of L. donovani were usedfilaments extending from axoneme microtubule in all the experiments.doublets 4-7 connect the proximal region to theIsolation of flagella: The culture medium containingaxoneme (Ismach et al., 1989). In Leishmania, thepromastigotes of L. donovani was centrifuged at 2100promastigotes possess a full-length flagellum with ax g for 20 min (Cunha et al., 1984). The pelletPFR, whereas amastigotes contain only an attenuated,containing the cells was washed twice in phosphatenon-emergent flagellum completely lacking a PFR.buffered saline (PBS; 10 mM, pH 7.2; Sigma) and thenThe biochemical composition of the PFR is quitein buffer A (25 mM TRIS-HCl , 0.2 mM EDTA, 5 mMcomplex and includes both major and minor PFRMgCl 2, 12 mM β-mercaptoethanol, 0.32 M sucroseproteins. Among them, the major PFR proteins havebeen extensively studied in the parasitic(all from Sigma; pH 7.4) and resuspended in buffer Asupplemented with 1% bovine serum albuminhaemoflagellates Trypanosoma cruzi, T. brucei, L.(Sigma), 0.1 mM CaCl 2 (Sigma), 0.5 mM phenylmexicana and L. amazonensis. In these organisms, themajor PFR proteins migrate in two bands on sodium methyl sulfonyl fluoride (Sigma) and 5 µg/mldodecyl sulphate polyacrylamide gel electrophoresis leupeptin (Sigma; Moreira-Leite et al., 1999). The(SDS-PAGE) with mol wt of about 68-75 kDa, and cells were subjected to three different centrifugationappear to be present in approximately equimolar speeds: 2600 x g, 3200 x g and 3700 x g, and theamounts (Deflorin et al., 1994; Schlaeppi et al., 1989).flagella were isolated from the cell bodies byThe major PFR components are conserved in thecentrifugation at an optimum speed of 3200 x g for 30trypanosomatidae family and form a doublet ofmin. The supernatant containing the flagella washomologous proteins in most species of the familyseparated from the pelleted and deflagellated cellbodies. This supernatant was then concentrated by(Araujo and Morein, 1991; Saborio et al., 1989). Bycentrifugation at 6780 x g for 20 min. The aboveSDS-PAGE analysis, PFR1 of L. amazonensisprocedure was carried out at 0-4° C. The pelletsmigrates at 74 kDa and PFR2 at 69 kDa (Bastin et al.,containing the flagella were finally suspended in1996). The PFR1 and PFR2 genes from T. cruzi, T.buffer A.brucei and L. mexicana are highly conserved acrossspecies (over 80% amino acid homology; Maga and Scanning electron microscopy: A scanning electronLebowitz, 1999). More complex patterns have been microscope (SEM) uses a fine beam of electrons todescribed in T. cruzi in which four major PFR proteins scan back and forth across the metal-coated surface.have been identified (Fouts et al., 1998). No The principal application of SEM is in the study of

32 Lahiri and Bhattacharyasurfaces such as those of cells. The aliquots of the Ultrastructural study of the flagellum bytreated cells were fixed with 2.5% glutaraldehyde transmission electron microscopy: Cells were fixed(Sigma) in 0.1M phosphate buffer (pH 7.2-7.4; Sigma) in a suitably buffered aldehyde fixative (2.5%for 24 h and washed in the same buffer for 15 min x 4. glutaraldehyde grade I; Sigma) in 0.1 M sodiumThe samples were rinsed thrice with double distilled cacodylate buffer (pH 7.4; Sigma) at 4° C for 1-4 h.water, each time for 5 min. Then they were dehydrated Then the cells were washed for 2 h or overnight at 4° Cin different grades of alcohol: 50, 70, 80, 90, 95 and in three changes of 0.1M sodium cacodylate buffer100% ethanol (20 min each). Gold coating of 200Å (pH 7.4). Cells were post-fixed in 1% OsO 4 (Sigma) inwas done at 5 mA using Giko Engineering-IB2 ion 0.8% potassium ferricyanide (Sigma) for 1-2 h at roomcoater. The cells were dried using vacuum pump and temperature and protected from light (Nakano et al.,observed under a SEM (model HITACHI S-530), and 2001). The above cells were washed for 5 min x 2 withthe photographs were taken by MAMIYA 6 x 7 camera distilled water. Dehydration was done using theusing NOVA 120 ASA films. following grades of alcohol: 50, 70, 90 and 95%PFR protein purification: To purify the proteinethanol, each for 15 min, and 100% ethanol for 15 mincomponent of PFR, it was necessary to remove thex 4. Finally, the treated cells were embedded in Eponflagellar membrane. The flagellar membranes werepolybed 820 epoxy resin. Ultrathin sections were cutremoved by non-ionic detergent treatment. Flagellarand stained with 5% aqueous uranyl acetate (Sigma)fractions of L. donovani promastigotes, obtained asand lead citrate (Sigma) and observed under adescribed above, were subjected to three rounds oftransmission electron microscope.treatment with 2% Nonidet P-40 (Sigma) in PBS(Sigma) at 0-4° C under constant shaking. Each 15 minRESULTSround of detergent treatment was followed by When observed under a SEM, it was revealed that thecentrifugation at 17,300 x g for 20 min at 4° C cells centrifuged at 2600 x g retained their flagella(Moreira-Leite et al., 1999). The pellet of the final (Fig. 1). The flagella were detached from the intact cellcentrifugation step was dissolved in PBS (Sigma) and bodies when centrifugation was carried out at 3200 x gsubjected to a brief treatment with 0.0015% trypsin (Fig. 2). However, the cells were ruptured when(type XIII, TPCK-treated, Sigma) for 90 s at 28° C. centrifuged at 3700 x g (Fig. 3).The trypsin treatment was stopped by adding 20-foldSDS-PAGE analysis showed two protein bands of molexcess soyabean trypsin inhibitor (Sigma). Thewt 76 kDa and 68 kDa that are presumed to be tworesultant protein fractions were subjected to SDSfractionsof the PFR proteins (Fig. 4).PAGE.SDS-PAGE: This method is based on the separation ofproteins according to mol wt and is particularly usefulfor monitoring protein purification. The sample to berun on SDS-PAGE was mixed with loading dye(protein:loading dye, 1:1) and boiled for 5 min in awater bath. The stock loading dye had the followingcomposition: double distilled water, 4.8 ml; TRIS (pH6.8), 1.2 ml; 10% SDS (Sigma), 2 ml; glycerol(Sigma), 1 ml and bromophenol blue (Sigma), 0.5 ml.Before use, 950 µl of stock solution was mixed with 50µl of β-mercaptoethanol. The sample was runsimultaneously with protein markers (Broad Range,Bangalore GENEI, Bangalore, India) in differentwells at a constant voltage of 100 V in 10% resolvinggel and 4% stacking gel. The gel was then fixed inmethanol and stained with Coomassie Brilliant BlueR-250 (Sigma) for a few h, and then washed indestaining solution until clear bands were visible(Laemmli, 1970).Transmission electron microscopy study revealed theultrastructural details of PFR. The longitudinalsection of a flagellum shows that the lattice-like PFRruns parallel to the axoneme even before theemergence of the flagellum from the flagellar pocket0002 15KV 5umFig. 1: SEM of a L. donovani cell centrifuged at 2600 x g.

The PFR proteins of Leishmania donovani330016 15KV 5umFig. 2: SEM of a L. donovani cell centrifuged at 3200 x g.0021 15KV 5umFig. 3: SEM of a L. donovani cell centrifuged at 3700 x g.MarkerFlagellaFPFRkD205ABBK97N682914Fig. 4: SDS-PAGE of PFR proteins of L. donovani. The markerproteins are shown on the left side.Fig. 5: Transmission electron microscope photograph of a L.donovani promastigote. [F, flagellum; PFR, paraflagellar rod; A,axoneme; BB, basal body; K, kinetoplast; N, nucleus](Fig. 5). It also shows the attachment of the flagellumto the basal body at the anterior end of the parasite. Theaxoneme of the flagellum showed typical eukaryotic

34 Lahiri and Bhattacharya9+2 microtubular arrangement. The basal body as well To isolate and purify the PFR proteins, the firstas the base of the flagellum remains ensheathed by a essential step was to successfully separate the flagellamembrane, and the central rod of the flagellum from intact cell bodies. The process of flagellaemerges freely. Centrifugation at 2600 x g detached isolation was monitored by using SEM to make surethe flagellum partially from the basal body. Complete that most of the cells remained intact and only lostdetachment was obtained at 3200 x g but the flagellum their flagella. Because the promastigotes werewas still surrounded by a membrane. Non-ionic obtained by centrifuging the culture medium at 2000 xdetergent treatment removed the flagellar g, the cells were subjected to three different highermembranes.centrifugation speeds starting from 2600 x g andDISCUSSIONincreasing gradually to 3700 x g to isolate the flagella.When observed under a SEM, it was observed thatAmong protozoans, the kinetoplastid trypanosomes most of the cells centrifuged at 3700 x g were rupturedhave always held an important position in the (Fig. 3), while those centrifuged at 2600 x g retainedscientific research scenario, not only for their medical their flagella (Fig. 1). Thus, the optimumimportance but also for possessing diverse cellular centrifugation speed for successful isolation ofprocesses. One such unique cytoskeletal structure is flagella was chosen to be 3200 x g (Fig. 2).PFR. The canonical 9+2 axoneme of the flagellumThe flagellar membranes were removed by treatmentinitiates beating of the latter in most eukaryotes, butwith the non-ionic detergent Nonidet P-40. It latterthe elaborate PFR structure has been observed in onlyappears to have no effect on the integrity of thekinetoplastids, euglenoids and some dinoflagellates.flagellar cytoskeleton and is, therefore, unable toTill date, the PFR and its proteins, namely PFR1 and break the attachment between the PFR and axonemePFR2 have been studied in T. cruzi, T. brucei, L. (Fig. 5). Beacuse it has been determined that the linksmexicana and L. amazonensis. These studies revealed between PFR and axoneme are highly sensitive tothat PFR is essential for proper parasite motility and trypsin (Moreira-Leite et al., 1999), the detachment ofviability, and both these functions are directly PFR from axoneme was accomplished by proteolyticattributable to the PFR proteins – PFR1 and PFR2 treatment.(Santrich et al., 1997; Bastin et al., 1998). Using newSDS-PAGE analysis showed two protein bands of molmolecular-genetic techniques, PFR1, PFR2 andwt 76 kDa and 68 kDa that are presumed to be twoPFR1/PFR2 null mutants of T. brucei and L. mexicanafractions of the PFR proteins (Fig. 4). Extensivehave been generated. The mutant cells lacked a nativestudies carried out till date on the major PFR proteinsPFR structure, showing that despite sharing over 60%in the parasitic haemoflagellates T. cruzi, T. brucei, L.amino acid homology and similar physical properties,mexicana and L. amazonensis have shown that thePFR1 and PFR2 are essential and significantlyPFR proteins migrate in SDS-PAGE as two bands withfunctional components of PFR. Mutants of L.mol wt of 70-75 kDa and 68-72 kDa, respectively, andmexicana having a PFR2-phenotype were unable toappear to be present in approximately equimolarcolonize in the vector's gut whereas PFR1 and/oramounts. So, this result is in conformity with thePFR2 mutants were viable in axenic culture. Thisresults reported in previous studies.indicates that the PFR plays a key role in the viabilityof Leishmania in a natural habitat (Hunger-Glaser and Transmission electron microscopy study revealed theSeebeck, 1997). Leishmania parasites lacking PFR ultrastructural details of PFR. The longitudinalwere found to display severe disturbances in flagellar section of a flagellum shows that the lattice-like PFRwaveforms e.g. reduced wavelength and amplitude runs parallel to the axoneme even before theand decrease in frequency as compared to the beating emergence of the flagellum from the flagellar pocketpatterns of normal parasites (Santrich et al., 1997). So, (Fig. 5). It also shows the attachment of the flagellumit is of prime importance to isolate and study the to the basal body at the anterior end of the parasite. Theultrastructure of the flagellum and PFR of L. axoneme of the flagellum showed typical eukaryoticdonovani, and to determine its role in the life cycle of 9+2 microtubular arrangement (Fig. 5). The basalthe parasite with special reference to PFR proteins. body as well as the base of the flagellum remainsThe present studies were carried out with an aim to ensheathed by a membrane and the central rod of thefulfill the above objectives.flagellum emerges freely. Centrifugation at 2600 x gdetached the flagellum partially from the basal body.

The PFR proteins of Leishmania donovani35Complete detachment was obtained at 3200 x g but theflagellum was still surrounded by a membrane. Nonionicdetergent treatment removed the flagellarmembranes.Bray RS. 1983. Leishmania: chemotactic responses ofpromastigotes and macrophages in vitro. J Protozool 30:322-329.Cachon J, Cachon M, Cosson M-P and Cosson J. 1988. Theparaflagellar rod: a structure in search of a function. BiolThe alternation of flagellate and nonflagellate forms Cell 63: 169-181.of the parasite is attributable to their needs forCunha NL, De Souza W and Hassón-Voloch A. 1984. Isolationinfection and survival within the host cells. The and characterization of the paraxial structure offlagellum is essential for attachment to macrophages, Herpetomonas megaseliae. J Submicrosc Cytol 16: 705-and subsequent penetration and stabilization within 713.macrophages. Apart from attachment, penetration andDe Souza W and Souto-Padron T. 1980. The paraxial structurestabilization within host cells, the main function of of the flagellum of trypanosomatidae. J Parasitol 66: 229-flagella is to confer motility and viability to cells so 236.that they can infect host cells. Once the infectionDeflorin J, Rudolf M and Seebeck T. 1994. The majorprocess is completed, the presence of flagella is nocomponents of the paraflagellar rod of Trypanosome bruceilonger required. So, the flagella become rudimentary are two similar, but distinct proteins which are encoded byand non-functional in the intracellular forms. two different gene loci. J Biol Chem 269: 28745-28751.The present study shows the successful isolation of Farina M, Attias M and Souto-Padron T. 1980. The paraxial rodflagella through scanning electron microscopy. It of trypanosomatids. J Protozool 33: 552-557.reports that PFR is composed of two major proteins Fouts DL, Stryker GA, Gorski KS, Miller MJ, Nguyen TV,(mol wt 76 kDa and 68 kDa) in L. donovani, which are Wrightsman RA and Manning JE. 1998. Evidence for fourunique to that structure and bear no significant distinct major protein components in the paraflagellar rod ofhomology to other known proteins. TransmissionTrypanosoma cruzi. J Biol Chem 273: 21846-21855.electron microscopy study of the flagellum and PFR Freymüller E and Camargo EP. 1981. Ultrastructuralprovides a relatively complete picture of the biology differences between species of trypanosomatids with andof these structures. It shows the position of the without endosymbionts. J Protozool 28: 175-182.axoneme and PFR within the flagellum, how they are Fuge H. 1969. Electron microscopic studies and theconnected to each other and their structural intraflagellar structure in trypanosomes. J Protozool 16:peculiarities. This study is an important guideline for 460-465.those who are carrying out researches on the flagellum Gallo JM and Schrevel J. 1985. Homologies betweenand PFR of L. donovani.paraflagellar rod proteins from trypanosomes andACKNOWLEDGEMENTSeuglenoids revealed by a monoclonal antibody. Eur J CellBiol 36: 163-168.The financial assistance from the Council of Scientificand Industrial Research, New Delhi, [scheme No. 60(0042)/01/EMR-II] is gratefully acknowledged.REFERENCESAraujo FG and Morein B. 1991. Immunization withTrypanosoma cruzi epimastigote antigens incorporated intoiscoms protects mice against lethal challenge in mice. InfectImmun 59: 2909-2914.Bastin P, Mattews KR and Gull K. 1996. The paraflagellar rodof kinetoplastida: solved and unsolved questions. ParasitolToday 12: 302-307.Bastin P, Sherwin T and Gull K. 1998. Paraflagellar rod is vitalfor trypanosome motility. Nature 391: 548.Beard CA, Saborio JL, Tewari D, Krieglstein KG, Henschen AHand Manning JE. 1992. Evidence for two distinct majorprotein components, PAR 1 and PAR 2, in the paraflagellarrod of Trypanosoma cruzi. J Biol Chem 267: 21656-21662.Hunger-Glaser I and Seebeck T. 1997. Deletion of the genes forthe paraglagellar rod protein PFR-A in Trypanosomabreecei is probably lethal. Mol Biochem Parasitol 90: 347-351.Hyams J. 1982. The Euglena paraflagellar rod: structure,relationship to other flagellar components and preliminarybiochemical characterization. J Cell Sci 55: 199-210.Imbodem M, Müller N, Hemphill A, Matioli R and Seebeck T.1995. Repetitive proteins from the flagellar cytoskeleton ofAfrican trypanosomes are diagnostically useful antigens.Parasitol 110: 249-258.Ismach R, Ciani CML, Caulfield JP, Langer PJ, Hein A andMcMahon-Pratt D. 1989. Flagellar membrane and paraxialrod proteins of Leishmania: characterization employingmonoclonal antibodies. J Protozool 36: 617-624.Killick-Kendrick R. 1979. Biology of Leishmania inphlebotomine sandflies. In: Biology of the Kinetoplastida.Lumsden WHR and Evans DA (Eds) Academic Press, NewYork. pp. 203-260.

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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 37–40J P DOnchocercosis in Benue state, Nigeria: comparativeepidemiological studies amongst the Etulo and Idomaethnic groupsE. A. Omudu and B. O. AtuDepartment of Biological Sciences, Benue State University, Makurdi.ABSTRACT. The prevalence of onchocercosis caused by Onchocerca volvulus amongst two distinctethnic groups in Benue State was investigated using Rapid Assessment Methods (RAMs) and skinsnip technique. Results showed that of the 2126 individuals examined in the Idoma-speakingOkpokwu Local Government Area, 16.6% were positive either for Leopard skin (LS) or palpablenodules. While of the 1005 individuals examined in the Etulo area, 5.6% had either LS or palpablenodules. The prevalence rate varied significantly (p < 0.05). Correlation analysis showed significantassociation between LS, palpable nodules and aging (r=0.43, p < 0.001). Skin snip results showedcommunities with higher LS and nodules had higher community microfilarial load. Correlationcoefficient r=0.64 was obtained when skin snip prevalence was compared with RAMs in the Etuloarea. The implications of these results were discussed especially in terms of prioritizing theimplementation of community-directed treatment with ivermectin.Keywords: microfilariae, Nigeria, onchocercosis, Rapid Assessment MethodsINTRODUCTIONOnchocercosis (river blindness) is a parasitic diseasecaused by the nematode Onchocerca volvulus andtransmitted by the bites of blackflies belonging toSimulium damnosum complex. It is estimated that over81 million people are at risk of infection, some 18million already infected and one million peoplevisually impaired of whom about 340,000 are blind(WHO 1995). Apart from ophthalmologicaldegeneration leading to blindness, the disease alsopresents bizarre dermatological, lymphatic andsystemic manifestations (Edungbola et al., 1990).Onchocercosis has been reported in some parts ofBenue State, (Gemade and Dipeolu, 1983; Amuta andOlusi, 2000), where it is affecting socio-economy ofCorresponding author: Dr. Edward Agbo Omudu, Departmentof Biological Sciences, Benue State University, P. M. B.102119, Makurdi, Nigeria. E-mail: eddieomudu@yahoo. comhuman population. The development and use of RapidAssessment Methods (RAMs) using prevalence ofLeopard skin (LS) and palpable nodules for diagnosisof onchocercosis and free distribution of ivermectinfor treatment has heightened expectation that thedisease can be contained. For this to happen, there is aneed to know the extent of the disease especially incommunities without baseline information. Thisstudy, using RAM, was undertaken to provide pretreatmentprevalence and identify communitieseligible for mass distribution of ivermectin.MATERIALS AND METHODSStudy areas: The study areas comprised ofnineteen Idoma speaking villages in Okpokwu LocalGovernment Area (LGA) and twelve Etulo speakingvillages in Buruku and Katsina-Ala LGA. There aredistinct cultural and occupational differences that mayinfluence exposure to biting blackflies and perceptionof disease manifestation. The vegetation and

38Edward and Bernardtopographical features of the study areas have been volvulus was carried out in whole population of thedescribed by Nwoke et al. (1998) as being favourable Etulo area; however, this was not possible in Okpokwuand suitable for onchocercosis transmission.LGA as a result of immunological and parasitologicalstudies just concluded in the same area by Amuta andOkpokwu: The local population of this area (8°00 andOlusi (2000). Two snips, one each from the right and8° 30 E and 6° 58 and 7° 25 N) practice peasantleft iliac crest were taken from each individual with theagriculture, fishing and hunting are secondaryaid of a 2 mm bite corneoscleral punch (E-2802, Holtoccupations. With numerous breeding sites on theStorz, Germany). Each snip was incubated in a well ofOkpokwu river system that drain these villages andmicrotitre plate containing normal saline. The platesoutdoor activities performed by the population, theywere then examined within 30 min to one hour byare continually exposed to bites of the vectors.microscopy for the presence of O. volvulusBuruku and Katsina-Ala: The Etulo community microfilariae (Wentworth, 1988).inhabits twelve villages which are bisected by riverKatsina-Ala, a major tributary of Benue River.RESULTS AND DISCUSSIONMajority of the villages are located along the bank of Out of 31 villages examined, all were found to havethe river with fishing and farming constituting the individuals with LS, whereas 27 (87%) villages weremajor occupation. carrying nodules of onchocercal origin. Out of 2126individuals examined from the Idoma speakingApart from river Katsina-Ala, another perennial rivervillages in Okpokwu LGA, 248 (11.6%) had LS, 105known as Ogaturu drains most of Etulo land. The two(4.9%) had nodules and 35 (1.6 %) had both nodulespredominant rivers are fast flowing and harbour larvaeand LS while 353 (16.6%) had either LS or nodulesand pupae of S. damnosum complex (Nwoke et al.,(Table 1). In 1005 Etulo-speaking individuals, 451998; Gemade and Dipeolu, 1983).(4.5%) had LS, 12 (1.2%) had nodules and 2 (0.2 %)RAMs for diagnosis: Based on the analysis of large had both LS and nodules while 57 (5.6%) had either LSvolumes of epidemiological data on onchocercosis, or nodules (Table 2). The number of cases with LS,depigmentation of skin LS and palpable onchocercal visible or palpable nodules observed in the Okpokwunodules were recommended as rapid assessment area varied significantly (p < 0.05) from that of Etuloprocedures to determine communities eligible for area.treatment with ivermectin. The merits and limitationsWhereas LS in Okpokwu area ranged fromof these methodologies are summarized by Edungbola7.3–28.8%, in Etulo area it ranged from 0.8–14.0%.et al. (1993).Only one village (Ashitenaku) had 14% LS, whereasCommunity: Only adults 18 years and above were all the other Etulo villages had LS below 7.3%. Inexamined for presence of nodules and LS. All persons Okpokwu, the prevalence of onchocercal nodulessampled were engaged in one or more rural occupation ranged from 2.2%–10.9% and in Etulo area it rangedand have resided in the community for at least five from 0.8–3.1% only one village (Ugye) in Etulo hadyears (Gemade, 1993). Communities were visited a nodule prevalence of 3.1%, whereas all other villagesday prior to the fixed date for examination in order to had the prevalence below 2.3%. LS and onchocercalmobilize people for the survey and free treatment. nodules were mostly noticed in individuals within40–69 years of age, highest (48.7%) being in betweenSearch for nodules: For search of nodules, the50–59 years. This same age group accounted forpatients were requested to strip down to the loin. As the20.5% of onchocercal nodules. Correlation analysis toperson stretched his arms above the head, the wholedetermine association between these onchocercalchest region was examined both visually and bymanifestations with age showed significantpalpation. Special attention was given to iliac crestcorrelation (r = 0.45, p < 0.001). The patientsaround knees, ankles, ribs (from and rear), chest wall,complained varying degrees of visual impairment but,shoulders, elbows, wrist and head.only five cases of total blindness were recorded. OneSearch for LS: The search for LS on patients was classical blind case was seen in a 61 years old man inmore specific in location and faster as typical Effa (Okpokwu), who had Leopard skin on both shinsonchocercal depigmentation commonly occurs on and a nodule on the knee. The Etulo area on the otherlower limb of adults above 30 years. Patients were hand had four cases of blindness (two in Ashitenaku,asked to pull up trousers and/or wrappers to the thigh one each in Oglazi and Agbou respectively).region.Skin snip examination in the Etulo area showed 536Skin snip examination: Skin snip parasitological (53.3%) persons were positive for O. volvulusprocedure for identification of microfilariae of O. microfilariae. The prevalence by villages varied with

Epidemiology of onchocercosis in Nigeria39Table I: Determination of the prevalence of onchocercosis in Okpokwu villages by using LS and palpable nodules asdiagnostic methodsVillages No. of persons No. positive No. positive for No. positive for bothexamined LS (%) nodules (%) nodules and LS (%)Ojoga 82 23(28.8) 9(10.9) 5 ( 6.1 )Ai-Ebiega 51 11(21.5) 3(5.8) 2 ( 3.9 )Ipole 49 10(20.4) 2(4.0) -Iwewe 45 3(6.6) 2(4.4) -Obotu 85 12(14.1) 8(9.4) 3 ( 3.5 )Opialu 132 13(9.8) 10(7.5) 4 ( 3.0 )Ollo 72 8(11.1) 5(6.9) 1 (1.3 )Ai-Ohida 72 6(8.3) 2(2.7) 1 (1.3 )Ai-Okpe 80 10(12.5) 3(3.7) -Ogene 102 15(14.7) 3(2.9) 1 (0.9 )Ijege 90 9(10.0) 2(2.2) -Ogege 250 22(14.6) 8(3.2) 1 (0.4 )Effa 92 21(14.6) 10(10.8) 5 (5.4 )Idobe 60 6(10.0) 6(10.0) 2 (3.3 )Oto-Oklenyi 52 6(11.5) 3(5.7) -Odokpo 68 5(7.3) 2(2.9) 1 (1.4 )Ai-Akpa 58 10(17.2) 2(3.8) 3 (5.1 )Aidogodo 418 36(8.6) 15(3.5) 4 (0.9 )Ede 268 22(8.2) 10(3.7) 2 (0.7 )Total 2126 248 (11.6) 105 (4.9) 35 (1.6)Table II: Determination of the prevalence of onchocercosis in Etulo villages by using LS, palpable nodules andskin snip as diagnostic methodsVillages No. of persons No. of positive No. of positive No. positive for No. of positiveexamined LS (%) nodules (%) both LS and skin snip (%)nodules (%)Agbou 64 2(3.1) - - 40(62.5)Agbatala 55 2(3.6) 1(1.8) - 44(80.0)Agia 87 1(1.1) 2(2.3) - 39(44.8)Angwauje 92 2(2.1) - - 44(47.8)Ashitenaku 100 14(14.0) 1(1.0) 1(1.0) 74(74.0)Oglazi 68 5(7.3) 1(1.4) - 46(67.6)Otsaazi 99 9(9.0) 2(2.0) - 59(59.5)Otsafu 47 3(6.3) 1(2.1) - 31(65.9)Ugye 95 2(2.1) 3(3.1) 1(1.05) 31(34.0)Ogurube 94 2(2.1) - - 38(40.4)Otanga 84 2(2.3) - - 29(34.5)Okpashila 120 1(0.8) 1(0.8) - 61(50.8)Total 1005 45 (4.5) 12 (1.2) 2 (0.2) 536 (53.3)

