Released August 2007 - The Indian Society for Parasitology

JOURNAL OF PARASITIC DISEASESVolume 31, Number 1, June 2007The official organ ofTHE INDIAN SOCIETY FOR PARASITOLOGYCentral Drug Research Institute, Lucknow-226 001, India.Editorial office: National Institute of Pharmaceutical Education and ResearchSector-67, Phase-X, S. A. S. Nagar-160 062, India.Phone: 0172-2214682-87; Fax: 0172-2214692; E-mail: drppsinghjpd@yahoo.co.in

Journal of Parasitic DiseasesCopyright © 2007 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 the copyright owner.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 3-13Invited reviewJ P DProblems confronting helminthic diseases of domesticanimals in India1 2M. C. Agrawal and P. S. Banerjee1College of Veterinary Science and Animal Husbandry, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur.2Division of Parasitology, Indian Veterinary Research Institute, Izatnagar.Received 26 March 2007; revised 8 June 2007; accepted 30 June 2007ABSTRACT. India is harbouring a large number of helminthic species in all varieties of animals.However, fasciolosis, schistosomosis, amphistomosis, metacestosis and haemonchosis are mostimportant helminthosis affecting almost all domestic animals. Voluminous work has been done toattend the problem of helminthosis in India, yet there are certain areas, which, though important,have not been given due attention. There is a need to solve taxonomic problems, hybrid formationand interaction between different species of same genera or different families sharing same hostand locations. Even confirmation of intermediate hosts of Fasciola hepatica, F. jacksoni andBivitellobilharzia nairi etc. is still desired. There is circumstantial evidence of Garole sheep beingresistant to fasciolosis but requires experimental confirmation and identification of other resistantanimal breeds as well as resistant genes for important helminthic species. Morbid pathology isneeded to be replaced by molecular pathology that should incorporate enzymatic, metabolic,hormonal and immunological changes which occur in helminthic infections. There are a number ofreports on the treatment of common helminthic infections but there is a need to correlate treatmentwith production traits and to identify drug-resistant helminths with reasons for such developments.Routine faecal examination has been found to be least sensitive in the diagnosis of hepaticschistosomosis, whereas hatching method and mesenteric examination have reported 10–20% and60–80% prevalence, respectively. Surprisingly, much attention has been paid on immunodiagnosis,albeit of doubtful field utility due to both blood invasion and cost of each test. Though the use ofhelminthic stages or their products confirmed development of resistance against re-infection;however, apparently, no work has been carried out towards the development of a vaccine(s) againstimportant helminths. Likewise, no serious attempt has been made to identify biological agents forthe control of helminthic infections. The observation of dragon fly nymphets devouring fresh watersnails, inhabiting same habitats may be explored as a control measure for trematode infections. Inour opinion, more attention should be paid on topics which have a direct bearing in the control ofhelminthic infections and may increase production of domestic animals.Keywords: control, diagnosis, helminth, helminthosis, India, strategyINTRODUCTIONHelminthic infections are wide spread in animals, menCorresponding author: Prof. M. C. Agrawal, ¾ Datt Arcade,Phase Three, South Civil Lines, Jabalpur-482 001, India. E-mail:mcagrawal@yahoo.comand plants. In India, considerable work has been doneto tackle this problem. There have been goodmonographs (Srivastava and Dutt, 1962; Dutt, 1980;Gupta, 1993), review articles (Mukherjee andChauhan, 1965; Chauhan et al., 1973; Baugh, 1978;Agrawal and Shah, 1984; Agrawal and Shah, 1989;

Problems confronting helminthic diseases of domestic animals5made from different parts of the country. For this carefully. Much work has been done on the bloodreason, Verma (1953) created a new species viz. F. flukes, where existence of natural hybrids has beenindica, which was not accepted by the scientific demonstrated in the African countries (Webster andcommunity. F. hepatica has been claimed to be present Southgate, 2002). Microscopic examination of bloodin Himachal Pradesh, Kashmir and Uttaranchal, albeit flukes, collected from mesentery of bovines atwithout the confirmation of its intermediate host Jabalpur, have at times showed heterologous mating(Agrawal, 2003).between S. spindale (atuberculated) and S. indicum(tuberculated ) with presence of fertile eggs. WithAmong three species of Haemonchus which occur in present knowledge, it is difficult to identifyIndia, H. contortus occurs in sheep, goat, cattle, heterologous mating among tuberculated blood flukesbuffalo and other ruminants, whereas H. longistipes (S. nasale x S. incognitum and S. indicum x S.occurs in camels and H. similis in cattle (Sood, 2003b). incognitum) or among atuberculated blood flukes (S.Thus, cattle may harbour two species together with spindale x O. dattai).possible interplay. Further, Haemonchus spp. sharesabomasum with different species of Trichostrongylus, What is the status of amphistomes, which areCooperia and Ostertagia, leading to competition for hermaphrodite with a number of species existingspace, food and immune evasion. It is not clear if drug- together in rumen? Or different species ofresistant H. contortus is comparatively more powerful Haemonchus which share same platform? The subjectin snatching food and space from the host, thereby requires attention as hybridization is a distinctreducing the population of susceptible strain.possibility because animals are known to harbourmore than one helminthic species in the same habitat.There are cases where either new helminth species or Recently, a case of trypanosomiasis with patentexisting species are reported from domestic animals trypanosomes in blood has been reported (Joshi et al.,for the first time from a new location of the country 2005) from a man from Chandrapur district of(Tandon, 2003). Importance of such findings also Maharashtra state that confirms the occurrence ofdeserves assessment with regards to animal health. significant changes in parasites and their vectors.Further, there are helminthic species, which have been Additionally, Shrivastava and Shrivastava (1974)reported from India infrequently but deserve attention reported two cases of Trypanosoma (Herpetosoma)due to public health importance. Paragonimus infection in man from Maharashtra.species, Angiostrongylosus cantonensis,Echinococcus multilocularis, Gnathostoma Unfortunately, taxonomic work has not been attendedspinigerumand Spirometra spp. (sparganosis) have all well in India, which has resulted in limitedbeen reported from humans (Parija, 1990; Agrawal, breakthrough discoveries on new helminths and the1998) and are awaiting detailed investigations. As diseases caused by them. Even discovery of S.these helminths survive in animal host species, their haematobium or S. indicum variant, incorporatinglife cycle and geographical distribution are yet to be Ferrissia tenuis from Gimvi village of Ratnagiriunderstood. Globalization, leading to increased district, has remained controversial with regards toexchange of personals between the countries, may species of the parasite (Gaitonde et al., 1981). Thecause entry of new helminths in any country. Here geo- status of Paramphistomum cervi vis-a-vis P. epiclitumhelminths may establish in a new surrounding with or spread of F. hepatica in geographical areas is notease though cyclo-helminths may also establish, beyond controversy. The correct identification ofpossibly in an alternative intermediate host. intermediate host species is worst where existence orTherefore, the subject requires a strategy for dealing absence of some intermediaries has been taken forwith such exotic infections.granted. Apathy towards taxonomy is despiteavailability of modern techniques including electronAnother area, which has remained unattended, is cross and fluorescence microscopes, development of DNAfertilization between closely related helminthic probes etc. which may resolve species problems morespecies. There is a controversy over the subject with accurately.one school of thought discarding such a possibility onthe grounds that definition of species itself suggests GENETIC FACTORSintra species reproduction, whereas others havedemonstrated mating of two distinct helminthic As helminths are multicellular organisms with aspecies (Tchuente et al., 1996). Obviously, this is one characteristic sexual reproduction, it is evident toform of evolution hence should be studied more record genetic variations within a given helminthic

6Agrawal and Banerjeespecies. This genetic make-up influences host range,pathogenicity and patency etc. of the parasite. Thegenetic variations in S. japonicum are wellestablished, which later led to creation of new specieslike S. mekongi, S. malayalensis and S. sinensium orstrains which differ in their snail susceptibilities(Attwood, 2001).with a variety of host species, there is all the possibilityof the existence of wide genetic variations amongsame helminthic species from different geographicalareas. However, this subject has failed to attract Indianscientists perhaps because of the complexity of subjectand requirements of more sophisticated equipments toprove genetic variations at molecular level.Attempts have been made by Dutt and Srivastava(1968)to differentiate S. nasale species/ strains whichinfect cattle and buffaloes, as only the former host wasfound to suffer clinically from nasal schistosomosis.The cross-transmission experiments revealed nodifference between S. nasale species from these twohost species. Nevertheless, no attempt was made tocompare S. nasale from different geographical areaseither from the same or different host species to recordgenetic variation.When nasal schistosomosis from differentgeographical areas was compared, indirect evidencewas obtained which suggested the existence ofdifferent parasite strains, which were found to differ intheir host susceptibility and clinical symptoms. Thus,Banerjee and Agrawal (1991)recorded the presence ofS. nasale at Jabalpur in buffaloes, whereas local cattledid not show any symptoms, though it was present incross-bred cattle where parasite was causing a mildform of the disease. In contrast, at Bhubaneswar localcattle did suffer from larger granuloma and profusewatery nasal discharge (Agrawal et al., 2003). Even S.nasale at Balaghat of Madhya Pradesh was differentfrom that of Bhubaneswar with regard to clinicalsymptoms etc. All these findings suggested geneticvariation among S. nasale belonging to differentgeographical areas.Variation in prevalence of fasciolosis, among differentdomestic animals and incrimination of different snailspecies (Choudhary, 1994) in different geographicalareas suggest that genetic factors play role in suchvariations.EPIDEMIOLOGYThe epidemiology consists of four components i.e.host, etiological agent, transmission factors andenvironment. Diagnosis, as fifth component, has beenincorporated by Agrawal (2003) as it influencesepidemiological findings to a great extent. Theepidemiological aspects of fluke infections in Indiahave been dealt with in greater details recently byAgrawal (2003), whereas details of haemonchosis inIndia have been covered by Sood (2003b). Therefore,these aspects are not discussed here.There is influx of research papers in recent pastdealing with prevalence of helminths in differentdomestic animals from different parts of the country.These works, some of which claim to be the first timework in a particular animal, helminth species orlocation or by a different technique, have notcontributed much except variation in the prevalencerates of a particular parasite in a host or in a givengeographical area. In these studies, no serious efforthas been made either to study biological behaviour ofthe helminth in nature or in different domestic animalspecies in the locality or correlating prevalence withecological changes, husbandry practices, introductionof new animals or with production traits or even withhealth condition of the animals. In the absence of thesedata, such repeated works do not add any newinformation on helminthic diseases.With regards to host, no serious attempt has been madeto identify resistant breeds of animals either for one ormore helminthic infections. However, there iscircumstantial evidence that Garole sheep ofThere are reports of buffalo-dog, camel-dog, goat-dog Sunderban area in West Bengal is resistant tostrains of Echinococcus granulosus from India but fasciolosis despite grazing in marshy land. A moreconfirmation on molecular basis is awaited (Das et al., detailed study, including experimental infection, is2003). Human susceptibility to these different strains needed for confirming the phenomenon with work forof E. granulosus has yet to be acertained.isolating-resistant gene.A new variety of helminthic population in the form ofdrug-resistant strains is emerging in different parts ofthe country, which sooner or later may replacesusceptible strains leading to a big problem. BecauseIndia is characterized by different agro-climatic zonesIndia is well known for its rich biodiversity, whereanimals are resistant to many pathogens (includinghelminths), and hence able to survive under adverseconditions. However, resistant animal breeds orresistant genes for important helminthic infections

Problems confronting helminthic diseases of domestic animals7have not yet been identified. Why the pigs are helminthic infections. A limited amount of work onresistant to all schistosome species except S. this subject has shown variations in populations ofincognitum? Why B. nairi develops only in elephants? host species of amphistome species in Bareilly districtThe answer may be present in the genes of elephants, with passage of time and change of ecologicalpigs and schistosomes. Further, epidemiology of conditions (Prasad and Verma, 1999). A perspectivehydatidosis with reference to susceptibility of man change has been observed in recent years in localitieswith different strains of E. granulosus also needs of dairies at Jabalpur with changes in husbandryattention.practices, not allowing animals to graze and disposalof animal wastes in local rivers or nalas. The waterIt is perturbing that our knowledge on intermediatebodies are also covered for construction of newhosts even of some important helminths is still elusivebuildings. Adoption of gobar gas plants,in Indian context. For instance, there is no proof of anvermicomposts, deworming by dairy men, possibleintermediate host(s) for F. hepatica though Lymnaeadrug resistance and rise in atmospheric temperature allauricularia sensu stricto was the only snail identifiedmight have changed transmission potentials offrom Srinagar, where the infection occurs inhelminthic infections, and need to be incorporated inabundance (Choudhary, 1994). None of thefuture epidemiological work on helminthic diseases.intermediate hosts for B. nairi, Orientobilharziaharsutai, F. jacksoni are known with conformity. STATUS OF DISEASEAgain, status of Ferrissia tenuis in transmittingschistosomosis has not been ascertained. It is taken forAll the above referred helminthic diseases are wide-granted that Indoplanorbis exustus or any other snailspread and have been reported from almost all thespecies from all geographical areas are identical anddomestic animals including pet ones. Because theseno studies on snail susceptibility with trematodes ofreports have been prepared by individual scientists,different areas have been undertaken. A deeper studythe reported work covered only limited geographicalon Gastropoda is required involving parasitologicalarea and limited number of animals. Therefore, it willand biochemical studies to understand their habitat,be a far-fetched idea to consider identical situation inbiological variations, susceptibility status which mayall parts of that particular state. Moreover,be influencing epidemiology of fluke infections in theconsiderable work has been done on slaughter housecountry (Subba Rao, 1989).materials, hence origin of the case remains doubtful. Itseems that, we do not have basic data on helminthicThe study of transmission factors is important for diseases of each animal species even from each districtunderstanding epidemiology of a particular parasite. of the country. Such work needs to be undertaken.Again, little attention has been paid, though important, However, there are chances of getting similar resultsto know prevalence rate, intensity of infection as per from similar agro-climatic zones hence future workfrequency, time, site and duration of grazing of may be undertaken in selected geo-climatic zones withanimals. Though buffalo is known to spend more time diversified environs. Attempts have been made toin water bodies, yet schistosomosis is more prevalent describe helminthic diseases of Jammu and Kashmir,in cattle, hence mere contact with infecting stage Himachal Pradesh, Haryana, North-East states(cercariae in this case) may not decide the prevalence (Choudhary, 1994; Parija, 1990; Sood, 2003a;rate. Likewise, ruminants show higher blood fluke Tandon, 2003), which also suffer from aboveprevalence (60–90%), whereas only 1–2% snails shed mentioned shortfalls. Nevertheless, these reports arecercariae (Agrawal, 2004). How such a lower important in reflecting helminthic diseases ofprevalence rate in intermediate host is sustaining so particular states. There is also a need to undertakehigh infection in ruminants is worth investigating. If detailed work on yak, Karakul sheep, Pashmina andthis is the case, it may be doubtful that control of Gaddi goats, which are specialities of some particularintermediate host may contain helminthic infections states.to a significantly lower level in domestic animals. Thisalso leads to another interesting study of populationMOLECULAR DIAGNOSISdynamics among the helminths and intermediate While dealing with diagnosis, the most critical issue,hosts, particularly when they are sharing same food often ignored, is the reason for diagnosis, which mighttable.be treatment of the diseased animal or accumulation ofTime is also influencing the epidemiology ofepidemiological data for control of helminthic

8Agrawal and Banerjeediseases. The clinical symptoms are vague, hence, if Earlier, all sorts of immunodiagnostic methods viz.animal is showing diarrhoea, anorexia, bottle jaw, intradermal test, complement fixation test, counteranaemia, loss of body weight, it is difficult to ascertain current immunoelectrophoresis, indirectif it is suffering from fasciolosis, schistosomosis or haemagglutination test, ring precipitation test andgastrointestinal nematodiosis, or some bacterial or double-immunodiffusion tests have been tried inviral infection. Therefore, a parasitological diagnosis helminthosis mostly in experimental cases, thoughis recommended to specify the etiological agent and to slaughter house and field materials have also beentreat the case accordingly. Here coprological incorporated in some of these studies (Agrawal andexamination, with more sensitive methods, is Shah, 1989; Parija, 1990; Agrawal and Alwar, 1992;sufficient to identify infected cases with possible Agrawal, 2003; Sood, 2003b). One interestingexception of schistosomosis or pre-patent, career or observation in these studies was the change oflarval infections.sensitivity and specificity of serological tests withchange of comparing parasitological tests due to vastImmunodiagnosis has played a pivotal role in viral anddifference in the sensitivity of these parasitologicalbacterial diseases owing to its ability to reflectdiagnostic methods. This has been shown by Banerjeeimmune status of the host and undertaking vaccinationet al. (1991) in bovine schistosomosis by comparingprogram with available vaccines. In contrast, there isresults of cercarian hullen reaction (CHR) andno available vaccine, which may be used againstmiracidia immobilization test (MIT) with faecalhelminthosis, in non-immune animals (even theexamination including hatching and liver digestionproduction of lung worm vaccine has beenmethods.discontinued). Hence immunodiagnosis is not ofmuch significance for controlling helminthosis. The crude worm homogenate, excretions andTherefore, it is difficult to justify all the efforts and secretions, sensitised RBCs or inert particles werefunds diverted towards immunodiagnosis in incorporated as antigens in these studies. In applyinghelminthosis. Again, it may be of doubtful MIT, CHR and COP (circum oval precipitation) insignificance to develop immunodiagnostic methods schistosomosis, live miracidia, cercariae and eggsagainst a hook worm (even marketing of an effective were required, respectively. The other tests employedvaccine was not successful) or stomach worm or higher quantity of antigens. Both alive and solubleascariid where eggs start appearing in large number, forms of antigens are difficult to procure due tonot later than a month of the infection. Nevertheless, complex life cycle of helminths. However, forimportance of immunodiagnosis in metacestodes and experimental work, larger quantity of wormother larval forms can not be denied and must be homogenate could be prepared with F. gigantica orattended looking to economy of the infections.amphistomes but collection of blood flukes in largernumber is a big problem, though attempts have beenIn recent years, a number of research schemes onmade by teasing mesentery or perfusion of domesticimmunodiagnosis are being funded by scientificanimals. Here also, cattle yielded highest numberorganizations including National Agriculture(50–200/200–300 g mesentery) of blood flukes (S.Technology Program of ICAR, which has beenspindale, S. indicum) followed by buffaloesoperational since July 2000 in seven centres, to(50–100/200–300 g mesentery); goat and sheepdevelop immunodiagnostic kits for fasciolosis,yielded lesser number (2–20/whole mesentery) ofschistosomosis and amphistomosis (first phase of thisblood flukes; collection of schistosomes from pigsproject has been completed in December 2004). Thewas difficult due to large amount of fat linked withproject was able to develop ELISA tests (Mishra,mesentery (Agrawal, 2004). Naturally, such2004) for all the three fluke infections, which areprocedures are difficult to follow for mass antigenhighly sensitive but with limited specificity (there areproduction. Realizing this fact, and for enhancingcross-reactions among fluke infections). Besidesensitivity and specificity of serological tests, workersspecificity problem, availability of antigens, cost ofhave tried developing different kinds of ELISA teststhe kits and reluctance of animal owners to provideincluding Dot-ELISA which required antigens inanimal blood for testing may deter their wider use. Itmicrogram quantities (Vohra et al., 2006).will be more prudent if immunodiagnostic research isassociated with development of vaccines against these To overcome availability of pure antigen in largerinfections.quantity, it is imperative to incorporate recombinant