40Edward and Bernardfour villages being meso-endemic (60–89%) while theother eight were hypo-endemic (< 60%; Table 2).Communities with higher prevalence of LS andonchocercal nodule also have proportionately higherprevalence of skin microfilariae. Correlationcoefficient (r) = 0.64 was obtained when skin snipprevalence was compared with RAMs; there was,however, no correlation between microfilarialprevalence and visual impairment (r) = 0.017 in theEtulo area.10%. It is also important to train communities on howto conduct rapid assessment procedures in order toprioritize community eligibility.ACKNOWLEDGEMENTSWe acknowledge with thanks the assistance andsupport of National Onchocercosis ControlProgramme Coordinators for Buruku, Katsina-Alaand Okpokwu LGAs in carrying out the presentstudies.The prevalence of onchocercosis varied significantly REFERENCES(p < 0.05) in the different villages. Others have also Amuta EO and Olusi TA. 2000. Sero-epidemiological study ofreported varying endemicity within same Onchocerca volvulus using eluate of blood collected onbiogeographical zones (Nwoke et al., 1994; Nocks et filter paper. Nigerian Journal of Parasitology 21:33-38.al., 1998). It may be due to differences in duration and Carme B, Samba Y, Ntsoumou MV and Yebakima A. 1993.degree of exposure of members from different Prevalence of depigmentation of skin: a simple and cheapcommunities to bites of infected vectors (Nwoke et al., way to screen for severe endemic onchocercosis in Africa.1994) and immunological factors (Murdock, 1992). Bulletin of the World Health Organization 70: 755-758.Another reason might be topographical differences, Edungbola LD, Babata AL, Asaolu SO, Duke BOI and Connorwhereas Okpokwu area is characterized by hilly DH. 1990. Leopard skin and onchocercosis. Nigerianterrain with many fast flowing streams, the Etulo area Journal of Parasitology 9:77-82.is predominantly flat plain with few fast flowing Edungbola LD, Nwoke BEB, Onwurili COE, Akpa AUC andstreams. Tayo-Mafe M. 1993. Selection of rapid assessmentmethods for community diagnosis of onchocercosis.A total of 479 (89.4%) individuals who had Nigerian Journal of Parasitology 12:45-50.microfilariae in their skin biopsy showed no clinicalGemade EI and Dipeolu OO. 1983. Onchocerciasis in Benuesign of nodules or LS. Many other studies in NigeriaState of Nigeria. II. Prevalence of the disease among the Tivswhere skin snip, LS and palpable nodules were living in the Kwande local government area. Annals ofcombined to determine onchocercosis prevalence Tropical Medicine and Parasitology 77:513-516.revealed similar findings where LS and visibleGemade EI. 1993. Training manual in Rapid Assessmentnodules varied significantly (p < 0.05) with the Methods using nodules and Leopard skin, treatmentcommunity microfilarial rate (Nwoke et al., 1994; schedule based on weight and height and health educationNock et al., 1998). Amuta and Olusi (2000) also for large-scale distribution of Mectizan. Revised andreported high prevalence rate using skin snip in some updated by sight savers international (Nigeria). pp 42.villages where we recorded lower prevalence using LS Murdock ME. 1992. The skin and the immune response inand onchocercal nodules.onchocercosis. Tropical Doctor (Supplementary) 1:44-62.The very low incidence of blindness recorded in both Nock IH, Ripiye P and Galadima M. 1998. Diagnostic value ofstudy sites agreed with Nwoke et al. (1994) and has anodules and Leopard skin in community assessment ofhuman onchocercal endemicity. Nigerian Journal ofcommon observation because rainforest region ofParasitology 19:19-24.Nigeria is endemic for O. volvulus strains that rarelycause blindness unlike to savanna types which are Nwoke BEB, Edungbola LD, Mencias BS, Njoku AJ, AbanobOC, Nwogu, FU et al. 1994. Human onchocercosis in rainmore invasive and more pathogenic to the eyesforest zone of southern-eastern Nigeria. 1: Rapid(Nwoke et al., 1994).assessment methods for community diagnosis in Imo riverbasin. Nigerian Journal of Parasitology 15:7-18.The use of LS and palpable nodules as diagnosticprocedures for onchocercosis are highly desirable as Nwoke BEB, Dozie INS, Gemade EI and Jiya JY. 1998. Thethey are cheap, fast, convenient and highly efficient present status of human onchocercosis in southeasternNigeria using rapid epidemiological mapping (REMO).(Carme et al., 1993; Edungbola et al., 1993; Nwoke etNigerian Journal of Parasitology 19:11-18.al., 1998; Nock et al., 1998). A combination of thesetwo methods as done in this study is of exceptional Wentworth BB. 1988. Diagnostic Procedures for Mitotic andparasitic infections. American Public Health Associationadvantage since it identifies community's endemicity.Publication Inc. Washington DC. 637 pp.There is a need to commence treatment withivermectin in some of the villages investigated, WHO. 1995. Expert Committee Report on Onchocercosis.especially those with LS prevalence of more thanFourth Report, Technical Report Series. 253 pp. Geneva.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 41–44J P DDistribution of iron in plasma, erythrocytes and tissuesof calves with the progression of Theileria annulatainfection1 2N. Sangwan and A. K. Sangwan1Department of Veterinary Biochemistry, CCS Haryana Agricultural University, Hisar.2Department of Veterinary Parasitology, CCS Haryana Agricultural University, Hisar.ABSTRACT. The distribution of iron in plasma, erythrocytes and tissues was studied in relation tothe progression of Theileria annulata infection in cross-bred, 4-6 months old male calves. Group-Ianimals were experimentally infected with Theileria annulata by attaching ten Theileria positiveticks on the ear of each calf and Group-II was kept as a healthy uninfected control. The blood andplasma samples were collected at 0, 10, 15, 20 and 25 days post-infection. The tissue samples of liverand spleen were collected immediately after the death of the infected calves. The ironconcentrations were estimated by using atomic absorption spectrophotometer. The infected calveshad significantly (p < 0.05) low levels of iron in whole blood and erythrocytes, whereas the plasmairon levels did not change much. The liver and spleen iron concentrations increased significantly (p< 0.05) as compared to the normal range reported in the literature. This study suggests that careshould be taken while treating theileriosis cases by not supplementing with iron, but byadministering agents which minimize oxidative damage and help in the better utilization of iron forerythropoiesis.Keywords: cattle, haematology, iron, liver, spleen, Theileria annulataINTRODUCTIONTropical theileriosis is a tick-borne disease of cattle,which causes morbidity and loss of productivity inzebu cattle and lethal disease in European and crossbredstock (Gill et al., 1977; Purnell, 1978).Theileriosis is characterized by fever andlymphoproliferative disorders, which may beassociated with leucopenia, wasting, anaemia andjaundice in susceptible cattle (Preston et al., 1992).The damage to the host is caused by both schizonts inlymphocytes/monocytes and piroplasms inerythrocytes, resulting in lymphadenopathy andCorresponding author: Dr. Nirmal Sangwan, Department ofVeterinary Biochemistry, CCS Haryana Agricultural UniversityHisar-125 004, Haryana, India. E-mail:ns@hau.ernet.inhaemolytic anaemia with icterus. Serum iron has beenfound to be reduced significantly in Friesian cattlenaturally infected with Theileria annulata (Omer etal., 2003), whereas, on the contrary, it increased inHolstein calves infected with T. sergenti;concentrations of non-haem iron and ferritin in liverand spleen were also significantly higher in infectedcalves (Watanabe et al., 1998). Therefore, to knowwhether the animals suffering from theileriosis shouldbe supplemented with iron or not, the present studieswere conducted to estimate the status of iron in blood,plasma, erythrocytes and tissues of T. annulata-infected calves.MATERIAL AND METHODSTwo groups of six cross-bred, 4–6 months old malecalves were de-wormed, sprayed with acaricides and

42Sangwan and Sangwanvaccinated against Foot and Mouth Disease. The initial mean value of 9.88 ± 0.14 (g%) and 30.71 ± 0.63animals were fed concentrate as per National Research (%) to 2.9 ± 0.5 (g%) and 9.0 ± 1.41(%), respectively,Council (1988). Wheat bhoosa (roughage) and green on day 25 post-infection. The clinical andfodder were provided ad libitum. After 15 days, haematological findings of bovine tropical theileriosisGroup-I animals were experimentally infected with T. reported here resembled those documented by otherannulata by attaching ten Theileria positive ticks (five authors (Preston et al., 1992; Sahu et al., 1996; Forsythpairs of females and males) on the ear of each calf, and et al., 1999).the other group was kept as a healthy uninfectedThe infected calves had significantly (p < 0.05) lowcontrol. Infection was monitored by daily clinicallevels of iron in whole blood and erythrocytes,examination (rectal temperature and lymph nodewhereas in plasma, the levels of iron did not changepalpation) and at 2-day intervals, Giemsa-stained thinmuch (Table II). The levels of iron in blood andblood and lymph node biopsy smears were examinederythrocytes reduced significantly (p < 0.05) fromafter the appearance of lymphadenopathy. The day of266.9 ± 7.38 to 67.9 ± 8.37 µg/ml and 384.3 ± 12.46 tothe death of infected calves was recorded. The blood72.7 ± 10.7 µg/ml, respectively, on day 25 postandplasma samples were collected in heparinised andinfection. Generally, the risk of iron inadequacy issterilized glass tubes on 0, 10, 15, 20 and 25 days postassessedby measuring blood hemoglobin and/or theinfection. The tissue samples of liver and spleen werehighly correlated PCV. However, in the study undercollected immediately after the death of infectedreport, the low levels of hemoglobin and PCV did notcalves (four animals). The iron concentrations incorrespond to the low levels of iron in the bodydifferent samples were estimated by using atomicreserves. The liver and spleen iron concentrationsabsorption spectrophotometery (Dunkan, 1976),were found to be 628.8 ± 78.23 [µg/g dry matter (DM)]following acid digestion of organic matter and byand 1647.7 ± 182.53 (µg/g DM), respectively. Theseusing calibration standards made in 0.1N HCl. Thevalues in infected group are much higher than thesamples of blood (1 ml) and plasma ( 2 ml) wererange reported by Georgievskii et al., 1982 i.e.digested separately with 10 ml of digest acid (4:1;180–376 µg/g DM for liver of adult cattle andnitric:perchloric acid ), dried, washed x 2 with 5 ml of200–400 µg/g on fresh basis for spleen, respectivelydeionised water, dried each time and reconstituted(Georgievskii, 1982). Therefore, it is important towith 10 ml and 5 ml of 0.1 N HCl, respectively. Thedistinguish anaemia associated with Theileriatissue samples of liver and spleen ( 0.25 g , dried andinfection from that caused by iron deficiency. Ironground ) were also digested as for plasma and thendeficiency is indicated by low levels of iron in thereconstituted in 10 ml of 0.1 N HCl. Simultaneously,liver, and a marginal band of 150–250 µg/g DM isblanks were also run. The iron concentrations intentatively proposed to separate deficient from normalerythrocytes were calculated by using the followingclaves (Green et al., 1993).formula: trace-element in erythrocytes/ml of blood =whole blood-plasma (1-packed cell volume/100). The high levels in liver and spleen could be attributedHaemoglobin and haematocrit were estimated by either to the increased haemolysis or animals' limitedusing cyanmethaemoglobin and microhaematocrit capacity to excrete iron (Kreutzer and Kirchgessner,methods, respectively (Schalm et al., 1975). The data 1991). If there would have been haemolysis that wouldwere subjected to standard error of means (SE) and have resulted in high plasma iron concentration. But inAnalysis of Variance (ANOVA) for statistical the present study, the plasma iron levels did notsignificance (Snedecor and Cochran, 1967).increase, which point towards the elimination ofRESULTS AND DISCUSSIONinfected erythrocytes by phagocytosis. Also, whilestudying the pathogenesis of anaemia in T. annulataDuring the course of Theileria infection, the calves infection, Hooshmand-Rad (1976) suggested that anshowed fever, anaemia, anorexia, cachexia, diarrhoea, autoimmune reaction was largely responsible for therespiratory distress and recumbancy. The clinical and development of anaemia and postulated that theparasitological findings are recorded in Table I. The production of antibodies was triggered by thedeath of infected calves occurred between 18–27 days development of schizonts; erythrocytic formspost-infection. The haematological responses of apparently were not involved. This contention hascalves are given in Table II. As the disease progressed, been supported by the finding that automarkedfall in hemoglobin and packed cell volume haemagglutinin antibodies were detected only in cases(PCV) were observed. The values for hemoglobin and of theileriosis due to a field or an agamogenous strainPCV were reduced significantly (p < 0.05) from the (lacking erythrocytic forms) but not in premune

Iron distribution in Theileriosis43Table I. Host responses (clinical and parasitological) in calves infected with Theileria annulataClinical observations Parasitological observations ResultThermal reaction Macroscizonts Piroplasms (died onday)Group Calf No. Lymph- Commencement Maximum Appearance Max. Appearance Max.adenopathy (day) (°F) (day) (%) (day) (%)(day)1 4 9 105.5 10 15 13 85 23rd2 5 9 104.8 10 10 13 14 29thI 3 4 9 105.8 10 20 13 25 27th4 4 8 106.0 9 10 11 20 18th5 5 9 104.4 10 25 13 15 19th6 4 8 106.0 10 10 13 20 20thII 7-12 - - - - - - - -Group-I: calves were experimentally infected with Theileria annulata by attaching ten positive ticks on the ear of eachcalf. Group-II:healthy uninfected control.Table II. Estimated values for haemoglobin, packed cell volume and iron in the blood, plasma and erythrocytes of1cross-bred calves infected with Theileria annulataParametersDays post-infection0 10 15 20 25Hb(g/dl)a9.88 ± 0.14b6.03 ± 0.48b5.88 ± 0.41c5.10 ± 0.42d2.90 ± 0.5PCV(%)a30.71 ± 0.63b21.42 ± 0.69b20.60 ± 0.36b20.00 ± 1.88c9.00 ± 1.41Blood(µg/ml)a266.9 ± 7.38b188.5 ± 12.7c165.9 ± 10.31cd151.1 ± 11.77e67.9 ± 8.37Plasma(µg/ml)a2.00 ± 0.05ab1.52 ± 0.21b1.33 ±0.18ab1.64 ± 0.05ab2.03 ± 0.35Erythrocytes(µg/ml of blood)a265.5 ± 7.39b187.3 ± 12.81cb164.8 ± 10.27c149.8 ± 11.83d66.1 ± 8.721Calves were experimentally infected with Theileria annulata by attaching ten positive ticks on the ear of each calf. Hb,hemoglobin; PCV, packed cell volume.2Values with common superscripts do not differ significantly (p < 0.05).splenectomised calves. Only mild anaemia and iron to erythropoietic tissues. At a time when the liverbilirubinaemia occurred in the premune and spleen iron levels are already high, additional ironsplenectomised calves in spite of the high level of supplementation in the Theileria-infected animalsparasite load and it was suggested that the infected could result in increase in the liver iron concentrationerythrocytes may be eliminated by augmented to toxic levels. The liver iron concentration of 1000phagocytosis. During infection, iron is redistributed µg/g DM is considered hepatotoxic (Underwood andby the host in an attempt to deplete the pathogen of iron Suttle, 1999) and sufficient reactive iron may be(Weinberg, 1984). The redistribution of iron by the available to cause peroxidative damage to liver (Kenthost may cause the secondary anaemia by depriving and Bahu, 1979), and this may be the underlying

44Sangwan and Sangwanpathogenic mechanism of peroxidative damage tolipid membranes (Gordeuk et al., 1987). The extent ofinjury depends on the antioxidant status of the animal,particularly its vitamin E status (Omara and Blakeley,1993; Ibrahim et al., 1997). Caeruloplasmin, a coppercontaining protein, has also been reported to provideantioxidant defences by scavenging free-iron andfree-radicals (Saneko et al., 1994).In summation, clinical signs in terms of anaemia, lowlevels of iron in whole blood and erythrocytes and thepresence of high concentrations of iron in liver andspleen suggest that T. annulata can harm its host bydisrupting the normal functioning of liver and spleen,upon which effective protective immune responsesand biochemical mechanisms depend. So, it isimportant not to supplement Theileria-infectedanimals with iron, but rather give them other nutrientswhich help in preventing the oxidative damage (suchas vitamin E) and better utilization of iron forerythropoiesis.ACKNOWLEDGEMENTSThis work was carried out at CCS HaryanaAgricultural University, Regional Research Station,Uchani, Karnal, and was financially supported by theNational Agricultural Research Project of the IndianCouncil of Agricultural Research.REFERENCESDunkan L (Edt.).1976. Chemical Analysis by AtomicAbsorption Spectrophotoscopy. (Third Ed). Lea andFebiger, Philadelphia.Forsyth LMG, Minns FC, Kirvar E, Adamson RE, Hall FR,McOrist S, Brown CGD and Preston PM.1999. Tissuedamage in cattle infected with Theileria annulataaccompanied by metastasis of cytokine-producing, schizontinfected mononuclear phagocytes. Journal of ComparativePathology. 120:39-57.Georgievskii VI, Annenkov BN and Samokin VI (Eds.).1982.Studies in the Agricultural and Food Sciences. MineralNutrition of Animals. Butterworths, London, pp 171-224.Gill BS, Bhattacharyulu Y and Kaur D.1997. Symptoms andpathology of experimental bovine tropical theileriosis(Theileria annulata infection). Annales de Parasitologie.52:597-608.Gordeuk VR, Bacon BR and Brittenham GM. 1987. Ironoverload: Cause and consequences. Annual Review ofNutrition. 7:485-508.Green LE, Berriatua E and Morgan KL.1993. Anaemia inhoused lambs. Research in Veterinary Science. 54:306-311.Ibrahim W, Lee V-S, Ye C-C, Szabo J, Bruckner G and ChowCK. 1997. Oxidative stress and antioxidant status in mouseliver: effects of dietary lipid, vitamin E and iron. Journal ofNutrition. 127:1401-1406.Hooshmand-Rad P.1976. The pathogenesis of anaemia inTheileria annulata infection. Research in VeterinaryScience. 20:324-329.Kent G and Bahu RM. 1979. Iron overload. In: MacSweenRNM, Anthony PP and Schewr PJ (Eds). Pathology of theliver. Churchill Livingstone, Edinburgh, pp 148-163.Kreutzer M and Kirchgessner M.1991. Endogenous ironexcretion: a quantitative means to control iron metabolism.Biology of Trace Element Research. 29:77-92.Naessens J, Grab DJ, Fritisch G.1996. Characterization ofbovine transferring receptor on normal activated andThieleria parva transformed lymphocytes by a newmonoclonal antibody. Veterinary Immunology andImmunopathology. 52:65-76.National Research Council.1988. Nutrient Requirements ofDairy Cattle. Sixth Ed. National Academy of Science.Washington, D.C., USA.Omara FO and Blakeley BR. 1993. Vitamin E is protectiveagainst iron toxicity and iron-induced hepatic vitamin Edepletion in mice. Journal of Nutrition. 123:1649-1655.Omer OH, El-Malik KH, Magzoub M, Mahmoud OM,Haroun EM, Hawas A and Omar HM. 2003. Biochemicalprofiles in Friesian cattle naturally infected with Theileriaannulata in Saudi Arabia. Veterinary ResearchCommunication. 27:15-25.Preston PM, Brown CGD, Bell-Sakyi LJ, Richardson W andSanderson A. 1992. Tropical theileriosis in Bos Taurus andBos indicus calves. Response to infection with graded dosesof sporozoites. Research in Veterinary Science. 53:230-243.Purnell RE. 1978. Theileria annulata as a hazard to cattle in thecountries on the northern Mediterranean littoral. VeterinaryResearch Communication. 2:3-10.Sahu PK, Misra SC, Panda DN and Mohapta M. 1996.Haematological and biochemical alterations in Theileriaannulata infected cross-bred cattle. Indian VeterinaryJournal. 73: 995-997.Saneko EL, Yaroplov AI and Harris ED. 1994. Biologicalfunctions of caeruloplasmin expressed through copperbindingsites. Journal of Trace Elements and ExperimentalMedicine. 7: 69-88.Schalm OW, Jain NC and Caroll EJ (Eds.). 1975. VeterinaryHaematology. Third Ed., Lea and Fabiger, Philadelphia.Snedecor GW and Cochran WJ.1967. Statistical Methods.Seventh Ed. Oxford and IBG Publishing Co., New Delhi.Underwood EJ and Suttle NF. 1999. The Mineral Nutrition ofLivestock. Third Ed., CAB International Publishing Co. p361.Watanabe K, Ozawa M, Ochiai H, Kamohara H, Lijima N,Negita H, Orino K and Yamamoto S. 1998. Changes in ironand ferritin in anaemic calves infected with Theileriasergenti . Journal of Veterinary Medical Science. 60:943-947.Weinberg ED. 1984. Iron withholding: a defence againstinfection and neoplasia. Physiological Reviews. 64:65-102.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 45–52J P DMosquito breeding in riceland agro-ecosystem nearChennai, Tamil Nadu, India1 2J. Ravindran and J. Williams1Integrated Disease Vector Control Project (National Malaria Research Institute), Field Station, Chennai.2P. G. and Research Department of Zoology, Loyola College, Chennai.ABSTRACT. Breeding ecology of mosquitoes was studied in riceland area near Chennai during theperiod 1992-1999. The study area was a transition zone with withdrawal of intense agriculturalactivities to facilitate urbanization. A myriad of mosquitoes including potential vectors of malariaand Japanese encephalitis (JE) were observed breeding in various habitats present. A longitudinalsurvey showed no malaria vector breeding in rice fields. Extensive breeding of Culextritaeniorhynchus (JE vector) was observed. Natural breeding pattern indicated peak anophelineand culicine density during first two weeks after transplantation. Pre- and post-monsoon surveysin irrigation wells indicated breeding of 16 species of mosquitoes. Both Anopheles stephensi and An.culicifacies, the urban and rural malaria vectors, respectively, were encountered breeding in thewells; An. stephensi breeding was predominant. Observations on species-specific habitatpreferences showed An. stephensi to breed extensively in open wells and An. culicifacies inpuddles/bed pools. Cx. tritaeniorhynchus was found breeding in almost all types of habitatssurveyed. In retrospective, the study revealed information on mosquito species breeding in varioushabitats, which may be helpful for optimizing species sanitation.Keywords: breeding habitats, immature stages, vectorsINTRODUCTIONRice is the staple food in India, and its cultivation hasbeen traditionally carried out under differentgeographical and climatic conditions (Singh et al.,1989). Modern irrigation facilities and plant breedingtechnologies have resulted in increased acreage andcultivation of rice crops throughout the year leading toits increased production. Unfortunately, along withincreased production it has also led to widespreadoutbreaks of mosquito borne diseases like malaria andJapanese encephalitis (JE; Lacey and Lacey, 1990).Corresponding author: Dr. K. John Ravindran, IntegratedDisease Vector Control Project (NMRI), 332-A, SpartanSchool Road, Paneer Nagar, Mogappair, Chennai-600 037,Tamil Nadu, India. E-mail: johnravindran@gmail.comFor their breeding, mosquitoes are known to exploitaquatic habitats present in riceland agro-ecosystem.Any change in this ecosystem, either naturally or manmade, tend to alter the biotic community includingmosquitoes, which may be advantageous ordisadvantageous for mankind. Therefore, informationon mosquito fauna and breeding ecology in atransitory environment is imperative for successfulintervention. Ongoing urbanization in agriculturalarea in the outskirts of Chennai, Tamil Nadu, India,resulted in an ecologically transitional region, whichreceived our attention. Information on breedingecology of mosquitoes in this environment may behelpful to ensure preparedness for effective vector anddisease control.