Problems confronting helminthic diseases of domestic animals9DNA technology, in future work, enhancing chancesof developing commercial kits against helminthosis.As antibody detection, though more sensitive, hasinherited disadvantage of not differentiating presentand past infections, attention has been diverted inidentifying coproantigens which will do away theneed of blood collection and antigens forimmunodiagnosis.PATHOGENICITYThis aspect has been attended well by many Indianscientists (Deo, 1964; Dutt, 1980; Bhatia andChauhan, 1984; Agrawal and Shah, 1989; Parija,1990; Agrawal and Alwar, 1992; Choudhary, 1994;Agrawal and Southgate, 2000; Sood, 2003a; Singh,2005). Detailed studies have been made onpathological changes at different time intervals duringschistosomosis, fascioliosis, immatureamphistomosis and haemonchosis. The pathologicalchanges have been co-related with differenthelminthic stages, which differed not only as per flukespecies but also as per host factors. Buffaloes havebeen shown to suffer more acutely during prepatentperiod of fasciolosis (Mishra, 2004). Biliaryamphistomosis was a problem of cattle with lesssuffering in buffaloes (Ghosh and Chauhan, 1994);immature amphistomosis occurred in clinical form insheep and goats. Sheep and goats suffered moreseverely with schistosomes; however, higher infectionrates were well tolerated by pigs. Though H. contortushas been reported from all the ruminants, its severity ismore marked in sheep linked with severe anaemia,diarrhoea, loss of body weight and death.Cysticercosis, though affects the body weight of pigs,is considered more pathogenic in human beings.Most of the above studies have established damagescaused by helminthosis in domestic animals but thesestudies could not be used for ante-mortem diagnosis,prognosis or treatment of animals as tissue changeswere studied after the death of animal. Therefore, inrecent years, much emphasis has been given on bloodbiochemical changes taking place in animals. Forinstance, Agrawal and associates (Gupta, 2002;Agrawal and Singh, 2000) showed development ofhypoglycaemia in schistosomosis in experimentallyinfected rabbits and goats. Severe anaemia withhaemoglobin as low as 4 mg/100 ml was noticed incaprine schistosomosis prior to the death of animals(Gupta, 2002). Such studies were important to pinpointchanges taking place, which may not only help indiagnosis but also in the treatment of animals.The advancement of science has revealed hormonal,metabolic and immunological changes taking place inhelminthosis, which are comparatively moreimportant in dealing with pathogenesis of any diseasecondition, rather than morbid tissue changes. This is anew field to be explored by Indian scientists withchances of achieving more spectacular results. Apreliminary work on porcine schistosomosis has triedto explain sudden death of experimentally-infectedpiglets within 1–7 days due to anaphylactic shockbecause of prenatal sensitization of animals withschistosome antigens (Agrawal et al., 2001). Mosthelminthic infections, including schistosomosis, havebeen characterized by the production of Th2associated cytokines (interleukins) and antibodies(IgG and IgE), which persisted for the duration ofinfection. When this immune response becamechronic, improperly controlled or exaggerated, it maylead to lethal consequences (Hoffmann et al., 2002).Such work in India is lacking and immunological workhas centred towards identification of antibodies orantigens for immunodiagnosis.There are reports that helminthosis is also responsiblefor delayed puberty or sterility in animals;nevertheless, this contention needs confirmationunder Indian conditions. Similarly, little work hasbeen undertaken to correlate helminthosis withproduction traits; detailed work is needed in thisdirection due to its direct bearing on the economy offarmers. So far, studies carried out have claimed lowermilk and wool production or lower body weight due tohelminthosis. Work along this line should be extendedincorporating more types of parasites, mixedinfections and different categories of animals. There isalso a need to correlate sub-clinical infections withproduction traits in different animal species underdifferent agro-climatic zones and with different feedconsumption rates. There is another field awaitingexploration to record trace element deficiencies whichmay be caused by helminthosis, as a classical work onDiphylobothrium latum has shown for vitamin B 12.Such work will greatly help in an early clinicalrecovery along with lower cost of therapy.ADVANCES IN VACCINE PRODUCTIONA right direction to research was given somewhere in1970 and onwards when Dutt and his group triedschistosome cercariae for protecting laboratoryanimals against schistosomosis. Both homologousand heterologous schistosome cercariae,schistosomulae, whole worm homogenate, and

10Agrawal and Banerjeeexcretions and secretions were found to protect the triclabendazole, recommended against Fasciolaanimals against re-infection with varying degree of species, killed S. incognitum to a significant (p < 0.05)success. Most of the work has been carried out using S. level. These works of Shrivastava and Agrawal (1999)incognitum as it is easy to grow this parasite in emphasized the need of the trial of drugs in differentlaboratory animals, though S. spindale has also been doses and combinations in other helminthic infectionsused in heterologous immunological trials (Agrawal as well.and Shah, 1989). There is a success story of usingvaccine containing irradiated larvae against lungSome recent work has shown the development ofworms of sheep produced by Indian Veterinarydrug-resistance by H. contortus against not onlyResearch Institute (IVRI) workers (Dhar and Sharma,benzimidazole group of drugs but also against1981). Some work was also carried out usinglevamisole and ivermectin etc. warranting moreirradiated larvae against H. contortus infection inattention on the subject and to discouragesheep (Sood, 2003b). However, the work could not beindiscriminate use of anthelmintics by commercialextended in domestic animals against schistosomosis;dairy farms (Yadav et al., 1995; Singh et al., 2002).production of vaccine against lung worms wasLikewise, there is little work on the use of combinationdiscontinued by IVRI. It seems that presently noof drugs for synergistic effect or for studyingIndian institute is continuing the work on vaccinepharmacokinetics of a particular drug against aproduction against any helminthic disease.common helminthic infection in two different hostspecies. Sanyal (1995, 1996) has demonstratedNo doubt, the future vaccine work cannot be continued different pharmacokinetics of triclabendazole inwith irradiated larvae or live cercariae but pure buffalo and cattle, thereby suggesting the requirementantigenic molecules have to be identified by of higher therapeutic dose in buffaloes for killing F.employing genetic engineering techniques, which will gigantica. Similar type of work is needed on otheralso help in their production in large quantities. Here it drug and host combinations.may be pertinent to mention that research onimmunodiagnosis of fluke infections has led toThere are some interesting efforts made in selfidentification of antigenic molecules responsible formedication system for integrated management ofproducing antibodies, which are helping in diseasegastrointestinal parasites in dairy animals and in-feeddiagnosis. It will be worthwhile to investigate if theseformulations for ovine and caprine parasiticantigens, some of which are common to other flukesgastroenteritis (Sanyal, 1998). However, research onalso, may be used as vaccine candidates. Unlike India,new drug molecules against helminthosis is lackingmore work is going on in other countries on thedue to apathy on part of pharmaceutical companies.development of sub-unit/molecular vaccines againstThough clinical research is taking shape in humanhelminths and it will be beneficial to collaborate withmedicine, it will take some more time to cash this ideasuch research groups.in veterinary practice in India.CONTROLA critical analysis of drug industry reflects either theyare not interested in drug research againstConsiderable attention has been paid to treat helminthosis due to lower market demand or have nohelminthosis and new drugs have been tried as soon as research facilities to that magnitude. Also, therethey become available. Work on the comparison of the appeared little social concern by the drug industryefficacy of different drugs has also been undertaken with no attention on molluscicides or even marketingwith different dosages. Whereas there are effective the well established molluscicides in India to combatdrugs against fasciolosis, amphistomosis, snail population, responsible for all the flukegastrointestinal nematodiasis, apparently, there is no infections.effective drug against Indian schistosomes as lithiumantimony thiomalate has proved to be of doubtfulControl of helminthosis has largely been dependent onefficacy. Even praziquantel, recommended by Worldchemotherapy. However, as it is confined to individualHealth Organisation for human schistosomosis couldtreatment or at the most treating a group of animals, itnot kill significant number of S .incognitum worms.has not influenced helminth population in theLooking at the doubtful efficacy and the cost of theenvironment. Control measures, besidedrug, it is difficult to recommend this drug for thechemotherapy, have still been confined to laboratorytreatment of bovine schistosomosis. Interestingly,and no organization has come forward either to

Problems confronting helminthic diseases of domestic animals11popularize the potential control measures or tocommercialize the important ones.REFERENCESAgrawal MC. 1998. Parasitology in India since independence.Indian J Anim Sci 68 (8, special issue):793-799.The helminth population in recent years has beenaffected adversely because of changes in agricultural Agrawal MC. 2002. Remembering Dr. S C Dutt. Thepractices and use of fertilizers and fungicides. The Parasitologist. Department of Parasitology, College oflimited work done has revealed that the use of Veterinary Science and A H, J. N. K. V. V., Jabalpur.fertilizers and fungicides has adverse effect on Agrawal MC. 2003. Epidemiology of Fluke Infections. In:nematode eggs and larvae, thus indirectly helping in “Helminthology in India”. (Ed. M. L. Sood). Internationaltheir control. Treatment of faeces in gobar gas plants Book Distributor, Dehradun.s 511-542.also kills eggs and larvae. Fungi have been isolated Agrawal MC. 2004. Final report “Diagnosis of parasiticfrom pastures, which are detrimental to nematode eggs diseases of domestic animals” Collaborating Center, NATPand larvae. Sanyal (2000) has tried fungi for the (MM), College of Veterinary Sciences and A.H., Jabalpur.control of gastrointestinal nematodes, which are selfAgrawal MC and Alwar VS. 1992. Nasal schistosomiasis: Apropagating and capable of killing nematode larvae. review. Helminth Abstr 61:373-384.However, it is still to confirm that prolong use of thesefungi in animal feed will not adversely affect animal Agrawal MC and Shah HL. 1984. Stephanofilarial dermatitis inhealth or their production. Obviously, management ofIndia. Vet Res Comm 8:93-102.pastures for the control of helminths has not received Agrawal MC and Shah HL. 1989. A review on Schistosomafavours and thus been confined to text books only. incognitum Chandler, 1926. Helminth Abstr 58:239-251.Computer programs have also been developed whichAgrawal MC and Singh KP. 2000. Effect of Schistosomacan monitor haemonchosis in a farm and may help in incognitum infection on serum biochemistry of rabbits. J Vetits control (Bandyopadhyay and Rajkhowa, 1997). Parasitol 14:31-34.Biological control has great potentials in India due to Agrawal MC and Southgate VR. 2000. Schistosoma spindalewide biodiversity and much more attention should be and bovine schistosomosis. J Vet Parasitol 14:95-107.paid on this issue. The ducks have been used to some Agrawal MC, Banerjee PS and Shah HL. 1991. Fiveextent for reducing snail population. A recent mammalian schistosome species in an endemic focus inencouraging report is that dragon fly nymphets share India. Tran R Soc Trop Med Hyg 85:231.the habitats of fresh water snails and devour youngAgrawal MC, Jain J and Rao KNP. 2001. Sudden death ofsnails voraciously reducing their number to a piglets during immature schistosome infection. Indian Jsignificant (p < 0.01) level (Agrawal, 2004). These Anim Sci 71:681-682.nymphets survive for one to two years, therebyAgrawal MC, Vohra S, Gupta S and Singh KP. 2003.removing the need of their frequent introduction.Schistosomosis and other intestinal helminths in domesticUnder laboratory conditions, Mesocyclops leuckarti animals in and around Bhubaneswar (Orrisa). Indian J Animaequatorialis, a water crustacean, has been found Sci 73:875-877.devouring on miracidia of S. incognitum and F.Attwood SW. 2001. Schistosomiasis in the Mekong Region:gigantica (Banerjee, 1996). Agrawal (2004) hasEpidemiology and Phylogeography. Adv Parasit 51:87-152.conclusively proved that snails carry mono-speciesfluke infections (with a few exceptions), hence, if they Bandyopadhyay S and Rajkhowa C. 1997. Haemonchus: Aare pre-infected with non-pathogenic fluke species,computer programme for haemonchosis in animals. Indian JAnim Sci 67:684.the snails may be resistant to pathogenic flukeinfections. However, this hypothesis has to be tested Banerjee PS. 1996. Mesocyclops leuckarti aequatorialisunder field conditions because only 1–2 % positive (Kiefer), a possible biocontrol agent of Schistosomasnails are capable of sustaining fluke infections in incognitum and Fasciola gigantica. Indian Vet J 73:1218-1221.nature.ACKNOWLEDGEMENTSBanerjee PS and Agrawal MC. 1991. Prevalence ofSchistosoma nasale Rao 1933 at Jabalpur. Indian J AnimThe authors are highly thankful to Dr. S. Vohra,Sci 61:789-791.Assistant Professor, Department of Parasitology, Banerjee PS, Agrawal MC and Shah HL. 1991. Application ofCollege of Veterinary Sciences, Jammu, in providing CHR and J-index in bovine schistosomiasis. Indian Vet J68:1022-1026.help during preparation of this manuscript.

12Agrawal and BanerjeeBaugh SC. 1978. A century of Schistosomiasis in India: Humanand Animal. Rev Iber Parasitol 38:435-472.Bhatia BB and Chauhan PPS. 1984. Parasitic Diseases ofBuffaloes in India. Cosmo Publications, New Delhi.Chauhan AS, Srivastava CB and Chauhan BS. 1973. Studies onthe trematode fauna of India, Part 6-Digenea:Schistosomatidae. J Zoo Soc India 25:83-127.Mishra AK. 2004. Final report Diagnosis of parasitic diseasesof domestic animals. Lead Center, NATP (MM), IndianVeterinary Research Institute, Izatnagar.Mukherjee RP and Chauhan BS. 1965. Studies on the trematodefauna of India. Part V. Subclass Digenea: ParamphistomidaeFishchoeder, 1901. J Zool Soc India 17:160-225.Parija SC. 1990. A Review on Parasitic Zoonosis. ATIBSPublishers, Delhi.Choudhary, N. 1994. Helminths of Domesticated Animals inIndian Subcontinent. In “Helminthology” (Eds N Prasad A and Verma TK. 1999. On the prevalence andChoudhury and N J Tada). Narosa Publishing House, New community dominance among paramphistomes infectingDelhi, pp. 73-120. domestic ruminants. J Vet Parasitol 13:129-133.Choudhary N and Tada NJ. 1994. Helminthology. Narosa Sanyal PK. 1995. Kinetic disposition of triclabendazole inPublishing House, New Delhi. buffalo compared to Cattle. J Vet Pharmacol Therap 18:370 -Das SS, Kumar D and Sreekrishnan R. 2003. Hydatidosis in374.animals and man. In: “Helminthology in India”. (Ed ML Sanyal PK. 1996. Kinetic disposition and critical efficacy ofSood). International Book Distributor, Dehradun pp. 425- triclabendazole against experimental bovine and bubaline451. fasciolosis. J Vet Parasitol 10:147-152.Deo PG. 1964. Roundworms of poultry. Animal HusbandrySeries No. 3. ICAR, New Delhi.Dhar DN and Sharma RL. 1981. Immunization with irradiatedlarvae against Dictyocaulus filaria in young lambs. Vet.Parasitol 9:125-131.Dutt SC. 1980. Paramphistomes and Paramphistomiasis ofdomestic animals of India. Punjab Agricultural University,Ludhiana.Dutt SC and Srivastava HD. 1968. Studies on Schistosomanasale, Rao, 1933. II. Molluscan and mammalian hosts ofthe blood fluke. Indian J Vet Sci and Anim Hus 38:210-216.Gaitonde BB, Sathe BD, Mukerji S, Sutar NK, Athalye RP,Kotwal VP and Renapurkar DM. 1981. Studies ofschistosomiasis in village Gimvi of Maharashtra. India JMed Res 74:352-357.Ghosh RC and Chauhan HVS. 1994. Occurrence and pathologyof naturally occurring biliary amphistomes in buffalo andcattle. Indian J Anim Sci 64:1332-1334.Gupta NK. 1993. Amphistomes: Systemics and Biology.Oxford and IBH Publishing company Private Limited, NewDelhi.Sanyal PK. 1998. The pharmacokinetics and efficacy of longterm level and split dose administration of albendazolethrough in-feed formulations against ovine and caprineparasitic gastroenteritis. Vet Res Comm 22:467-477.Sanyal PK. 2000. Screening for Indian isolates of predaciousfungi for use in biological control against nematodeparasites of ruminants. Vet Res Comm 24:55-62.Shah HL and Agrawal MC. 1990. Schistosomiasis: In: AReview on Parasitic Zoonosis (Ed. S. C. Parija). ATIBSPublishers, Delhi-143-172.Shrivastava KK and Shrivastava GP. 1974. Two cases ofTrypansosoma (Herpetosoma) species infection of man inIndia. Trans Roy Soc Trop Med Hyg 68:143-144.Shrivastava MK and Agrawal MC. 1999. Effect oftriclabendazole and oxyclozanide on Schistosomaincognitum infection in mouse. Indian Vet J 76:493-496.Singh D, Swarnkar CP and Khan F. 2002. Anthelminticresistance in gastrointestinal nematodes of livestock inIndia. J Vet Parasitol 16:115-130.Singh KRS. 2005. Veterinary Helminthology. Indian Councilof Agricultural Research, New Delhi.Gupta S. 2002. Clinical, biochemical and parasitological Sood ML. 2003a. “Helminthology in India”. Internationalstudies and prevalence of caprine schistosomosis in and Book Distributor, Dehradun.around Jabalpur. Ph.D. Thesis, Rani Durgavati VishwaVidyalaya, Jabalpur, India.Sood ML. 2003b. Haemonchosis in India. In: “Helminthologyin India”. (Ed. M. L. Sood). International Book Distributor,Hoffmann KF, Wunn TA and Dunne DW. 2002. Cytokines- Dehradun pp. 575-617.mediated host responses during schistosome infections;Walking the fine line between immunological control andSrivastava HD and Dutt SC. 1962. Studies on Schistosomaimmunopathology. Adv Parasit 52:265-307.indicum. Research Series Bulletin no. 34 Indian Council ofAgricultural Research, New Delhi.Joshi P, Shegokar VR, Powar RM, Herder S, Katti R, SalkarHR, Dani VS, Bhargava A, Jannin J and Truc P. 2005.Subba Rao NV. 1989. Hand Book: fresh water molluscs ofHuman trypanosomiasis caused by Trypanosoma evansi inIndia. Zoological survey of India, Kolkatta.India: The first case report. Am J Trop Med Hyg 73:491-495. Tandon V. 2003. Spectrum of helminthic parasites in north-east

Problems confronting helminthic diseases of domestic animals13India. In: “Helminthology in India”. (Ed. M. L. Sood).International Book Distributor, Dehradun 57-76.Tandon V and Dhawan BN. 2005. Infectious diseases ofdomestic animals and zoonoses in India. Proc Nat Acad SciIndia 75 (B), Special issue. The National Academy ofSciences, India, Allahabad.Tchuente LAT, Southgate VR, Combes C and Jordane J. 1996.Mating behaviour in schistosome: Are paired worms alwaysfaithful?. Parasit Today 12:231-236.Verma AK. 1953. On Fasciola indica n. sp. with someobservations on Fasciola hepatica and Fasciola gigantica. JHelm 27:67-76.Vohra S, Agrawal MC and Malik YP. 2006. Dot ELISA indiagnosing caprine schistosomosis. Indian J Anim Sci76:988-991.Webster BL and Southgate VR. 2002. Evolution of Schistosomahaematobium, S. intercalatum and their natural hybrids atLoum, Cameroon. In Remembering Dr. S. C. Dutt: TheParasitologist (Ed. M. C. Agrawal) Department ofParasitology, College of Veterinary Science and AnimalHusbandry, Jabalpur. pp 88-93.Yadav CL, Kumar R, Uppal RP and Varma SP. 1995. Multipleanthelmintic resistance in Haemonchus contortus in a sheepfarm in India. Vet Parasitol 60:355-360.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 14-21Invited reviewJ P DAntimalarial agents: the present conceptVarsha Gupta and Nidhi SinglaDepartment of Microbiology, Government Medical College and Hospital, Chandigarh.Received 13 April 2007; revised 10 July 2007; accepted 13 July 2007ABSTRACT. Malaria has afflicted humankind for millennia and continues to do so. It not onlyaffects the health of nations but is also an economic burden. The emergence and spread of drugresistantmalaria parasites poses a considerable challenge to malaria control. Although the burden ofmalaria and the main focus of its control are in Africa, it is an important public health problem intropical parts of the world viz. India, Indonesia, Papua New Guinea and the Amazon region of LatinAmerica. The chemotherapy continues to be a major element of malaria control, and has beenadopted by the World Health Organization as a sustainable and realistic approach. Current effortsfocus on research into the discovery and development of new compounds with novel mechanisms ofaction, and on measures to prevent or delay the emergence of resistance, whenever they areintroduced. Drug discovery and development is a long, expensive and risky venture. Therefore, whilstwaiting for the drugs of tomorrow, there is pressing need to use the available antimalarials in aneffective way. Although quinine, chloroquine and antifolate compounds have been the mainstay ofchemotherapy for years, it's the advent of artemisinins that has led to newer strategies in antimalarialtreatment. Artemisinin combination therapies offer effective, easy to use and affordable alternatives.These are the only compounds now-a-days which, apparently, have not yet developed significantresistance. This review summarizes the old as well as current antimalarial drug options under way.Keywords: antimalarials, artemisinins, combination therapy, newer approachesMalaria is a serious global health challenge that affects400–600 million cases each year (Snow et al., 1999). Itremains an important public health concern not only incountries where transmission occurs regularly, but alsoin areas where malaria transmission has been largelycontrolled or eliminated. Malaria is a complex diseasethat varies widely in epidemiology and clinicalmanifestations in different parts of the world. Thisvariability is the result of many factors but the mostimportant is the development of drug-resistance inPlasmodia and the resistance of anopheles mosquitoesto DDT. Resistance to antimalarial drugs, by and large,is seen in two of the four species of malaria parasite thatCorresponding author: Dr. Varsha Gupta, H. No.1154A, Sector-32-B, Chandigarh-160 031, India.E-mail:varshagupta_99@yahoo.comnaturally infect humans: Plasmodium falciparum and P.vivax. P. falciparum has developed resistance topractically all the antimalarials used currently (Bloland,2001), and continues to increase in both intensity andgeographic distribution.Almost half-a-century after the first global malariaeradication programme was started, the Roll BackMalaria (RBM) initiative was launched in 1998 by theWHO, UNICEF, UNDP, and the World Bank. Its aimhas been to provide a coordinated internationalapproach to fight malaria, with the goal of halving theburden of malaria by 2010. RBM is a global partnershipestablished to enable countries and communities to takeeffective and sustainable action against malaria. TheWHO strategy to RBM includes: prompt treatment witheffective drugs, effective use of insecticide-treatedmaterials, and other vector control methods;

Antimalarial agents15intermittent preventive treatment in pregnancy; and • Sesquiterpine lactones: artemisinin derivativesemergency epidemic preparedness and response (Shiff, artesunate, artemether, arteether2002; Gardiner et al., 2005). Presently, RBM is underimplementation, especially, in Africa with the aim of • Phenanthrene-methanol: halofantrinereducing the malaria mortality in African children byhalf, by the year 2010 (Remme et al., 2001).• Hydroxy napthoquinone: atovaquoneIn 2002, the malaria genome-sequencing project was • Ribosomal inhibitors: tetracyclines, clindamycincompleted (Gardner et al., 2002). In addition, the• Antifolate combination drugs: dihydrofolategenomes of both the human host (Venter et al., 2002)reductase inhibitors (pyrimethamine,and the most important mosquito vector Anophelestrimethoprim) and sulphonamides (sulfadoxine,gambiae (Holt et al., 2002) have also now beendapsone, sulfalene, sulfamethoxazole, sulfadoxinecompleted. The information on genomes is providingand others)new insights that may lead to novel strategies to combatthis disease. Already, this information has been • Newer drugs: pyronaridine, lumefantrine (fluoroinstrumentalin the development of new approaches for methanol), piperaquine (bisquinoline), G25,malaria control.fosmidomycin, trioxanes, bulaquine, diamidinesThe basic aims of using drugs in relation to malarialand various plant molecules.infection are: to prevent and treat a clinical attack of Chloroquine: It is a rapidly acting erythrocyticmalaria, to completely eradicate the parasite from the schizonticide that is active against all species ofpatient's body and to reduce the human reservoir of Plasmodium. It acts by accumulating inside the acidicmalaria parasites to cut-down their transmission by vesicles of the parasite. It raises the vesicular pH and somosquitoes.interferes with degradation of hemoglobin by parasiticClassification of antimalarial drugs can be done underlysosomes. Its oral absorption is excellent and it is safethree broad headings: biological, clinical andeven during pregnancy. Commercial preparations arepharmacological (Tripathi, 2003).available as Resochin, Cloquin, Lariago and Nivaquin.Although development of resistance againstA. Biologically, antimalarials are primary tissue- chloroquine has been slow, P. falciparum showsschizonticides, secondary tissue- schizonticides or significant resistance against this drug. Resistant strainshypnozoitocides, blood-schizonticides, are prevalent worldwide, especially in eastern parts ofgametocides and sporontocides.India, Southeast Asia, Africa and South America.Chloroquine-sensitive strains of P. falciparum areB. Clinically, antimalarials are drugs which are usedconsidered to be confined mainly in areas of Centralfor causal prophylaxis, anti-relapse and for clinicalAmerica, Dominican Republic and Haiti (Bloland,or suppressive cure and the ones used to eliminate2001; Olliaro and Taylor, 2004). Chloroquine-resistantsexual stages.P. vivax strains were first reported from Papua NewC. Pharmacologically, antimalarials are:Guinea in 1989, and are now prevalent in Burma,Colombia, Brazil, Indonesia, focal areas of India and• 4 aminoquinolines: chloroquine and amodiaquine Solomon islands (Baird et al.,1995; Olliaro and Taylor,2004). Emergence of chloroquine-resistant P. malariae• Quinoline-methanol: mefloquine has also been reported from Indonesia (Maguire et al.,• Acridine: mepacrine2002).• 8-aminoquinolines: primaquineAmodiaquine: It is identical to chloroquine in itsproperties but acts somewhat faster than chloroquine.Serious side effects like toxic hepatitis and• Cinchona alkaloids: quinine and other structurallyagranulocytosis have been reported after its use, so,related compoundsalthough it has been used for treatment of clinical• Biguanides: proguanilattacks of uncomplicated malaria, it is notrecommended for prophylaxis.• Sulphonamides and sulfone: sulfadoxine anddapsoneMefloquine: It is also a relatively rapid acting blood

16 Gupta and SinglaPyrimethamine: It is also a slow-acting bloodschizonticide like proguanil, but its action iscomparatively faster and more potent than that ofproguanil. It does not eliminate the pre-erythrocyticphase of P. falciparum. Therefore, it is not a radicalcurative. However, extended treatment causesexhaustion of secondary tissue phases of P. vivaxleading to suppressive cure. In combination withsulphonamides, it is used to treat clinical attacks ofmalaria.Primaquine: Primaquine is the most important drugused for the radical cure of relapsing malaria. It is a poorblood schizonticide, but has marked efficacy againstprimary as well as secondary tissue phases of malarialparasite. Primaquine differs from all other antimalarialsin being highly active against gametocytes andhypnozoites. Its mechanism of action is not clearlyknown. It is readily absorbed orally and the mostsignificant side-effect is dose-related hemolysis andmethemoglobinemia, especially in persons with G-6PD deficiency. As foetus is also G-6 PD deficient, itsuse is not advised during pregnancy.Artemisinin compounds: Artemisinin is the activeprinciple of the plant Artemisia annua, used in Chinesetraditional medicine as 'Quinghaosu.' It is asesquiterpine lactone active against P. falciparum-resistant to all other antimalarial drugs as well assensitive strains. The mechanism of action ofartemisinin is not definitely known. The endo-peroxidebridge in its molecule appears to interact with heme inmalaria parasite, which results in the release of a highlyreactive free-radical that binds membrane proteins,causes lipid peroxidation, damages endoplasmicreticulum and ultimately leads to parasite lysis.It is a potent and rapid blood schizonticide. Severalderivatives of artemisinin have been produced, ofwhich three are marketed in India: water solubleartesunate, oil soluble artemether and arteether(developed in India). Artesunate preparations areavailable as Falcigo, Falcyanate and Arnatecommercially, whereas artemether is available asPaluther and Larither. These compounds are used totreat severe malaria and show very rapid parasiteclearance. However, the duration of action is short andrecrudescence rate is high, when these are used alone inshort courses. Therefore, monotherapy needs to beextended beyond the disappearance of parasite toprevent recrudescence. So far, no artemisinin resistanceamong P. falciparum has been noted. Arteether is thecompound developed in India, and has been releasedschizonticide, but its action is slower than chloroquine.It is effective against both chloroquine-sensitive and -resistant strains of malaria parasites. It is also aneffective suppressive prophylactic for multi-drugresistant P. falciparum and other types of malarias.Mefloquine-resistance among P. falciparum strains hasbecome common in Thailand, Combodia andMyanmar, but is sporadic in Africa, South America andMiddle East. Because it is not used extensively in India,mefloquine-resistance is not a problem here yet.Resistance to mefloquine causes cross-resistance toquinine and halofantrine also (Ter Kuile Fo et al., 1993;Mockenhaupt, 1995).Mepacrine: It is a blood schizonticide that is moretoxic and less effective than chloroquine. It is obsoleteas an antimalarial, but is still marketed.Quinine: The first known effective antimalarial wasquinine, often referred to as the Jesuit's bark. It is thelevo rotatory alkaloid obtained from the bark of theSouth American Cinchona sp. plant. It first rose tothprominence in the 17 century when, in the apocryphalthstory the 4 Condessa (Countess) de Chinchon wascured from malaria while staying in Lima (Gardiner etal., 2005). It is a blood schizonticide for all species ofPlasmodium. It is also less effective and more toxic thanchloroquine. It is effective in terminating an acuteattack of falciparum malaria, but does not preventrecrudescence completely, so, there is incompleteclearance of parasites from the body. It also does not acton pre-erythrocytic stages and on hypnozoites ofrelapsing malaria, but kills the gametes of P. vivax. Itsuse primarily is restricted to the chloroquine-resistantstrains of Plasmodia where it is used orally. Intravenouspreparation is used for complicated/cerebral malariaeven for chloroquine-sensitive strains. Its main sideeffectis cinchonism, in which patient exhibits ringingin the ears, nausea, vomiting, headache, mentalconfusion, vertigo etc. Signs and symptoms ofcinchonism gradually disappear as the drug is stopped.Quinine still remains an effective antimalarial drug,although resistance to it has been reported in Indochina,South East Asia and Brazil (Tripathi, 2003).Proguanil: Proguanil, also known as chloroguanide, isa slow-acting blood schizonticide. It is also causalprophylactic for P. falciparum. Proguanil inhibits theplasmodial dihydrofolate reductase (DHFR) enzyme.Currently, it is used in combination with chloroquinefor malaria prophylaxis in areas with low levelchloroquine-resistance against P. falciparum. It canalso be given during pregnancy.

Antimalarial agents17for institutional use only. It is used for efficacious agents is the need of the hour, optimizationcomplicated/cerebral malaria in areas with of existing agents by new dosing regimen as well aschloroquine-resistant P. falciparum (Tripathi, 2003). combination therapy with pre-existing agents has goneOther derivatives available are dihydroartemisinin, a long way. Combination therapy offers variousartelinate and artemotil (injectable artemether). Major advantages: it improves the antimalarial efficacy anddrawback with artemisinin compounds is their cost. provides synergistic antiparasitic action along withTherefore, many synthetic derivatives of artemisinin decreasing the chances of the development ofare under development and production. Artemisone is resistance. Combination therapy also shortens theone such metabolically stable, semi-synthetic duration of treatment, and in turn increases compliancederivative of artemisinin (Olliaro and Taylor, 2003). (Kremsner and Krishna, 2004).B. Lumefantrine is a fluoromethanol compound, whichis being tried as a fixed combination tablet withartemether and is marketed under the name of Riamet orCoartem. Riamet is available in developed, non-endemic countries for use by travellers, whereasCoartem is marketed in malaria endemic areas. The sixdoseregimen is highly efficacious against MDR P.Halofantrine: It is a phenanthrene methanol bloodschizonticidehaving activity comparable tomefloquine. It has been especially recommended foruse in areas with multi-drug resistant (MDR) P.falciparum infections. It is also effective againstresistant strains of P. vivax. But it is not active againstgametocytes or hepatic stages of malarial parasite.Serious side-effects in the form of severe, potentiallyfatal cardiac conduction abnormalities have beendocumented (Nosten et al., 1993).Atovaquone: This recently produced synthetichydroxy naptha-quinone is a rapidly acting bloodschizonticide for P. falciparum and other Plasmodia. Itcollapses plasmodial mitochondrial membranes andinterferes with ATP production. When used singly,development of resistance is rapid due to a single point-mutation in cytochrome b gene and so, presently, it islicensed for use as an antimalarial, only in combinationwith proguanil as 'Malarone'. As this drug is costly, costfactor limits its wide-spread use and is predominantlyused by the travellers to Southeast Asia, confrontedwith MDR parasites (Hyde, 2005). It can be givenduring pregnancy, and is being distributed throughdonation programmes (Bloland et al., 1997; Foege,1997).Tetracyclines: Tetracyclines are ribosomal inhibitors.They are weak blood schizonticide and also act on preerythrocyticstages of P. falciparum. Tetracyclines arebasically used for chloroquine-resistant falciparummalaria, in combination with quinine orpyrimethamine- sulfadoxine (Bloland, 2001).Clindamycin: Clindamycin is given to patients unableto take tetracyclines. In combination with quinine, itcan increase the efficacy of treatment and alsodecreases the duration of treatment, which furtherreduces the likelihood of the development of resistanceand quinine associated side-effects.Combination drugs: Although, for successfulantimalarial therapy, development of newer andAntifolate combination drugs: Such drugs are variouscombinations of dihydrofolate reductase inhibitors(proguanil, chlorproguanil, pyrimethamine,trimethoprim) and sulfa drugs (dapsone, sulfalene,sulfamethoxazole, sulfadoxine). They exhibit supra-additive synergistic effect due to sequential block ofDHFsynthetase and DHFRase, and are primarily usedfor clinical cure of P. falciparum, especially the strainswhich are chloroquine-resistant. Typical combinationsinclude sulfadoxine/pyrimethamine (Fansidar),sulfalene/ pyrimethamine (Metakeflin) andsulfamethoxazole/trimethoprim (Co-trimoxazole).However, these next generation antimalarial drugsfared even worse. Resistance to Fansidar developedwithin a year of its introduction into Thailand and isnow widespread (White, 1992). Resistance to sulfapyrimethaminehas been found to a limited extent inIndia, and that too mainly in North-East states.However, resistance to sulfa-pyrimethamine is sohighly prevalent in South East Asia, South America andSouthern Africa that it precludes its clinical use(Bloland, 2001). Fansimef or MSP, a combination ofmefloquine, sulfadoxine and pyrimethamine, wasdeveloped but not used eventually due to possiblesevere adverse reactions, this combination can cause.Newer drug combinations: A. A new antifolate drugcombination, which is currently used, primarily inAfrica, is a combination of chlorproguanil and dapsone(Lapdap). It has been found to be a much more potentdrug, with efficacy more than that of Fansidar. Itsbenefits include a greater cure rate, less chances of thedevelopment of resistance and comparatively less cost(Watkins et al., 1997).