46Ravindran and WilliamsMATERIALS AND METHODStest of independence was performed to establishChennai (longitude: 80° 15'E, latitude: 13° 05'N) isthepreferences towards breeding at various well depths.capital city of the state of Tamil Nadu, India, and is Habitat specific preferences were studied in fewsituated in the northeastern part of the state. Minimum selected habitats namely open wells, bed pools,mean and maximum mean temperatures recorded puddles and hoof-prints present in outskirts of periduring1990–2000 were 19.8° C and 38.5° C, urban localities with or without ongoing agriculturalrespectively. Maximum rainfall is during Northeast activity. Any water stagnation with a surface area ofmonsoon (October–December). The study was2less than 1 m was classified as a puddle and those withundertaken in Chennai metropolitan area in western2 2more than 1 m but less than 50 m as bed pool.outskirts of the city during the period 1992–1999. Random surveys were undertaken and immaturesAgricultural activities have been totally withdrawn in were collected using well nets (wells), ladles (bedareas bordering the city due to ongoing urbanization pools and puddles) and spoons (hoof-prints).and partially withdrawn in areas away from the Interspecific association and index of association inoutskirts of the city. The area represents an ecological relation to breeding habitats of various mosquitotransition zone.vector species were calculated using the method ofCole (1949) and Whittaker and Fairbanks (1958).Studies on mosquito breeding in rice fields wereDuring sampling, immatures collected were countedcarried out in agricultural fields located atinstar-wise. Fourth instar larvae and pupae werePoonamallee and Thirukandalam areas, which are 10brought to laboratory and reared until emergence forkm and 20 km away from the outskirts of Chennai.identification.Observations were carried out from September 1992to September 1993, covering one long-duration and RESULTS AND DISCUSSIONtwo short-duration crops. Five fields were selectedand regularly observed for mosquito breeding and A total of 11 Anopheles, 10 Culex and four Aedespopulation structure on a weekly basis during each species were collected from various breeding habitatscrop. A quadrat with a dimension of 33 x 33 x 20 cm in the study site (Table I). Among important disease2enclosing an area of 0.1m was used as a sampler vectors, An. stephensi and An. culicifacies, the urban(Chandrahas, 1990). Ten samples were taken/plot. and rural malaria vectors, respectively, Cx.Sampling was confined to previously earmarked tritaeniorhynchus, Cx. vishnui and Cx. pseudovishnui,sampling sites, which included four corner, four the potential vectors of JE in peninsular Indian regioncentral and two random sites within 0.5 m from the and Ae. aegypti, vector of dengue were present. In ricebund. During each sampling occasion, height of rice fields, no malaria or filaria vectors were observedplant, depth of standing water and peak noon water breeding. Similar results were obtained at Maduraitemperature were recorded with other information (John Victor and Reuben, 1999) and Pondicherrysuch as fertilizer used.(Chandrahas and Rajagopalan, 1979). Among vectorsof JE, Cx. tritaeniorhynchus was predominant beingIn Irrigation wells, study period covered a pre- encountered during all the 25 weeks of observationmonsoon (August–October) and post-monsoon carried out over a period of three rice growing seasons,(November–February) period during 1998–1999. whereas Cx. vishnui and Cx. pseudovishnui wereObservations were carried out in irrigation wells in observed breeding during seven and one occasionsriceland areas in Vanagaram area near Poonamallee, only. Breeding pattern of Cx. tritaeniorhynchuswhere irrigation wells suitable for the study were differed from observations of Reuben (1971) andfound. The conditions for selection of a well were Rajendran and Reuben (1991) who reported intenseabsence of fishes, no floating vegetation and breeding at the latter stages of plant growth. In Tamilaccessibility for sampling from all sides. Weekly Nadu, JE virus isolations have also been reported inobservations were carried out in ten irrigation wells. A An. subpictus, Cx. infula, Cx. whitmorei and Cx.well net with a 20 cm diameter was used as a sampler. fuscocephala, all of which breed in ricefields (PhilipImmature samples were collected by pulling well nets Samuel et al., 2000). Except Cx. infula and Cx.through a distance of 1 m along the edges of the wall. whitmorei, all other species were found breeding inRepeated sampling in the same area was avoided. rice fields.Depth of water table and peak noon water temperaturewas recorded during each visit. Chi square analysis for The density of immatures during the three cultivation

Mosquito breeding in riceland agro-ecosystem near Chennai47seasons is given in Fig. 1 and 2. Anopheles and Culexdensity was high during first week aftertransplantation. Thereafter, intensity in breedingdecreased. In Culex species, another peak in thedensity of immatures was observed during fourth tosixth week after transplantation. Standing watermaintained for a period of two weeks aftertransplantation provided opportunity for intenseexploitation of the habitat for breeding. Reduction inthe density of immatures after this period, is due toalterations in daily irrigation practices that arecontrolled by various factors like soil texture, acreageand the number of crops raised in a year, erratic powersupply and rainfall. Fields are usually irrigatedimmediately after cessation of standing water tomanage adequate water supply. Such enforcedintermittent irrigation due to lack of sufficientirrigation water has also been reported by Rajagopalanet al. (1990) and Russell et al. (1942) in areas in theadjacent erstwhile South Arcot district. In addition,rice cultivation practices such as deweeding,application of fertilizer and pesticides also reduce thedensity of immatures. In the present study, nopesticides were applied though application of urea as afertilizer was undertaken before and three weeks aftertransplantation.50004500400035003000Density/m 2 250020001500100050000 I II III IV V VI VII VIII IX XWeeksTransplantationAug/Sep Jan/Feb Jun/JulFig. 1. Anopheles immature density in different rice growing seasons.50004500400035003000Density/m250020001500100050000 I II III IV V VI VII VIII IX X2Aug/Sep Jan/Feb Jun/JulTransplantationWeeksFig. 2. Culex immature density in different rice growing seasons.

48Ravindran and WilliamsTable I. Mosquito species observed breeding in different habitats in the study areaSpecies Rice Irrigation Draw wells Puddles Hoof- Bedfields wells prints poolsAnophelinesAn. subpictus + + + + + +An. vagus + + + + + +An. peditaeniatus + - - - - -An. nigerrimus - + + + + +An. barbirostris + + + + + +An. stephensi - + + + + +An. aconitus - + + - - -An. splendidus - + - - - -An. annularis - - + + - +An. pallidus - - - + + +An. culicifacies - + + + + +CulicinesCx. tritaeniorhynchus + + + + + +Cx. vishnui + + - + + -Cx. pseudovishnui + - - - - -Cx. bitaeniorhynchus + - - - - +Cx. gelidus - + + - - -Cx. fuscocephala + - - + - -Cx. (Lutzia) fuscanus + + + + + -Cx. (Lophoceraomyia) sp. - + + - - -Cx. vegans - - - - + -Cx. quinquefasciatus - + + + + +Ae. vittatus - + - - - -Ae. scathophagoides - - - + - -Ae. aegypti - - + - - -Ae. vexans - - - + - -Irrigations wells supported breeding of 15 species of monsoon season and An. culicifacies, An. nigerimus,mosquitoes. An. barbirostris, An. subpictus, An. Cx. vishnui, Cx. (Lophoceraomyia) species and Ae.vagus, Cx. quinquefasciatus and C x . vittatus were found breeding exclusively in posttritaeniorhynchuswere potential breeders and were monsoon season. All other mosquitoes were foundencountered breeding for more than 20 weeks. An. breeding in both seasons. An. stephensi, An.aconitus, An. splendidus, Cx. vishnui, Ae. vittatus, subpictus, An. splendidus, An. Aconitus, Cx.were very poor breeders and were obtained in less than quinquefasciatus and Cx. gelidus showed preferencefive weeks during the course of observation. Among to breed at a well-depth greater than 15 feet, whereasmalaria vectors, An. stephensi was predominant being An. barbirostris, An. vagus, An. nigerrimus, Cx.observed in 18 of the total 30 weeks of observation tritaeniorhynchus and Cx. (Lophoceraomyia) sp. atwhen compared to An. culicifacies, which was shallow levels with depth of less than five feet. Chiobserved breeding only in two weeks and during post- square analysis performed on species obtained at allmonsoon season. Rapid urbanization in rice well depths indicated preference of An. stephensi, An.cultivation area adjoining extended areas may be the barbirostris, An. vagus, Cx. quinquefasciatus and Cx.reason for prolific breeding of An. stephensi in these gelidus to breed at different well depths (Table III).irrigation wells. Seasonal breeding preference in Irrigation wells may act as foci for mosquito breedingirrigation wells was noticed (Table II). An. aconitus during non-monsoon seasons and non-rice cultivationand An. splendidus were found breeding in pre- periods.

Mosquito breeding in riceland agro-ecosystem near Chennai49Table II. Details of the density of immatures and larval emergence in irrigation wellsParticulars Pre-monsoon Monsoon /Post-monsoonImmature density/dip-Anopheles 10.216.6 12.722.2Immature density/dip-Culex 5.843.3 5.323.9Well Depth (Min.-Max.) 1417.3 2.623.2Anophelines-No. emerged 918 1775An. stephensi 210 (22.9) 19 (1.1)An. barbirostris 431 (46.9) 1094 (61.6)An. vagus 34 (3.7) 322 (18.1)An. subpictus 238 (25.9) 285 (16.1)An. aconitus 2 (0.2) 0 (0)An. splendidus 3 (0.3) 0 (0)An. culicifacies 0 (0)2 (0.1)An. nigerrimus 0 (0) 53 (3)Culicines-No. emerged 2544 1282Cx. quinquefasciatus 1584 (62.3) 437 (34.1)Cx. tritaeniorhynchus 531 (20.9) 145 (50.3)Cx. gelidus 253 (9.9) 15 (1.2)Cx. fuscanus 176 (6.9) 34 (2.7)Cx. vishnui 0 (0) 41 (3.2)Cx. (Lophoceraomyia) sp 0 (0) 106(8.3)Ae. vittatus 0 (0) 4 (0.3)(Figures in parenthesis denote percent)Table III. Mosquito breeding at different well depthsParticularsWell depth (ft)< 5 515 > 15 X2No. of wells withmosquito breeding 80 62 14 -AnophelinesAn. stephensi 9 (11.3) 18 (29) 5 (35.7) 8.95*An. barbirostris 60 (86.3) 41 (66.1) 9 (64.3) 9.04*An. vagus 23 (28.8) 7 (11.3) 1 (7.1) 8.25*An. subpictus 19 (23.8) 9 (14.5) 4 (28.6) 2.44An. aconitus 0 (0) 0 (0) 1 (7.1) NPAn. splendidus 0 (0) 0 (0) 1 (7.1) NPAn. culicifacies 1 (1.3) 1 (1.6) 0 (0) NPAn. nigerrimus 12 (15) 1 (1.6) 0 (0) NPCulicinesCx. quinquefasciatus 27 (33.8) 31 (50) 9 (64.3) 6.62*Cx. tritaeniorhynchus 30 (37.5) 20 (32.3) 4 (28.6) 0.67Cx. gelidus 2 (2.5) 8 (12.9) 2 (14.3) 6.27*Cx. fuscanus 8 (10) 10 16.1) 1 (7.1) 1.59Cx. vishnui 0 (0) 1 (1.6) 0 (0) NPCx. (Lophoceraomyia) sp 5 (6.3) 0 (0) 0 (0) NPAe. vittatus 0 (0) 4 (6.5) 0 (0) NP(Figures in parenthesis denote percent; NP - not performed, * p > 0.05)

50Ravindran and WilliamsTable IV. Details of species specific habitat positivity and survey particularsParticulars Domestic wells Bed pools Puddles Hoof-printsNo. surveyed 225 175 275 217No. positive forAnopheles breeding 87 (59) 93 (56) 136 (74) 62 (35)No. positive forCulex breeding 49 (27) 51 (33) 79 (45) 88 (65)Habitat positivity for breeding (%):An. subpictus 1.7 82.1 55.4 40An. vagus 8.5 32.1 32.4 65.7An. nigerrimus 3.4 3.6 10.8 5.7An. barbirostris 11.9 16.1 21.6 3An. stephensi 88.1 1.8 1.4 5.7An. aconitus 1.7 0 0An. annularis 1.7 17.9 4.1 0An. pallidus 0 1.8 5.4 2.9An. culicifacies 5.1 17.9 13.5 8.6Cx. tritaeniorhynchus 77.8 15.1 26.7 13.9Cx. vishnui 0 0 6.7 7.7Cx. bitaeniorhynchus 0 3 0 0Cx. gelidus 3.7 0 4.4 0Cx. fuscocephala 0 0 2.2 0Cx. (Lutzia) fuscanus 3.7 0 6.7 4.6Cx. (Lophoceraomyia) sp. 18..5 0 0 0Cx. vagans 0 0 0 3.1Cx. quinquefasciatus 77.8 15.1 26.7 13.9Ae. scathophagoides 0 0 4.4 0Ae. aegypti 3.7 0 0 0Ae. vexans 0 0 4.4 0(Figures in parenthesis denote the number of habitats where adult mosquitoes emerged)Various mosquitoes sp. found breeding in puddles, bed was most commonly encountered in all breedingpools, hoof-prints and domestic wells are shown in habitats, whereas Cx. pseudovishnui was notTable I. Puddles with rainwater were found to harbour encountered in any of the breeding habitats. Cx.the maximum number of species (16 nos.). Except for vishnui was collected from puddles and hoof-printsAe. scathophagoides, all mosquitoes breeding in only. Reuben (1971) reported breeding of Cx.irrigation wells were also observed breeding in these tritaeniorhynchus and Cx. vishnui in wells, ponds,habitats. An. stephensi was found to breed profusely in ditches, irrigation channels, borrow pits and from ricedomestic wells and An. culicifacies in bed pools and fields (fallow or planted) in North Arcot district ofpuddles (Table IV). Breeding of An. stephensi in bed Tamil Nadu.pools, hoof-prints and puddles is rather unusual butsimilar observations have been reported (Yadav et al.,Interspecific association (Table V) with regard to use1989). Breeding of An. stephensi in hoof-printsof habitats for breeding of vectors (An. culicifacies,with rainwater collections in Chennai outskirts hasAn. stephensi, Cx. tritaeniorhynchus and Cx.been reported (Vasanthi, 1996). An. stephensi wasquinquefasciatus) existed between An. stephensi andpredominant breeder in domestic wells, An.An. culicifacies, and Cx. quinquefasciatus and Cx.subpictus in bed pools and puddles and An. vagus intritaeniorhynchus in bed pools, indicating bed pools tohoof-prints. Cx. tritaeniorhynchus, among JE vectorsbe a very favourable source where all vector speciesof various diseases co-existed. In canal irrigated area

Mosquito breeding in riceland agro-ecosystem near Chennai51Table V. Interspecific associations among important vector mosquitoes in study areaHabitat Species Interspecific Index ofassociation (C ) association (I)Domestic An. stephensi An. culicifacies - 0.008 ± 0.314 -0.970well An. stephensi - Cx. quinquefasciatus -0.060 ± 0.146 -An. stephensi - Cx. tritaeniorhynchus -0.093 ± 0.167 -An. culicifacies - Cx. quinquefasciatus 0.036 ± 0.377 -An. culicifacies - Cx. tritaeniorhynchus 0.75 ± 1.445 -Cx. tritaeniorhynchus - Cx. quinquefasciatus 0.012 ± 0.071 -0.860Bed Pools An. stephensi An. culicifacies 1.000 ± 0.393 -0.954An. stephensi - Cx. quinquefasciatus -0.833 ± 3.690 -An. stephensi - Cx. tritaeniorhynchus -0.970 ± 1.259 -An. culicifacies - Cx. quinquefasciatus 0.035 ± 0.079 -An. culicifacies - Cx. tritaeniorhynchus 0.737 ± 0.372 -Cx. tritaeniorhynchus - Cx. quinquefasciatus 0.461 ± 1.526 -0.848Puddles An. stephensi An. culicifacies -0.909 ± 2.795 -1.000An. stephensi - Cx. quinquefasciatus -0.923 ± 2.519 -An. stephensi - Cx. tritaeniorhynchus -1.000 ± 0.786 -An. culicifacies - Cx. quinquefasciatus -0.462 ± 0.64 -An. culicifacies - Cx. tritaeniorhynchus -0.420 ± 0.370 -Cx. tritaeniorhynchus - Cx. quinquefasciatus 0.180 ± 0.216 -0.505Hoof-Prints An. stephensi An. culicifacies 0.600 ± 3.561 -1.000An. stephensi - Cx. quinquefasciatus 0.818 ± 1.98 -An. stephensi - Cx. tritaeniorhynchus 0.111 ± 0.616 -An. culicifacies - Cx. quinquefasciatus 0.75 ± 1.591 -An. culicifacies - Cx. tritaeniorhynchus 0.938 ± 0.507 -Cx. tritaeniorhynchus - Cx. quinquefasciatus 0.205 ± 0.277 -0.762ABin Gujarat, An. culicifacies was positively associated surveyed. Intensive exploitation of rice fieldswith An. stephensi only in paddy fields (Bhatt et al., throughout the rice growing season for breeding by1990). Negative association was noticed in puddles, this species is a cause of concern. However, naturalwhich was also similar to present findings. Likewise, enforced intermittent irrigation due to acute waterin domestic wells a positive association existed shortage and withdrawal of rice cultivation maybetween An. culicifacies, Cx. quinquefasciatus and relatively contribute towards decreased risk inCx. tritaeniorhynchus. In puddles and hoof-prints, the outbreak of JE in this area. Investigations on mosquitocoexistence of vectors was relatively low.breeding in habitats in other extended areas mayprovide information on other species that may bePlanned and systematic extension of Chennai city isprevalent in areas adjoining the city. Due to theunderway resulting in rapid environmental changes.prevalence of disease factor and high labourBoth An. stephensi and An. culicifacies have beenmovement and activity, careful monitoring isobserved in extended regions of the city. An.indispensable for prevention of disease outbreaks.stephensi, responsible for intense malariaBioenvironmental control, found to be successful intransmission in Chennai dominated indicating speciesmany areas, can be planned and adopted in thisreplacement on account of environmental change. Cx.transition region before complete urbanization totritaeniorhynchus, among potential vectors of JE wasachieve effective species sanitation.ubiquitous and was found to breed in all habitats

52Ravindran and WilliamsACKNOWLEDGEMENTSLacey LA and Lacey CM. 1990. The medical importance ofrice land mosquitoes and their control using alternatives toThe authors wish to express their sincere thanks to Dr. chemical insecticides. J Amer Mosq Contr Assoc 6:1-93.V. P. Sharma and Dr. Sarala K. Subbarao, formerPhilip Samuel P, Hiriyan J and Gajana A. 2000. JapaneseDirectors of National Institute of Malaria Research encephalitis virus infection in mosquitoes and its(NMRI), Prof. Dr. A. P. Dash, Director, NMRI and Shri epidemiological implications. ICMR Bulletin 30:No.4.Alex Eapen, Officer-in-charge, Integrated DiseaseRajagopalan P K, Das PK, Panicker KN, Reuben R,Vector Control Project (IDVC; NIMR), Field Station,Raghunatha Rao D, Self LS and Lines JD. 1990.Chennai, for encouragement and facilities provided. Environment and water management for mosquito control.The authors also thank Dr. R. C. Dhiman, Deputy In: Appropriate Technology in Vector Control. C F CurtisDirector, NMRI, for his critical review of the (Ed.) CRC Press, USA. pp 121-138.manuscript. The technical assistance rendered by staff Rajendran R and Reuben R. 1991. Evaluation of the waterof IDVC (NIMR), Field Station, Chennai, is also fern Azolla microphylla for mosquito populationgratefully acknowledged.management in the rice-land agro-ecosystem of south IndiaMed & Vet Entomol 5:229-310.REFERENCESReuben R. 1971. Studies on the mosquitoes of North ArcotBhatt RN, Sharma, RC and Kohli VK. 1990. InterspecificDistrict, Madras State, India. Part 5: Breeding places of theassociations among anophelines in different breedingCulex vishnui group of species. J Med Ent 8:363-366.habitats of Kheda district Gujarat: Part ICanal Irrigatedarea. Indian J Malariol 27:167-172.Russell P F, Knipe FW and Rao HR. 1942. On the intermittentirrigation of rice fields to control malaria in South India. JChandrahas RK. 1990. Host parasite interaction in ricefieldsMal Inst India 4:321-340.and feasibility of mosquito control in urban areas. Ph. D.thesis, University of Berhampur, Orissa.Singh Neeru, Singh OP and Soan V. 1989. Mosquito breedingin rice fields and its role in malaria transmission in MandlaChandrahas RK and Rajagopalan P K. 1979. Mosquitodistrict, M. P. Indian J Malariol 26:191-198.breeding and the natural parasitism of larvae by a fungus,Coelomomyces a n d a m e r m i t h i d n e m a t o d e , Vasanthi V. 1996. Field and laboratory studies on selectedRomanomermis, in paddy fields in Pondicherry. Indian J ecological and behavioural aspects of variants of AnophelesMed Res 69:63-70.stephensi Liston from South India. Ph. D. thesis, Universityof Madras, Chennai.Cole LC. 1949. Measurements of interspecific association.Ecology, 30:411-424.Whittaker R H and Fairbanks CW. 1958. A study of planktoncopepod communities on the Columbian basin,Dev V. 1994. Breeding habitats of anopheline mosquitoes inSoutheastern Washington. Ecology 39:46-65.Assam. Indian J Malariol 31: 31-34.Yadav R S, Sharma RC, Bhatt RM and Sharma VP. 1989.John Victor T and Reuben R. 1999. Population dynamics ofStudies on the Anopheline fauna of Kheda district andmosquito immatures and the succession in abundance ofspecies-specific breeding habitats. Indian J Malariol 26:65-aquatic insects in rice fields in Madurai, South India. Indian74.J Malariol 36:19-32.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 53–57J P DRandom amplified polymorphic DNA of Trichomonasvaginalis isolates from Tarbiz, Iran1 1 2 2 2R. Jamali, B. Zareikar , A. Kazemi , M. Asgharzadeh , S. Yousefee , R. Estakhri , S. Montazer and A.1Ghazanchaei12Tarbiz University of Medical Science, Faculty of Medicine, Parasitology Department.Tarbiz University of Medical Sciences, Faculty of Medicine.ABSTRACT. Trichomonas vaginalis, the causative agent of human trichomoniasis, is the mostcommon nonviral sexually transmitted disease. The infection may be asymptomatic or may causesevere vaginitis and cervicitis in women. Despite its high prevalence, little is known about its geneticvariability and factors leading to asymptomatic infections. The random amplified polymorphicDNA (RAPD) technique is a simple method to detect DNA polymorphism. RAPD was performed byusing four different random primers (OPD1, OPD2, OPD3 and OPD5) for the typing of 120 isolatesof T. vaginalis from Tarbiz. Phylogenetic analysis was performed using SPSS program, anddendrogram with two distinct clusters was constructed. The asymptomatic isolates tended to form acluster, separate from symptomatic isolates. Further studies for better understanding therelationship are suggested.Keywords: polymorphism, random amplified polymorphic DNA, Trichomonas vaginalisINTRODUCTIONamplification assays are highly desirable alternativesto culturing, having both sensitivity and specificity forTrichomonas vaginalis is a prevalent vaginal detecting T. vaginalis DNA (Schee et al., 1999). Thepathogen, affecting 180 million persons worldwide random amplified polymorphic DNA (RAPD)annually (Wang 2000). Evidence from published technique represents as an efficient tool for the studystudies exists that T. vaginalis is independently of genetic polymorphism of DNA. It involves theassociated with a variety of adverse health amplification of random segments of genomic DNAconsequences in both women and men, including by polymerase chain reaction (PCR) using short singleincreased human immunodeficiency virus (HIV) primers of arbitrary sequences (Fraga et al., 2002).transmission, infertility, cervical intraepithelial Different studies suggest that RAPD providesneoplasia (CIN) development in women, and powerful markers to analyze the genetic diversity in T.nongonoccocal urethritis and chronic prostatitis in vaginalis (Rojas et al., 2004; Kaul et al., 2004;men (Jane and Edward, 2003; Soper, 2004).Vanacova et al., 1997). The aim of this study was toDetection of T. vaginalis has traditionally relied ongenetically characterize by RAPD a collection of T.wet-mount microscopy or culture. These methods arevaginalis isolates from patients with clinical signs andhighly specific but lack sensitivity. Nucleic acidsymptoms.Corresponding author: Dr. Rasoul Jamali, ParasitologyDepartment, Faculty of Medicine, Tarbiz University of MedicalSciences, Daneshgah St, Tarbiz, Iran. Telefax : 0411-3364665,E-mail: rasooljamali@yahoo.comMATERIALS AND METHODSA total of 2630 women visiting the health care centersof Tarbiz with and without symptoms of T. vaginalis

54 Jamali et al.(vaginal discharge, itching, dysuria and dyspareunia) asymptomatic patients, axenically. DNA extractedwere selected and examined for the presence of T. from these isolates was subjected to RAPD analysisvaginalis. From each individual two samples were and amplified with 4 different random primerscollected from the posterior vaginal fornix by using (Fig. 1a-d) show the RAPD patterns obtained with thetwo sterile cotton swabs. First swab was used for wet primers used. All the primers provided distinctmount preparation and the second one was used to patterns. For each primer, the banding pattern wasinoculate the Kupferberg medium (Quelab scored as presence (1) or absence (0) for each isolateLaboratories, Canada). Culture tubes were incubated and matrix table was constructed by SPSS 11.0at 37° C up to 7 days, and examined microscopically software. Dendrogram was built based on RAPD-PCRon days 2, 3, 5 and 7 after inoculation.results for each primer, and for four primers, usingWards method and SPSS 11.0 program (Fig. 2). TheDNA extraction: Log phase T. vaginalis cultures wereisolates with similar banding pattern were assigned aswashed with phosphate buffered saline (pH 7.4) anda single type. OPD1 had the least typing ability as itthe cell pellet was suspended in 400 µl TE (10 mM gave 32 types for typing of 120 strains, whereas OPD5Tris, 1 mM EDTA) buffer (pH 8). To this suspension, 5 had the highest typing ability that gave 58 types. OPD2µl proteinase K (20 mg/ml) and 60 µl of 10% sodium gave 56 and OPD3 gave 43 types. A total of 62dodecyl sulphate solutions were added, and incubated different types were obtained from 120 T. vaginalisovernight at 55-65° C. Following incubation, 100 µl of specimens analyzed. There was one cluster consistingNaCl and 80 µl of pre-warmed (at 65° C)CTAB/NaCl of four patients, seven with three patients, 41 with twosolution were added, vortexed well and incubated at patients and 13 with one patient. According to the tree,65° C for 10 min. DNA was cleaned by adding 700 µl the isolates fell into two major groups (theof chloroform-isoamyl alcohol (24:1) solution and classification results based on discriminant analysisvortoxed for 20 s, and precipitated by 1ml of cold are shown in Table II). The upper branch consisted ofethanol (70%) and centrifugation at 12,000 x g for 5 65 isolates out of which 24 were from symptomaticmin (×2) at 10° C. Finally, after air-drying, the DNA patients and 41 isolates belonged to asymptomaticpellet was dissolved in 50-100 µl TE buffer (pH 8). patients. The lower branch of tree consisted of 55isolates, 50 of them from symptomatic patients andRAPD PCR: Four different 10 base pair primers were only five from asymptomatic ones. The 11 isolates thatused for RAPD analysis (their sequence is shown in were from patients with the history of treatmentTable I; Snipes et al., 2000). The DNA amplification failure, showed a scattered format in the tree (isolateswas performed at final volume of 25 µl containing: 2.5 99, 83, 84, 70, 107, 27, 14, 7, 74, 9 and 36).µl of 10 x PCR reaction buffer (500 mM KCl and 200Table I. The sequence of four primers used for RAPDmM Tris-HCl, pH 8.4), 1.25 µl MgCl 2 (50 mM), 1 µl ofanalysiseach primer (Cinnagen, Iran), 0.5 µl of mixed dNTP(10 mM), 4 µl of template DNA, 15.35 µl of doubleSize (mer) Squence (5' to 3')distilled water and 0.4 µl of Taq DNA polymerase (5OPD 10 ACCgCgAAggunit/µl; Cinnagen, Iran). Negative controls for each of OPD2 10 ggACCCAACCfour primers used contained all components except OPD3 10 gTCgCCgTCAtemplate DNA. The amplification protocol consisted OPD5 10 TgAgCggACAof an initial denaturation step at 94° C for 5 minfollowed by 40 cycle's repetitions of 1 min at 94° C, 1 Table II. Classification result based on discriminantamin at 36° C and 2 min at 72° C. The final cycle “the analysisextension step” was of 15 min at 72° C. The PCRPredicted groupproducts were analyzed by electrophoresis in 1.2%membershipagarose gel in TBE buffer. The gels were then stainedwith ethidium bromide (0.5 µg/ml) and visualizedWard method 1 2 Totalunder the UV transilluminator.Original count 1 62 3 65RESULT2 3 52 55Overall, 4.6% (120/2630) of specimens yielded apositive T. vaginalis culture. Seventy four isolateswere obtained from symptomatic patients and 46 from% 1 95.4 4.6 100.02 5.5 94.5 100.0a. 95% of original grouped cases correctly classified.