18 Gupta and Singlafalciparum and is well tolerated (Olliaro and Trigg, tafenoquine, an 8-aminoquinoline, offers more activity1995; Vugt et al., 1999). against hepatic stages of malarial parasites than parentcompound primaquine. Similarly, lumefantrine wasC. Piperaquine is another resurgent antimalarial drug. It developed as an analogue of halofantrine, which hasis a bisquinoline that was first synthesized and used in more toxicity (Rosenthal, 2003).China in 1960's. Due to development of resistance, itsuse was abandoned. But now, it is being tried again Compounds active against newer targets: Whereasalong with artemisinin as artemisinin combination artemisinin derivatives, particularly in combinationtherapy (ACT) to provide inexpensive, short course with other drugs, offer the best hope for the treatment oftreatment regimen with a high cure rate. Piperaquine malaria, especially MDR parasite strains, there havebased ACT's are CV4 (China Vietnam 4) which has been a number of exciting new developments.dihydroartemisinin, trimethoprim, piperaquine Information gleaned from the malaria genomephosphate and primaquine phosphate, CV 8 same sequencing project has led to the identification of adrugs with increased concentration (this product is still number of novel drug targets in the parasite that can bea part of national policy in Vietnam), Artecom without exploited for the discovery of new antimalarial drugs.primaquine and ArtekinTM or Duo- Cotecxin, which This is the most innovative approach of antimalarialhas dihydroartemisinin and piperaquine phosphate only drug discovery. Many of the enzymatic pathways in(Denis et al., 2002; Davis et al., 2005).plasmodia are unique or differ significantly from thehuman host. Targets could be of various types viz.D. Artesunate-amodiaquine (ASAQ) and artesunate- among cytosolic targets folate metabolism, purinemefloquine. These are two non-fixed drug salvage and pyrimidine synthetic pathways arecombinations which are being tried in Thailand, some potential drug acting sites. A recent report showed that aregions of South East Asia, Brazil and France. These are number of aspartic protease inhibitors of HIV are alsohighly effective against MDR P. falciparum and are effective antimalarials (Skinner-Adams et al., 2004).well tolerated. Artesunate-sulphadoxinemetabolismSimilarly, in parasitic membrane targets, phospholipidpyrimethamine is another combination drug that isoffers a potential drug target site.available as blister pack using age-based dosing and is Phospholipid synthesis is important for producingcurrently used in clinical trials in Africa (von Seidlein et membranes for parasitophorous vacuoles, cytosol andal., 2000). The other drug combination under multiple sub-cellular compartments. A new compoundevaluation is artesunate, chlorproguanil and dapsone G25 has even been found to be effective in vitro (Calas(Lapad plus). It is being tried where sufadoxinewhichet al., 2000). Another site of action is food vacuoles,pyrimethamine has failed. It offers the advantage ofare acidic in malarial parasite. The food vacuolecombining rapid potency of artesunate with slow appears to be the site of action of a number of existingcurative efficacy of recovery of chlorproguanilagainstantimalarials and also offers opportunities for therapiesdapsone (Alloueche et al., 2004).newer targets. In the food vacuole, haemoglobinis degraded into haeme, which is polymerised intoE. Artesunate along with pyronaridine is in early stages insoluble haemozoin pigment and globin. Globin inof development. Pyronaridine is an antimalarial which turn, is hydrolysed to individual amino acids.was synthesized and developed in China and has proven Antimalarial drugs appear to act by preventingits efficacy against drug-resistant falciparum malaria haemozoin pigment, producing free radicals in the food(Ringwald et al., 1996).vacuole or, in the case of experimental compounds,preventing globin hydrolysis. Globin hydrolysis isF. Naphthoquine and dihydroartemisinin. Fewmediated by a number of proteases and these enzymespreliminary studies indicated a cure rate of 100%offer potential targets for chemotherapy (Rosentahl,obtainable with a two-dose, one-day regime, leading to 2001). Other targets could be mitochondrial pathwaysinterest in this product but lack of pre-clinical data, (e. g. atovaquone acts on electron transport system)especially toxicology data on naphthoquine, makes it and apicoplasts involved in fatty acid and amino aciddifficult to determine the future status of this product. metabolism. Most of the antibiotics like tetracyclines,Miscellaneousclindamycin, macrolides, chloramphenicol etc. act bytargeting only apicoplast or mitochondrial sitesDevelopment of analogue drugs: Research is in (Ringwald et al., 1996).progress to develop newer antimalarials by chemicalmodification of the existing compounds. For example,Fosmidomycin: It is another novel compound which

Antimalarial agents19inhibits 1-deoxy-D-xylulose 5-phosphate Iron chelators have also some antimalarial activityreductoisomerase, an enzyme of the nonmevalonate (Loyevsky and Gordeuk, 2001). Similarly, vitamin andpathway of isoprenoid biosynthesis, which is absent in mineral supplements have been found to reduce clinicalhumans but present in many pathogens and plants. attacks of malaria especially vitamin A and ZincFosmidomycin has been well tolerated but produced (Greenwood et al., 2005).modest cure rates when used alone (Missinou et al.,2002; Lell et al., 2003). However, it can be used Drug resistance reversers: These are the agents whicheffectively in combination with other antimalarials like can reverse the parasitic resistance to knownclindamycin (Olliaro and Taylor, 2004).antimalarials, rendering these antimalarials effectiveagain. Important example is antihypertensiveTrioxane antimalarials: Several structural analogues verapamil (Martin et al., 1987) and antidepressantof artemisinin with crucial 1, 2, 4 trioxane activity are desipramine (Bitonti et al., 1988), both of which canbeing synthesized and screened for antimalarial reverse the resistance of P. falciparum to chloroquineactivity. Ferozan-50F is one such compound and is when used in high concentrations. Antihistaminefound to be effective, orally, against P. berghei infection chlorpheniramine has also been found to reversein mice. It exerts some action against pre-erythrocytic resistance even at safe dosing levels (Sowunmi et al.,schizogony and has a potent gametocidal effect in 1997). Similarly, chlorpromazine helps in the reversalinfected mosquitoes. Synthetic trioxanes are of resistance to quinine, and penfluoridol inconsidered as effective as arteether against P. mefloquine-resistance reversal (Dutta, 1995).falciparum-resistant to chloroquine, pyrimethamineand quinine. These compounds are cheaper to The medicines for malaria venture (Gardiner et al.,synthesize and might prove to be a viable alternative to 2005), a non-profit organization, created to facilitateartemisinins (Posner et al., 1994).discovery, development, and delivery of new affordableantimalarial drugs, is currently supporting theBulaquine, a new drug that has been discovered by evaluation of the effectiveness of many new classes ofCentral Drug Research Institute, Lucknow, India potential antimalarial compounds. If or when these new(CDRI compound 80/53), is being marketed under the drugs will become widely available, is still far fromTMname Aablaquin by Nicholas Piramal India Limited certain; however, they do hold the promise that drug-(Kshirsagar, 2006). An analogue of primaquine, resistant malaria can be combated in a foreseeablebulaquine appears to be a relatively safer substitute for future. Unfortunately, as with all the other newlythe radical cure of relapsing form of vivax malaria. Its developed drugs, the cost per treatment may be theadvantage is that it is 8-times less toxic than primaquine important rate-limiting step to their general use.(Dutta et al., 1989; Puri et al., 1989).REFERENCESPlant molecules: From time to time, plant productsAlloueche A, Bailey W, Barton S, Bwika J, Chimpeni P, Faladehave been evaluated for antimalarial activity. CinchonaCO et al. 2004. Comparison of chlorproguanil-dapsone withand Artemisia are such plants providing quinine and sulfadoxine-pyrimethamine for the treatment ofartemisinins, respectively, which are presently the uncomplicated falciparum malaria in young African children:mainstay for treatment of malaria. Some other plants double-blind randomised controlled trial. Lancet 363:1843-also have key molecules, which exhibit antimalarial 1848.activity e. g. Alstonia plant with indole alkaloids, Baird JK, Basri H, Subianto B, Fryauff DJ, McElroy PD,Dichroa febrifuga with febrifugine, Simaba cedron Leksana B et al. 1995. Treatment of chloroquine-resistantwith cedronin and Brucea javanica with brucein. Out of Plasmodium vivax with chloroquine and primaquine orthese, febrifugine especially has been found to be halofantrine. J Infect Dis 171:1678-1682.effective. It is 50- to 100-times more active than quinine Bitonti AJ, Sjoerdsma A, McCann PP, Kyle DE, Oduola AM,against P. vivax, but its use is limited due to its side- Rossan RN, Milhous WK and Davidson DE Jr. 1988.effects (Guru et al., 1996).Reversal of chloroquine resistance in malaria parasiteDiamidines, which have been used asPlasmodium falciparum by desipramine. Science 242:1301-1303.antitrypanosomal and antileishmanial drugs, are alsoBloland PB, Kazembe PN, Watkins WM, Doumbo OK,being evaluated for their antimalarial actionNwanyanwn OC and Ruebush TK. 1997. Malarone-donation(Werbovetz, 2006). programme in Africa. Lancet 350:1624-1625.

20 Gupta and SinglaBloland PB. 2001. Drug resistance in malaria. Resistance, and New Directions in Drug Discovery.WHO/CDS/CSR/DRS 4:1-27. Rosenthal. PJ, Totowa. NJ (Eds.) Humana Press, pp 307-324.Calas M, Ancelin ML, Cordina G, Portefaix P, Piquet G, Vidal- Maguire JD, Sumawinata IW, Masbar S, Laksana B,Sailhan V and Vial H. 2000. Antimalarial activity of Prodjodipuro P, Susanti I et al. 2002. Chloroquine-resistantcompounds interfering with Plasmodium falciparum Plasmodium malariae in south Sumatra, Indonesia. Lancetphospholipid metabolism: comparison between mono- and 360:58-60.bisquaternary ammonium salts. J Med Chem 43: 505-516.Martin SK, Oduola AM and Milhous WK. 1987. Reversal ofDavis TM, Hung TY, Sim IK, Karunajeewa HA and Ilett KF. chloroquine resistance in Plasmodium falciparum by2005. Piperaquine: a resurgent antimalarial drug. Drugs verapamil. Science 235:899-901.65:75-87.Missinou MA, Borrmann S, Schindler A, Issifou S, AdegnikaDenis MB, Davis TM, Hewitt S, Incardona S, Nimol K, Fandeur AA, Matsiegui PB et al. 2002. Fosmidomycin for malaria.T et al. 2002. Efficacy and safety of dihydroartemisinin- Lancet 360:1941-1942.piperaquine (Artekin) in Cambodian children and adults withuncomplicated falciparum malaria. Clin Infect Dis 35:1469-Mockenhaupt FP. 1995. Mefloquine resistance in Plasmodium1476.falciparum. Parasitol Today11:248-253.Dutta GP, Puri SK, Bhaduri AP, Seth M. 1989. Radical curativeactivity of a new 8-aminoquinoline derivative (CDRI 80/53)against Plasmodium cynomolgi B in monkeys. Am J TropMed Hyg 41:635-637.Dutta GP. 1995. Challenges of drug resistance reversal inmalaria. J of Parasit Dis19:5-8.Foege WH. 1997. Malarone-donation programme. Lancet350:1628-1629.Gardiner DL, McCarthy JS and Trenholme KR. 2005. Malaria inthe post-genomic era: light at the end of the tunnel or justanother train? J Postgrad Med 81:505-509.Gardner MJ, Hall N, Fung E, White o, Berriman M, Hyman RWet al. 2002. Genome sequence of the human malaria parasitePlasmodium falciparum. Nature 419: 498-511.Greenwood BM, Bojang K, White CJ and Targett GA. 2005.Malaria. Lancet 365:1487-1498.Guru PY, Singh SN and Chatterjee RK. 1996. Traditionalremedies in the management of parasitic diseases. Proc Nat Trop Med Hyg 41:638-642.Acad Sci Ind 66:245-273.Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab Rand Nurskern DR et al. 2002. The genome sequence of the Med Hyg 64: 76-84.malaria mosquito Anopheles gambiae. Science 298:129-149.Nosten F, ter Kuile FO, Luxemburger C, Woodrow C, Kyle DE,Chongsuphajaisiddhi T et al. 1993. Cardiac effects ofantimalarial treatment with halofantrine. Lancet 341:1054-1056.Olliaro PL and Trigg PI. 1995. Status of antimalarial drugs underdevelopment. Bull W H O 73:565-571.Olliaro PL and Taylor WRJ. 2003. Antimalarial compounds:from bench to bedside. J Exp Biol 206:3753-3759.Olliaro PL and Taylor WR. 2004. Developing artemisinin baseddrug combinations for the treatment of drug resistantfalciparum malaria: A review. J Postgrad Med 50:40-44.Posner GH, Oh CH, Webster HK, Ager AL Jr and Rossan RN.1994. New, antimalarial, tricyclic 1, 2, 4 trioxanes:Evaluation in mice and monkeys. Am J Trop Med Hyg 5: 522-526.Puri SK, Srivastava R, Pandey VC, Sethi N and Dutta GP. 1989.Methemoglobin toxicity and hematological studies onmalaria anti-relapse compound CDRI 80/53 in dogs. Am JRemme JH, Binka F and Nabarro D. 2001. Towards a frameworkand indicators for monitoring Roll Back Malaria. Am J TropRingwald P, Bickii J and Basco L. 1996. Randomised trial ofHyde JE. 2005. Drug-resistant malaria. Trends in Parasitology pyronaridine versus chloroquine for acute uncomplicated21:494-498. falciparum malaria in Africa. Lancet 347:24-27.Kremsner PG and Krishna S. 2004. Antimalarial combinations. Rosenthal PJ. 2001. Protease inhibitors. In: AntimalarialLancet 364:285-294.chemotherapy: Mechanisms of Action, Resistance and NewDirections in Drug Discovery. Humana Press,Totawan, NJ,Kshirsagar NA. 2006. Malaria: antimalarial resistance andpp 325-345.policy ramifications and challenges. J Postgrad Med 52:291-293. Rosenthal PJ. 2003. Antimalarial drug discovery: old and newLell B, Ruangweerayut R, Wiesner J, Missinou MA, Schindler A,approaches. J Exp Biol 206: 3735-3744.Baranek T et al. 2003. Fosmidomycin, a novel Shiff C. 2002. Integrated Approach to Malaria Control. Clinchemotherapeutic agent for malaria. Antimicrob Agents Microbiol Rev 15:278-293.Chemother 47:735-738.Skinner-Adams TS, McCarthy JS, Gardiner DL, Hilton PM andLoyevsky M and Gordeuk VR. 2001. Iron chelators. In: Andrews KT. 2004. Antiretrovirals as antimalarial agents. JAntimalarial Chemotherapy: Mechanisms of action, Infect Dis 190:1998-2000.

Antimalarial agents21Snow RN, Craig M, Deichmann U and Marsh K. 1999. Von Seidlein L, Milligan P, Pinder M, Bojang K, Anyalebechi C,Estimating mortality, morbidity and disability due to malaria Gosling R et al. 2000. Efficacy of artesunate plusamong Africa's non-pregnant population. Bull W H O 77: pyrimethamine-sulfadoxine for uncomplicated malaria in624-640. Gambian children: a double-blind, randomised, controlledSowunmi A, Oduola AM, Ogundahunsi OA, Falade CO,trial. Lancet 355:352-357.Gbotosho GO and Salako LA. 1997. Enhanced efficacy of Vugt MV, Wilairatana P, Gemperli B, Gathmann I, Phaipun L,chloroquine-chlorpheniramine combination in acute Brockman A et al.1999. Efficacy of six doses of artemetheruncomplicatedfalciparum malaria in children. Trans R Soc lumefantrine (Benflumetol) in multidrug-resistantTrop Med Hyg 91:63-67.Plasmodium falciparum malaria. Am J Trop Med HygTer Kuile FO, Dolan G, Nosten F, Edstein MD, Luxemburger C,60:936-942.Phaipum L et al. 1993. Halofantrine versus mefloquine in Watkins WM, Mberu EK, Winstanley PA and Plowe CV. 1997.treatment of multidrug resistant falciparum malaria. Lancet The efficacy of antifolate antimalarial combinations in341:1044-1049. Africa: a predictive model based on pharmacodynamic andTripathi KD. 2003. Antimalarial Drugs. In: Essentials of Medicalpharmacokinetic analyses. Parasitol Today 13:459-464.Pharmacology. Jaypee Brothers, Medical Publishers Pvt. Ltd. Werbovetz K. 2006. Diamidines as antitrypanosomal,New Delhi. pp 735-748.antileishmanial and antimalarial agents. Curr Opin InvestigVenter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GGDrugs 7:147-157.et al. 2002. The sequence of the human genome. Science White NJ. 1992. Antimalarial drug resistance: the pace quickens.291:1304-1351. J Antimicrob Chemother 30:571-585.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 22-28Original paperJ P DDot-ELISA for the detection of preclinical Haemonchuscontortus infection in sheep by using an adult somaticantigen and an immunoaffinity-purified fractionA. Prasad, A. Nasir and Nem SinghDivision of Parasitology, Indian Veterinary Research Institute, Izatnagar.Received 5 March 2007; accepted 28 April 2007ABSTRACT. Haemonchosis is a major cause of production losses in livestock, particularly sheepand goat, due to the blood-feeding habit of L 4 larvae and adult parasites. In this study, dot-ELISAwas used for the detection of infection in sheep, experimentally-infected with 10,000 L 3 ofHaemonchus contortus. Serum samples from infected sheep were collected at weekly intervals.Adult somatic antigen was prepared from the worms collected from the local abattoir at Bareilly(U.P.). Nitrocellulose membranes (0.2 µm) were dotted with 2–6 µl antigen that contained 2–6 µgprotein, and were incubated at 37°C for 2 h. Non-specific binding sites were blocked with blockingagent casein in phosphate-buffered saline. Positive, negative and uninfected control sera fromsheep, both experimentally- and naturally-infected, were used. Positive reaction with brown soliddot formation was obtained with the sera, as early as first week post-infection, withimmunoaffinity- purified adult somatic antigen.Keywords: dot-ELISA, Haemonchus contortus, immunoaffinity chromatography, SDS-PAGE,somatic antigenINTRODUCTIONsudden death (Soulsby, 1982) within a week (Fraser,1991). During prepatency, the clinical symptoms arenot noticed. Therefore, in the present study, dot-enzyme-linked immunosorbent assay (ELISA) hasbeen utilized to detect the infection during prepatentperiod. The importance of preclinical diagnosisfurther increases because the L 4 stage of this nematodeis pathogenic being a blood sucker and till theinfection is detected by coprological examination,considerable damage is done to the host. Dot-ELISAbeing a field-oriented, simple and easy test has alsobeen used to detect several other helminthicinfections, particularly Fasciola hepatica, F.gigantica and Paramphistomum epiclitum.Gastro-intestinal parasitism represents a severe healthproblem in small ruminant production system, and itsconsequences can be extensive ranging from reducedproductivity to mortality (Skykes, 1994). In India,parasitic gastro-enteritis due to nematode infectionhas been reported as a major constraint to profitablesheep production. Haemonchus contortus is one of themost pathogenic, blood-sucking abomasal nematode,which primarily infects sheep and is responsible foreconomic loss to sheep industry. Due to its blood-feeding habit, H. contortus causes severe anaemia andCorresponding author: Dr. A. Prasad, Network Programme(GIP), Division of Parasitology, Indian Veterinary ResearchInstitute, Izatnagar-243 122, India.E-mail: drarvind@ivri.up.nic.inMATERIALS AND METHODSCoproculture of H. contortus larvae: Coproculture