Random amplified polymorphic DNA analysis551 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Fig. 1-a: RAPD banding pattern of T. vaginalis isolates using OPD1 primerLane 1: Size marker 100 bp DNA ladderLane 2-23: Banding pattern of T. vaginalis isolates No.6 - 27Lane 24: Negative controlLane 25: Size marker lambda DNA/EcoRI+HindIII1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Fig. 1-b: RAPD banding pattern of T. vaginalis isolates using OPD2 primerLane 1: Size marker 100 bp DNA ladderLane 2-29: Banding pattern of T. vaginalis isolates No.75-102Lane 30: Size marker lambda DNA/EcoRI+HindIII1 2 3 4 5 6 7 8 9 10 11 12 13 14Fig. 1-c: RAPD banding pattern of T. vaginalis isolates using OPD3 primerLane 1: Size marker lambda DNA/EcoRI+HindIIILane 2-13: Banding pattern of T. vaginalis isolates No.52-63Lane 14: Size marker 100 bp DNA ladder

56Jamali et al.1 2 3 4 5 6 7 8 9 10 11 12 13 14Fig. 1-d: RAPD banding pattern of T. vaginalis isolates using OPD5 primerLane 1: Size marker lambda DNA/EcoRI+HindIIILane 2-13: Banding pattern of T. vaginalis isolates No.52-63Lane 14: Size marker 100 bp DNA ladderDISCUSSIONTrichomoniasis is an important sexually transmitteddisease which may manifest with a wide range ofsymptoms ranging from an asymptomaticpresentation to severe sequel. It is `not clear yet as towhy only a proportion of individuals infected with T.vaginalis become symptomatic, whereas the restothers remain asymptomatic. Strain variation and hostfactors may play a role in leading to symptomatic orasymptomatic infections (Kaul et al., 2004). Recentstudies have shown the ability of DNA fingerprintingtechniques in differentiating strains of variousorganisms (Tibayrenc, 1998). Vanacova et al. (1997)for the first time used RAPD technique forphylogenetic analysis of T. vaginalis and found it auseful method in epidemiological analysis. Theirresults suggested a concordance between the geneticmarkers with resistance to metronidazole and clinicalfindings, but they found no concordance with thepresence of T. vaginalis virus (TVV) and the virulenceof strains.association between genetic polymorphism oforganism and its clinical characters. Their resultsemphasize that the severity of infection depends on thegenetic type of T. vaginalis involved.In the present study, we used RAPD method for thegenetic analysis of 120 clinical isolates of T. vaginalis,and we investigated the association of T. vaginalisgenetic polymorphism and its clinical classification assymptomatic or asymptomatic. In conclusion, ourresults show that the isolates from asymptomaticpatients tend to form a distinct cluster separate fromsymptomatic isolates, and that T. vaginalis isolatesfrom patients with or without symptoms aregenetically different. Further studies are necessary tobetter understand the relationship between geneticmarkers and the pathogenicity of the organism.REFERENCESFraga J, Rojas L, Sariego I and Sarria CA. 2002. Optimizationof random amplified polymorphic DNA technique for its usein genetic studies of Tricomonas vaginalis isolates. InfectGenet E 2: 73-75.Hample et al. (2001) assayed the relationship between Hample V, Vanacova S, Kulda J and Flegr J. 2001.20 strains of T. vaginalis from eight countries using Concordance between genetic relatedness and phenotypicRAPD analysis, and they found that the phylogenic similarities of Trichomonas vaginalis strains. BMC Evoltree reflects the pattern of virulence, geographic origin Biol 48:1-11.or infection by TVV. Rojas et al. (2004) used RAPD Jane RS and Edward W. 2003. High rate of Trichomonastechnique in 40 isolates of T. vaginalis to find an vaginalis among men attending a sexually transmitted

Random amplified polymorphic DNA analysis57Rescaled Distance cluster CombineCASELabel Num0 5 10 15 20 25899091396832115991011009498959693808387102888182841111135511910645701031101041051091071071141204661116971122829244460271215212142610830667149863437118111334675101659172181596777626964587273745456766375485150235738404142472543657879936853353313519202292disease clinic: implications for screening and urethritismanagement. J Infect Dis 188: 465-468.Kaul P, Gupta I, Sehgal R and Malla N. 2004. Trichomonasvaginalis: random amplified polymorphic DNA analysis ofisolates from symptomatic and asymptomatic women inIndia. Prasitol Int 53: 255-268.Rojas L, Fraga J and Seriego I. 2004. Genetic variabilitybetween Trichomonas vaginalis isolates and correlationwith clinical presentation. Infect Genet E 4:53-58.Schee C, Belkum A, Zwijgers L, Brugge E, Oneill EL, LuijedjkA et al.1999. Improved diagnosis of Trichomonas vaginalisinfection by PCR using vaginal swabs and urine specimencompared to diagnosis by wet mount microscopy, cultureand fluorescent staining. J Cli Microbiol 37:4127-4130.Snipes LJ, Gamard PM, Narcisi EM, Ben Beard C, Lehman Tand Secor EW. 2000. Molecular epidemiology ofmetronidazole resistance in a population of Trichomonasvaginalis clinical isolates. 38: 3004-3009.Soper D. 2004. Trichomoniasis:under control orundercontrolled. Am J Obstet Gynecol 190: 281-290.Tibayrenc M. 1998. Beyond strain typing and molecularepidemiology: integrated genetic epidemiology ofinfectious disease. Parasitol Today 14: 323-329.Vanacva S, Tachezy J and flegr J. 1997. Characterization oftrichomonad species and strains by PCR fingerprinting. JEukayot Microbiol 44: 545-552.Wang J. 2000. Trichomoniasis. Prim Care Update Ob/Gyns7:148-153.Fig. 2. Dendrogram for 120 isolates ofTrichomonas vaginalis based onRAPD-PCR data.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 58–63J P DTwo new species of Trypanosoma from freshwater fish(Heteropneustes fossilis and Channa punctatus) fromBareilly, India1D. K. Gupta, N. Gupta and R. GangwarDepartment of Zoology, Bareilly College, Bareilly.1Department of Animal Science, M. J. P. Rohilkhand University, Bareilly.ABSTRACT. Haematological examination of fresh water fish Channa punctatus (115 specimens)and Heteropneustes fossilis (163 specimens) revealed 10.6% and 14.6% infection, respectively, ofdimorphic species of Trypansoma. Both in the former and latter hosts, the parasite concentrationindex was 112.4 and 110.5 Trypanosomes/100 RBCs, respectively. The flagellate from C. punctatus ischaracterized by its dimorphic nature [small (18-33 µm) and large (30-52 µm) forms], and wasfound to be distinct from the earlier reported species in total body length, flagellar length,kinetoplast dimensions and granulation pattern. The trypanosome species from H. fossilis was alsoobserved to be dimorphic [small (23.5-30.5 µm) and large (33.1-43.8 µm) forms], and differencesexisted in cell breadth, nuclear size, flagellar length and cytomorphological features from theearlier recorded species. These two dimorphic species have been described herein as Trypanosomasaulii n. sp and Trypanosoma heteropneusti n. sp from C. punctatus and H. fossilis, respectively.Keywords: Channa, dimorphic, division, Heteropneustes, TrypanosomaINTRODUCTIONThe freshwater fish, Channa punctatus andHeteropneustes fossilis have served as favourablehosts of Trypanosoma: T. ophiocephali Pearse, 1933;T. striati Qadri, 1955; T. punctati Hasan and Qasim,1962; T. elongatus Raychaudhuri and Misra, 1973; T.bareilliana Gupta et al., 1987 and T. rohilkhandaeGupta and Saraswat, 1991 have been reported fromvarious species of Channa. The dimorphic speciesrecorded are T. gachuii Misra et al., 1973 from C.gachua and T. aligaricus Gupta and Jairajpuri, 1982afrom C. punctatus. On the other hand, T. saccobranchiCastellani and Willey, 1905; T. danilewskyi Qadri,1962; T. singhii Gupta and Jairajpuri, 1981; T.Corresponding author: Prof. Neelima Gupta, Department ofAnimal Science, M.J.P. Rohilkhand University, Bareilly 243006, U.P., India. E-mail: guptagrawal@rediffmail.comkargenensis Gupta and Gupta, 1994 and T. karelensisGupta et al., 2001 have been recorded from H. fossilis;however, T. mukundi Raychaudhuri and Misra, 1973 isthe only dimorphic species. Herein, two new speciesof Trypanosoma have been recorded from C.punctatus and H. fossilis.MATERIALS AND METHODSLive specimens of C. punctatus and H. fossilis,collected from fresh waters of Bareilly weretransported to the laboratory and maintained inseparate aquaria under optimum conditions of foodand aeration. Blood was collected from their caudalvein and examined immediately (hanging droppreparation and micro-haematocrit) for the presenceof parasites. When positive, smears were made, airdried, fixed in methanol and stained in Leishman +phosphate buffer (pH 6.6) in the ratio of 1:7 for 40 min.

Two new species of Trypanosoma59After thorough washing, the films were air dried, Kinetoplast: Well developed structure and takes deepmounted in DPX, sealed and observed and stain upon fixation. Shape may be conical or spherical.photographed at 1000 x magnification under LEICA Division of the kinetoplast not observed.DMLB photoautomat. Camera lucida drawings weremade for statistical measurements of the parasites.Flagellum and undulating membrane: Flagellumoriginates from kinetoplast, runs anteriorly borderingNuclear index (NI), flagellar index (FI) and the undulating membrane and finally extends beyondkinetoplast index (KI) were calculated as follows: body as a free flagellum. Flagellum well-developed inNI = PN/NA; FI = CL/FL; KI = PN/KNlarger forms than smaller forms. Undulatingmembrane conspicuous and takes a light stain. Itwhere PN = posterior end to nucleus ; NA = anterior extends throughout length of cell body. In smallerend to nucleus ; CL = body length ; FL = flagellar forms, it has about 1-2 folds, whereas in larger forms itlength and KN = kinetoplast to nucleus.has 1-3 folds. Width of undulating membrane greaterin larger forms.RESULTSIndicesTrypanosoma saulii n.sp. (Fig. 1 A-D; Table 1)FI = 1.92Host - Channa punctatus Bloch KI = 1.72Locality - Bans Mandi, Bareilly, India NI = 1.62Percentage ofinfection/intensity - 10.6%/1-12.4DISCUSSIONTrypanosomes/100 RBCsThe genus Channa (=Ophiocephalus) has, from time(n = 115)to time, been reported as a suitable host for protozoanThe live parasite in hanging drop preparation showed parasites (Haldar and Mukherjee, 1979). Variousan active wriggling movement amongst the blood trypanosome species have been put on record and acorpuscles. Out of the infected fish, 6.4% werecomparison indicates that T. ophicephali, T. punctatimoderately infected (parasitaemia 5-12.4and T. elongatus are monomorphic forms and,Trypanosomes/100 RBCs), the remaining had scantinfection (parasitaemia 1-5 Trypanosomes/100ARBCs). In stained smears, two forms of the parasiteBcould be differentiated : minuta and magna.C1. MorphologyDShape: Trypanomastigote elongated, anterior andposterior ends tapering, the latter may assume a beaklikeappearance. Configuration of parasite usually ofletter 'C', sometimes elongated 'S' and in rare cases,almost a straight line. Posterior end extremity in both(small and large) cases, more or less rounded.Cytoplasm: Granular cytoplasm stains lightly. In thesmaller forms, the granules more concentrated in theanterior region of the body commencing from justanterior tip of the nucleus to approximately half thedistance from their commencement to the origin of theflagellum. In large forms, granules occur along bothsides of nucleus.Nucleus: Deep red stained nucleus oblong or beanshapedin middle of cell body, occupying almost entirecell breadth.EIGFig. 1. A-D T. saulii n. sp. (A, B: minuta froms; C, D: magnaforms), E-K T. heteropneusti n. sp. (E, F: smaller forms; G, H:larger forms; I-K: diving forms)J10 µmKFH

60 Gupta et al.therefore, the present form is distinct from them. The infected fish, 4.2% were moderately infectedpresent species also can not be compared to T. striati, (parasitaemia examined 5-10.5 trypanosomes/100T. channai and T. bareilliana which are polymorphic RBCs) and the remaining had scant infectionspecies.(parasitaemia 1-5 trypanosomes/100 RBCs). Instained smears, the smaller and larger forms could beThe dimorphic species recorded from C. punctatus areclearly identified showing dimorphism.T. gachuii and T. aligaricus (Table I) and the presentform warrants comparison with these species. A MORPHOLOGYcritical examination indicates that the trypanosomedescribed herein is not comparable to both of these Shape: 'C' or 'S' shaped body somewhat elongated butspecies in the total body length (CF) as the present usually takes twisted conformation upon fixation.form is a much smaller species, the larger formCytoplasm: Lightly stained granular cytoplasm;approximates the smaller form of the above mentionedgranules more prominent in posterior part of parasite.species. Again, the length of the cell body of theVacuoles were absent.parasites is also not comparable as also the flagellarlength (FL). Moreover, the present form is also not Nucleus: Deeply stained oval or bean-shaped nucleuscomparable with T. gachuii in breadth of cell body situated in the middle of body. Nucleolus not(CB) although it may be comparable to T. aligaricus. observed.Interestingly, although the parasite is much smallerthan T. gachuii and T. aligaricus but the nuclear length Kinetoplast: Oval-shaped kinetoplast posteriorly(NL) is comparable to T. aligaricus indicating a placed, takes a deep stain upon fixation.relatively larger nuclear size in relation to its totalFlagellum and undulating membrane: Flagellumlength. The kinetoplast dimensions of T. gachuii haveoriginates from kinetoplast. Free-flagellumnot been provided by the authors but the kinetoplast ofmoderately well developed. Light pink stainedthe present form is again comparatively larger than inT. aligaricus. Apart from the above statisticalundulating membrane has 2-4 folds; 1-2 major foldsdifferences, the present parasite also differs from themay also be present.compared species in the granulation and vacuolationpattern.Although two other species have been described fromthe same ecological niche, the present form cannot besynchronized with them due to its dimorphic nature.The above discussion indicates that the present form isdifferent from any species reported either from thesame ecological niche or elsewhere. It is, therefore,proposed that based on the geographical location,morphometrics, cytological pecularities and hoststatus, the species discovered from the blood of C.punctatus collected from Bareilly region, India,should be given a new species status and the name T.saulii n. sp is proposed.Trypanosoma heteropneusti n.sp. (Fig. 1 E-H;Table II)mgmiIasmHost- Heteropneustes fossilisLocality- Local fish market, BareillyPercentage of - 14.6%/1-10.5infection/intensity Trypanosomes/100 RBCs(n = 163)The live parasites showed an active wrigglingmovement amongst the blood corpuscles. Out of theEFig. 2. Photomicrographs of Trypanosoma. A. T. saulii n. sp. (miminutaform; mg - magna form), B-F. T. heteropneusti n.sp. B.dimorphic forms (sm-small form, la - large form), C.differentiating nucleus, D. longitudinal kinetosome division, Eand F. migrating nuclei.

Two new species of Trypanosoma61Indicesforms. Although CL of both parasites are comparablebut conspicuous difference in CB and nuclear size areFI = 1.82evident. Moreover, the flagellar lengths of both theKI = 1.72parasites also differ. The granulation pattern and theNI = 1.16vacuolation of both the parasites cannot be treated atDISCUSSIONpar. It is, therefore, proposed that the parasitediscovered from the blood of H. fossilis collected fromH. fossilis has been considered to be an ideal host for the fresh waters of Bareilly be designated as a newtrypanosomes. A dimorphic form, T. mukundi was species and, accordingly, the name T. heteropneustidiscovered by Raychaudhri and Misra (1973) from the n.sp is proposed to accommodate the parasite with thefresh waters of Kolkata, India existing as “slender” specific characters as given in this account.and “stumpy” forms. Table II shows a comparison ofdimorphic parasites recorded from the test fish. T. DIVISION IN T. heteropneusti N.SP.mukundi Raychaudhuri and Misra (1973) is aSome forms undergoing division showed peculiardimorphic species and the present form warrants afeatures in the blood of H. fossilis. However, only twocritical examination of the said species. The contour offish out of 163 specimens exhibited multiplyingthe parasites differ, the present form being “slender”forms. Division commenced in the nucleus showingand “elongated”, whereas T. mukundi has beenwell differentiated chromatin prior to division (Fig.reported as “slender” and “stumpy” form. The present1I). The two centrally-placed, round to ovoid nucleiforms have therefore contrastingly been described asand the single, undivided kinetoplast was placed“small” (23-30.5 µm) and “large” (33.1-43.8 µm)Table I: Dimorphic trypomastigotes from Channa (= Ophiocephalus; all measurements in µm)Host Parasite O. gachua C. punctatus C. punctatusAuthor(s) T. gachuii T. aligaricus T. saulii : n.sp (N=50)Component Misra et al., 1973 Gupta and Jairajpuri, 1982 Present studyparts/forms*Short Slender Small Large Minuta MagnaCF 36.4 53.9 34.37 54.68 24.0 35.5(34.25-35.5) (53.25-55.3) (18.0-33.0) (30.2-52.0)CL 24.9 42.1 23.50 46.18 18.5 28.2(23.01-25.0) (44.95-46.8) (12.2-24.4) (26.3-36.4)CB 2.3 3.9 1.65 2.45 1.9 2.6(1.5-1.8) (2.1-2.6) (1.0-2.5) (2.0-3.6)FL 11.1 10.9 10.87 8.50 6.6 8.8(10.25-13.5) (7.82-9.4) (4.0-8.2) (8.0-12.0)NL 2.4 2.7 3.00 3.73 3.2 3.7(2.5-3.5) (3.6-3.8) (1.0-6.0) (1.0-6.0)NB 2.2 2.7 1.12 2.02 1.6 2.0(1.0-1.2) (1.8-2.2) (1.0-3.0) (1.0-4.0)KB - - 0.45 0.25 1.2 1.25(0.4-0.5) (0.21-0.3) (0.5-2.0) (0.5-2.0)KL - - 10.5 0.51 1.6 1.9(0.5-1.6) (0.4-0.6) (1.0-2.0) (0.5-2.0)* CB= Breadth of cell body; CF = Total length; CL = Length of cell body; FL = Flagellar length;KB = Kinetoplast breadth; KL = Kinetoplast length, NB = Nuclear breadth; NL = Nuclear length

62 Gupta et al.Table II: Dimorphic trypomastigotes from Heteropneustes fossilis (all measurements in µm)Parasite T. mukundi T. heteropneusti n.spAuthor(s) Raychaudhuri and Misra Present studyLocality 1973, India India (n = 50)ComponentParts/formsSlender Stumpy Small LargeCF 32.5-43.5 22.0-28.0 27.9 37.83(23.5-30.5) (33.1-43.8)CL 24.0-28.5 18.0-23.5 16.7 22.6(14.4-19.0) (20.1-24.2)CB 2.0-2.5 1.9-2.5 1.68 1.40(1.45-1.95) 1.3-1.5)FL 9.0-15.5 4.0-6.5 8.48 16.05(6.95-11.4) (12.05-18.6)NL 2.25-3.75 2.25-3.75 2.64 2.63(1.41-3.35) (1.51-3.30)NB 1.75-2.25 1.75-2.25 1.14 1.23(1.1-1.2) (0.95-1.25)KB 0.75-7.5 0.75 0.93 0.90(0.90-0.95) (0.8-0.95)KL - - 1.18 1.16(1.07-1.35) (0.8-1.35)* Abbreviations same as in Table Iposteriorly with a single, long free-flagellum and a process slowed down by increasing (30° C) ornormal undulating membrane (Fig. 1J). The decreasing (10° C) the temperature (Lom, 1973; Wookinetoplast usually divided subsequent to nuclear et al., 1983; Islam and Woo 1992). Qadri (1955), Dalydivision but in some instances, it was the first to and Degiusti (1971), Khan (1972), Grogl et al. (1980)divide. Interestingly, both longitudinal as well as and Mukerjee and Haldar (1982) reported the absencetransverse division occurred during the division of of divisional stages in the fish blood. Longitudinalkinetoplast although the former was more frequent binary fission in culture form of T. striati (Qadri,(Fig. 2D). The two nuclei now migrated, one in either 1955); binucleated trypomastigotes in T. batrachiarm (Fig. 2E) and the parasite stretched between the Qadri (1962), T. maguri and T. vittati (Tandon andtwo kinetoplasts prior to cytokinesis (Fig. 2F). One Joshi, 1973), T. mrigali (Joshi, 1976) and T. attiiabnormal trypanosome with three kinetoplasts (Fig. (Gupta and Jairajpuri, 1982b) have also been1K), dividing prior to nuclear division, could also be observed.observed which does not fit into the sequence ofdivisional events of trypanosome multiplication and isTwo small trypanosomes possessing two nuclei andapparently an aberrant form.two kinetoplasts (Baker, 1960), and two oppositenuclei and a central kinetoplast (Raychaudhuri andDivision in trypanosomes has been reported rarely. Misra, 1973) have also been put on record. WenyonLaveran and Mesnil (1907) reported absence of (1926) and Lewis and Ball (1981) reported kinetoplastdivision in T. remarki but occasionally observed forms division prior to nuclear, whereas Burreson (1982)with two nuclei in the parva variety. T. danilewskyi recorded a reverse condition in Trypanoplasmadivided at 20° C satisfactorily in gold fish but the bullocki. In T. heteropneusti n.sp, both instances have

Two new species of Trypanosoma63been recorded but prior nuclear division dominatesprior kinetoplast division. The nuclear division in ourspecimens indicate only the transverse division. Thecytokinesis and flagellar division were not observed.ACKNOWLEDGEMENTThanks are expressed to the University GrantCommission, New Delhi, India, for providingfinancial grant in the form of a research project.REFERENCESBaker JR. 1960. Trypanosomes and dactylosomes from theblood of freshwater fish in East Africa. Parasitology. 50 :515 - 526.Burreson EM. 1982. The life cycle of Trypanoplasma bullocki(Zoomastigophorea: Kinetoplastida). Journal ofProtozoology. 29 : 72 - 77.Castellani A and Willey A. 1905. Observations on haematozoain Ceylon. Quarternary Journal of Microscopic Science. 49 :383 - 402.Daly JJ and Degiusti DL. 1971. Trypanosoma catostomi n. sp.from the white sucker Catostomus C. commersoni. Journalof Protozoology.18 : 414 - 417.Grogl M, Marinerlle CJ, Suarez MF, Sanchez NH and Guhl F.1980. Trypanosoma magdalenae n. sp. (Protozoa:Kinetoplastida) from a fresh water teleost, Petenia kraussiiin Columbia. Journal of Parasitology. 66 : 1022 - 1026.Gupta DK and Gupta N. 1994. A comparative study of culturemedia for Trypanosoma kargenensis n. sp. fromHeteropneustes fossilis and its effect on the biochemicalcomposition of fish. In: Proc. Third Asian Fish. Forum.Chou et al. (Eds) A. F. S. Philippines. pp320-323.Gupta DK, Gupta N and Yadav P. 1987. New polymorphictrypanosomes from two freshwater fishes. HimalayanJournal of Environment and Zoology. 1 : 93 - 97.Gupta N, Gupta DK and Saraswat H. 2001. Hypoglycemia inHeteropneustes fossilis parasitized by two new species ofparasites Trypanosoma karelensis n.sp and Myxosomafossilii. n.sp. In: Proceedings of the National Symposium onFish Health, Management and SustainableAquaculture.pp179-186.Gupta N and Jairajpuri DS. 1981. Trypanosoma singhii n. sp.from a fresh water teleost, Heteropneustes fossilis. IndianJournal of Parasitology. 5 : 251 - 253.Gupta N and Jairajpuri DS. 1982a. Trypanosoma aligaricus n.sp. from the freshwater murrel, Ophiocephalus punctatusBloch. Archiv fur Protistenkunde. 125 : 109 - 114.Gupta N and Jairajpuri DS. 1982b. A new polymorphictrypanosome from an Indian freshwater fish Wallago attu(Bl. and Schn.) Rivista di Parassitologia. 43: 287-292.Gupta N and Saraswat H. 1991. Trypanosoma rohilkhandae n.sp. from fresh water teleost fish Channa (= Ophiocephalus)punctatus. Himalayan Journal of Environment and Zoology.5 : 29 -33.Haldar DP and Mukherjee M. 1979. An annolated list ofprotozoan parasites from Ophicephalus punctatus Bloch, acommon food - fish. Archiv fur Protistenkunde. 121 : 392 -400.Hasan R and Qasim SZ. 1962. Trypanosoma punctati n. sp.from the fish Ophiocephalus punctatus Bloch a commonfresh water murrel of India. Zeitschrift fur Parasitenkunde.22 : 118 - 122.Islam AKMN and Woo PTK. 1992. Effects of temperature onthe in vivo and in vitro multiplication of Trypanosomadanilewskyi, Laveran and Mesnil. Folia Parasitologica. 39 :1 - 12.Joshi BD. 1976. Two new species of trypanosomes from freshwater teleosts. Indian Journal of Zootomy. 17 : 5 - 10.Khan RA. 1972. On a Trypanosoma from the Atlantic cod,Gadus morhua. Canadian Journal of Zoology. 50 : 1051 -1054.Laveran A and Mesnil F. 1907. Trypanosomes andTrypanosomiasis. Ist Ed. Bailliere, Tindall and Cox(Translated into English by D. Nabarro) London.Lewis JW and Ball SJ. 1981. Observation on the division ofepimastigotes of Trypanosoma cobitis. Archiv furProtistenkunde. 124 : 337 - 344.Lom J. 1973. Experimental infection of gold fish with bloodflagellates. In : Progress in Protozoology, Proceedings of theInternational Congress in Protozoology. pp 255.Misra KK, Chandra AK and Choudhury A. 1973. Trypanosomagachuii n. sp. from a fresh water teleost fish Ophicephalusgachua Ham. Archiv fur Protistenkunde. 115: 18 - 21.Mukherjee M and Haldar DP. 1982. Host susceptibilityexperiments on a new fish trypanosome in India.Angewandte Parasitology. 23 : 69 - 73.Pearse AS. 1933. Parasites of Siamese fishes and crustaceans.Journal of the Siamese Siena Society of Natural History,Supplement. 9 : 179 - 191.Qadri SS. 1955. The morphology of Trypanosoma striati n. sp.from an Indian freshwater fish. Parasitology. 45 : 79 - 85.Qadri SS. 1962. An experimental study of the life cycle ofTrypanosoma danilewskyi in the leech, Hemiclepsismarginata. Journal of Protozoology. 9 : 254 - 258.Raychaudhuri S and Misra KK. 1973. Two new fishtrypanosomes from India. Archiv fur Protistenkunde. 115 :10 - 17.Tandon RS and Joshi BD. 1973. Studies on the physiopathologyof blood of freshwater fishes infected with two new forms oftrypanosomes. Zeitchrift fur Wissen schaftliche Zoologie.185 : 207 - 221.Wenyon CM. 1926. Protozoology. Bailliere, Tindall and Cox.London. pp1563.Woo PTK, Wehnert DS and Rodgers D. 1983. Thesusceptibility of fishes to haemoflagellates at differentambient temperatures. Parasitology. 87 : 385 - 392.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 64–67J P DImpact of anthelmintic therapy on live weight gain ingastrointestinal nematode-infected goats*A. K. Jayraw and Y. V. RaoteDepartment of Parasitology, College of Veterinary and Animal Sciences, Parbhani.ABSTRACT. A trial was conducted to assess the effect of anthelmintic therapy on live weight gainin gastrointestinal (GI) nematode-infected goats using three anthelmintics, viz. tetramisolehydrochloride, morantel citrate and albendazole. Twenty four goats with heavy natural infection ofStrongyle, Strongyloides papillosus and Trichuris spp. were selected and divided into four groups ofsix animals each. Group-I served as untreated control group, whereas Group-II, III and IV were-1treated with tetramisole hydrochloride @ 15 mg kg body weight (b.w.), morantel citrate @ 5.94 mg-1 -1kg b.w. and albendazole @ 7.5 mg kg b.w., respectively. Animals were weighed at weekly intervalsand their live weight was recorded for a period of 28 days post-treatment (PT). On 28 day PT,overall live weight gain recorded was 9, 7.44 and 8.78% in animals of Group-II, III and IV,respectively, whereas only 3.73% weight gain was recorded in goats of untreated control group. Thenet profit obtained by the end of experimental study was Rs. 206, 138 and 174 in animals of Group-II, III and IV, respectively, as against a profit of Rs. 94 obtained in animals of untreated controlgroup.Keywords: anthelmintic therapy, gastrointestinal nematode, goat, weight gainINTRODUCTIONOwing to the growing demand for high quality animalproteins for human consumption, small ruminantsoccupy a special place as they are extremely efficientin converting the indigestible cellulose andhemicellulose to animal protein. India has apopulation of 120 million goats, which contributegreatly to rural economy (Abraham, 2001). Control ofgastrointestinal (GI) nematodosis is one of the mostserious challenges, as it poses a major constraint in thegrowth of highly profitable animals due to the chronicand insidious nature of parasitism (Sanyal, 1996). In*Part of M.V. Sc. thesis by first author, Marthwada AgriculturalUniversity, Prabhani.Corresponding author: Dr. A. K. Jayraw, Department ofParasitology, Nagpur Veterinary College, Nagpur-440 006,Maharashtra, India.the absence of a potent prophylactic agent,chemotherapy continues to play a vital role against GInematodosis. Therefore, the present investigation wasundertaken to evaluate the live weight gain response ingoats following chemotherapy with commonlyavailable anthelmintics, viz. tetramisolehydrochloride, morantel citrate and albendazole.MATERIALS AND METHODSThe experimental study was conducted at the Collegeof Veterinary and Animal Sciences, Parbhani duringmonsoon. A total of 49 female goats were maintainedunder a semi-intensive system and each animal wasprovided with 250 g concentrate daily. The helminthicburden of each animal was ascertained after countingeggs per gram (EPG) of faeces using Stoll's dilutionmethod (Soulsby, 1982). Out of 49 goats, 1-2 year oldnaturally infected 24 animals were selected, showing

Effect of antihelmenthic therapy on live weight gain65the mean EPG counts of 3783 ± 1106.96; 404.16 ± albendazole (Rs.174) and morantel citrate (Rs. 138)127.54 and 212.5 ± 55.01 for strongyle species, S. treated groups. On the other hand, the net profitpapillosus and Trichuris spp., respectively. The recorded in infected untreated control group wasexperimental animals were equally divided into four Rs. 94 in comparison with the treated groups (Table I).groups (Gr.), where, Gr.-I served as infected, untreatedcontrol group, Gr.-II animals were treated withDISCUSSIONtetramisole hydrochloride (Nilverm soluble powder, GI nematode infection is recognized as a major-1M/S ICI India Ltd., Kolkata) @ 15 mg kg body constraint to small ruminant production worldwide. Itweight (b.w.) and Gr.-III animals with morantel citrate is well known to cause huge economic losses due to(Banminth Tab., M/S Pfizer Ltd., Ahmedabad) @ 5.94 mortality incidental to the severe parasitism and high-1mg kg b.w. The animals in Gr.-IV were drenched with morbidity, reduction and/or spoilage of wool, meatalbendazole (Albomar suspension, M/S Glaxo India and milk production in chronic cases (Chowdhury,-1Ltd., Mumbai) @ 7.5 mg kg b.w. The experimental 1994). The continued presence of GI nematodes isgoats were denied access to water and feed for responsible for anorexia, reduced feed intake,approximately 18 h, prior to recording their live alterations in protein metabolism, low levels ofweight (weekly) using a weigh-bridge. The net profit minerals, depressed activity of some intestinalin terms of rupees (Rs.) was calculated taking into enzymes and diarrhoea (Soulsby, 1982). Theaccount the final difference in body weight of the parasitism is also associated with loss of blood andexperimental goats before and after chemotherapy, plasma proteins into the GI tract (Jayraw and Raote,cost of medication including cost of drug (tetramisole 2004a) thereby lowering the weight gain in untreatedhydrochloride - Rs. 10, morantel citrate - Rs. 29 and control group. Reduced skeletal growth brought aboutalbendazole - Rs. 24) and labour cost (Rs. 60 per head by mineral deficiency affects growth rate, becausefor one day), except for the animals belonging to Gr.-I, skeletal size ultimately determines the capacity ofwhich served as infected untreated control and were growing animal to accumulate muscle (Sykes et al.,not treated with any anthelmintic. The average rate of 1977). In addition, reduced levels of amino acid-1chevon was considered as Rs. 120 kg at Parbhani incorporation in muscle protein results in reduceddistrict of Maharashtra. The data obtained were weight gains (Soulsby,1982). Reduced weight gain inanalysed following standard statistical procedures GI nematode-infected goats was also reported by(Snedecor and Cochran, 1994).Howlader et al. (1997) and Githigia et al. (2001). TheRESULTShigher weight gain recorded in tetramisole treatedanimals is attributable to its higher efficacy againstAnimals were weighed at weekly intervals and their Trichuris spp. and strongyle species when comparedlive weight recorded for a period of 28 days. The mean to efficacy of albendazole and morantel citrate againstEPG counts recorded at 28 days post-treatment were these parasites (Jayraw and Raote, 2004b). Thefound to be 345.84 ± 290.47, 4.165 ± 4.165 and 154.16 present finding is in agreement with Sakhawat et al.± 37.50 for strongyle species, S. papillosus and (1997), who also observed that levamisole treatedTrichuris spp., respectively. A maximum of 9% (mean sheep gained more weight than albendazole and1.97 kg) live weight gain was recorded in tetramisole morantel treated animals. The mean weight gain ofhydrochloride treated animals (Gr.-II) followed by 1.97 kg recorded in the present study corroborates well8.78% enhancement (mean 1.82 kg) in experimental with the findings of Sathianesan and Peter (1972), whogoats belonging to albendazole treated group (Gr.-IV). recorded 1.7 kg weight gain in tetramisole treatedThe morantel citrate treated group (Gr.-III) exhibited goats over 30 days of observation. The 8.78% weight7.44% weight gain (mean 1.56 kg). The lowest weight gain observed in albendazole-treated animals is ingain of 3.73% (mean 0.79 kg) was evident in animals general agreement with Faizal et al. (1999) andof infected untreated group (Gr.-I). The mean daily Githigia et al. (2001). Rajangam and Balchandranweight gain recorded is presented in the descending (1989) earlier reported a mean weight gain of 38.3 g-1 -1-1order as 70 g day in animals of Gr.-II, 65 g day in Gr.- day in morantel citrate treated goats.-1 -1IV, 55 g day in Gr.-III and 28 g day in animals of Gr.- Correspondingly, a mean daily weight gain of 55 g-1I. The net profit obtained at the end of experimental day in morantel treated group was also evident in thestudy was higher (Rs. 206) in tetramisole present study. Das et al. (2004) recorded the per headhydrochloride treated group, when compared with loss of Rs. 351 and 377 in 3-6 and 6-12 months old GI

66 Jayraw and RaoteTable I. Effect of anthelmintic medication on live weight gain in natural GI nematode infection in goatsSl.No.Group 0 day IweekIIweekIIIweekIVweekMeanweightgain (kg)PercentweightgainMeandailyweightgain(g day -1 )Gross profit(Rs.) followingchemotherapy(except Gr.-I)Cost ofmedicationNet profit(Rs.)followingchemotherapy(except Gr.-I)1 I 21.18±4.0121.38±3.721.56±3.4522.05±3.4421.95±3.210.79 3.73 28 94 - 942 II 21.87±1.8522.25±1.8922.79±2.0723.26±1.8923.84±2.211.97 9.00 70 236 30 2063 III 20.95±2.8321.36±2.9621.57±2.421.95±2.422.51±2.181.56 7.44 55 187 49 1384 IV 20.72±3.0821.05±3.0921.73±3.0722.09±3.1622.54±2.971.82 8.78 65 218 44 174Gr.-I: infected untreated control; Gr.-II: treated with tetramisole hydrochloride; Gr.-III: treated with morantel citrate and G r.-IV: treated withalbendazole

Effect of antihelmenthic therapy on live weight gain67nematode-infected Bengal goats, respectively, over aperiod of nine months. Our findings are also consistentwith the findings of above workers, as a net gain of Rs.206 was recoded in tetramisole treated group followedby Rs. 174 and 138 in albendazole and morantel citratetreated groups, respectively. However, the net profitrecorded in infected untreated control group was onlyRs.94, which may be attributed to the worm burden,resulting in weight loss. The effective elimination ofGI nematodes in all treated groups has a direct bearingon the improved feed utilization ratio and gain in bodyweight by preventing blood, as well as, protein lossinto the GI tract. Tetramisole hydrochloride appears tobe superior to albendazole and morantel citrate ineffective elimination of GI nematodes and subsequentimprovement in weight gain.ACKNOWLEDGEMENTSgoats in Kenya. Small Ruminant Research 42: 21-29.Howlader M M R, Capitan S S, Eduardo S L, Roxas N P andSevilla C C.1997. Performance of growing goatsexperimentally infected with stomach worm (Haemonchuscontortus). Asian Australasian Journal of Animal Sciences10: 534-539.Jayraw A K and Raote Y V. 2004a Effect of anthelmintictreatment against gastrointestinal nematodes with a note onhaematology and plasma proteins in goats. Journal ofVeterinary Parasitology 18: 51-54.Jayraw A K and Raote Y V. 2004b Comparative efficacy oftetramisole hydrochloride, morantel citrate and albendazoleagainst gastrointestinal nematodes in goats. Journal ofVeterinary Parasitology 18: 131-134.Rajangam R K and Balchandran S. 1989. Efficacy of morantelcitrate (Banminth- Pfizer) against gastrointestinal parasitesand its effect on body weight gain in stall-fed goats. IndianVeterinary Journal 66: 919 -922.The authors are thankful to the Associate Dean, Sakhawat A, Anwar A H, Hayat B, Zafar I and Hayat C S. 1997.Field evaluation of anthelmintic efficacy of levamisole,College of Veterinary and Animal sciences, Parbhanialbendazole, ivermectin and morantel tartrate againstand Farm Superintendent for providing necessarygastrointestinal nematodes of sheep. Pakistan Veterinaryfacilities to do this work. Journal 17: 114-116.REFERENCESAbraham J. 2001. Processing of meat for enhancing its qualitythand value. Towards Food Security in India, 88 sessionISCA, New Delhi.Chowdhury N. 1994. Helminths of domesticated animals inIndian subcontinent. In: Helminthology. Chowdhury. N andTada. I (Edt.) Narosa and Springer, New Delhi, pp 73-120.Das R, Datta S and Ghosh JD. 2004. Economic impact ofgastrointestinal nematodosis in Bengal goat. FifteenthNational Congress of Indian Association for theAdvancement of Veterinary Parasitology, Pantnagar,Uttaranchal, India, 25 - 28 October.Sanyal P K. 1996. Gastrointestinal parasites and small ruminantproduction in India. Sustainable parasite control in smallruminants. Australian Center of International AgricultureResearch Proceedings Series 74: 109-112.Sathianesan V and Peter C T. 1972. Comparative efficacy ofthiabendazole, Nilverm and Promintic againstgastrointestinal nematodes of goats. Kerala Journal ofVeterinary Sciences 3:143-157.Snedecor G W and Cochran W G. 1994. Statistical Methods. 8ed., Iowa State University Press, Ames, Iowa, pp 135-170.Soulsby E J L. 1982. Helminths, Arthropods and Protozoa ofthDomesticated Animals. 7 Edition, ELBS and BailliereFaizal A C M, Rajapakse R P V J, Jayasinghe S R andTindall, London, pp 239 - 240.Rupasinghe V. 1999. Prevalence of Eimeria spp. and Sykes A R, Coop R L and Angus K W. 1977. The influence ofgastrointestinal nematodes versus weight gains in treated chronic Ostertagia circumcincta infection in the skeleton ofgoats raised in the dry areas of Sri Lanka. Small Ruminant growing sheep. Journal of Comparative Pathology 87:521-Research 34: 21-25. 529.Githigia S M, Thamsborg S M, Munyua W K and Maingi N.2001. Impact of gastroinestinal helminths on production inth

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 68–75J P DUltrastructure, differential density and distributionpattern of polymorphic microtriches in tegument ofStilesia globipunctata infecting Ovis aries (sheep)C. Venkatesh, K. Ramalingam and V. VijayalakshmiP.G. and Research Department of Zoology, Govt. Arts College, Chennai.ABSTRACT. Scanning electron and transmission electron microscopic study of tegument ofStilesia globipunctata revealed a complex pattern of microtrichial brush border. They show a widerange of morphological variations. S. globipunctata shows species specific pattern of papilliform,filiform, spiniform and blade-like microtriches. The scolex shows the presence of papilla-like,spine-like and blade-like microtriches. The immature proglottid region is densely covered byposteriorly directed filiform, spiniform and papilliform microtriches. In mature proglottid region,papilla-like and filament-like microtriches are predominantly seen. The microtriches appear to benon-uniform in density and size. In gravid segments, there is obviously an increasing degree ofdisorder in the arrangement of microtriches. It increases the absorptive surface. It helps to resistthe peristaltic movement of intestine and maintains the parasite in position. Its absence in thegravid region is due to the morphological changes in the tegument and the interaction of luminalenvironment.Keywords: density, distribution, microtriches, polymorphism, Stilesia globipunctataINTRODUCTIONThe external surface organization of the cestodeparasite not only confers protection to the parasite, butalso functions as a metabolically active tissue takingup nutrients from the luminal environment and as anoutlet for elimination of its extracellular secretionsand the excretion phenomenon. It has been noticedthat in cestodes, the basic structure may be comparedwith a gut turned inside out, with the external bodycovering serving the absorptive functions normallyassociated with the intestinal mucosa (Smyth andMcManus, 1989).Corresponding author: Dr. V. Vijaylakshmi, P. G. and ResearchDepartment of Zoology, Govt. Arts college, Men (Autonomous),Nandanam, Chennai - 600 035, Tamil Nadu, India.Ultrastructural studies have shown that the externalbody covering of the adult tapeworm consists of anaked cytoplasmic layer. There is no resistant cuticle.This outer layer is a syncytium with no partitions ofcell walls. It is referred to as a tegument distinguishingit from non-living cuticles of the type that occur innematode species. Before discussing the functionalmorphology of the tegument, it is essential to havesome understanding of the structural properties ofabsorptive surfaces in general. The structure,physiology and biochemistry of the tegument are,therefore, of fundamental importance inunderstanding cestode physiology as a whole.In cestodes, the tegument grows, not by cell division,but by recruitment. Cells form the parenchyma comeup and attach to the tegument and vesicles pass up intothe tegument. However, the amplification of the

Ultrastructure of microtriches in Stilesia globipunctata69surface is achieved by the presence of delicate a. Immature proglottides containing scolex andcytoplasmic extensions called microtriches, anterior region.reminiscent of mucosal cell microvilli. Themicrotriches on the tapeworm surface increase theb. Mature proglottides with functionalsurface area of the parasite by about 20-times. Thereproductive organs.most dominant feature of the cestode tegument is thecovering by microtriches, which are thought to bec. Gravid proglottides containing eggs.responsible for nutrition and protection and possibly Scanning electron microscopic study : The scanningalso the mechanical functions of anchoring and electron microscopic (SEM) studies of the soclex,traction. They show a wide renge of morphlogical immature, mature and gravid proglottides of S.variations.globipunctata were undertaken to understand itsultrastructure. For this purpose, the specimens wereMicrotriches are unique in the cestodes and are dissected in chilled glutaraldehyde (2.5%) and fixedevident on the tegument and other epithelia that open for 16 h at 4° C. The tissues were subsequently washedinto the tegument. Microtriches have been widely thrice at an interval of 15 min each in phosphate bufferrepeated among all major orders of Eucestoda. These at pH 7.0 and then dehydrated by passing through anare present on the scolices and strobila of ascending series of alcohol from 30 to 100% an h inrepresentatives of most groups that have been each concentration. They were ultimately kept inexamined with SEM. Papilliform, spiniform and 100% alcohol overnight. Following dehydration, thefiliform structures have been variously reported tissues were air-dried in a desiccator for 7 to 10 days.among the Pseudophyllidea (Anderson, 1975, The dried samples were mounted on an aluminiumGranath et al., 1983) Cyclophyllidea (Berger and stub and gold sputtered in vacuum for 10 min, using anMettrick, 1971; Ubelaker et al., 1973; Sampathkumar, Eiko IB-2 ion coater. The samples were observed2001; Vijayalakshmi, 2001; Radha, 2003). eventually on a Hitachi, S-415A scanning electronIn the present study the characterization ofmicroscope, scanned at 25 KV and micrographed atmicrotrichial structure, density, distribution and theirdifferent magnifications (Hayat, 1977).functional significance in S. globipunctata has been Transmission electron microscopic study: Theattempted as the species of cestode has so far not been scolex, mature and gravid proglottid regions of S.exclusively studied due to its inconspicuousness in the globipunctata for transmission electron microscopygut of sheep.(TEM) were immersed in 2.5% glutaraldehyde inMATERIALS AND METHODSMillong's phosphate buffer (pH 7.3, 380 mOsm/L),where they were diced into small pieces. After 3-4hCollection of tapeworms: The tapeworm S. fixation at room temperature, the tissue was rinsed inglobipunctata (Rivolta, 1874) were collected from the Millong's buffer. The tissue was then post-fixed in 1%intestine of naturally infacted sheep autopsied in the osmium tetroxide in Millong's buffer for 1.5 h, rinsedslaughterhouse at Perambur, Chennai. The sheep quickly in distilled weter, dehydrated in an ethanolintestines were transported to the laboratory within series, infiltrated with propylene oxide, embedded inhalf an hour of collection. In the laboratory, each Spurr's low-viscosity epoxy resin and polymerized atintestine was carefully dissected and the tapeworms 60° C. Thin sections were cut at 70-90 nm with awere collected. Then the worms were washed in diamond knife, mounted on uncoated copper grids,distilled water to render them free from intestinal stained with uranyl acetate/ethanol and aqueous leadcontents and rinsed quickly 3-4 times in normal saline. citrate, and examined under a Philips 204 TEM at anThe tapeworms were then observed through aaccelerating voltage of 40 or 60 kV (Conn, 1993).compound microscope to confirm their taxonomic RESULTScharacters. The entire worm was spread out on a boardand the length was measured.The immature, matureSEM observations: Specimens of S. globipunctataand gravid proglottid region of the worm was locatedwere found to have species-specific patterns ofand separated as follows and dried on moist blottingpapilliform, filiform, spiniform and blade-likepaper and used for scanning and transmission electronmicrotriches that are restricted to particular regions ofmicroscopic studies.scolex and strobila. The central to peripheral regionsof the scolex are covered with papilliform and filiform

70 Venkatesh, Ramalingam and Vijaylakshmistructures. The adherent surfaces of the sucker andtheir cavities have a dense uniformly distributedcovering of spiniform and blade-like microtriches ofconsistent structure (Fig. 1a). The tegument on themargins and outer surface of the sucker is denselycovered with relatively long blade-like microtriches.Similarly, different types of microtriches can also beseen in neck and strobila. The microtriches seem to bedirected posteriorly. Microtriches in the center ofscolex measure about 2.0 µm and in the sucker region,it measures 2.1µm.The immature proglottid region is also denselycovered by posteriorly directed filiform, spiniformand papilliform microtriches (Fig.1b). But themicrotriches are not distributed uniformly. This trendcontinues as segments become older. Thepolymorphic nature of the microtriches increases asthe segments become older. The mature proglottidregions of S.globipunctata clearly reveal the decreasein density and non-uniformity of microtriches(Fig.1c). On gravid segments, however, there isobviously an increasing degree of disorder in thearrangement of microtriches (Fig.1d). The surface ofthe posterior most part in the majority of wormsexamined is in a stage of dissolution with onmicrotriches being apparent. Thus the scanningelectron micrographs of the microtriches and theirdistribution in the tegument of scolex, immature andmature strobilar regions of the parasite revealed thepolymorphic nature of the microtriches.MtMtSb(a)(b)TfMtMt(c)(d)Fig. 1. Scanning electron micrographs of scolex, immature, mature and gravid regions showing microtriches of S. globipunctata.a. Adherent surfaces of sucker showing spiniform and blade-like microtriches (x80).b. Tegumental surface of the immature region showing filiform, spiniform and papilliform microtriches (x15).c. Mature region showing posteriorly directed microtriches (with less density; x25).d. Microtriches of gravid region showing non-uniform and less density (x15).Mt - Microtriches, Sb - segental boundary, Tf - tegumental folding.

Ultrastructure of microtriches in Stilesia globipunctata71can be seen in the immature proglottid region (Fig.2d). Whereas in the mature proglottid region, papillalikeand filament-like microtriches are predominantlyseen (Fig. 3a). The microtriches appear to be non-uniform in density and size. A decreased microtrichialdensity down the length of strobila and morphologicalchanges in the tegumental surface of the gravidsegments can be clearly observed (Fig. 3b). Thispicture clearly reveals the stages of disintegration ofmicrotriches. Such changes involve surfacesculpturing accompanied by loss of all microtrichesand erosion of folds in the posterior region of theparasite (Fig. 3c). The disintegrated microtriches canalso be seen in Fig. 3d.TEM observations: In addition to the light and SEMfindings, ultrastructural observations were made byTEM. The TEM picture of scolex (Fig. 2a-c) shows thepresence of papilla-like, spine-like and blade-likemicrotriches. The microtriches may be divided intothree anatomical regions (Fig. 2b) viz. 1)microfilament containing a base, 2) a dense cap and 3)a complex junctional region between the base and cap.Each base is found to contain an inner sleeve of densematerial, the core tunic. Distally, the core is foundconnected individually to slightly curved tubule, thejunctional tubule.As observed in SEM pictures, posteriorly directedfilament-like, spine-like and papilla-like microtrichesCpCrBJrCpJrSplGxBCtSplGx(a)(b)BmMtMtGxSpl(c)(d)Fig. 2. Transmission electron micrographs of tegument brush border of scolex and immature regions of S. globipunctata.a. T.S. of the tegument showing different kinds of microtriches (x45,000).b. Higher magnification of architecture of blade like mictothrix (x1,000,000).c. L.S. of the margin of suckers showing microtriches (x7000).d. Brush border of tegumental folds of immature region showing different types of microtriches (x20,000).B - Base, Bm - basement membrance, Cp - cap, Ct - core tunic, Gx- glycocalyx, Jr - junctional region, Mt - microtriches, spl - subplasmalemmal layer.