Dot-ELISA for diagnosis of preclinical haemonchosis23was performed in the laboratory as per the methoddescribed by Sahai (1960). The source of faeces forculture was donor animals which were not allowed tograze, and were maintained in the sheds of Division ofParasitology, Indian Veterinary Research Institute.The larvae (L 3) were stored in distilled water at 4°C.Collection of seracollected and filtered through 0.2 µm filter membrane.The filtrate was stored at -70°C withphenylmethylsulphonylfluoride for further use.Protein estimation: The protein concentration ofadult somatic antigen and immunoaffinity purifiedsomatic antigen was estimated as described (Lowry etal., 1951).Positive control sera: Two rabbits were Immunoaffinity chromatography of somatichyperimmunized with H. contortus somatic antigen. antigen of adult H. contortus: ImmunoaffinityInitially, 500 µg protein of antigen was emulsified purification of the somatic antigen of H. contortus waswith Freund's complete adjuvant, and was injected performed utilizing prepacked column containinginto rabbits by i–m route. After 15 days, first booster amino link as matrix (Pierce). The protocol for affinitydose (750 µg of antigen with incomplete Freund's purification of protein was modified as per theadjuvant) was given to immunised rabbits. Similarly, requirements of immunoaffinity chromatography.second, third and fourth boosters were given along Anti H. contortus antibodies were collected from thewith increasing doses of antigens at the rate of 250 sera of sheep infected with L 3 of H. contortus. Theµg/booster. Finally, test bleed was performed five infection was confirmed on autopsy as well as bydays after the last booster dose, and then blood was obtaining larvae from the coproculture of faeces ofcollected by cardiac puncture. Sera were separated infected animal. The antibody was dissolved inand tested for antibody titer by direct coupling buffer (5 mg antibody/ml gel). The columnimmunodiffusion (DID).was equilibrated to room temperature and washedwith five ml of coupling buffer loaded with antibodyNegative control sera: Zero day lamb serum and and incubated at 4°C with gentle shaking. Theuninfected control animal sera were used as negative supernatant was collected and its O. D. value was readcontrols. at 280 nm. The column was washed with PBS (pH 7.2)Experimental sera of sheep: Eight sheep of same age and the somatic antigen was loaded to the column atgroup (each one year old) and weight were selected for the rate of 5 mg protein/ml gel. The column wasthe production of experimental sera. Six sheep freebykeeping at 4°C with gentle shaking. Afterallowed to embed the antigen into the bed completelyfrom helminthic infections were infected with 10,000L of H. contortus and two sheep were kept as control. incubation, the antigen was eluted. Further, 40 ml3Sera, from experimental as well as control animals,elution buffer (glycine buffer 100 mM, pH 2.5–3.0)strdwas added to the column and allowed to drip throughwere collected at weekly (from 1 week–3 week postslowlyunder gravity. Twenty five aliquots of 1.5 mlinfection) intervals, and were stored frozen at -20°Ceach of unbound and bound fractions were collected.and utilized for dot-ELISA.The collected fractions were neutralized by addingSera of sheep naturally-infected with H. contortus: 100 ml of 1M Tris, (pH 7.5) and the O. D. was taken atSera were collected from local abattoir at Bareilly (U. 280 nm. Each fraction was subjected to sodiumP., India). The sera samples as well as the abomasums dodecyl sulphate-polyacrylamide gel electrophoresisof the same animal were collected. The abomasums (SDS-PAGE) analysis and the bound fractions ofwere examined in the laboratory to check worm interest were pooled.burden.Dot-ELISA: Dot-ELISA was performed with adultNormal sheep sera: Normal, uninfected sheep sera somatic antigen and immunoaffinity purified adultwere collected from abattoir at Bareilly.somatic antigen, with sera of sheep bothexperimental- and naturally-infected with H.Preparation of adult somatic antigen: Adult H. contortus. Positive sera (hyperimmune sera raised incontortus were collected in phosphate-buffered saline rabbits) and negative sera (0 day lamb sera), and(PBS; pH 7.2) from the abomasums of sheep, and were control animals tested through DID were utilized.washed with PBS three-times. The worms were first Antigens containing 2–4 µg H. contortus protein werehomogenized in PBS, sonicated and centrifuged at blotted on 0.2 µm nitrocellulose membrane strips and12,000 rpm at 4°C. The clear supernatant was incubated at 37°C for 1 h. Non-specific sites were

24 Prasad et al.blocked with blocking solution. After washing with contortus: Dot-ELISA was performed with adultphosphate buffered soline-tween (PBST), these stripsst, nd rdsomatic antigen and 1 2 and 3 week experimentalwere incubated with primary antibody (1:100) for 2 h sera, as well as with positive, negative and controland washed with PBST. The strips were then animal sera. No dot formation was observed with 1 stincubated with donkey anti-sheep horse-reddishndand 2 week sera, as well as with negative control andperoxidase conjugate. After washing, finally diamino control animal sera. A solid dot formation wasrdbenzidine (DAB) substrate was added and dot observed with positive control sera. When 3 weekformation was observed.sera was allowed to react with adult somatic antigen, asolid dot was formed which indicated the recognitionSDS-PAGE: The adult somatic antigen andof antibodies at this stage (Fig. 1 A, B, C).immunoaffinity purified adult somatic antigen wereanalyzed through SDS-PAGE (Laemmli, 1970) byAusing mini vertical slab gel electrophoresis system.1 2 3 4Tris glycine gel (12%) under non-reducing conditionwas used at constant voltage (100 V). The proteinsamples were mixed with the sample buffercontaining (0.05M Tris pH 6.8, 5% SDS, 20% glyceroland 0.02% bromophenol blue) in the ratio of 1:1, and10–20 µg protein/lane was loaded. The gel was runapproximately for 90 min. After complete run, the gelwas stained with 0.1% Coomassie Brilliant Blue G-B250, and destained with destaining solution (30%methanol and 10% acetic acid). Molecular weights ofthe fractionated polypeptides were determined byusing molecular weight markers.1 2 3 4RESULTSCoproculture of H. contortus larvae: L larvae of H.3contortus were obtained from coproculture and wereidentified by the presence of kinky tail and tubercleson the body surface as described by Levine (1980). Allthe larvae were confirmed to be H. contortus.Positive control sera: Hyperimmune sera raised inrabbits were used as positive controls in dot-ELISA,previously tested in DID which showed precipitin linewith adult somatic antigen.Experimental sera: Experimental sera werecollected after infection with H. contortus larvae fromstrd1 week to 3 week post-infection. Coprologicalexaminations of faeces, when conducted up to threeweek post-infection, were found negative whichindicated prepatency.Dot-ELISA with adult somatic antigen and sera ofsheep naturally-infected with H. contortus: Dot-ELISA performed with adult somatic antigen and seraof sheep naturally-infected with H. contortus revealedsolid dot formation with five out of ten serum samplesat 1:100 serum dilution, whereas solid dot formationwas observed with positive control and no dotformation occurred with control animal sera (Fig. 2 A,B, C).Protein estimation: In the adult somatic antigen,protein concentration was estimated to be 6.3 mg/ml,whereas in the pooled immunoaffinity purifiedfraction of adult somatic antigen, the proteinconcentration was estimated to be 0.2 mg/ml.Dot-ELISA with adult somatic antigen andexperimental sera of sheep infected with H.C1 2 3 4Fig. 1: Dot-ELISA with the 1st, 2nd and 3rd week experimentalsera and somatic antigen of H. contortus.A. 1: Somatic antigen and positive control; 2: Somatic antigenand negative control; 3: Somatic antigen antigen and controlanimal sera; 4: Somatic antigen and 1st week sera.B. 1: Somatic antigen and positive control: 2: Somatic antigenand negative control: 3:Somatic antigen and control animalsera;4: Somatic antigen and 2nd week sera.C. 1: Somatic antigen and positive control:2: Somatic antigenand negative control:3:Somatic antigen and control animalssera; 4: Somatic antigen and 3rd week sera.

Dot-ELISA for diagnosis of preclinical haemonchosis25A1 2 3 4 5B1 2 3 4 5CFig. 2: Dot-ELISA with the Dot-ELISA with naturally infectedsera and somatic antigen of H. contortus.A. (1-5) Somatic antigen and Naturally infected sera.B. (6-10) Somatic antigen and Naturally infected sera.C. Positive control, negative control, and control animal sera.Dot-ELISA with immunoaffinity-purified adultsomatic antigen and sera of sheep experimentallyinfectedwith H. contortus: Sera (1:100) ofst nd rdexperimentally-infected sheep at 1 , 2 and 3 weekpost-infection, when allowed to react withimmunoaffinity-purified somatic antigen revealedst nd rdsolid dot formation with 1 , 2 and 3 week sera whichindicated recognition of the antibodies against thepurified antigen. Solid dot formation was observedduring pre-patent infection. Dot formation was notobserved with negative control and control animalsera (Fig. 3 A, B, C).Dot-ELISA with sera of sheep naturally-infectedwith H. contortus and immunoaffinity-purifiedadult somatic antigen: Dot-ELISA performed withnaturally infected sera of sheep and immunoaffinitypurifiedadult somatic antigen revealed formation ofsolid dot with seven out of ten sera tested, whereas dotformation did not take place with control animal seraand negative control sera (Fig. 4 A, B, C).SDS-PAGE of adult somatic antigen: On SDS-PAGE analysis, the adult somatic antigen revealed thepolypeptides in the range of 10–200 kDa. Out of thesepolypeptides, 15, 26, 40, 50, 60 and 120 kDapolypeptides were more prominent. Two polypeptides> 120 kDa were also observed (Fig. 5).SDS-PAGE of immunoaffinity-purified adultsomatic antigen: SDS-PAGE analysis ofimmunoaffinity-purified antigen revealed only fourpolypeptides: two faint bands of 26 and 32 kDa andtwo sharp bands of 60 and 120 kDa (Fig. 6).DISCUSSIONFor the detection of H. contortus infection in sheepduring prepatency/preclinical stage, Dot-ELISA hasnot been tried extensively; however, some workers(Schallig et al., 1994), by using ELISA, have detectedimmune response to H. contortus during prepatent aswell as patent period. Because detection of infectionduring prepatency is of greater importance from thecontrol point of view, a simplified field-oriented Dot-ELISA test has been used in the present study.Immunodiagnostic tests based upon serodiagnosishave not been tried in haemonchosis becauseprepatent period of H. contortus is 18–21 days.Nevertheless, Sahai (1960) has reported that in sheepA1 2 3 4B1 2 3 4C1 2 3 4Fig. 3: Dot-ELISA with the 1st, week 3rd week PI experimentalsera and immunoaffinity purified somatic antigen of H.contortus.A. 1-Purified antigen and positive control, 2-Purified antigenand negative control, 3, Purified antigen and control animalsera, 4-Purified antigen and 1st week sera.B. 1-Purified antigen and positive control, 2 -Purified antigenand negative control, 3-Purified antigen and control animalsera, 4-Purified antigen and 2nd week sera.C. 1-Purified antigen and positive control, 2-Purified antigenand negative control, 3-Purified antigen and control animalsera, 4-Purified antigen and 3rd week sera.

26 Prasad et al.12060261 2 3 4 M170130100725540kDa1206032261 MkDa20012085706050403025201517111510Fig. 5: SDS-PAGE of adult somatic antigen of H. contortus.Lane 1-4. Adult somatic antigen of H. contortus (prominentbands are 15, 26, 60 and 120 kDa)Lane M. MarkerA1 2 3 4 5B1 2 3 4 5Fig. 6: SDS-PAGE of affinity purified bound fractions (pooledand concentrated of adult somatic antigen of H. contortus.Lane 1: Pooled fraction.Lane M. Markeranimals were treated with albendazole; coprologicalstthexamination was conducted from 1 week to 4 week.The animals were treated as having mono-specificinfection with H. contortus. Dot-ELISA wasperformed with mono-specific sera, because theanimals were infected with L 3 larval stage of H.contortus that was raised through coproculture in thelaboratory.Fig. 4: Dot-ELISA with the naturally infected sera andimmunoaffinity purified somatic antigen of H. contortus.A. (1-5) Purified antigen and naturally-infected sera.B. (6-10) Purified antigen and naturally-infected sera.CThe test was performed with both adult somaticantigen and immunoaffinity-purified somatic antigen,to detect antibodies in experimental sera duringstrdprepatency (1 week to 3 week post-infection) as wellas sera from sheep that were naturally-infected andhad confirmed infection of H. contortus. In dot-ELISA in which adult somatic antigen was used,rdantibodies could be detected only in 3 week sera andpositive control (by formation of solid dot) but not inst nd1 and 2 week sera, negative and uninfected control.rdC. Positive control, negative control, and control animal sera. However, a solid dot was formed with the 3 week seraand adult somatic antigen which indicated thepresence of sufficient antibodies against H. contortusinfection becomes patent by 27–28 days, and thatin the infected animal. When dot-ELISA waswhen the L 4 larvae and immature worms are present,performed with the same antigen and sera of naturallynoeggs are present in faeces. The L 4 larvae cause infected sheep, solid dot was formed with 50% of thesufficient damage, and before the infection becomes sera which indicated the presence of infection.patent, the animal may die suddenly. Other gastrointestinalnematode infections, except Dot-ELISA performed with immunoaffinity-purifiedoesophagostomosis, have less intensity of infection, somatic antigen gave different results in terms ofand the degree of pathogenicity is lower in earlier detection of infection. Solid dot formation tookst nd rdcomparison to H. contortus (Soulsby, 1982).place with 1 , 2 and 3 week sera as well as positivecontrol, and no dot formation was observed in case ofDuring the present study, care was taken that the negative controls. It indicated that immunoaffinityexperimentalsheep do not graze outside the shed purified antigen contained antigenic polypeptidespremises, and before the experiment started, all reactive to anti H. contortus antibodies in sheep as

Dot-ELISA for diagnosis of preclinical haemonchosis27stearly as 1 week post-infection. Similarly, when results during the present work. Further studies wereimmunoaffinity-purified antigen was allowed to react also conducted to characterise the somatic antigen andwith sera of naturally-infected sheep, solid dot immunoaffinity-purified antigen. In SDS-PAGEformation occurred with 80% of the sera tested, which analysis of somatic antigen of H. contortus, prominentagain indicated reactivity of antigenic polypeptides to bands were of 15, 26, 60 and 120 kDa, and severalanti -H. contortus antibodies.bands were between 3040 kDa. Immunoaffinity-purified antigen revealed four polypeptides whichDot-ELISA has not been extensively utilised for the may be utilised for the detection of H. contortusdiagnosis of prepatent infection in sheep. Kaur et al. infection in sheep or for immunoprophylaxis. These(2002) and Sood (2006) utilised it for the detection of ploypeptides have been detected in either somaticantibodies in rabbits immunised with H. contortus antigen or ES antigen, which may be the reason forantigen. Sood et al. (1996) utilised competitive detection of antibodies in H. contortusinhibition dot-ELISA for the detection of H. contortus experimentally-/naturally-infected sheep. Lowerantigen. However, Schallig et al. (1994) utilised sensitivity of this test with sera of naturally-infectedELISA, using somatic antigen, to detect antibodies in sheep may be due to lower intensity of infection orboth experimentally- and naturally- infected sheep, individual variation among animals.which could detect antibodies as early as one weekpost-infection. During the present study, dot-ELISA ACKNOWLEDGEMENTScould detect antibodies as early as one week postinfection,utilising immunoaffinity-purified somaticAuthors are thankful to the Indian Council ofantigen. Moreover, as compared to ELISA, dotfinancialsupport for the present work in the NetworkAgricultural Research, New Delhi, for providingELISA is simpler to perform.Programme on Gastrointestinal parasitism. We thankDot-ELISA for diagnosis of parasitic infections has Director, Indian Veterinary Research Institute,been utilised by several workers including protozoan Izatnagar, for providing necessary facilities.and helminth parasites. Pappas et al. (1983) utiliseddot-ELISA for the rapid diagnosis of human visceralREFERENCESleishmaniasis. Zimmerman et al. (1985) utilised dot- Dixit AK, Yadav SC, Saini M and Sharma RL. 2003.ELISA for the diagnosis of ovine fasciolosis in Purification and characterization of 28 kDa cysteineproteinase for immuno diagnosis of tropical fasciolosis. Jexperimentally-infected sheep using FasciolaVet Parasitol 17:5-9.hepatica ES antigen, and the infection could bedetected four weeks post-infection. Dixit et al. (2003) Fraser CM. 1991. The Merek Veterinary Manual. A handbookutilised this test for the diagnosis of F. gigantica of diagnosis, therapy and disease prevention and control forinfection in experimentally-infected buffalo calves atthe veterinarians. Marek and Co. Inc. Rahway, USA. pp205-215.eight weeks post-infection. Saxena et al. (2006)utilised dot-ELISA for the detection of F. gigantica Kaur K, Kapur J, Parmar A and Sood ML. 2002. Kinetics ofinfection in buffalo utilising crude antigen antibody response by Dot-ELISA in rabbits immunizedfractionated with gel-exclusion chromatography and with adult Haemonchus contortus antigen. Parasite 9:363-365.naturally-infected buffalo serum.Laemmli UK. 1970. Cleavage of structural proteins during theThe results of dot-ELISA performed with assembly of bacteriophage T 4. Nature 227:680-685.immunoaffinity-purified somatic antigen of H.contortus were comparable to ELISA, and could Lowry OH, Rusbrought NJ, Farr AL and Randall RJ. 1951.stdetect infection as early as 1 week post-infection Protein measurement with Folin phenol reagent. J BiolChem 193:265-275.during prepatency. However, un-purified somaticrdantigen could detect the infection as early as 3 week Levine ND. 1980. Nematode parasites of domestic animalspost-infection, which may be considered early patent and of man. Burgess Publishing Company Minneapolis,period. The reason for these observations may be Minnesota.lower concentration of polypeptides available in the Pappas G, Michael Hajkowski R and Hockmeyer TW. 1983.un-purified antigen to antibodies. Because it is well Dot-enzyme-linked immunosorbent assay (Dot-ELISA): aknown that nanogram quantities of antibodies could micro technique for the rapid diagnosis of visceralbe detected by dot-ELISA (Pappas, 1983), leinlimaniasis. J Immun 84:205-214.immunoaffinity-purified antigen could give better