72 Venkatesh, Ramalingam and VijaylakshmiSplBmSpl(a)(b)DbSplMt(c)(d)Fig. 3. Transmission electron micrographs of tegument brush border of mature and gravid regions of S. globipunctata.a. Tegument under higher magnification of mature proglottid region showing different kinds of microtriches (x30,000).b. Tegument (T.S.) of gravid region showing the dissolution of microtriches (x20,000).c. L.S. of tegument of the gravid region showing smooth margin with complete lack of microtriches (x20,000).d. Transmission electron micrographs showing disintegrated microtriches (x70,000).Bm - basement membrane, Db - dense bodies, Mt - microtriches, Spl - subplasmalemmal layer.DISCUSSIONThe highly active outer surface of cestodes viz. thetegument is a multifunctional entity serving forabsorption, digestion, protection, excretion(Featherston, 1972; Thompson et al., 1980; Hayunga,1991), anchoring (Rothman, 1963; Morseth, 1966;Thompson et al., 1980) and traction for locomotion(Rothman, 1963; 1966; Berger and Mattrick, 1971).At the parasite host interface, it additionally serves forchemical and tactile reception (Featherston, 1972;Webb and Devey, 1974; Jones, 1988; Hass andGuggenheim, 1977; Granth et al., 1983). Althoughthe tegument contains specific systems for thetransport of molecules and ions, especially the aminoacids, hexose sugars, vitamins, purnes, pyrimidines,nucleotides and lipids, it also serves a number of othervital functions as suggested by Podesta (1980): (a) it isa major site of catalytic activity and contains enzymesof parasite and possibly of host origin, (b) it may be asite of volume regulation, (c) it serves a protectivefunction both against the host's digestive enzymes andthe host's immune reactions, (d) it may also function asa site of matabolic transfer (Posdesta, 1982; Pappas,1983; Threadgold, 1984).Such diverse functions necessitate a high degree ofmorphological specialization. Jones (1998) hasdescribed three features for cestode epithelia, namely,the occurrence of regional specialisation, microtrichesand secretory components. In addition he has alsosuggested that some cestode epithelia are involved in

Ultrastructure of microtriches in Stilesia globipunctata73developmental processes such as the nurture of polymorphic modification confers not onlyembroyos and maintnance of surrounding tissues. advantages to the parasitic species, but also representsas an adaptation for multiple parasitisms. The host'sThe microtriches of cestodes are more complex thanimmunological reactions would have also acted as amammalian microvilli. An excellent model of theirlimiting mechanism to the multiple parasite loads withstructure is given by Holy and Oaks (1986). TEMdifferential organization.studies have, however, revealed that all cestodespecies hitherto examined possess microtriches and Although the scolex is generally regarded largely as anthat they are probably of universal occurrence organ of attachment, in some cestodes such as(Morseth, 1966; Yamane, 1986). However, there are Echinococcus sp., may also have a 'placental' functiondifferences in shape and density of microtriches and absorb nutrients directly form the mucosal wall, abetween larvae and adult worms (Yamane, 1968). condition which occurs in some trematodes (SmythNovak and Dowsett (1983) have observed that during and Halton, 1983). The root like projection withasexual reproduction of T. crassiceps the metacestode rootlets increases the nutritional surface. Such ategumental microtriches differentiate into at least nutritional function has been widely reported bythree morphologically distinct types. Berger and Lumsden (1975a) and Lumsden and Hildreth (1983).Mettrick (1971) have described the size, shape and The microtriches covering the scolex region revealednumber of microtriches in different parts of the worm a structure different from that of the strobila, aof three Hymenolepis species. The study of Anderson situation presumable related to the topography of the(1975) shows that in Diphyllobothrium dendritucum host mucosa.and D. ditremum there is a change in the shape andlength of microtriches during development fromThe present study revealed marked difference in theplerocercoid to adult worms. In H. diminuta,distribution pattern and density of microtrichialmicrotriches have been quoted as having maximumstructures between scolex, and immature and maturediameter of 0.14-0.19 µm and maximum length of 0.9-segments. The socolex revealed more dense1.08 µm (Threadgold, 1984).distribution and uniform distribution of microtriches.The immature proglottid region showed dense andThe presence of microtriches in S. globipunctata has non-uniform distribution of microtriches, whereas inbeen established in the present study by SEM and the mature region, the microtriches appeared to beTEM. These ordered structures have been linked by non-unitorf in density and size. A decreasedmany authors to mictovilli of mammalian brush microtrichial density down the length of strobila hasborder (Read, 1955; Lumsden and Specian, 1980). been noticed.There is not only morphological but also functionalresemblance, as the parasite absorbs nutrients of lowmolecular weight through its body surface. In additionto the absorptive functions, the parasite epithliumperforms the function of body protection (Odland,1966). This protective function of the tegument is alsoattributed to a complicated structure as suggested byJha and Smyth (1969). The higher level ofarchitectural complexity may reflect a more complexlevel of function for these surface specialisations ofcestodes.The comparison of microtriches among differentspecies thus reveals that the ubuquitous nature ofmicrotriches in all cestodes, which have occupied theluminal niche of the lost animals also have evolvedstructural and functional features of homology in thesedifferent species. The above homology of tegumentalstructure in different cestode species thus reveals thatthe distribution microtriches and their furtherThe scolex has also revealed differential nature of themicrotriches. The above microtrichial complexity inthe scolex region is of parasitic sognificance as itrepresents the anchoring region and nodular region inthe host tissue.In this context, the species S. globipunctata differsfrom other cestode specieses, which show a simpleanchoring device over the intestinal mucosal region ofthe host. The deep association of the Stilesia sp. ingroups, forming nodular regions in the host mucosaltissue is of parasitic importance. Such groupassociation may not have only adverse consequencesto the host but it is also difficult to eliminate suchgroup associations than individual parasitesanchoring in the host lumen.Berger and Mettrick (1971) have describedpolymorphism of microtciehes all along the stobilarlength. Braten (1968a) has indicated that microtriches

74 Venkatesh, Ramalingam and Vijaylakshmiform an almost continuous covering of the worm.Earlier ultrastructural studies of T. hydatigena(Featherston, 1972) have revealed three differenttypes of microthrix each associated with a particulararea of the strobila. Jha and Smyth (1971) haveexamined the rostellum of E. graunlosus and reported,"The microtriches and their branches are curved invarious directions to form a criss-cross pattern". Thesurface of the scolex of Silurotaenia siluri is coveredwith filiform microtriches and giant spine-like andblase-like microtriches. They are also present on theneck region and posterior margins and internalcavities of the suckers (Scholz et al., 1999). Caira andRuhnke (1991) noted substantial changes in thepattern and distribution fo microtriches duringontogeny of the scolex in Calliobothriumverticillatum. Vijayalakshmi and Ramalingam (2005)observed filament-like, blade-like and intermediatetypes of microtriches on the tegument of A. lahorea byusing SEM and TEM studies.The present SEM and TEM study also clearly revealedthe existence of microtrichial polymorphism all alongthe strobilar length of S. globipunctata. It revealed acomplex pattern of microtrochial brush bordershowing wide range of morphological variations.Species specific pattern of papilliform, filiform,spiniform and blade- like microtriches were alsoobserved in S. globipunctata.In the light of the observations, results and discussionsof previous studies, the results on TEM studies on thetegument of S. globipunctata thus infer that theadhesive and absorptive microtriches of the tegumentnot only allow the diffusion and intake of variousnutrients, micro/trace elements and electrolytesindispensable for the growth of the parasites but affordfirm positions inside the intestinal cavity wall againstthe immune factors of the host which could reject theparasite's holdfast. The above dense distribution ofmicrotriches in the scolex region of S. globipunctataalso strengthens the above suggestion. Its absence inthe gravid region is due to the morphological changesin the tegument and the interaction of luminalenvironment.REFERENCESAnderson KI. 1975. Comparison of surface topography of threespecies of Diphyllobothrium (Cestoda, Pseudophyllidea) byscanning electron microscopy. Int J Parasitol 5:293-300.Braten T. 1968. An electron microscopy study of the tegumentand associated structures of the plercercoid ofDiphyllobothrium latum (L). Z Parasitenk 30:95-103.Caira JN and Ruhnke TR. 1991. A comparison of soclexmorphology between the plerocercoid and the adult ofCalliobothrim verticillatum (Tetraphyllidea:Onchobothridae). Can J Zool 69:1484-1488.Conn DB. 1993. Ultrastructure of the gravid uterus ofHymenolepis diminuta (Platyhelminthes: Cestoda). JParasitol 794: 584-590.Featherston DW. 1972. Taenia hydatigena. IV Ultrastructurestudy of the tegument. Z Parasitend 38:214-232.Granath WO, Lewis JC and Esch GW. 1983. An ultrastructuralexamination of the scolex and tegument of Bothriocephalusacheilognathi (Cestoda: Pseudophyllidea). Trans AmMicrosc Soc 102:240-250.Hayat MA. 1977. Principal and Techniques of ElectronMicroscopy. Biological applications. Vol. 1-9. New York.Van Nostrand Reinhold.Hayunga EG. 1991. Morphologica adaptations of intestinalhelminthes. J Parasitol 77:865-873.Hess E and Guggenheim R. 1977. A study of microtriches andsensory processes of the tetrathyridium of Mesocestoidescorti hoeppli, 1925, by transmission and scanning electronmicroscopy. Z Parasitenk 53:189-199.Holy JH and Oaks JA. 1986. Ultra structure of tegumentalmicrovilli (Microtriches) of Hymenolepis diminuta. CellTissue Res 244:457-466.Jha RK and Siyth JD. 1969. Echinococcus granulosus:Ultrastructure of Microtriches. Exptl Parasitol 25:232-24.Jha RK and Siyth JD. 1971. Ultrastructure of the rostellartegument of Echinococcus granulosus with specialreference to biogenesis of mitochondria. Int J Parasitol169-177.Jones MK. 1998. Structure and siversity of cestode epithelia.Int J Parasitol 28:913-123.Lumsden RD. 1975. Parasitological review: Surface andcytochemistry of parasitic helminthes. Exptl Parasitol37:267-339.Lumsden RD and Hildreth MB. 1983. The fine structure ofadult tapeworms. Biology of the Eucestoda. Vol. 1 pp. 177-233. Academic Press London.Lumsden RD and Specian R. 1980. The morphology, histology,and fine structure of the adult stage of cyclophyllidentapeworm Hymenolepis diminuta In: The Biology ofHymenolopis diminuta. 147-280. Academic Press, NewYork.Morseth DJ. 1966. The fine structure of tegument of adultEchinococcus granulosus, Taenia hydatigena and TaeniaBerger J and Mettrick DF. 1971. Microtrichial polymorphismamong hymenolepid tapeworms as seen by scanning pisiformis. J Parasitol 52:1074-1085.electron microscopy. Trans Am Microsc Soc 90:393-403.

Ultrastructure of microtriches in Stilesia globipunctata75Novak M and Dowett JA. 1983. Scanning electron microscopyof the metacestode of Taenia crassiceps. Int J Parasitol 13:383-388.Smyth JD and Mc Manus, DP. 1989, The physiology andbiochemistry of cestodes. Cambridge University.Thompson RCA, Hayron AR and Sue LPJ. 1980. AnOdland GF. 1966. Skin. In Histology (ed. R.O. Greep), pp. 420- ultrastructural study of the microtriches of adults425, New York, London, Sydney, Toronto: McGraw-Hill Proteocephalus tidswelli (Cestoda: Proteocephalidae). ZBook Company. Parasitenk 64: 95-111.Pappas PW. 1983. Host - parasite interface. In: Biology of theEucestoda. Vol. 2 (ed. Arme. C. and Pappas P.W.) 297-334.Academic Press New York.Threadgold LT. 1984. Parasitic platyhelminthes, In: Biology ofthe tegument (ed. J. Hereiter - Hahn, Maltolsty, A.G. andRichards, K.S.) 132-191. Springer - Verlag, Berlin.Podesta RB. 1980. Concepts of membrance biology in Ubelaker JE Allison VF and Specian RD. 1973. SurfaceHymenolepis diminuta. In Biology of the tapeworm topography of Hymenolepis diminuta by scanning electronhymenolepis, ed. H.P. Arai, pp. 505-509. Academic Press: microscopy. J Parasitol 59: 667-671.New York.Sampathkumar. 2001. Studies on the implications of metabolicPodesta RB. 1982. Adaptive features of the surface epithelial and biochemical parameters of the larval growth andwyncytium favouring survival in an immunologically differentiations of Cysticercus tenuicollis (Cestoda) insidehostile environment. In Parasites-their world and ours, ed. the sheep host (intermediate host). Ph. D., Thesis.D.F. Mettrick and S.S. Desser, pp. 149-155. Elsevier University of Madras.Biomedical Press: Amsterdam.Vijayalakshmi V. 2001. Studies on the significance ofRadha, T. 2003. Studies on the ultrastructure, biochemical and ultrastructure, bichemical and metabolic parameters inmetabolic aspecits of Raillietina echinobothrida (Megnin, proglottization oriented growth and differentiation of1881): (Davaineidae), cestode parasite of the fowl host Avitellina lahorea (Woodland, 1927): (Anoplocephalid(Gallus domesticus) in relation to host-parasite interaction. cestode) inside the sheep host (final host). Ph. D., Thesis.Ph.D. Thesis, University Press: New Jersey.University of Madras.Rivolta 1874. Referred from The Fauna of British India Vijayalakshmi V and Ramalingam K. 2005. RegionalIncluding Ceylon and Burma. Cestoda. Vol. II by Southwell, difference, ultrastructure and adaptive significance ofT. 1930. tegumentary microtrichial polymorphism in Avitellinalahorea (Anoplocephalid cestode) infecting sheep's (OvisRothman AH. 1963. Electron microscope studies ofaries) intestine. J Parasitic Dis 29 (1): 9-16.tapeworms. The surface structure of Hymenolepis diminuta.Trans Am Micros Soc 82: 22-30.Webb RA and Davey KG. 1974. Ciliated sensory receptors ofthe unactivated metacestode of Hymenolepis diminuta andRothman AH. 1966. Ultra-structural studies of enzyme activityH. microstoma. Tissue cell 6: 587-598.in the cestode cuticle. Exptl Parasitol 19: 332-338.Yamane Y. 1968. On the fine structure of DiphyllobothriumScholz T, Zdarska Z, de Chambrier A and Drabek R. 1999.eriacei with special reference to the tegument. Yanago ActaScolex morphology of cestode Silurotaenia siluri (Batsch,Med 12: 169-181.1786) (Proteocephalidea; Gangesiinae) a parasite ofEuropean wells (Silurus glanis). Parasitol Res 85: 1-6.Smyth JD and Halton DW. 1983. The physiology of trematodes,2nd edn. Cambridge University Press: Cambridge.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 76–80J P DThe protozoan fauna living in the digestive system ofPeriplaneta americana in Kolkata, West Bengal, IndiaJ. Ghosh and A. GayenPost Graduate Department of Zoology, Maulana Azad College, Kolkata.ABSTRACT. The protozoan fauna living in the digestive system of Periplaneta americana in Kolkata,West Bengal, India, was studied. Two ciliates, one amoeba and one mycetozoan were recognized. Ofthese, Nyctotherus ovalis was the most common and prevalent species (frequency index 92.31,concentration index 4.79). The other ciliate was Balantidium (frequency index 35.55, concentrationindex 2.57) whose species designation was not very clear. This first study demonstrates the presenceof Endamoeba blattae from the hindgut (frequency index 60, concentration index 2.75) and ofCoelosporidium periplanetae from Malpighian tubules (frequency index 14.71, concentration index2.8) that are new records for the protozoan fauna of Kolkata. Additionally, this study highlightsvarious morphological details as well as population distribution pattern of N. ovalis.Keywords: distribution, gut, morphology, Periplaneta americana, protozoaINTRODUCTIONCockroach is a household insect that acts as amechanical carrier as well as vector to a large numberof pathogens. They harbour a variety of protozoans intheir digestive system wherein most of them live asendocommensals. The major groups of protozoansreported in the lumen of the gut of cockroaches areciliates, amoebas, flagellates and apicomplexans(Kudo, 1926a; Kudo, 1926b; Kudo, 1926c; Kudo andMeglitsch, 1938; Hoyte, 1961). Researchers haveexamined various aspects of parasites includingmorphological, cytological and ecological details.Roth and Willis (1957) discussed the importance ofcockroaches as vectors of various vertebratepathogens. In the recent past, the contribution(s) ofanaerobic protozoans and methanogens to hindgutmetabolic activities of American cockroach has beenCorresponding author: Mrs. Jayati Ghosh, Post GraduateDepartment of Zoology, Maulana Azad College,Kolkata-700 013, W.B., India. E-mail: jayati1973@yahoo.comstudied in detail (Gijzen and Barugahare, 1992).Surprisingly, in India, especially in West Bengal,Kolkata, only a few studies of this kind have beenreported. In 1922, two new ciliates Balantidiumovatum and B. blattarum (Ghosh, 1922a; Ghosh,1922b) were reported. In a description of Indianciliophorans, Nyctotherus ovalis was reported to be apart of protozoan fauna of Periplaneta americana(Bhatia and Gulati, 1927). But afterwards, apparently,no detailed studies were undertaken on this aspect.Therefore, presently, it is difficult to draw a list of thefauna of cockroach gut for these areas without makingproper investigations. We, therefore, considered itexpedient to study the fauna of the digestive system ofP. americana in Kolkata. Such studies can be expectedto generate a new list of species, and the data related totheir prevalence (number of host infected) andintensity (average number of parasites/host).The goal of present investigation, therefore, was tostudy the protozoan fauna of P. americana in Kolkata,and to determine their morphology and distribution.Our findings suggest that N. ovalis is the most

Protozoan parasites of Periplaneta americana77common and dominant specimen, which showed haemocytometer following Petroff-Hauser countingconsiderable variation in its nuclear structure. Large method (Prescott et al., 1999). The gut smears werenumbers of N. ovalis were observed in the colon of P. first examined under a light microscope and thenamericana with only a few specimens of Balantidium, permanent preparations were made. Fixation wasbut its species status was not very clear because the done by Schaudinns' fixative and the smears wereorganisms had some morphological similarities and stained with iron-alum haematoxylin (Cable, 1963).dissimilarities with Balantidium blattarum. This is For the study of nuclear characters, Feulgen nuclearthe first study wherein we report the presence of technique (Feulgen and Rossenbeck, 1924) wasEndamoeba blattae and Coelosporidium periplanetae utilized. An ocular micrometer calibrated with anin considerable numbers in the hindgut and objective micrometer was used for all the cellMalpighian tubules, respectively, of P. americana measurements. All measurements given in the studycollected from West Bengal, India. Nevertheless, are in micrometers, and the figures were drawn bythese two protozoan species have already been using a camera lucida.reported earlier in P. americana from some othercountries (Kudo, 1926a, Sprague, 1940).RESULTS AND DISCUSSIONMATERIALS AND METHODSData in Table I show the population distributionpattern of the parasites obtained from the gut of P.The cockroaches used in this study were collected americana. A comparative population distributionfrom Beliaghata, East Kolkata households and pattern of the parasite species showed that meanBelgachia market area during June 2004 to February density range of N. ovalis was highest (4.79) followed2006. The cockroaches were dissected within 24 h of by C. periplanetae (2.8), E. blattae (2.75) andtheir collection. The mid-gut, ileum, colon and rectum Balantidium sp. (2.57). These data corroborate withof P. americana were then taken out separately and the previously published results by Gijzen andkept in different watch-glasses containing saline Barugahare in 1992. But the maximum SD value was(0.6% NaCl in distilled water) solution. The attained by N. ovalis (2.83) and the lowest waspopulation counts were made through observed in E. blattae (1.55).Table I: Comparative population distribution pattern of protozoan fauna in the hindgut of P. americanaName of Number of Number of Frequency Density Mean Standard Standardspecimen host studied host infected index range deviation (SD) error (SE)N. ovalis 26 24 92.314(1-12) x 10 4.79 2.83 0.58Balantidium sp. 90 32 35.554(1-7) x 10 2.57 1.73 0.31E. blattae 40 24 604(1-5) x 10 2.75 1.55 0.32C. periplanetae 34 5 14.714(1-6) x10 2.8 1.72 0.77Table II: Data of various morphological characters of N. ovalisNo. of Range Mean Standard StandardSpecimens (in µm) (in µm) deviation (SD) drror (SE)Body length (L) 25 57-123.5 87.21 11.93 2.39Body width (W) 25 47.5-95 62.32 12.83 2.57L/W 25 1.13-1.8 1.43 0.20 0.04Macronucleus length (ML) 25 19-47.5 25.46 6.97 1.39Macronucleus width (MW) 25 9.5-23.75 14.63 4.64 0.93ML/MW 25 1-3 1.923 0.56 0.11

78 Ghosh and GayenDescription of the specimens:N. ovalis Leidy, 1849Habitat: Maximum number of specimens was foundin the colon of hind-gut.Morphology: Various morphological characteristicsof N. ovalis have been studied including body length(L) and width (W) and their ratios (L/W),macronucleus length (ML) and width (MW) and theirratios (ML/MW; Table II).stains lightly. Also variable forms of macronucleus arecommon in N. ovalis. Available forms here arespherical, ovoid, cylindrical, club shaped, semi-ovoidforms. Light micrograph of N. ovalis showingvariation in nuclear shape (Fig. 2).Genus Balantidium Claparède and Lachmann,1858Habitat: Colon of the hind-gut.The body observed to be oval, comparatively wider inposterior than anterior. Elongated macronucleussituated at anterior one-third. Body length variedfrom 57-123.5 µm and width from 47-95 µm.Cytopyge terminal and oval or slit like in shape.Peristome begins at the anterior end turns slightly tothe right and ends in cytostome located midwaybetween the ends. Nuclear length varied from 19-47.5µm and width from 9.5-23.75 µm. In our study, bodydimensions of N. ovalis were relatively smaller thanthose described by Leidy, 1849. When the L/W ratiowas considered, it can be said that the specimens were E. blattae Bütschli, 1878slightly oval whereas ML/MW ratio indicated thatnuclear shape varied from spherical to ovoid andelongated.Morphology: Body was pear shaped, anterior endtapering, slightly bent to the side opposite to theperistome. Small peristome observed about onefourththe body length. Endoplasm was coarselygranular. Macronucleus was spherical (diameter 9.5-14.25 µm) and placed behind the peristome in thecenter of the body. Body length varied from 57-76 µmand width from 38-57 µm. Fig. 3 shows generalmorphology of Balantidium sp. under a light,microscope, stained with Heidenhain s ironhematoxylin. A large contractile vacuole was presentat the posterior end in B. blattarum (Ghosh, 1922b)that was lacking in this specimen.Habitat: Colon and ileum of hind-gut.Morphology: Amoeba with few, broad pseudopodiaFig. 1 shows the general morphology of N. ovalis and showed distinct ectoplasm and endoplasm.under a light microscope and stained with Endoplasm was clear and homogeneous. Nucleus,Heidenhain s iron hematoxylin (a) and using Feulgenwith characteristic round or ovoid shape of thisnuclear stain (b). Compact macronucleus gave strongparasite can be easily distinguished. Dark granules arepositive reaction in Feulgen test where micronucleusalso found along its peripheral region with lightAnVMaMaCPP9.5 µm(a)PCyAn,Fig. 1. General morphology of N. ovalis under a light microscope: (a) stained with Heidenhains iron hematoxylin, (b) using Feulgennuclear stain. An. anterior, P. posterior, V. vestibular opening, CP. cytopharynx, Ma. macronucleus, Cy. cytopyge.(b)9.5 µm

Protozoan parasites of Periplaneta americana79VAnAnAnMa19 µm AnMaMaVMaCyCPP 19 µm19 µm19 µmPPP(a) (b) (c) (d)CyFig. 2. Light micrograph of N. ovalis showing variation in nuclear shape: (a) ovoid, (b) club-shaped, (c) cylindrical , (d) semi-ovoid. An.anterior, P. posterior, V. vestibular opening, CP. cytopharynx, Ma. macronucleus, Cy. cytopyge.central part. Length of the amoeba varies from 66.5-152 µm and width 47.5-104.5 µm. Nuclear diameterwas about 19-47.5 µm. Fig. 4 shows generalmorphology of E. blattae under a light microscope,following staining with Heidenhain s ironhematoxylin.C. periplanetae Lutz and Splendore, 1903Habitat: Malpighian tubule.Morphology: Trophozoite stages are common withdeveloping spore inside. Shape of the trophozoite maybe spherical, ovoid or cylindrical. Diameter ofspherical trophozoites varies from 28-31.5 µm and inAn19 µmPSEcNEnVFig. 4. General morphology of E. blattae under a light,microscope, stained with Heidenhains iron hematoxylin. Ps.pseudopodia, N. nucleus, En. endoplasm, Ec. ectoplasm.P19 µmFig. 3. General morphology of Balantidium sp. under a light,microscope, stained with Heidenhains iron hematoxylin. An.anterior, P. osterior, V. vestibular opening, Mi. micronucleus, Mamacronucleus.MiMacase of ovoid ones observed length is 42 µm and width35 µm. Fig. 5(a), (b) and (c) show light micrograph of,C. periplanetae stained with Heidenhain s ironhematoxylin showing trophozoites containing variousstages of developing spore.The above findings indicate that the fauna of P.americana collected from Kolkata is quite rich invarious species of protozoan parasites. For the lastfifteen years, ciliates in the cockroach gut havereceived special emphasis due to the discovery ofhydrogenosome, a double-membrane sub-cellularorganelle, present in some anaerobic protist includingN. ovalis (Müller, 1993). Biochemical and moleculargenetic evidences argue that hydrogenosome share acommon ancestry with mitochondria (Embley et al.,1997). However, all hydrogenosomes studied so far

80 Ghosh and Gayen30 µm30 µmCBCBCBTDS30 µm(a) (b) (c),Fig. 5. Light micrograph of C. periplanetae stained with Heidenhains iron hematoxylin showing trophozoites containing various stagesof a developing spore: (a), (b) and (c), CB. chromatoid bodies, T. trophozoite, DS. developing spore.lack a genome. Recently Akhmanova et al. (1998) Feulgen R and Rossenbeck H. 1924. Mikroskopisch-isolated hydrogenosomal DNA from N. ovalis. chemischer Nachweis einer Nucleinsarure von Typus derThymonucleinsaure und die darauf beruhende electiveHydrogenosomes in N. ovalis are intimatelyFarbung von Zellkernen in mikroskopischen Praparaten.associated with endosymbiotic, methanogenic Ztschr. Physiol. Chem. 135:203.bacteria that may have a major role in cockroachGhosh E. 1922a. On a new ciliate, Balantidium ovatum sp. nov.,metabolism. In vitro studies also suggest a major role an intestinal parasite in the common cockroach (Blattafor hind-gut protozoa in cockroach metabolic americana). Parasitology 14:371.activities, especially during the insect growth period Ghosh E. 1922b. On a new ciliate, Balantidium blattarum sp.(Gijzen and Barugahare, 1992).nov., an intestinal parasite in the common cockroach(Blatta americana). Parasitology14:15-16.ACKNOWLEDGEMENTSGijzen HJ and Barugahare M. 1992. Contribution of anaerobicThe authors would like to acknowledge University protozoa and methanogens to hindgut metabolic activitiesof the American cockroach, Periplaneta americana. Appl.Grants Commission, New Delhi, for providingEnviron. Microbiol. 58: 2565-2570.financial support for this work [Minor project No.Hoyte HMD. 1961. The protozoa occurring in the hind-gut ofPSW/032/04-05(ERO)]. The authors would also likecockroaches I. Parasitology 51:415-436.to thank Prof. Dipankar Sengupta, Head, Dept. ofZoology, Maulana Azad College, Kolkata, for Kudo R.1926a. Observations on Endamoeba blattae. Am. J.Hyg. 6: 139-52.providing departmental facilities and to othercolleagues for continuous cooperation. The authors Kudo R.1926b. Observations on Lophomonas blattarum, aflagellate inhabiting the colon of the cockroach, Blattaare especially thankful to Prof. Biswapati Dasguptaorientalis. Arch. Protistenk. 53: 191-214.and Dr. Tarak Khan for their valuable suggestions andKudo R. 1926c. A cytological study of Lophomonas striatacomments.Bütschli. Arch. Protistenk. 55: 504-515.REFERENCESKudo RR and Meglitsch PA.1938. On BalantidiumAkhmanova A, Voncken F, van Alen T, van Hoek A, Boxma B, praenucleatum n.sp. inhabiting the colon of BlattaVogels G, Veenhuis M and Hackstein JHP. 1998. Aorientalis. Arch. Protistenk. 91:111-124.hydrogenosome with a genome. Nature 396:527-528.Müller M. 1993. The hydrogenosome. J. Gen. Microbiol.139:2879-2889.Bhatia BL and Gulati AN. 1927. On some parasitic ciliates fromthIndian frogs, toads, earthworms and cockroaches. Arch. Prescott LM, Harley JP and Klein DA. 1999. Microbiology.4Protistenk. 57:85-120. Edn. WCB/McGraw-Hill, USA. pp 117.Cable RM. 1963. An illustrated laboratory manual ofparasitology, Allied Pacific Private Limited, Bombay, India,pp133-134.Embley TM, Horner DA and Hirt RP. 1997. Anaerobiceukaryote evolution: hydrogenosomes as biochemicallymodified mitochondria? Trends Ecol. Evol. 12: 437-441.Roth LM and Willis ER. 1957. The medical and veterinaryimportance of cockroaches. Smithson. misc. Collns.134:no. 10Sprague V. 1940. Observations on Coelosporidiumperiplanetae with special reference to the development ofthe spore. Trans. Am. Microsc. Soc. 59: 460-474.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 81–84J P DChelatrema neilgherriensis n. sp. (Trematoda:Gorgoderidae) infecting the freshwater fishes fromNoolpuzha river in Wynad district, Kerala, IndiaK. T. Manjula and K. P. JanardananDepartment of Zoology, University of Calicut, Calicut.ABSTRACT. A new species of digenetic trematode Chelatrema neilgherriensis (Gorgoderidae) wasrecovered from the freshwater fishes Danio neilgherriensis and Labeo rohita, collected fromNoolpuzha river in Wynad district, Kerala. Herein, we have described this new species in detail,discussed its systematic position and have compared it with the only other species in the genus C.smythi Gupta and Kumari, 1973. We found C. neilgherriensis n. sp. to be different from C. smythi inbody measurements, size of oral and ventral suckers, sucker ratio, extent of uterine coils, nature oftestes and seminal vesicle, and shape and size of eggs. Apparently, this first study reports a secondspecies of the genus Chelatrema, a gorgoderid from Kerala.Keywords: Chelatrema, Gorgoderidae, Kerala, trematodaINTRODUCTIONthe only species in the genus Chelatrema, and isreported here as a new species. This forms the secondThe genus Chelatrema was erected by Gupta andspecies of the genus Chelatrema and the first report ofKumari (1973) with C. smythi as the type speciesa gorgoderid from Kerala.which infect the freshwater fish, Chela bacala (Ham.)from Satluj River at Ropar, Punjab, India. The authors MATERIALS AND METHODSincluded the genus under the subfamily ArnolinaeYamaguti, 1958 of the family Hemiuridae Luhe, 1901.The fresh water fishes Danio neilgherriensis (Day,But Gibson and Bray (1979) stated that it is not an=16) and Labeo rohita (Hamilton, n=6), collectedhemiurid, and Gibson (2002) considered it as afrom Noolpuzha river in Wynad district of Kerala,gorgoderid.from April 2004 to May 2005, were examined fortrematode infections. The flukes recovered from theDuring an explorative study on the trematode fauna of intestine of D. neilgherriensis and the intestine andfreshwater fishes in the hill streams of Wynad district body cavity of L. rohita were observed under phase-in Kerala, we came across a species of Chelatrema in contrast microscope, with or without vital staining.Danio neilgherriensis and Labeo rohita , collected Those used for permanent preparations were fixed forfrom Noolpuzha river. Detailed studies proved that approximately 1 h in 10% formalin under cover glassthe trematode is significantly different from C. smythi,pressure at room temperature, and stained with alumcarmine as described by Cantwell (1981). TheCorresponding author: Dr. K. P. Janardanan, Department ofZoology, University of Calicut, Calicut-673 635, Kerala, India.E-mail: kpjanardanan@yahoo.co.indescriptions are based on 20 whole mounts, andmeasurements of 10 mature flukes fixed in 10%formalin. Measurements are presented in

82Manjula and Janardananmicrometres, unless otherwise stated; the range is 390-625 (490) in diameter. Ventral sucker round,followed by mean values in parentheses. The figures muscular, large; 510-1,035 (801) in diameter. Suckerwere drawn with the aid of a camera lucida, and details length ratio 1: 1.69-1.82 (1.78). Anterior body 900-added free-hand from observations made on live 2,192 (1,962) long; 30-37 (34)% of body length.specimens.Prepharynx distinct, 20-72 (64) in length. PharynxRESULTSelongate, ovoid; measured 125-232 x 94-215 (163 x136). Oral sucker/pharynx width ratio 1: 0.30-0.38Infections with adult flukes were observed in the (0.34). Oesophagus 18-42 (29) long. Many glandintestine of 10 out of 16 (62.5%) Danio cells found scattered around oesophagus and anteriorneilgherriensis, and intestine and body cavity of one region of caeca. Intestinal bifurcation in anterior body;out of six (16.7%) Labeo rohita. The intensity of 800-1,205 (1,016) anterior to ventral sucker. Caecainfection varied from one to three.narrow, 3.0-4.985 (3.872) mm long; terminate blindly,320-456 (389) from posterior extremity.Description (Fig. 1)Testes two, symmetrical, spherical to ovoid, entire;Body large, light red, elongate, ovoid, narrowing in left testis 250-432, 209-382 (311 x 246); right testisanterior body; measured 2.7146.5 x 1.0112.602 (4.413 249-382 x 197-309 (305 x 243). Post-testicular regionx 1.747) mm; width 30-45 (40)% of body-length. 820-1,198 (1,006) long; 18-27 (22)% of body-length.Tegument unarmed. Oral sucker round, sub-terminal; Vas deferens traverses along left side of ventral sucker.800 µm75 µmBA300 µm30 µmCFig. 1. A-D. C. neilgherriensis n. sp. A, Entire worm. B,Terminal genitalia. C, Egg. D, Miracidium.D

A new species of Chelatrema from fishes in Wynad83Cirrus-sac large, elongate, post-bifurcal, medially 232 (1,309 x 201), extends to midlevel of testes.placed; 279-415 x 107-193 (366 x 159) in size.Seminal vesicle saccular, bipartite; 212-384 x 51-97Taxonomic summaryth(348 x 77), reaches about ¾ of cirrus-sac, narrows Genus: Chelatrema Gupta and Kumari,distally forming pars-prostatica. Pars-prostatica 1973thick-walled, ensheathed in numerous gland cells.Ejaculatory duct long, tubular, with narrow walls; Species: Chelatrema neilgherriensis n. sp.opens into base of genital atrium. Cirrus not visible.Type host: Danio neilgherriensis (Day),Genital pore submedian, post-bifurcal; 162-283 (246)Cyprinidaefrom ventral sucker.Additional host: Labeo rohita (Hamilton),Ovary immediately posterior to ventral sucker,Cyprinidaeequatorial, dextral, round to ovoid; 301-439 x 376-483(345 x 406). Ventral sucker to ovary distance 84-129 Site: Intestine and body cavity(113); 2-3.1 (2.5)% of body-length. Oviduct leadsfrom lateral margin of ovary. Seminal receptacle Type locality: India, Kerala, Wynad district,large, saccular, ovoid, placed lateral to ovary; 300-452Noolpuzha riverx 223-327 (361 x 255) in size. Vitellarium in the formHolotype: Deposited in the Department ofof a compact mass, situated behind seminal receptacle,Zoology, University of Calicut,on the left side of median line; 180-362 x 150-286 (276Kerala, India. No: Z./Par./Digx225) in size. Uterus fills the whole post-testicular2005-1aposterior body, extends into entire extra-caecal space,up to the level of caecal bifurcation. Distal end of Paratypes: Z./Par./Dig-2005- lb-duterus passes along left side of ventral sucker to formdistinct metraterm and opens at genital pore. Uterus Date of collection: 13 April 2005filled with numerous, small, oval, thin-shelled, non- Etymology: Named after the species name ofoperculate, embryonate, and fragile eggs, measuringthe type host.78-86 x 70-81 (83 x 76).Excretory bladder long, I-shaped; 1,190-1,570 x 104-Table I. Comparison of C. neilgherriensis n. sp. with C. smythi Gupta and Kumari, 1973Character C. smythi Gupta and Kumari, 1973 C. neilgherriensis n. sp.1. Body size 6.9-7.0 x 2.5-2.75 mm 2.714-6.5 x 1.011-2.602 (4.413 x 1.747) mm2. Oral sucker Round; 600 x 500-550 Round; 390-625 (490)3. Ventral sucker Round; 1,000 x 900 Round; 510-1,035 (803)4. Sucker length ratio 1 : 1.6 1 : 1.785. Testes Entire or slightly lobed Entire, round to ovoid6. Seminal vesicle Ovoid; 66 x 88 Bipartite; 212-384 x 51-97 (348 x 77)7. Ovary Entire; 500 x 400 Entire, round to ovoid; 301-439 x 376-483(345 x 406)8. Uterine coils Fill the space behind ventral sucker Fill the space behind ventral sucker, and extendextra-caecally up to the level of caecalbifurcation.9. Eggs Small, round; 45-60 Small, ovoid; 78-86 x 40-49 (83 x 46)10. Host (s) C. bacala (Hamilton) D. neilgherriensis (Day) and L. rohita(Hamilton)Note: Measurements are in micrometres unless otherwise mentioned.

84Manjula and JanardananMiracidiumFreshly laid eggs hatched in 2-3 h. Miracidiapyriform, with conical papilla at anterior end;posterior end round; measured 132-176 x 46-73 (151 x64). Cilia long, uniformly distributed. Eyespots two,oval and conjoined; 19-23 x 8-13 (20 x 11.5) in size. Alarge apical gland and a pair of lateral penetrationglands present. A pair of flame cells present behindthe eyespots. Miracidia swim actively in water,changing direction intermittently. They exhibitednegative phototaxis.ratio, extent of uterine coils which lead extra-caecallyup to caecal bifurcation, nature of testes and seminalvesicle and the shape and size of eggs. A comparisonof the characters of C. smythi and of C.neilgherriensis, presented in Table I, showed that thetwo species were different from each other. Further,they infect different hosts too. Therefore, the flukereported herein has been considered as a new speciesand named Chelatrema neilgherriensis after thespecific name of the type host, Danio neilgherriensis.ACKNOWLEDGEMENTSDISCUSSION The authors express their sincere thanks to Dr. D. I.Gibson, Department of Zoology, The Natural HistoryIn the present study, the fluke is characterised by theMuseum, London, for taxonomic identification of thepresence of large ventral sucker, well-developedfluke to generic level, and to the Head of thepharynx, short oesophagus, symmetrical testes, pre-Department of Zoology, University of Calicut, fortesticular ovary, single compact vitellarium, stronglyproviding facilities.convoluted uterus and embryonated eggs, and hence itis included in the genus Chelatrema Gupta andKumari, 1973. These authors included the genus under REFERENCESfamily Hemiuridae Luhe, 1901, but Gibson and BrayCantwell GE. 1981. Methods for invertebrates. In: staining(1979) treated it as a genus inquirendum and Gibson inprocedures. Clark G (Ed.) Williams and Wilkins, Baltimore.2002 considered it a gorgoderid. As the characters ofpp 255-280.the genus Chelatrema agree fully with that ofGorgoderidae, we agree with the arrangement made Gibson DI. 2002. Family Derogenidae Nicoll, 1910. In: Keys tothe Trematoda. Volume 1. Gibson DI, Jones A & Bray RAby Gibson and place the genus under the family(Eds.) CAB International. pp 351-368.Gorgoderidae.Gibson DI and Bray RA. 1979. The Hemiuroidea: terminology,The new species reported here needed to be compared systematics and evolution. Bull. Br. Nat. Hist. (Zool.). 36:with the only species in the genus, C. smythi which 35-146.infects the freshwater fish Chela bacala. It resembledGupta N K and Kumari A. 1973. Chelatrema smythi n. gen. n.C. smythi in the extent of caeca, position of gonads, sp. (Trematoda, Hemiuidae: Arnolinae) from a fresh-waternature and position of vitellarium and position of fish, Chela bacala, from Ropar. Res. Bull. (N.S.) of thegenital pore. But it differed greatly in body Panjab Univ. 24:109-112.measurements, size of oral and ventral suckers, sucker

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 85–88Short communicationJ P DHaemato-biochemical studies on fowl coccidiosis inlayer birds*N. D. Hirani, J. J. Hasnani, R. S. Joshiand K. S. PrajapatiDepartment of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.ABSTRACT. This study on haemato-biochemical profile of 48 coccidia-infected and 24 healthybirds from commercial layer farms revealed that the infected-birds had significantly (p < 0.05)lower mean haemoglobin concentration (7.62 ± 0.14 vs 10.59 ± 0.19 g%) and total erythrocyte count(1.63 ± 0.04 vs 3.10 ± 0.05 million cells/cmm), apparently lower packed cell volume (23.00 ± 0.71 vs31.46 ± 0.70 unit %) and significantly (p < 0.05) higher mean total leucocyte count (16.41 ± 0.37 vs10.40 ± 0.16 thousand cells/cmm) than the healthy ones. The mean values of blood glucose (221.01 ±2.66 vs 176.03 ± 0.75 mg%) and serum total cholesterol (271.71 ± 4.71 vs 112.68 ± 0.60 mg%) weresignificantly (p < 0.05) higher and serum total protein was lower (2.98 ± 0.08 vs 4.42 ± 0.10 g%) incoccidia-infected birds as compared to healthy birds, whereas the activities of serum aspartateaminotransferase (77.84 ± 1.10 vs 65.62 ± 0.63 U/L), alanine aminotransferase (9.20 ± 0.28 vs 7.07 ±0.25 U/L) and alkaline phosphatase (902.23 ± 9.30 vs 816.58 ± 25.95 KAU%) were non-significantlyhigher in infected-birds than the healthy ones. Almost a similar trend was observed for all theparameters in layer birds managed under both deep litter as well as cage system. These findingsreflected anaemic condition with depressed metabolism due to tissue damage and increasedimmune response in infected-birds.Keywords: coccidiosis, deep litter vs cage system, haematology, layer birds, serumCoccidiosis is a parasitic disorder, which can have anacute or chronic course of infection in birds. Itproduces a deviation in the various haematological,biochemical and enzymatic components of the body(Padmavathi and Muralidharan, 1986a,b).Therefore, measurement of biochemical and enzymeactivities is useful in determining the pathologicconditions in the tissues. Serological profiles ofcertain enzymes are altered greatly in cell membranedegeneration, and inflammatory and diffuse tissue* A part of M. V. Sc. thesis of the first author; approved byA A V, Anand.Corresponding author: Dr. N. D. Hirani, Department ofVeterinary Parasitology, College of Veterinary Science andAnimal Husbandry, Anand Agricultural University, Anand-388001, Gujarat, India.degeneration and loss (Deger et al., 2002). Scantyliterature is available on haemato-biochemicalalterations due to coccidiosis in layer birds from roundthe globe.This study was, therefore, conducted on 99 layer farmsin the middle of Gujarat, involving fecal sampling of594 birds managed under deep litter and/or cagesystem. All the samples were examined by thestandard methods. From all coccidia positive farms,two infected and one healthy birds were selected forhaemato-biochemical studies. In all, 48 blood samplesfrom coccidia-infected birds and 24 samples fromnormal healthy birds were studied. Data thusgenerated were statistically analyzed using unpaired 't'test (Snedecor and Cochran, 1980).In this study, significantly (p < 0.05) lower mean

86 Hirani et al.haemoglobin concentration and total erythrocyte the intestinal mucosa, which was necessary for thecount, apparently lower packed cell volume, and passage of plasma protein at the height of the disease.significantly (p < 0.05) higher mean total leucocyte These lower protein levels might be due to reducedcount were found in coccidia-infected birds than the absorption of amino acids derived from the proteinhealthy ones (Table I). These findings were in constituents of feed due to reduced feed consumptionconformity with the earlier reports (Joshi et al., 1974; (Padmavathi and Muralidharan, 1986b). The resultsPadmavathi and Muralidharan, 1986a; Jaipurkar et al., on cholesterol are in agreement with the observations2004). The reduction in the value of haemoglobin and of Singh et al. (1976), Padmavathi and Muralidharantotal erythrocyte count observed in the infected-birds (1986b) and Basith et al. (1998), whereasmight be attributed to haemorrahges in the intestine Constantinescu (1976) could not see significantfollowed by development of intestinal lesions. There changes in serum cholesterol values in both infectedmay be injury to tissue and liberation of large and healthy birds. The hypercholesteremia observedquantities of histamine, which increase the local blood in the present study among the infected-birds might beflow and also increase the permeability of capillaries due to disturbed fat metabolism and loss of fluidand venules allowing large quantities of fluid to come resulting in apparent increase (Padmavathi andout (Padmavathi and Muralidharan, 1986a). The Muralitharan, 1986b) or due to impaired liver functionreduction observed in packed cell volume during the consequent to injury to intestinal epithelium inacute-phase of infection might be due to severe blood coccidiosis (Basith et al., 1998).loss resulting in anaemic condition (Joshi et al., 1974).The increased total leucocyte count in coccidiaalanineaminotransferase and alkaline phosphataseMean activities of serum aspartate aminotransferase,infected birds might be due to immune suppression ofinfection. This was suggestive of increasedobserved in infected-birds were non-significantlyleucopoiesis as a first step of defense mechanism tohigher as compared to the healthy ones. Singh et al.infection (Padmavathi and Muralidharan, 1986a).(1976) reported comparable findings, while Kumarand Rawat (1975) found no significant difference, butIn the present study, significantly (p < 0.05) higher values were slightly less in infected group asblood glucose and serum total cholesterol and lower compared to normal group. The increase in SGOT andserum total proteins were found in coccidia-infected SGPT activities observed in coccidia-infected birdsbirds as compared to healthy ones. Joshi et al. (1974) under study might be due to extensive damage toreported similar findings on blood glucose. However, intestine and liver by the parasites and thereby loss ofPadmavathi and Muralidharan (1986b) recorded appetite. Constantinescu (1976) reported significantserum hypoglycaemia in birds with experimental increase in serum AKP activity among infected-birds.Emeria tenella infection, whereas Basith et al. (1998) On the contrary, Kumar and Rawat (1975) reportedfailed to see any change in plasma glucose. This high significant decrease in serum AKP of E. necatrix- andlevel of blood glucose observed in coccidia-infected E. acervulina-infected 3-4 months old cockerels.birds may be either due to stress conditions leading tothe liberation of adrenal corticoids which induceIt can be concluded that haemoconcentration, reducedhyperglycaemia or disturbed carbohydratehaemoglobin and total erythrocyte count and anmetabolism due to interference with phosphorylativeincrease in total leucocyte count in coccidia-infectedcarbohydrate dissimilation by unidentified materialbirds suggest haemorrhage and increased leucopoiesispresent in the intestine of infected-fowls. The apparentas a first step of defense mechanism to infection. Thehyperglycaemia was undoubtedly due to loss ofhigh level of blood glucose observed in coccidia-erythrocytes. The observations on protein were ininfected birds may be either due to stress leading to theconformity with those of Turk (1972), who reportedliberation of adrenal corticoids which inducemarked reduction in protein during the acute-phase ofhyperglycaemia concomitant with loss ofE. necatrix infection. The significant (p < 0.05)erythrocytes. Reduced serum total protein andreduction in serum total protein observed in infectedbirdsindicate damage to intestine and vital organs,increased serum enzyme activity in coccidia-infectedbirds might be due to reduced feed intake and/orhaemorrahges through the gut and formation ofincreased cell membrane permeability and loss ofinflammatory exudates rich in blood proteins (Basithintracellular contents.et al., 1998). The decrease might also be due to leakageof protein resulting from the increased permeability of

Haemato-biochemical studies on fowl coccidiosis87Table I. Haemato-biochemical profile of coccidia-infected and healthy layer birds managed under deep litter and cage system in GujaratParameter Deep litter Cage system Overall totalInfected birds Healthy birds Infected birds Healthy birds Infected birds Healthy birds 't' valueN 12 6 36 18 48 24Haemoglobin (gm %) 7.87 ± 0.39 10.93 ± 0.16 7.54 ± 0.13 10.48 ± 0.24 7.62 ± 0.14 10.59 ± 0.19 2.22*Packed cell volume (%) 24.68 ± 1.69 32.67 ± 1.61 22.44 ± 0.76 31.06 ± 0.77 23.00 ± 0.71 31.46 ± 0.70 1.41nsErythrocytes count 1.70 ± 0.08 3.30 ± 0.11 1.61 ± 0.05 3.03 ± 0.04 1.63 ± 0.04 3.10 ± 0.05 3.79**(million cells/cmm)Leukocytes count 15.33 ± 0.94 10.20 ± 0.17 16.77 ± 0.37 10.47 ± 0.20 16.41 ± 0.37 10.40 ± 0.16 2.24*(thousand cells/cmm)Blood glucose (mg %) 230.53 ± 6.15 178.50 ± 1.70 217.84 ± 2.74 175.21 ± 0.74 221.01 ± 2.66 176.03 ± 0.75 2.40*Serum total protein 3.22 ± 0.20 4.26 ± 0.14 2.90 ± 0.08 4.48 ± 0.13 2.98 ± 0.08 4.42 ± 0.10 1.93*(gm %)Serum cholesterol 278.55 ± 7.11 113.40 ± 1.70 269.43 ± 5.82 112.43 ± 0.59 271.71 ± 4.71 112.68 ± 0.60 4.85**(mg %)SGOT (U/L) 76.13 ± 2.61 65.58 ± 1.52 78.41 ± 1.19 65.63 ± 0.69 77.84 ± 1.10 65.602 ± 0.63 1.49nsSGPT (U/L) 9.49 ± 0.53 6.77 ± 0.19 9.10 ± 0.33 7.18 ± 0.32 9.20 ± 0.28 7.07 ± 0.25 0.93nsAlkaline phosphatase 884.58 ± 15.79 761.57 ± 52.57 908.11 ± 11.18 834.91 ± 29.41 902.23 ± 9.30 816.58 ± 25.95 0.60ns(KAU %)N = number of birds , * Significant at p < 0.05, ** Significant at p < 0.01, ns = non-significant.

88Hirani et al.ACKNOWLEDGEMENTSJoshi HC, Singh BP, Prasad B and Prasad RK. 1974. Variationsin certain blood constituents during caecal coccidiosis inWe thank the Principal and Dean of the College, and poultry. Indian J Parasitol. 9:22-24.the poultry farmers of the region for the facilities,Kumar A and Rawat JS. 1975. A note on the effect ofsupport and co-operation extended for this work on coccidiosis on serum enzymes, blood glucose andtheir birds. The facility of autoanalyzer provided by cholesterol in chicken. Indian J Anim Sci 45:154-156.ADIO, Navsari, Gujarat State is also gratefullyPadmavathi P and Muralidharan SRG. 1986a. Alteration inacknowledged.haematological parameters in chicken during EimeriaREFERENCEStenella infection. Indian Vet J 63:716-722.Basith Abdul S, Rajavelu G and Murali Manohar B. 1998.Biochemical studies in experimental Eimeria necatrixinfection in chickens. Indian Vet J 75:876-878.Constantinescu V. 1976. Biochemical and histoenzymicchanges in coccidiosis in chickens. Buletinul InstitutuluiAgronomic Cluj Napoca 30:115-117.Deger Y, Dedo S and Deger S. 2002. Enzyme activity changes inthe sera of chickens treated with coccidiostatic agents.Indian Vet J 79:912-916.Jaipurkar SG, Deshpande PD, Narladkar BW, Rajurkar SR andKulkarni GB. 2004. Caecal coccidiosis in broiler chicks:haematological, pathological changes during treatmentwith herbal antidiarrhoels. J Vet Parasitol 18:135-138.Padmavathi P and Muralidharan SRG. 1986b. Studies on thealteration in the serum metabolites during the Eimeriatenella infection in chicks. Indian Vet J 63:530-536.Singh CV, Joshi HC and Shah HI. 1976. Biochemical studies inintestinal coccidiosis of poultry. Pantnagar J Res 1:63-66.Snedecor GW and Cochran WG. 1980. Statistical Methods. 8Edn. Iowa State Univ. Press, Ames, Iowa, USA.Turk DE. 1972. Protozoan parasitic infections of the chickintestine and protein digestion and absorption. J Nutr102:1217-1222.th

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 89–91Short communicationJ P DRe-redescription of Dissurus farrukhabadi Verma, 1936(Digenea - Echinostomatidae) with a discussion of thegenus Dissurus Verma, 1936P. C. Gupta and R. B. SinghDepartment of Zoology, D. A. V. College, Kanpur.ABSTRACT. Dissurus farrukhabadi Verma, 1936 from the large intestine of a White-neckedstork, Dissurus episcopus (Boddaert) from Unnao, India is re-redescribed. The validity of thespecies of Dissurus, Verma, 1936 is discussed. D. xenorhynchi Wahid, 1962 is considered asynonym of Stephonoprora gigantica Gupta, 1962 and Psilocollaris guptai Gupta and Saxena,1986 is considered a synonym of D. farrukhabadi.During the survey of digenetic termatode parasites of 0.58 long. Intestinal caeca simple, extending to hindbirds, nine specimens of Dissurus farrukhabadi end of body. Ventral sucker sub-spherical, much largerVerma, 1936 were collected and described.than oral sucker, 0.40-0.46 x 0.43-0.50 at 0.87-1.14The trematodes were fixed in 70% alcohol under slightfrom anterior extremity.cover glass pressure for 24 h, stained with acetic alum Excretory bladder Y-shapped; arms bifurcation justcarmine, dehydrated in graded series of alcohols, behind posterior testis and their extension beyondcleared in clove oil and mounted in Canada balsam. anterior testis not traceable due to vitallariaumThe drawings were made with the aid of a camera follicles, excretory pore subterminal.lucida, and unless otherwise stated, all measurementsare in mm.Testes oval, entire or notched, subequal, tandem,apart, in last fifth of body. Anterior testis 0.30-0.55 xDESCRIPTION0.23-0.28 at 3.61-4.30 from anterior extremity.Dissurus farrukhabadi Verma, 1936Posterior testis, 0.47-0.60 x 0.31-0.32 at 0.48-0.52from posterior extremity. Vasa efferentia arising fromBody long, slender, delicate, spinose, 5.34-5.71 xtestis, join together to form vas deferens, running0.57-0.69, with rounded extremities. Head collaranteriorly to form vesicula seminalis. Cirrus sac small,reniform, 0.30-0.34 x 0.41-0.45; with 24 spines,heart shaped, 0.21-0.27 x 0.14-0.17, extendinginterrupted dorsally, arranged in a single row with noposteriorly either upto anterior margin of ventralend group. Oral sucker terminal, avoid 0.10-0.12 xsucker or overlapping anterior third of it. Vesicula0.08-0.09. Prepharynx 0.06-0.09 long. Pharynxseminalis bipartiate, 0.12-0.15 x 0.09-0.16. Parssubglobular0.11-0.13 x 0.09-0.16. Oesophagusprostatica small, 0.03-0.06 long surrounded by largetubular, with thick irregularly sinuous walls, 0.48-number of prostate gland cells. Cirrus muscular, 0.01-0.02 long. Genital pore median, between intestinalCorresponding author: Dr. P.C. Gupta, 8/200-A, Arya Nagar,Kanpur-208 002, U. P., India.bifurcation and ventral sucker at 0.72-0.99 fromanterior extremity.