28 Prasad et al.Pappas MG. 1988. Recent application of Dot-ELISA inimmunoparasitology. Vet Parasitol 29:105-129.Sahai BN. 1960. Studies in host specificity of Haemonchusspecies in sheep and buffaloes. M. V. Sc. thesis, IVRI,Izatnagar.Saxena MK, Ram H, Banerjee PS, Adam CL and Garg R. 2006.Immunodiagnosis of fasciolosis by Dot-ELISA. J VetParasitol 20:77-79.Sood ML. 2006. Histochemical, biochemical andimmunological studies in Haemonchus contortus(Nematode: Trichostrongyleidea) an Indian perspective. JParasit Dis 30:4-15.Sood ML, Kaur G, Parmar A and Kapur J. 1996. Developmentof Dot-ELISA for detection of Haemonchus contortus.Helminthologia 33:73-75.Soulsby ELJ 1982. Helminth, arthropods, and protozoa ofdomesticated animals. 7th Ed. Bailliere Tindal, London.Schallig HDF, Leeuven Van MAW and Hendrikx WML. 1994.Immune responses of Texel sheep to excretory/secretory Waller PJ. 1999. International approaches to the concept ofproducts of adult Haemonchus contortus. Parasitol integrated control of nematode parasites of livestock. Int J108:351-357. Parasitol 29:155-164.Skykes AR. 1994. Parasitism and production in farm animals.Animal Production 59:155-172.Zimmerman GL, Nelson ML and Clark CRB. 1985. Diagnosisof ovine fasciolosis by Dot-ELISA: A rapid diagnostictechnique. American J Vet Res 43:1513-1515.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 29-32Original paperJ P DTrypanosoma evansi infection in rabbits: studies on thedevelopment of anaemia and copper and iron levels inplasma and tissues1 2 3N. Sangwan , A. K. Sangwan and S. S. Chaudhri1Department of Veterinary Biochemistry, CCS Haryana Agricultural University, Hisar.2Department of Veterinary Parasitology, CCS Haryana Agricultural University, Hisar.3CCS Haryana Agricultural University, Regional Research Station, Uchani, Karnal.Received 24 January 2007; revised 13 April 2007; accepted 4 May 2007ABSTRACT. The present study was undertaken to determine the effects of Trypanosoma evansi5infection (5x10 trypanosomes/animal; intraperitoneally) on haematology and levels of copper andiron in plasma and tissues of New Zealand white rabbits. Plasma copper and iron levels reducedsignificantly from 1.38 ± 0.07 µg/ml to 1.01 ± 0.05 µg/ml and 1.92 ± 0.04 µg/ml to 0.87 ± 0.06 µg/ml,respectively, towards the end of the experiment. Liver copper ( 5.32 ± 0.66 µg/g fresh tissue) andspleen copper ( 1.65 ± 0.23 µg/g fresh tissue) levels were far below the deficiency levels, while the spleenand liver iron levels were reduced but not to the extent that anaemia may develop. Brain and heartcopper levels were also reduced, while iron levels did not change much. It is concluded that underpresent circumstances, it was the deficiency of copper, and not of iron, that was responsible for thedevelopment of anaemia and other clinical signs in rabbits suffering from trypanosomosis.Keywords: anaemia, copper, iron, rabbit, Trypanosoma evansiINTRODUCTION1997). Copper is responsible for incorporation of ironinto haeme. Trypanosomiasis, caused byIron and copper are required in all organisms forTrypanosoma evansi, a disease of economicgrowth and crucial metabolic pathways. The redoximportance in most of the domestic animals, is2+ 3+potential of Fe /Fe favours its use in a number of characterized by progressive loss of condition,protein-complexes, especially those involved in anaemia, emaciation, loss in milk yield, abortions andelectron transfer. A number of proteins require iron immunosuppression (Kreier, 1977; Radostits et al.,and copper for their activity in the form of haeme or 2000). Loss of trace-metals, if any, can have profoundiron-sulphur clusters to transfer electrons and effects on host and in aggravating clinical symptoms.ceruloplasmin for loading of iron onto transferrin, The laboratory animals (albino mice, rabbits, guinearespectively. Iron-complexes are not only necessary pigs and monkeys) are all susceptible to experimentalin the electron transport chain to supply cells with T. evansi infection. Guinea pig has been suggested toenergy, but they are also affected by oxygen radicals be a useful laboratory model to study trypanosomosis- 2+(O 2 ), and free-Fe is part of the Fenton reaction to (Monzon and Villavicencio, 1990). The T. evansigenerate reactive-oxygen species (Henle and Linn, infection in rabbits is similar to that in guinea pigs(Gill, 1991), and can be used as an alternate laboratoryCorresponding author: Dr. Nirmal Sangwan, Department of model. Therefore, the present study was undertaken toVeterinary Biochemistry, CCS Haryana Agricultural study the effects of trypanosomosis on blood, andUniversity, Hisar -125 004, India. E-mail: ns@hau.ernet.in copper and iron levels in tissues, so that these findings

30Sangwan et al.could provide a basis for understanding the adverse trace-minerals. At the end of the experiment, the rabbitseffects of trace-elements imbalances due to the were sacrificed and tissue samples viz. liver, spleen, heartdisease and the potential for predicting metabolic and brain were collected for the analysis of traceevents, which could be promoted by knowing the elements. The haemoglobin (Hb; g %), packed cellsignificant loss or redistribution of trace-metals in volume (PCV;%) and other haematological indices weretissues of animals suffering from trypanosomosis. estimated (Schalm et al., 1975). Plasma and tissueMATERIALS AND METHODSsamples were digested with diacid (HNO 3:HClO 4::4:1)and then after making suitable dilutions, samples wereTwo groups of five New Zealand white rabbits each, analysed for copper (Cu) and iron (Fe) by using atomiceither sex, aged 4–5 months, weighing 1.62–2.50 kg absorption spectrophotometer. The data obtained were(average 2.02 ± 0.16 kg) were used to determine the subjected to students't test and ANOVA for statisticalconsequences of T. evansi infection on the level of significance (Snedecor and Chochran, 1967)copper and iron in plasma and tissues of the animals.The rabbits, which had been reared under worm-freeRESULTS AND DISCUSSIONconditions since birth, were fed concentrated mixture The clinico-parasitological responses in rabbits,consisting of (g/kg): crushed wheat, 620; black gram, following experimental infection of T. evansi, showed280; refined oil, 46; casein, 10; mineral mixture, 40; fluctuations in parasitaemia following a prepatentvitamin mixture, 2; and choline chloride, 2 as per the period of 6–12 days (average 9 days). Amongrecommendations of National Institute of Nutrition, individual rabbits, there were considerable variationsHyderabad , and green fodder ad libitum. The diet, as in their parasitaemic intensities, and prepatent periodsper analysis, contained 2.91 µg/g copper and 219.64 ranged from 6–11 days (average 8 days). Animalsµg/g iron. The rabbits were kept in individual cages. showed an increase in body temperature of up to 107.2After a 15-day acclimatization, measurements were °F between days 6–12 post-infection and, thereafter,made at intervals of 15 days over 75 days. The there were lots of fluctuations in body temperatureanimals were divided into two groups of similar mean with no clear-cut correlation with the intensity oflive weight (2.02 kg). Each animal of Group-I was parasitaemia. All the infected rabbits exhibited5given a single dose of 5x10 trypanosomes, clinical signs like anaemia, wasting, partial anorexia,intraperitoneally, previously isolated from a cross- lacrymation, dullness, alopecia etc. The T. evansibred cow and maintained by regular passage in mice, infection proved fatal for 67% of the infected rabbits.and group II was maintained as uninfected control. Chand and Singh (1971) and Avasathi et al. (1985)The parasitaemia in rabbits was monitored at regular also observed similar clinical symptoms of the diseaseintervals by using haematocrit centrifugation during experimental T. evansi infection in rabbits.technique. Body temperature (°F) was also recorded.The levels of Hb, PCV and total erythrocyte countBlood samples were collected from ear vein at fortnightly reduced significantly (p < 0.05; Table I) with theintervals, in sterile and heparinised test tubes. The progression of the disease. However, the percentageplasma was separated and was used for the analysis of of circulating reticulocytes in infected animals first heTable I. Haematological parameters in rabbits infected with T. evansiDays post-infectionParameters Groups 0 15 30 45 60 75Hb I 13.55 ± 0.30 11.06 ± 0.60 11.47 ± 0.34 9.88* ± 0.84 9.00* ± 0.64 8.08* ± 0.74(g%) II 12.59 ± 0.53 12.25 ± 0.50 12.72 ± 0.72 13.43 ± 0.69 13.52 ± 0.65 13.27 ± 0.21PCV I 41.00 ± 2.27 34.00 ± 1.60 34.75 ± 1.00 33.60 ± 2.80 31.25* ± 2.43 27.00* ± 1.70(%) II 40.33 ± 0.83 38.66 ± 2.03 38.75 ± 2.67 41.25 ± 2.49 41.00 ± 1.47 39.00 ± 0.51TEC I 5.68 ± 0.20 4.51 ± 0.26 4.45 ± 0.11 4.33* ± 0.31 4.06* ± 0.23 3.60* ± 0.286 3(x10 /mm ) II 5.39 ± 0.18 5.22 ± 0.06 5.38 ± 0.20 5.59 ± 0.31 5.59 ± 0.24 5.31 ± 0.04Reticulocytes I 0.81 ± 0.65 6.52* ± 1.00 4.68* ± 0.57 4.02* ± 0.43 2.70* ± 0.28 2.35 ± 0.11(%) II 1.17 ± 0.32 2.24 ± 0.35 2.12 ± 0.37 1.88 ± 0.28 1.57 ± 0.03 1.90 ± 0.36Group-IGroup-II Non-infected healthy control.* Values differ significantly from control at p < 0.05.Data are mean + S.E.5Rabbits infected experimentally with 5x10 trypanosomes/animal, intraperitoneally.

Trace-metals and trypanosomosis in rabbits31increased and then started decreasing inspite of have resulted in the less incorporation of iron intoanaemia, which points towards the failure of ferritin in liver and spleen. Catecholamineerythropoiesis. The values of iron and copper in metabolism might have been critically impaired inplasma and tissues are shown in Tables II and III. With brain because of low levels of copper (Gross andthe progression of the disease, iron and copper plasma Prohaska, 1990). Clinical symptoms in infectedlevels decreased, while in tissues, the values of iron in animals viz. difficulty in locomotion and ataxia mightliver, heart and brain did not change significantly, be due to defects in myofibrillar protein synthesis inexcept in spleen where the levels reduced copper deficiency as low levels of copper have beensignificantly (p < 0.05). In liver, spleen and brain, the found to be linked to low cytochrome C oxidaselevels of copper reduced significantly, which might concentrations (Chao et al., 1994). This ubiquitoushave contributed to the reduction of Hb synthesis as and important enzyme responsible for the terminalcopper is required for blood formation. Copper electron transfer in the respiratory chain may becatalyses the incorporation of iron into the structure of involved in other dysfunctions associated with copperthe haeme and assists in maturation of erythrocytes in deficiency. Values for liver copper below the marginalthe early developmental stages. Iron deficiency band i.e. 2.13 µg/g to 6.4 µg/g fresh tissue indicateresults in microcytic hypochromic anaemia; with a high probability of current or future dysfunction andcopper deficiency the number of erythrocytes impairment of health and production (Underwood anddecreases without any change in the haemoglobin Suttle, 1999). Serum iron values below the marginalconcentration. The copper-containing protein band i.e. 0.192–0.576 µg/ml indicate that the animal isceruloplasmin loads iron onto transferrin (Cousins, at a relatively higher risk. Anaemia of copper1985; Evans and Halliwell, 2001). It also promotes the deficiency is caused by a break-down in intracellularincorporation of iron into the storage protein, ferritin iron metabolism in liver (Williams et al., 1983).(Saenko et al., 1994). The low levels of copper might Impairment of iron transport for erythropoieticTable II. Copper and iron in plasma of rabbits infected with T. evansiDays post-infectionParameters Groups 0 15 30 45 60 75Copper I 1.38±0.07 1.37 ± 0.05 1.16 ± 0.06 1.06 ± 0.06 0.97* ± 0.09 1.01* ± 0.05II 1.35 ± 0.07 1.30 ± 0.06 1.27 ± 0.03 1.28 ± 0.03 1.30 ± 0.04 1.29 ± 0.02Iron I 1.92 ± 0.04 1.62 ± 0.14 1.79 ± 0.11 1.15* ± 0.25 1.13* ± 0.21 0.87** ± 0.06II 1.94 ± 0.03 1.92 ± 0.05 1.91 ± 0.04 1.92 ± 0.04 1.94 ± 0.04 2.08 ± 0.04Group-I,5Rabbits infected experimentally with 5x10 trypanosomes/animal, intraperitoneally.Group-II, Non-infected healthy control.*, ** Values differ significantly from control at p < 0.05 and p < 0.01, respectively.Data are mean + S.E. µg/ml.Table III. Copper and iron in tissues of rabbits infected with T. evansiTissuesParameters↓ Groups ↓ Liver Spleen Heart BrainCopper I 5.32** ± 0.66 1.65** ± 0.23 3.09 ± 0.28 2.32** ± 0.19II 9.67 ± 0.39 4.49 ± 0.27 3.98 ± 0.38 3.22 ± 0.12Iron I 90.22 ± 19.28 142.58* ± 5.91 41.06 ± 2.30 19.75 ± 1.50II 132.5 ± 13.56 235.40 ± 32.30 56.70 ± 7.00 23.37 ± 0.88Group- IGroup-II, Non-infected healthy control.*, ** Values differ significantly from control at p < 0.05 and p < 0.01, respectively.Data are mean + S.E. µg/ml.5Rabbits infected experimentally with 5x10 trypanosomes/animal, intraperitoneally.

32Sangwan et al.function of copper has been suggested (Suttle et al., levels of ATP-synthase and cytochrome C oxidase subunit1987). On the basis of above findings, anaemia can not peptide from hearts of copper deficient rats are not alteredbe due to iron deficiency as liver iron concentration by the administration of dimethyl sulphoxide. J Nutrition24:789-903.was normal and indicated the failure in properutilization of Fe for Hb synthesis as infected animals Cousins RJ. 1985. Absorption, transport and hepatichad significantly low levels of copper in their tissues metabolism of copper and zinc: special reference toand plasma. Also, in an earlier study (Sangwan et al., metallothionein and caeruloplasmin. Physio Reviews65:238-296.1993), significantly low levels of folacin andcobalamin have been reported in buffaloes suffering Evans P and Halliwell B. 2001. Micronutrient:from T. evansi, which might have additionally oxidant/antioxidant status. British J Nutrition 65 suppl.contributed to the further reduction in Hb synthesis as S67-S74.these two vitamins are involved in maturation and Gill BS. 1991. Trypanosomes and Trypanosomiasis of Indiandevelopment of erythrocytes. So, in addition to Livestock. Publications and Information Division, ICAR,folacin and cyanocobalamin, copper deficiency may PUSA, New Delhi. pp 192.also be contributing to the development of anaemia in Gross AM and Prohaska JR. 1990. Copper deficient mice havetrypanosomosis, and iron is not the limiting factor for higher cardiac epinephric turnover. J Nutrition 120:88-96.the development of anaemia in the infected animals as Henle ES and Linn S. 1997. Formation, prevention and repairis the general view about this disease (Mwangi et al., of DNA damage by iron/hydrogen peroxide. J Bio Chem1995). 272:19095-19098.Saneko EL, Yaroplov AI and Harris ED. 1994. Biologicalfunctions of caeruloplasmin expressed through copper-binding sites. J Trace Elements and Exp Med 7: 69-88.Iron deficiency could also be indicated by low levelsof iron in liver and a marginal band of 150–250 mg/kgdry matter is tentatively proposed to separate thedeficient from the normal. However, in the presentstudy, liver and spleen iron values were reduced butnot to the extent that anaemia may develop. It is opinedthat it is the deficiency of copper, and not of iron, to bethe major factor responsible for the development ofanaemia and other clinical symptoms in rabbitssuffering from T. evansi infection. This study has alsohelped in understanding the pathophysiology ofmetal-ions in trypanosomosis, and the properremedial measures like copper supplementationshould be introduced to reduce the convalescentperiod by providing suitable supportive treatmentmeasures.ACKNOWLEDGEMENTSThis work was done at CCS Haryana AgriculturalUniversity, Regional Research Station, Uchani,Karnal, and financially supported by the NationalAgricultural Research Project of the Indian Council ofAgricultural Research, New Delhi.REFERENCESAvasathi BL, Kathuria IG and Parjapati KS. 1985. Course ofinfection in rabbits experimentally infected withTrypanosoma evansi. Indian J Parasitol 9:281-282.Chand K and Singh RP. 1971. A study on the clinical course oftrypanosomiosis in goats, donkeys, dogs and rabbitsexperimentally infected with Trypanosoma evansi. J Res8:270-274.Chao JCJ, Medeiros DM, Davidson J and Shiry L. 1994. LowKreier JP. 1977. Parasitic Protozoa. Vol. I Academic Press, NewYork, San Francisco, London. pp 277-288.Radostits OM, Gay CC, Blood DC and Hinchcliff. 2000.Veterinary Medicine. Ninth Ed. W.B. Saunders CompanyLtd., London. P. 1335.Monzon CM and Villavicencio VI. 1990. Serum proteins inguinea pigs and horses infected with Trypanosoma evansi(Steel, 1885). Vet Parasitol 36:295-301.Mwangi SM, McOdimba F, Logan-Henfrey L. 1995. The effectof Trypanosoma brucei brucei infection on rabbit plasmairon and zinc concentrations. Acta Tropica. 59:283-291.Sangwan N, Chaudhri SS, Rao AR, Sangwan AK and Gupta RP.1993. Folacin and cyanocobalamin in relation to naturalTrypanosoma evansi infection in buffaloes. Trop AnimalHealth and Production. 25:79-84.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.Suttle NF, Jones DG, Woolliams C and Woolliams JA. 1987.Heinz body anaemia in lambs with deficiencies of copperand selenium. British J of Nutrition 58:539-548.Underwood EJ and Suttle NF. 1999. The Mineral Nutrition ofLivestock. Third ed. CAB International Publishing Co. pp312, 361.Williams DM, Kennedy FS and Green BG. 1983. Hepaticiron accumulation in copper-deficient rats. British J ofNutrition. 50:653-660.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 33-37Original paperJ P DGlutathione synthetase in Plasmodium bergheiS. K. Sharma and H. S. BanyalDepartment of Biosciences, H. P. University, Shimla.Received 1 August 2006; revised 7 May 2007; accepted 22 May 2007ABSTRACT. Cell-free Plasmodium berghei (NK-65) contained 5.6-times more (around0.443±0.001 U/mg protein) glutathione synthetase (E.C. 6.3.2.3) enzyme activity as compared tonormal mouse erythrocytes. Subcellular fractionation revealed that enzyme activity is mainlyconfined to the cytosolic part of parasite. Around 41-fold purification of enzyme was achieved byusing a combination of ammonium sulphate fractionation and Sephadex G-200 gel filtration. Thepurified enzyme had a molecular weight of 70 kDa.Keywords: cell-free parasite, erythrocytes, glutathione, glutathione synthetase, PlasmodiumbergheiINTRODUCTIONand glutathione synthetase (GS). GS (γ-L-glutamate-L-cysteine: glycine ligase, E.C. 6.3.2.3) catalyzes theThe ubiquitous tripeptide glutathione (L-γ-glutamyl- ATP-dependent conversion of dipeptide γ-L-cysteinylglycine; GSH), usually the most prevalent glutamylcysteine to glutathione.intracellular thiol, is known to function directly orindirectly in many important biological phenomena GS was first demonstrated by Snoke and Bloch (1952)including synthesis of proteins and DNA, their in pigeon liver extracts and showed γ-transport, enzyme activity, metabolism and protection glutamylcysteine, an intermediate in the synthesis ofof cells against oxidative stress (Anderson et al., 1985; glutathione. The enzyme has subsequently beenSies, 1989). Plasmodium species invade and survive studied in Escherichia coli (Samuels, 1953), yeastinside erythrocytes by overcoming the stress exhibited (Mooze and Meister, 1967), human erythrocytesby the host cell. In malaria parasite, pathways for (Majerus et al., 1971), rat kidney (Oppenheimer et al.,detoxification of xenobiotics, antimalarial drugs and 1979) and P. falciparum (Meierjohann et al., 2002).maintenance of intracellular reducing environment Characterization of E. coli GS has demonstrated thatrely on glutathione (Becker et al., 2003; 2004). All the enzyme is functional as a tetramer, whereasthese functions are performed by various enzymes of mammalian and yeast GS are active as dimers, whichglutathione metabolic pathway like glutathione shows that there are two families of GS. GS deficiencyreductase, glutathione peroxidase and glutathione-S- in mammals is an autosomal recessive disorder andtransferases.leads to 5-oxoprolinuria manifested by either mild ordebilitating diseases (Dahl et al., 1997), whichGlutathione is synthesized intracellularly by thesuggests that GS is essential for survival in mammals.consecutive action of γ-glutamylcysteine synthetase In P. falciparum, GSH is essential for the survival ofparasite and GSH level in infected red blood cells areCorresponding author: Prof. H. S. Banyal, Laboratory of maintained through the synthetic pathway (AtamnaParasitology and Immunology, Department of Biosciences, H.and Ginsburg, 1997). Meierjohann et al. (2002)P. University, Shimla-171 005, India.isolated gene that encodes GS in P. falciparum andE-mail: hsbanyal@yahoo.co.in

34Sharma and Banyalcarried out recombinant expression and min; 10,000 g, 25 min; 24,000 g, 35 min and 1,05,000characterization of the protein. P. berghei, a rodent g, 60 min. The pellets were suspended, separately, inmalaria parasite, maintains sustainable GSH homogenization buffer, homogenized if required andconcentration and survives in host erythrocytes. The assayed along with cytosolic fraction.parasite possesses significant GS activity mainlyconfined to its cytosolic part, which in the presentPurification of GS: P. berghei GS was purifiedstudy, has been purified through columnaccording to a slightly modified method of Meisterchromatography and characterized.(1985). Homogenate of cell-free P. berghei parasiteswas centrifuged at 450 g for 10 min, and theMATERIALS AND METHODSsupernatant first subjected to ammonium sulphateprecipitation at 0-35%, 35-70% and 70-100%Cell-free parasites: Plasmodium berghei (NK-65)saturation with solid ammonium sulphate. Thewas maintained in albino Swiss mice (Banyal et al.,fractions were suspended in buffer A and anlaysed for1991). Blood, collected from normal or infected mice,GS activity.was passed through CF-11 cellulose column and lysedwith 0.2% (w/v) saponin in 0.01M PBS (pH7.2). Total Gel filteration chromatography on Sephadex G-erythrocytes and their fractions haemolysate, 200: Dried Sephadex G-200 (2 g; Sigma) was used forerythrocyte membranes and cell-free parasites were a 50 ml bed volume. The gel was swollen in distilledcollected as previously described (Kumar and Banyal, water at room temperature overnight. After swelling, it1997). was loaded onto a glass column (2 cm x 16 cm). Flowrate was adjusted to 15 ml/h. The column equilibratedGS (γ-L-glutamate-L-cysteine: glycine ligase EC with 50 mM imidazole-HCl buffer (pH 7.5) until the6.3.2.3) assay: Normal and P. berghei-infected final absorbance difference became zero at 280 nmerythrocytes, their fractions and cell-free P. berghei and pH value similar to that of equilibration buffer.were suspended in buffer A containing 50 mM Ammonium sulphate precipitated fraction containingimidazole HCl (pH 7.5), 10 mM magnesium chloride, the enzyme activity was loaded onto the column,0.1 mM phenylmethylsulphonylfluoride, eluted with imidazole-HCl and 45 fractions of 1 ml°homogenized, centrifuged (450 g, 10 min, 4 C) and the each were collected.supernatants used as enzyme extracts. GS was assayedaccording to Meister (1985). The standard 1 ml assay Sodium dodecyl sulphate-polyacrylamide gelmixture contained 100 mM tris HCl (pH 8.2), 1 mM γ- electrophoresis (SDS-PAGE): Purified fractionsL-glutamyl-L-cysteine, 15 mM glycine, 10 mM were characterized by SDS-PAGE according to theadenosine triphosphate (ATP), 10 mM magnesium method of Laemmli (1970) as given by Banyal andchloride, 1 mM phosphoenolpyruvate, 0.24 mM Inselberg (1985) by using 3% stacking and 10%nicotinamide adeninedinucleotide (NADH), 1 unit separating gels. Gels were stained overnight in 0.2%lactate dehydrogenase, 5 units pyruvate kinase, 100 Coomassie brilliant blue R-250.mM potassium chloride and appropriate volume ofenzyme extract. Reaction was initiated by the additionRESULTSof enzyme extract, and decrease in absorbance at 340 P. berghei contained significant amount of GS activitynm was monitored spectrophotometrically (Perkin (Table I). Parasitized host erythrocytes possessedElmer UV/VIS spectrometer Lambda 12). Protein was about three-times higher enzyme activity thandetermined using folin phenol reagent (Lowry et al., erythrocytes from normal mice. Lysis of erythrocytes1951) and bovine serum albumin was used as a revealed that the enzyme was mainly confined to thestandard. One unit of GS activity is the amount of haemolysate fraction compared to erythrocyteenzyme that catalyzes the formation of one µ mol of membranes. However, the values of GS activity were-1glutathione/hour (ε=6.22 mM ). Specific activity was higher in fractions of parasitized red cells than theexpressed as enzyme units/mg of protein.fractions of normal red cells. Cell-free parasitesdemonstrated approximately 0.443 U/mg of GSSubcellular fractionation: Freshly isolated cell-free activity, which was about 5.6-fold higher than theP. berghei parasites were suspended in 0.25 M sucrose normal host erythrocytes.in 0.01 M PBS and fractionated according to themethod of Banyal et al. (1979). Differential Differential centrifugation of P. berghei resulted incentrifugation of homogenate was carried at 600 g, 15 various sediments and cytosolic (supernatant of g

Plasmodium berghei GS35105,000 g) fractions. Almost entire activity of parasite Fig. 2, the two fractions giving maximum activity ofenzyme was detected in the cytosolic fraction parasite enzyme contained only a single band protein(3.416±0.002 U/mg), whereas traceable amount of of similar molecular weight.enzyme was detected in various sediments of 600 g to1, 05,000 g centrifugations. GS was purified first usingDISCUSSIONammonium sulphate precipitation. 0-35%, 35-70% GS catalyses the ATP-dependent formation ofand 70-100% ammonium sulphate concentrations glutathione, that is a ubiquitous compound involved inwere used to precipitate enzyme protein (Table II). cellular protection. There exists a parasite GSProteins precipitated by 35-70% ammonium sulphate independent of host enzyme known as cloned GS thatcontained maximum GS activity and > 9-fold has been reported from P.falciparum (Meierjohann etpurification of the enzyme was observed in this al., 2002). P. berghei contained significant amount offraction. This fraction was further loaded onto GS activity. The parasite enzyme activity is somewhatSephadex G-200 column and eluted with buffer A. GS similar to or within range of that has been reported forwas mainly eluted in fractions 28–34, and maximum other prokaryotic or eukaryotic cells and tissuesenzyme activity exhibited in fraction 31 (Fig. 1). (Samuels, 1953; Mooze and Meister, 1967; Majerus etAbout 41-fold purification of GS was achieved in this al., 1971; Oppenheimer et al., 1979). The increasedfraction, whereas 22 % of the total enzyme activity activity in parasitized host erythrocytes, as comparedof P. berghei was isolated in combined fractions 30, 31 to normal mouse erythrocytes, indicated that theand 32. The purified GS exhibited a molecular weight parasite possessed its own machinery to challenge theof 70 kDa by SDS-PAGE analysis (Fig. 2). As given inTable I. Gluathione synthetase activity in normal and P. berghei-infected erythrocytes, their fractions and cellfreeP. bergheiGluatthione symthetaseEnzyme Normal (U/mg) P. berghei-infected (U/mg)Toral erythrocytes 0.079 ± 0.005 0.234 ± 0.004Haemolysate 0.088 ± 0.005 0.118 ± 0.012Membranes 0.004 ± 0.007 0.443 ± 0.001Cell-free P. berghei --------------- 0.443 ± 0.001Values are Mean ± SD of three independent experiments each run in duplicate.Table II. Purification of glutathione synthetase from cell-free P. bergheiFraction (%) Volume Activity Protein Specific activity Purification(ml) (U/ml) (mg/ml) (U/mg) (x-fold)Crude 16 1.781 4.110 0.433 1Ammonium sulphate fractionation0-35% 3 0.267 12.36 0.021 0.48035-70% 4 5.972 1.521 3.931 9.07070-100% 1 0.924 9.772 0.094 0.212Supernatant 8 0.000 1.142 0.000 0.001Sephadex G-200 column chromatographyF281 0.786 0.162 4.852 11.201F291 1.163 0.143 11.181 25.811F301 2.019 0.129 16.201 37.412F311 2.211 0.123 17.962 41.472F321 2.142 0.125 17.121 39.531F331 1.481 0.117 12.641 29.191F341 1.0831 0.109 9.901 22.862

36Sharma and Banyal0.62.50.5ProteinActivity20.4Protein (mg/ml)0.30.21.51Activity (Units/ml)0.10.501 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45Fractions0Fig.1. Purification of P. berghei glutathione synthetase on Sephadex G-200.host related oxidative stress.The significant GS activity in malaria parasite wasevidently connected with the existence of anautonomous and efficient glutathione system,independent of host metabolism (Ayi et al., 1998). Theparasite is capable of de novo synthesis of glutathionewith GS: 0.443 U/mg as compared to 0.079 U/mg GSby non-parasitized RBC.Subcellular fractionation revealed that GS is confinedto cytosolic fraction of the parasite. The cytosoliclocation of GS may be responsible for protectionagainst damage induced by various oxidative speciesin the cytosol. GS involved in reduced glutathione(GSH) synthesis may be activated in response toutilization of GSH in initial response (Bozdech andGinsburg, 2004).Sephadex G-200 gel filtration chromatography leadsto a preparation having specific activity around 17.96U/mg .The homogenous P. berghei GS preparation isstriking in that the fold purification obtained isapproximately double than the fold purificationobtained in purification of GS from rat kidney(Oppenheimer et al., 1979) using gel filteration on1 2 3 kDaFig.2. SDS-PAGE of purified glutathione synthetase from P.berghei.Lane 1, Purified fraction 31; Lane 2, Purified fraction 32; Lane3, Protein molecular weight standards.20597.5664529

Plasmodium berghei GS37Sephadex G-100. However, further comparison of the Becker K, Rahlfs S, Nickel C and Schirmer RH. 2003.data showed a considerable difference in specific Glutathione-functions and metabolism in the malarialparasite Plasmodium falciparum. Biol Chem 384:551-566.activity between the two preparations. Oppenheimeret al. (1979) reported a value of 33.8 U/mg, which is Becker K, Tilley L, Vennerstorm JL, Roberts D, Rogerson S andabout two-fold higher than our homogenous enzyme. Ginsburg H. 2004. Oxidative stress in malaria-infectedDifferences in assay systems are sufficient to provide erythrocytes: Host parasite interactions. Int J Parasitol34:163-189.explanation for such differences. GS was reported as asingle band protein of molecular weight 70 kDa under Bozdech Z and Ginsburg H. 2004. Antioxidant defence inreducing and denaturing conditions. In contrast, Plasmodium falciparum: data mining of the transcriptome.Meierjohann et al. (2002) reported GS a homodimeric Malaria Journal 3:23-25.protein with subunit molecular weight 77 kDa. Dahl N, Pigg M, Ristoff E, Gali R, Carlsson B, Mannervik B,Similarly, GS reported from yeast (Mooz and Meister, Larsson A and Board P. 1997. Missense mutations in the1967), human erythrocytes (Majerus et al., 1971) and human glutathione synthetase gene result in severerat kidney (Oppenheimer et al. 1979) was also dimeric metabolic acidosis, 5-oxoprolinuria, hemolytic anemia andneurological dysfunction. Hum Mol Genet 6:1147-1152.with subunit molecular weight of 61.5, 75 and 59 kDa,respectively. Therefore, it is possible that P. berghei Kumar S and Banyal HS. 1997. Purification andGS is also a homodimer with subunit molecular mass characterization of the hexokinase of Plasmodium berghei,of 70 kDa. Further characterization of this enzyme anda murine malaria parasite. Acta Vet Hung 45:119-126.its possible role in the survival of parasite may be Laemmli UK. 1970. Cleavage of structural proteins during theelucidated. assembly of the head of bacteriophage T 4. Nature 227:680-685.ACKNOWLEDGEMENTSLowry OH, Rosebrough NJ, Farr AL and Randall RJ. 1951.S. K. Sharma is grateful to the Council of Scientific Protein measurement with folin phenol reagent. J Bioland Industrial Research, New Delhi, for providing a Chem 193:265-275.Junior Research Fellowship. Majerus PW, Brauner MJ, Smith MB and Minnich VJ. 1971.REFERENCESGlutathione synthesis in human erythrocytes II. Purificationand properties of the enzymes of glutathione biosynthesis. JAnderson ME, Powrie F, Puri RJ and Meister A. 1985. Clin Invest 50:1637-1643.Glutathione monoethylester: Preparation uptake by tissuesMeierjohann S, Walter RD and Muller S. 2002. Glutathioneand conservation to glutathione. Arch Biochem Biophyssynthetase from Plasmodium falciparum. Biochem J239:538-548.363:833-838.Atamna H and Ginsburg H. 1997. The malaria parasite suppliesMeister A. 1985. Glutathione synthetase from rat kidney.glutathione to its host cell-investigation of glutathioneMethods Enzymol 113:393-399.transport and metabolism in human erythrocytes infectedwith Plasmodium falciparum. Eur J Biochem 250:670-679. Mooz ED and Meister A. 1967. Tripeptide (glutathione)synthetase. Purification properties and mechanism ofAyi K, Cappadoro M, Baranca M, Turrini F and Arese P. 1998.action. Biochemsitry 6:1722-1734.Plasmodium falciparum glutathione metabolism andgrowth are independent of gluthione system of host Oppenheimer L, Wellner VP, Griffith OW and Meister A. 1979.erythrocyte. FEBS Lett 424:257-261.Glutathione synthetase: Purification from rat kidney andmapping of the substrate binding sites. J Biol ChemBanyal HS and Inselberg J. 1985. Isolation and characterization254:5184-5190.of parasite inhibitory Plasmodium falciparum monoclonalantibodies. Am J Trop Med Hyg 34:1055-1064.Samuels PJ. 1953. The assimilation of amino acids by bacteria.17. Synthesis of glutathione by extracts of Escherichia coliBanyal HS, Kumar S and Upma. 1991. The course ofBiochem J 55:441-444.Plasmodium berghei in Laca strain of mice. Bioved 1:197-200. Sies H. 1989. Biochemistry of thiol groups: the role ofglutathione. Naturwissenschaften 76:57-64.Banyal HS, Pandey VC and Dutta GP. 1979. Subcellularfractionation and localization of marker enzymes of Snoke JE and Bloch K. 1952. Formation and utilization of γ-erythrocytic stages of Plasmodium knowlesi. Ind J Parasitol glutamylcysteine in glutathione synthesis. J Biol Chem3:9-14. 199:407-414.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 38-43Original paperJ P DParasitological and pathological analysis of trematodecyst infestations in Tilapia nilotica, T. zilli and Mugilcephalus1 2 3 4 3 3S. I. Shalaby , S. I. A. Shalaby , M. El-Mahdy , Olafat Anter , Nabila Hassan , Iman Mohamed and5Neelima Gupta1Department of Complementary Medicine, National Research Centre, Dokki, Cairo, Egypt.2Department of Animal Reproduction, National Research Centre, Dokki, Cairo,Egypt.3Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.4Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.5Department of Animal Science, M. J. P. Rohilkhand University, Bareilly, India.Received 7August 2006; revised 15 December 2006; re-revised 21 May 2007; accepted 13 June 2007ABSTRACT. Two hundred seventy two Tilapia and 37 Mugil species harboured 65.37% incidence(highest in summer and lowest in winter) of larval trematodes (T. zilli, 59.4%; T. nilotica, 89.7% andM. cephalus, 59.7%). The small-sized fishes were the maximally infected and their tail region wasthe most favoured (98.11%) site of heterophyid, haplorchid, prohemistomatid and diplostomatidcysts. Pathologically, these cysts caused pressure atrophy and degenerative changes, and theinduced reaction was represented by inflammatory cells in earlier stages which later on gotconverted to more fibrous tissue.Keywords: metacercaria, Mugil, Tilapia, trematodeINTRODUCTIONAlexandria provinces, and each fish was weighed.The present study was aimed at the commonly Skin, scales and gill arches were pressed between twooccurring encysted metacercariae in edible fishes slides and examined for the presence of attached( Tilapia and Mugil spp.) that might cause public health metacercariae. Eye lens and surrounding tissues wereproblems, if these fishes were eaten improperly fragmented with needle and pressed between twocooked. In addition to the parasitological studies, the slides and examined microscopically. The branchialsubsequent hazardous pathological lesions in fish cavity was dissected for detection of encystedmuscles were also investigated.metacercariae. Small pieces of muscles were takenMATERIALS AND METHODSfrom the head, trunk and tail regions, from differentdepths and also from the right and left sides of fishA total of 309 fishes (272 Tilapia spp. and 37 Mugil body. The pieces of muscles were dissected, mixedspp.) were collected from Abbassa experimental fish with a few drops of saline, compressed between twostation and from fishermen on the Nile from Giza to glass slides and examined microscopically.Corresponding author: Prof. Neelima Gupta, Department ofAnimal Science, M. J. P. Rohilkhand University, Bareilly-243006, India. E-mail: guptagrawal@rediffmail.comRESULTSGross examination of fish did not reveal anydetectable lesions. Out of 309 fishes, 202 (65.37%)