90 Gupta and SinghOvary rounded, entire, submedian, postequatorial, Verma (1936) erected the genus Dissurus with D.pretesticular, intercaecal, 0.14-0.18 x 0.16-0.18 at farrukhabadi as its type, recovered form the intestine2.73-3.22 from anterior extremity. Receptaculam of a White-necked stork. D. espicopus fromseminalis oval, 0.044 x 0.035. Laurer's canal present. Farrukhabad, U.P. Wahid (1962) added D.Vitellaria follicular, dense extending from a level xenorhynchi from the large intestine of Black-neckedanterior to ovary up to hind end of body. Transverse stork. Xenorhynchus asiaticus at London zoo, andvitelline ducts from either side meet and form a yolk Dwivedi (1967) described D. setheae from the typereservoir to open at ootype, surrounded by a large host at Chindwara (M. P.). Srivastava (1974) renumberof Mehlis' gland cells. Uterus long, examined Verma's specimens and gave illustrationsintercaecal lying between ootype and venteral sucker, together with an emended generic diagnosis.Egg large, oval, few, 0.09-0.10 x 0.04-0.05.Singh (1954) established another genus PsilocollarisHost : White-necked stork with P. indicus as its type for his specimens from theDissurus episcopus (Bodd.) intestine of D. episcopus from Lucknow, U.P. TheLocation : Large intestinegenus Psilocollaris was characterized by an aspinouscollar.Locality : Unnao, U.P., IndiaGupta (1980) critically studied P. indicus Singh, 1954Material : Nine specimens (incidence in detail and raised question on its validity, doubting2/3)its conspecificity with D. farrukhabadi. Srivastava0.2mm240.05mmLCRCMG0.25mm3 560.05mm1Fig. 1-6. Dissurus farrukhabadi Verma, 1936.1. entire ventral view; 2. head collar, (enlarged); 3. extension of cirrus sac up to anterior third of ventral sucker; 4. cirrus sac,enlarged; 5. female genital complex, enlarged; 6. showing entire testes.OV - ovary, U - uterus, OO-ootype, RS - receptaculum seminalis, LC - Leurar's canal, YR - yolk reservoir.

Re-redescription of Dissurus Farrukhabadi Verma91(1982) re-examined the type specimen of P. indicus Gupta PD. 1980. Further observations on Psilocolaris indicusand found the presence of collar spines and accepted Singh, 1954 with a note on its systematic position(Trematoda: Psilostomatidae). Bulletin of the Zoologicalthe views of Gupta (1980). Further, he considered P.Survey of India. 2: 217-218.singhi Pandey, 1975 as species inquirenda and alsotransferred D. setheae to the genus Stephanoprora Gupta R. 1962. Studies of trematode parasites of Indian birds.Odhner, 1902, due to presence of 22 collar spines, II. On Stephanoprora gigantics sp. nov. from the Blackneckedstorck, Xenorhynchus asiaticus (Latham).tandam testes lying in mid-third of body, vitellariaProceeding of the National Academy of science, India.extending form posterior testis to hind end and short 32:381-386.uterus. Srivastava (1982) further observed that D.setheae is probably a synonym of S. nigerica Gupta, Gupta R. 1963. On Stephanoprora nigerica sp. nov. with a briefreview of the genus Stephanoprora Odhner, 19051983. The authors are in agreement with the views of(Trematoda : Echinostomatidae). Zoological Anazica.Gupta (1980) and Srivastava (1982). 170:117-130.In the opinion of present authors D. xenorhynchiWahid, 1962 better fits under the genus StephanoproraOdhner, 1902 and shares most of its characters viz. theextension of the vitellaria, 22 collar spines, theposition of the testes and thickening in the wall of theoesophagus with S. gigantica Gupta (1962). Theformer, therefore, is considered a synonym of later.Gupta V and Saxena AM. 1986. On Psilocollaris guptai sp. nov.(Psilostomatidae : Trematoda) from the intestine of anIndian Stork, Leptonhilos dubius (Gmelin) from Lucknow.Indian Journal of Helmonthlogy. 37: 1-3.Pandey KC. 1975. Studies on some known and unknowntrematode parasites. Indian Journal of Zootomy. 14: 197-219.Singh KS. 1954. Psilocollaris indicus n.g., n. sp.Gupta and Saxena (1986), overlooking the synonymy(Psilostomatidae) from an Indian stork, Dissurus episcopusof Psilocollaris, described P. guptai form the intestine episcopus. Journal of the Washington Academy society. 44:of an Indian stork, Leptoptilos dubius form Lucknow 24-26.and differentiated it from the type species on suchSrivastava CB. 1974. A critical study of Verma's "Notes oncharacters as the muscular pharynx, position of the trematode parasites of Indian birds" based on his trematodetestes and the ovary. These characters are merely collection on Part 2. Family Echinostomatidae Dietz, 1909.intraspecific variations and, therefore, P. guptai is a Journal of the Zoological society of India. 24:160-191.synonym of D. farrukhabadi.Srivastava CB. 1982. The fauna of Indian and the adjacentThe present specimens of D. farrukhabadi differedcountries. Platyhelminthes Vol. I (Supplement) Trematoda -Digenea. Addition to Prof. H. R. Mehra's volume onfrom the previous descriptions in having a smallertrematoda - Digenea. Publ. by the Director, Zoolgicalbody, in the extensions of vitellaria from a little Survey of India, Calcultta, 163pp.anterior to overy instead of from the level of it, and inVerma SC. 1936. Notes on trematode parasites of Indian birds.having a smaller post-testicular space. ThesePart I Allababad University Studies. 12: 148-188.differences are considered as intraspecific variations.Wahid S. 1962. On a new trematode from Black-necked stork,REFERENCESXenorhynchus asiaticus. Journal of Helminthology. 36:Dwivedi MP. 1967. A new species of the genus Dissurus Verma, 211-214.1936 (Trematoda Echinostomatidae). Natural and AppliedScience Bulletin, 20: 267-275.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 92–93Short communicationJ P DA case of vaginal bleeding due to leech biteR. P. Ganguly, M. S. Mukhopadhyay and K. K. PatraDepartment of Obstetrics and Gynaecology, R. G. Kar Medical College and Hospital, Kolkata.Medically important leeches belong to Phylum evidence of trauma anywhere. Per rectal examinationAnnelida and Class Hirudinea. It is a worm like revealed a boggy mass of about 5 cm x 4 cm which wascreature that attaches to their hosts by means of appreciated anteriorly. On milking the swellingchitinous cutting jaws and draws blood through towards introitus blood clot came out along with amuscular suckers. Haemadipsa sylvastris,H. zelanica leech and the swelling disappeared. Under intraand H. montana, H. ornata etc. are land leeches venous sedation vaginal douching was done withavailable in India. The most prevalent species is H. normal saline. The hysteroscope was introducedsylvestris. Hirudo birmanica and Limnatis nilotica are inside vagina which revealed a bleeding point on thewater leeches. L. nilotica is notorious for internal lateral wall of vagina about 1.5 cm from introitus. Nohirudiniasis. Internal hirudiniasis may involve other trauma in the vagina could be seen.pharynx, larynx, vocal cord, trachea, oesophagus andgenitourinary tract. Attachment is usually painless.A paediatric Foley's Catheter (No.10) was introducedContinued bleeding from the site takes place after theinside vagina and the bulb of Foley's Catheter wasleech has detached. Death due to exhaustion has beeninflated to 20 cc with distilled water. Three hundred mlreported. Leech gets dislodged from its site of(300 ml) of blood was transfused to the patient.attachment if saline water, strong vinegar or matchCatheter was kept in vagina for 4 h. During the periodflame is applied.there was no bleeding from vagina. Routine bloodexamination and coagulation profile showed normalCASE REPORTpicture. Patient was kept admitted for 1 week beforeshe was discharged as leech bite patient often getsA six year old girl was admitted as an emergency caserecurrent episodes of bleeding. This patient too hadin the Department of Gynaecology and Obstetrics atlittle trickle on the third day but did not require anyR. G. Kar Medical College and Hospital, Kolkata, withintervention .a complaint of bleeding per vagina for 4 h. Patient'smother gave history that her daughter had gone to a We present this unusual case of vaginal bleeding in alocal pond in the evening to wash herself after six-year old girl because this is a rare cause of vaginaldefecation. She came back home and after sometime bleeding and its management by Foley's bulbnoticed that her undergarments were wet with blood. tamponade avoids unnecessary and complicatedPatient denied any history of local trauma. She was intervention under general anaesthesia in a child. Itthen brought to the hospital for medical help.also maintains anatomical integrity of local parts.On examination, the patient was conscious but verypale. On local examination, fresh blood was seencoming out through vagina. The bleeding point couldnot be seen as it was inside vagina. There was noCorresponding author: Dr. R. P. Ganguly, BC 45/7, BichitraAbason, Sector 1, Saltlake, Kolkata-700 064, W.B., India.The leech is a parasite which has a habit of enteringanatomical orifice of human being and animals. Itreleases an anticoagulant (heparin like substance,hirudinin) during the process of sucking blood whichcauses bleeding from bite site. Deka and Rajeev(2001) have reported leech bite as an unusual cause ofhaematuria. Severe rectal bleeding from leech bite

case of vaginal bleeding93was reported by Raj et al. (2001), and vaginal bleedingresulting from leech bite was reported by Hernandezand Ramirez (1998).REFERENCESHernandez M and Ramirez RE. 1998. Vaginal bleedingresulting from leech bite. Ginecol Obstet Mex 66:246-248.Raj S M, Radzi M and Tee M H. 2000. Severe rectal bleedingdue to leech bite. Gastroenterol 95:1607.Deka P M and Rajeev T P. 2001. Unusual cause of haematuria.Urol Int 66:41-42.

Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 94–97Short communicationJ P DField evaluation of a rapid immunochromatographictest kit for the diagnosis of Plasmodium falciparum andnon-falciparum malaria parasites from Sonitpurdistrict, AssamC. Rajendran and S. N. DubeBiotechnology Division, Defence Research Laboratory, Tezpur.ABSTRACT. The sensitivity and specificity of ICT Parascreen test kit device (rapid test formalaria pan/Pf) was compared with conventional microscopic method as the gold standard. A totalof 126 patients were tested with ICT and microscopic blood-smear examination. Sixty one(48.41%) patients were found to be positive for malaria infection in microscopic examination. Withthe ICT kit, the sensitivities and specificities for Plasmodium falciparum and non-falciparum (P.vivax) parasites were 96.30 and 88.88%, and 98.48 and 98.48 %, respectively. The ICT kit gave alittle lower sensitivity as compared to microscopic examination.Keywords: HRP II, ICT Parascreen test kit, malaria, pLDHGlobally, malaria still remains a major parasitic microscopic method (WHO, 1996). The ICT is a RDTdisease with high morbidity and mortality. In the for P. falciparum specific HRP II antigen (Parra et al.,Northeast region of India, the endemicity of malaria 1991) and a pan malaria antigen (Garcia et al., 1996).infection and the predominance of Plasmodium RDT is very much useful where microscopy is lackingfalciparum over P. vivax is now well documented (Dev and where the malaria is severe (WHO, 2000b). Theand Phookan, 1996; Kamal and Das, 2001). Accurate conventional microscopy is the gold standard test todiagnosis and proper treatment, which are important diagnose malaria infections worldwide. Even thoughto the control of malaria, are the main aims of the it is sensitive and economical, several disadvantagesglobal malaria control strategy (WHO, 2000a). Based make it inconvenient for use in the field viz.,on clinical symptoms alone, malaria and non-malaria requirement of electricity, trained microscopyfebrile cases and the species of Plasmodium can not be personals and difficulties in accurate speciesdistinguished (Chandramohan et al., 2001). Further, identification as evidenced by sensitive molecularaccurate diagnosis of malaria is essential to make a methods. Currently, rapid ICT cards are available torational choice of drug for the treatment. The detect malaria infection upto species leveldevelopment of Rapid Diagnostic Tests (RDT) for (Wangsrichanalai, 2001). The first generation ofmalaria based on immunochromatographic test (ICT) detection kits was designed to diagnose only P.strips provides a valid alternative to conventional falciparum, but now the newer devices have beendesigned to diagnose both P. falciparum specificCorresponding author: Dr. Rajendran, Biotechnology Division, antigens as well as Plasmodium genus specificDefence Research Laboratory, Post Bag No. 2, Tezpur-784 001, antigens. Early detection and differentiation ofAssam, India. E-mail: chellairajendran@rediffmail.com malaria is of paramount importance due to the

Immunochromatographic test kit evaluation for malaria diagnosis95incidences of cerebral malaria and drug resistance smear examination. Out of these 61 positive samples,associated with falciparum malaria, and due to the 52 were found positive for P. falciparum (85.24 %) andmorbidity associated with the other malaria forms. So, 9 were positive for non-falciparum parasite (P. vivax;ICT kits are considered for malaria diagnosis as 14.75 %), whereas the Parascreen ICT kit showed 54microscopic method has limitations. So far, field samples positive for P. falciparum and eight for P.evaluation of ICT kits has not been done for its use in vivax. A summary of the findings by these two tests hasimmediate clinical management of malaria in Sonitpur been given in Table I. The sensitivity and specificitydistrict, Assam. Therefore, the present study was values of the ICT were calculated using the results ofundertaken to study the performance of the ICT kit in microscopic examination as the gold standard. As perthe field by comparing it with the gold standard the Parascreen ICT kit, the sensitivity and specificitymicroscopy method.for P. falciparum and non-falciparum (P. vivax)parasites were 96.30 and 88.88 %, and 98.48 and 98.48The study was conducted at seven different places of%, respectively.Sonitpur district, Assam, between August 2004 andMay 2005. Each patient, who was suffering from The present field study revealed that the ICT kit couldfever, was finger pricked by using a sterile lancet. detect P. falciparum from 54 cases, whereas lightThick and thin blood-smears were prepared and microscopy showed 52 positive cases. Among the 54stained with Giemsa according to standard cases, two blood-smears were found negative for P.procedures. The thick smear was used to detect the falciparum but the ICT was positive for them. Theinfection, whereas the thin smear was used for species possible reason could be due to the persistence ofidentification. A blood sample was considered PfHRP II following the clearance of P. falciparumnegative, when no parasite could be detected in 100 (Wongsrichanalai et al., 1999). But for the case of nonfieldsof an oil immersion (x1000 magnification) falciparum (P. vivax) parasite, the ICT kit could detectobjective lens of a microscope (Fernando et al., 2004). only eight cases out of nine found by microscopicalAt the same time, approximately 5 µl of whole blood method. Generally, irrespective of the manufacturers,from finger prick of the patient was transferred the sensitivity and specificity for non-falciparum,directly to a sample pad. For testing the sample, ICT with the available ICT kit, is varying from 50-70% andParascreen test kit (Zephyr Biomedicals, India) was 37.580%, respectively, as compared to microscopy.used as rapid diagnostic device (Lot No: 101003, Mfg But the present study revealed 88.88% sensitivity andDt: 08-2004 and Exp. Dt: 07-2006). Parascreen 98.48% specificity. This study was conducted basedutilizes the detection of P. falciparum specific on qualitative assessment but not quantitative one ashistidine rich protein II, which is water soluble protein the accurate counting of parasites was not carried outthat is released from parasitized-erythrocytes of to assess the correlation of the sensitivity. Butinfected individuals, whereas for the detection of pan increasing sensitivity of the test with increasingmalaria, Parascreen detects the presence of pan parasite densities is one of the main factors inmalaria specific pLDH released from the parasitised detecting parasites (Mason et al., 2002). Variouserythrocytes. Then, a drop of buffer was added and authors have reported different degrees of sensitivityallowed to react for 2 min. This buffer was added to and specificity of P.f/P.v test kit manufactured frominduce cell lysis and allow PfHRP II and pan-malarial different countries and from different localities.antigens to bind to colloidal gold-labeled monoclonal Palmer et al. (1998) evaluated OptiMAL test for theantibodies. All tests were considered as valid if a diagnosis of P. falciparum and P. vivax malaria, and itcontrol line was observed. The sensitivity and uses a monoclonal antibody to the intracellular antigenspecificity was calculated as per the formula given by parasite lactate dehydrogenase (pLDH). ItMason et al. (2002). The sensitivity was calculated as differentiates species by the use of a P. falciparumthe number of true positives by the test divided by total specific and a genus specific antibody. It has shown 88positives by Giemsa [TP/(TP+FN)], and the and 94% sensitivities on symptomatic Honduranspecificity was determined as true negatives divided patients and specificities of 100 and 99% for theby the false positives (TN/TN+FP).diagnosis of falciparum and vivax malarias. Beatriz Eferro et al. (2002) evaluated OptiMAL and gave aA total of 126 blood samples were collected. Amonghigher efficiency of 98.1% for P. vivax than 94.9% forthese, 61 (48.41 %) samples were found positive forP. falciparum, in a malaria referral center in Colombia.malaria infection by standard microscopical blood-But Mason et al. (2002) evaluated two test kits viz,

96 Rajendran and DubeOptiMAL and ICT of which both gave lower is required especially for P. falciparum cases whichsensitivities than the earlier report. ICT gave 86.2 and may develop cerebral complications. But for non-2.9% sensitivity for P. falciparum and P. vivax and falciparum malaria cases, the present study has given aspecificity of 76.9 and 100%, but OptiMAL gave 42.6 moderate percentage of sensitivity and specificity butand 47.1% sensitivity and, 97 and 96.9% specificity it is difficult to suggest on this aspect, as the number offor P. falciparum and non-falciparum parasites, cases studied was small. With the ICT P.f/P.v therespectively. Kolaczinski et al. (2004) evaluated sensitivity for the detection of P. vivax was 96% atOptiMAL for the diagnosis of P. vivax and P. parasitaemias greater than 500 parasites/µl blood but,falciparum with 34% as compared to 36% by gold at parasitaemias lower than this the sensitivity wasstandard microscopy. For OptiMAL 48 test, cross reduced to only 29% (Tjitra et al., 1999; Hunt-Cookechecking of the corresponding smears at the reference et al., 1999). The ICT can not replace the microscopylaboratory gave a sensitivity of 79.3% and a specificity method for the determination of parasitaemia. Severalof 99.7% for P. falciparum, and the corresponding factors may be responsible for a little lowervalues of 86.1% and 98.7% for P. vivax infections. The performance of the test kit viz, human error though theperformance of the field microscopy was better, with a manufacturer's instructions were strictly followed.sensitivity and specificity of 85.2 and 99.7% for P. The information regarding the history of selffalciparum,and 90.4 and 98.7% for P. vivax, medication by the patients and the storage condition ofrespectively. According to Tjitra et al. (1999), the ICT the kits also need consideration. Moreover, increasingmalaria P.f /P.v test was 96% sensitive and 90% sensitivity of the test with increasing parasite densitiesspecific for P. falciparum, and 75% sensitive and 95% is one of the main factors in detecting parasites at lowspecific for P. vivax even though the blood samples densities rather than the human error (Mason et al.,were collected in an anticoagulant, EDTA, from the 2002). So, therefore, to assess the sensitivity andveins but not from the finger prick. Forney et al. specificity of ICT for non-falciparum parasites,(2000) conducted a study in Thailand and Peru with further studies should be done with larger number ofP.f/P.v test kit and recorded 95% sensitivity and 85% samples for the evaluation of its detection limit.specificity for P. falciparum, and 68% sensitivity and87% specificity for P. vivax and concluded that the Table I. Showing the number of cases studied for thesensitivities were closely correlated with parasiteevaluation of ICT with standard microscopic methoddensities. Richter et al. (2004) evaluated ICT (Malaria Parasite Giemsa ICT (Parascreen)ICT Now, Binax, Portland, USA) for its performance method kiton 2547 patients, and ICT was positive in all of 204patients with symptomatic P. falciparum infections, P. falciparum 52 54whereas microscopy revealed parasites in 202 of 204 Non-falciparum 9 8of these patients. But, from these two cases, (P. vivax)trophozoites were detected by microscopy only insamples taken after 6 and 12 h. So ICT showed a Negative 65 -specificity of 99.74%, whereas for non-falciparummalaria, the sensitivity and specificity of ICT was onlyTotal 126 -50 and 37.5%, respectively, as compared tomicroscopy. In our present study, one out of nine caseswas found negative for non-falciparum (P. vivax) byACKNOWLEDGEMENTSICT. Moreover, this particular blood smear had a The authors are grateful to The Director, Defencecomparatively very low density of parasites (2 Research Laboratory, Tezpur, Assam, for providingparasites/100 microscopic fields). Even though the necessary facilities and constant encouragement. Theparasite could be seen on blood smear, the ICT missed study was not supported by the manufacturer of theto detect the infection. Moody et al. (2000) conducted ICT kit.a study and concluded that though parasite may lookhealthy in the blood smear but it may be non-viableREFERENCESand not producing pLDH. It is known that pLDH Beatriz E Ferro, Iveth J Gonzalez, Fanny de Carvajal, Gloria Iactivity gets declined with therapy. However, the ICT Palma and Nancy G Saravia. 2002. Performance ofkit may be a useful device where microscopy is not OptiMAL in the diagnosis of Plasmodium vivax andavailable, and immediate clinical diagnosis of malariaPlasmodium falciparum infections in a malaria referral

Immunochromatographic test kit evaluation for malaria diagnosis97center in Colombia. Mem Inst Oswaldo Cruz, Rio de Janeiro immunochromatographic tests to expert microscopy in the97:731-735. diagnosis of malaria. Acta Tropica 82:51-59.Chandramohan D, Carneiro I, Kavishwar A, Brugha R, Desai V Moody A. 2002. Rapid diagnostics tests for malaria parasites.and Greenwood B. 2001. A clinical algorithm for the Clinical Microbiology Reviews 15:66-78.diagnosis of malaria results of an evaluation in an area oflow endemicity. Tropical medicine and InternationalPalmer C J, Lindo J F, Klaskala W I, Quesada J A, Kaminsky R,Health. 6: 505-510.Baum M K and A L Ager. 1998. Evaluation of the OptiMALtest for rapid diagnosis of Plasmodium vivax andDev V and Phookan S. 1996. Malaria prevalence in Tea Estates Plasmodium falciparum malaria. Journal of Clinicalof Brahmaputra valley of Assam, India. Journal of Microbiology 36: 203-206.Parasitic Diseases 20:189-192.Parra M, Evans C and Taylor D W. 1991. Identification of P.fForney J R, Wongsrichanalai C, Magill A J, Sorichaisinthop J histidine rich protein 2 in the plasma of human with malaria.and Gasser R A. 2000. Performance characteristics of the Journal of Clinical Microbiology 26: 1629-1634.ICT Malaria P.f, P.v (TM) rapid diagnostic device for thedetection of P.f and P.v. Abstract No: 239, Presented at theRichter J, Harms G, Stover I M, Gobels K and Haussinger D.th2004. Performance of an immunochromatographic test for49 Annual Meeting of the American Society of Tropicalthe rapid diagnosis of malaria. Parasitology ResearchMedicine and Hygiene, Houston.Tx, USA, Oct 29 Nov92:518-519.2,2000. American Journal of Tropical Medicine andHygiene 62:235-236. Tjitra F, Suprianto S, Dyer M, Currie B J and Anstery N M.1999. Field evaluation of the ICT Malaria P.f /P.vFernando D, Karunaweera N D, Fernando W P, Attanayake Nimmunochromatographic test for detection of Plasmodiumand Wickremasinghe A R. 2004. A cost analysis of the use offalciparum and Plasmodium vivax in patients with athe rapid, whole blood, immunochromatographic P.f/P.vpresumptive clinical diagnosis of malaria in Easternassay for the diagnosis of P. vivax malaria in a rural area ofIndonesia. Journal of Clinical Microbiology 37:2412-2417.Srilanka. Annals of Tropical Medicine and Parasitology98:5-13. Wangsrichanalai C, Chuanak N, Tulyayon S, Thanoosingha N,Laoboonchai A, Thimasarn K, Brewer TG and Heppner DG.Garcia M, Kirimoama S, Marlborough D, Leafasia J and1999. Comparison of a rapid field immunochromatographicRickmann K H. 1996. Immunochromatographic test fortest to expert microscopy for the detection of Plasmodiummalaria diagnosis. Lancet 347:1549.falciparum asexual parasitemia in Thailand. Acta Tropica.Hunt-Cooke A, Chiodini P, Doherty T, Moody A, Reis J and M 73: 263-273.Pinder.1999. Comparison of parasite lactate dehydrogenseWangsrichanalai C. 2001. Rapid diagnostic techniques forbased immunochromatographic antigen detection assaymalaria control. Trends in Parasitology (formerly(OptiMAL) with microscopy for the detection of malariaParasitology Today) 17:307-309.parasites in human blood samples. American Journal ofTropical Medicine and Hygiene 60:173-176.World Health Organization, 1996. A rapid dipstick antigencapture assay for the diagnosis of falciparum malaria.Kamal S and Das S C. 2001. Epidemiological observations onWHO informal consultation on recent advances inmalaria in some parts of Darrang district, Assam. Indiandiagnostic techniques and vaccines for malaria. Bulletin ofJournal of Malariology 38:25-31.the World Health Organization 74:47-54.Kolaczinski J, Mohammed N, Ali I, Khan N, Ezard N and MWorld Health Organization, 2000a. WHO Expert CommitteeRowland. 2004. Comparison for the detection ofon Malaria. Twentieth Report. Technical Report SeriesPlasmodium vivax and Plasmodium falciparum:No.892. Geneva.WHO.Considerations for the application of the rapid test inAfghanistan. Annals of Tropical Medicine and Parasitology World Health Organization, 2000b. Report of a joint98:15-20. ththWHO/USAID. Informal consultation 25 to 27 OctoberMason D P, Kawamoto F, Lin K, Laoboonchai A and1999. WHO/CDS/RBM/200.14. Geneva.WHO.Wongsrichanalai C. 2002. A comparison of two rapid field

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THE INDIAN SOCIETY FOR PARASITOLOGYExecutive CommitteePresidentProfessor Veena TandonDepartment of ZoologyNorth-Eastern Hill UniversityShillongVice-PresidentDr. J. MahantaRegional Medical Research CentreDibrugarhSecretaryDr. J. K. SaxenaDivision of BiochemistryCentral Drug Research InstituteLucknowJoint SecretaryDr. Vas DevMalaria Research CenterSonpurTreasurerDr. L. M. TripathiDivision of ParasitologyCentral Drug Research InstituteLucknowMembersDr. Wasim Ahmed, AligarhDr. P. Prakash Babu, HyderabadDr. S. C. Dutta, KolkataDr. S. K. Ghosh, BangaloreDr. Neena Goyal, LucknowProf. Neelima Gupta, BareilleyDr. B. V. Jadhav, AurangabadProf. P. D. Juyal, LudhianaDr. A. M. Khan, DibrugarhDr. Ashwani Kumar, GoaProf. G. G. Mani, VisakhapatnamDr. S. K. Puri, LucknowProf. R. Kaleysa Raj, TrivandrumDr. K. K. Saxena, BareilleyProf. Prati Pal Singh, S. A. S. NagarStatements and opinions expressed in Journal of Parasitic Diseases or in the presentations during the regular meeting of theIndian Society for Parasitology are those of the author(s) and do not necessarily reflect the official position of the Society. TheEditorial Board, Publisher and the Society disclaim any responsibility for the accuracy of statements made by thecontributors.Copyright ©2006 The Indian Society for Parasitology

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