Parasitological and pathological analysis of trematode cyst39were infested with encysted metacercariae, the highestinfection rate occurred in Tilapia nilotica (88.65%),followed by T. zilli (60.0%), whereas the lowest ratewas recorded in Mugil cephalus (29.7%). The seasonwiseanalysis revealed that the infestation rate washighest (32.4%) in summer, followed by spring(22.3%) and autumn (17.23%), and was at its lowest inwinter (11.5%). The distribution of metacercarialcysts in the different regions of the fish is shown inTable I. The highest rate of infestation was found in thetail region (88.11%). On the other hand, the trunkregion of Tilapia was least infected.The relationship between the body weight of fish andprevalence revealed highest in comparatively smallerfish M. cephalus (83.33% in those > 25 g in weight and100% in < 50 g category). The higher prevalence wasalso recorded in medium weight class fishes, T. zilli (>OSOSES0 .1 m mVSTCW0.3 m mBITExCxAOSPS0.5 mm0 . 4 mmVSOVTDOSTRCWCCWFig. 1. : A. Heterophyidae encysted metacercaria, B. Haplorchid encysted metacercariaC. Prohemistomatid encysted metacercaria, D. Diplostomatid encysted metacercaria(CW = Cyst wall; Es = Eye spot; Ex = Excretory bladder; I = Intestinal caeca; OS = Oral sucker; OV = Ovary; PS = Pseudosucker; T = Testis; TR = Tribocytic organ; VS = Ventral sucker)

40 Shalaby et al.Fig. 2. Muscles of fish showing oval parasitic cyst with mild host reaction. A. surrounded by inflammatory cells mainly macrophases andlympocytes. B. Odema and eosinophilia and loss of striations of the muscle bundles. H&E x 12550 < 150 g weight group: 70.0985.7%) than lighter cyst was spherical, with double wall; the outer one wasfishes (< 50 g weight group: 33.3–40.8%) and the fragile and could be easily ruptured, whereas the innerheavier ones (150 < 250 g weight group: nil and 1 out one was difficult to remove. The body of theof 2 examined in > 250 g weight group). T. nilotica of metacercaria was surrounded with a membranous wallmedium weight group (> 50 < 150 g) also showed containing fluid. Rows of spines were seen on thehigher prevalence (87.19–0.9%). Heterophyid narrow part of the metacercarial body. (Fig. 1 C).metacercariae were detected in muscles of M.cephalus, T. zilli and T. nilotica. Morphologically, thecyst was globular to elliptical in shape having a bilayerwall, the outer one being thin and transparent,whereas the inner was homogenous, bright bluish incolour and had pigment granules in the body (Fig.1 A).Haplorchid metacercariae were found in the musclesof T. zilli and T. nilotica. The cyst wall was oval inshape, thick and composed of two layers. Clearrefractive granules were present inner to the cyst wall.The body of the metacercaria was folded with clearspines present in the anterior part, where the oralsucker was present. (Fig.1 B). Prohemistomatidmetacercaria were seen in T. zilli and T. nilotica. TheFig. 3. Muscles of fish showing empty parasitic cyst surroundedby inner layer of fibroblast and outer layer of few strands ofcollagen fibres with numerous inflammatory cells. H&E x 125.Fig. 4. A. Muscles of fish showing round parasitic cyst insubcutis causing pressure atropy of the surrounding musclebundles. H&E x 50.B. Higher magnification showing details of centrally locatedparasitic elements surrounded by thick fibrous connective tissuecapsule, considerable numbers of inflammatory cells togetherwith melanin carrying cells. H&E x 200.

Parasitological and pathological analysis of trematode cyst41Fig. 5. Muscles of fish showing dermal parasitic cyst withpositive melanomacrophage cells replacing the necrotizedepidermal layer - Fontana stain x 100.Diplostomatid metacercaria were recorded from T.zilli and T. nilotica. The cysts were elliptical to oval inshape, double walled; the outer one being fragile andcould be easily ruptured, whereas the inner wasdifficult to remove. The metacercaria was clearlydivided into coliform fore-body and more or lesscylindrical hind-body. (Fig. 1 D). Variable parasiticcysts were seen in the hypodermis between fatty cellsor deep in perimycium in between the muscle bundles.Each cyst consisted of a centrally located faintbasophilic parasitic element and homogeneousinternal structure, as seen after haematoxylin andeosin staining. Other cases revealed empty parasiticcysts. The parasitic element was surrounded by emptyspace and then surrounded by very thin fibrousconnective tissue capsule, which in some parasiticcysts was represented by tissue debris. The hostreactions differed according to the age of parasiticcysts. Some cysts were found with mild host reactionaround them, whereas in others, especially in acutestages, the reaction consisted of an aggregation of afew inflammatory cells, mainly lymphocytes andmacrophages (Fig. 2 A and B). In chronic stages, somecysts were surrounded with inner cellular layers,which appeared thick, and composed of youngfibroblasts and some inflammatory cells, mainlylymphocytes and macrophages, followed by outer thinlayer formed of fibrocyte collagen fibers, surroundedwith macrophages and lymphocytes (Fig. 3). Othercysts had thick fibrous connective tissue capsule andwere surrounded by a few lymphocytes. Macrophagesand melanin carrying cells (Fig.4 A and B) gavepositive results with Fontana stain (Fig. 5). Theparasitic pathway was elucidated by dilated bloodvessels and numerous inflammatory cells, mainlylymphocytes and macrophages. Adjacent musclebundles showed pressure atrophy and sometimesrevealed various degrees of degenerative changes(granulation and vacuolation of the sarcoplasm,especially under sarcolemal sheath). Some musclebundles showed hyaline degeneration in which themuscles loose their striation, swelling of myofibers, itappeared eosinophilic with pyknosis of their nucleus.Some bundles were completely destroyed anddisappeared (myolysis) and/or replaced byaggregation of macrophages and lymphocytestogether with melanin carrying cells. In some cases,Table I. The prevalence of encysted metacercariae in different regions of bodyType of fish Head region Trunk region Tail region1 2 3 1 2 3 1 2 3Mugil cephalus 11 11 100 11 8 72.72 11 7 63.63Tilapia zillii 104 71 68.26 104 28 26.92 104 84 80.76Tilapia nitotica 87 56 64.36 87 29 33.33 87 87 100Total 202 138 68.3 202 65 32.17 202 178 88.111Total number of fishes examined.2Number of infested fishes.3Incidence

42 Shalaby et al.there was an aggregation of a large number of accordance to Mahdy et al. (1995). The cysts wereinflammatory cells in perimycium in between the found in the dermis and between muscle bundles inmuscle bundles, mainly lymphocytes and histopathological examinations. These findings aremacrophages forming a tunnel of cellular elements in consonance with those reported by Mahdy (1991)between muscle bundles.and Mahdy et al. (1995). The tissue reaction in the skinDISCUSSIONand muscles of infested fish varied according to thestage of maturation. During the early stage ofRecent reports suggest that fishes act as a source of maturation, it was represented by lymphocytic andserious human parasitic infectious diseases (Dubois macrophage inflammatory cells, whereas in laterand Pearson, 1963; Schnurrenberger, 1975). stages, the tissue reaction became more fibrous andHeterophyidae constitute a public health problem less cellular with the presence of melanin carryingwhere people eat raw, salted or otherwise improperly cells. The tissue reaction in the skin and muscles wereprepared infested fish. This is a common infestation in possibly due to mechanical irritation effect of theNorthern Nile Delta, where, from one village near lake parasitic cysts during its migration and /or due to theirManzala, up to 90% of school children and 22% of toxic products .These results are in agreement to thoseadults were infested (Paperna, 1980). The prevalence of Mahmoud et al. (1989), El Reid (1994) and Mahdywith encysted metacercariae was found to be 65.37%. et al. (1995). Degenerative and necrotic changesIn T. zilli, it was 59.4% and in T. nilotica, it was 89.7%. observed in muscle bundles could be attributed toThis appeared to be higher than that recorded by pressure atrophy induced by the encystedMahdy et al. (1995) who recorded that they were metacercariae, in addition to the effect of toxic41.43% and 41.18%, respectively. Moreover, in M. parasitic metabolites in muscle cells as observed bycephalus, the prevalence was slightly higher than that Mahdy et al. (1995).recorded in marine fishes by El Reid (1994) who REFERENCESmentioned that the prevalence was 22.76%. But it was Dubois G and Pearson J. 1963. Les Streigedia (Trematoda) de l'obviously lower than that recorded in M. cephalus by Egypt. Ann Parasitol 38:91-92.Mahmoud (1983) being 87% and ElDally (1988) El-Dally K. 1988. The role of fish as intermediate host forbeing 82.5%. These data indicated that water pollution transmitting some parasites of zoonotic importance, inwith human excreta was more in fresh and brackish Behaira Governorate. M. Sc. Zoonoses, Diss. Faculty ofwaters than in Mediterranean Sea water (El Reid, Veterinary Medicine, Alexandria, Egypt.1994). The higher prevalance was seen in summer El-Reid A. 1994. Role of marine fish in transmission of some(33.4%), followed by spring (22.3%), autumn parasites to animals and birds. Ph. D. Thesis (Parasitology),(17.53%) and the lowest was in winter (11.5%).Faculty of Veterinary Medicine., Zagazig University, Egypt.Almost similar results were reported by Shalaby Evans H and Mickiewicz I. 1985. The incidence and location of(1982) for T. nilotica and Bagrus bayad. On the metacercarial cysts (Stigieda) on 35 species of central NewYork fishes. J Parasitol 44:23-35.contrary, Synodontis schall indicated a reversecondition by the same author in the investigation on Mahdy O. 1991. Morphological studies on the role of somefresh water fishes in transmitting parasitic heminthes offresh water fishes. The highest prevalence was in tailsome avian hosts. Ph. D. Thesis. (Parasitology), Faculty ofregion (32.17%) among the investigated Tilapia spp. Veterinary Medicine, Cairo University, Egypt.and M. cephalus fishes. Similarly, Evans andMahdy O, Manal A, Essa A. and Easa M El S. 1995.Mickiewicz (1985) reported that the caudal fin had Parasitological and pathological studies on heterophyidmore stringed metacercarial cysts in 35 species of infections in Tilapia spp. from Manzala lake, Egypt.central New York fishes. This observation disagreed Egyptian J Comparative Pathol and Clinic Pathol 8:131-with Shalaby (1985), where he found that the trunk 145.region of Clarias lazera recorded the highest Mahmoud N. 1983. Parasitic infestations of some nativeprevalence. Malhotra and Banerjee (1989) observed species of fishes in Cairo markets with special reference topathological interactions of metacercaria in the tailparasites transmissible to man and animals. M. V. Sc. Diss.(Hygiene and Food Control), Faculty of Veterinaryregion of Schizothorax richardsomii from Garhwal Medicine, Cairo University, Cairo, Egypt.Himalayan riverine ecosystem from India. HigherMahmoud N, Yousssef H, Khalifa R and Nassar A. 1989.prevalence was seen in young fishes (< 50 g)Studies on metacercarial infection in fresh water fish inespecially in M. cephalus. This might be related to the Assuit province. Egyption J Comparative Pathol and Clinicimmunity of fish and that young fishes were more Pathol 2:213-224.susceptible than the older ones. These results are in

Parasitological and pathological analysis of trematode cyst43Malhotra SK and Banerjee S. 1989. A review of environmentalcorrelates and systematics of the organisms of black spotdisease in Indian fishes. Dr. B. S. Chauhan CommemorationVolume 87-101.Shalaby SI. 1982. Studies on the role played by some Nile fishesin transmitting trematodes. M. Sc. Thesis (Parasitology),Cairo University, Cairo, Egypt.Shalaby SI.1985. Further studies on the role played by cat fishPaperna J. 1980. Parasitic infections and diseases of fish of in transmitting some trematodes to fish eating mammalsAfrica for inland fishes of Africa. CIFA Technical papers: with special reference to the morphobiology of M.51-62. appendiculatous. Ph. D. Thesis (Parasitology), CairoSchnurrenberger P. 1975. Diseases transmitted from animals toUniversity, Cairo, Egypt.man. Sprinffield, Illinois, USA 646-677.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 44-48Original paperJ P DThe prevalence of intestinal helminth infections inprimary school children in Owerri municipality, Imostate, NigeriaF. N. Opara, A. A. Udoye, P. U. Okere, F. O. U. Osuala and M. O. E. IwualaDepartment of Biotechnology, Federal University of Technology, Owerri.Received 2 November 2006; revised 20 May 2007; accepted 20 June 2007ABSTRACT. Intestinal helminth infections in primary school children was surveyed in eightrandomly selected primary schools in Owerri Urban, Imo State, Nigeria, between October 2003 andMarch 2004. Stool samples of 578 pupils (244 males, 334 females), aged 5–16 years, were examinedmicroscopically by using wet mount (normal saline) and concentrated saturated sodium chloridefloatation techniques. Four intestinal helminths, Ascaris lumbricoides, hookworm, Trichuristrichiura and Trichostrongylus sp. were identified with 122 (21.1%) of the 578 pupils infected withone or a combination of the worms. A. lumbricoides had the highest prevalence (65.6%) followedby hookworm (35.2%), T. trichiura (14.8%) and Trichostrongylus sp (6.6%). Pupils aged 11–13 and5–7 years had the highest (25.1%) and lowest (14.4%) rates of infection, respectively.Trichostrongylus sp. infection was highest and lowest among the 14–16 and 8–10 year old pupils,respectively. Though there was no significant (p > 0.05) sex related difference in the prevalence ofhelminth infection, A. lumbricoides and hookworm infections were relatively higher in females,whereas T. trichiura and Trichostrongylus sp. infections were higher in male pupils. Mixedinfections were recorded, with Ascaris and hookworm, and with Ascaris, hookworm and Trichurisbeing the two most commonly occurring combinations. The finding of Trichostrongylus sp.infection in this study is significant and of public health importance. Improvement of personalhygiene, avoiding ingestion of contaminated food, restricting sheep, goat and cattle from straying ininhabited areas are recommended intervention approaches to control human intestinal helminthinfections.Keywords: children, helminth infections, intestinal, NigeriaINTRODUCTIONThe health of school age children in developingcountries is a concern that has received increasingattention in the recent past, following high morbidityrates due to parasitic diseases which are preventable(Bundy and Guyatt, 1995). Much of this morbidityCorresponding author: Dr. F. N. Opara, Department ofBiotechnology, Federal University of Technology, Owerri, ImoState, Nigeria. E-mail: ofineopara@yahoo.co.ukhas been attributed to parasitic helminth infections(Etim and Akpan, 1999; Etim et al., 2002).Helminthic infestation is a serious public healthproblem, especially in areas of low environmentalquality and of people of low nutritional status (Kuttyet al., 2003). In the growing stage, children are moresusceptible to the ill-effects of parasitic attacks, astheir need for nutrients is high. Although death ratesamong these children are low, it has recently beenestimated that school age children experience aconsiderable worm burden, which may have both

Intestinal helminth infections in school children in Nigeria45immediate and long term consequences for their School Codehealth, growth and education (Bundy and Guyatt,1995; Etim et al., 2002).Shell Camp Primary School(SCPS)S1More than one billion of the world's populations,including at least 400 million school children, areCollege of Education Demonstrationchronically infected with Ascaris lumbricoides,Primary School (COEDPS)S2Trichuris trichiura, hookworms, Enterobiusvermicularis and Taenia sp. (Adeyeba and Akinlabi,Model Primary School (MPS) S32002; Braide, 2004). Various prevalence rates of Orlu Road Primary School I (ORPS I) S4infection of these helminths in school children indifferent parts of Nigeria have been reported byOrlu Road Primary School II (ORPS II) S5several workers including Adeyeba and Akinlabi Amakohia Primary School (APS) S6(2002). Etim et al. (2002), for instance, obtained aprevalence rate of 53.2%, 31%, 27.0% and 5.5% for Manns Street Primary School (MSPS) S7Ascaris sp., Ancylostoma sp, Trichuris sp andSchistosoma mansoni, respectively, from primaryIkenegbu Primary School (IPS)S8school children aged 5–13 years in Calabar, Nigeria. Collection and examination of faecal samples:The prevalence of these helminths varies not only Wide mouthed specimen bottles were given to thefrom one locality to the other, but also among randomly selected pupils who were asked to returnindividuals, age, standard of sanitation, socio- them the following morning with a small quantity ofeconomic status of parents, with children of parents in their early morning faeces for examination. The namethe low income group having the highest prevalence (optional), age and sex of each pupil were noted afterof infection, and sex, with males being more infected the faecal sample had been collected and accordinglythan females. Of interest in intestinal helminthic labelled. The specimens were taken to the laboratoryinfection is multiple infection or polyparasitism, for examination with a Nikkon compound microscopeoccurring in various combinations and rates of using x10 and x40 objectives. The normal saline (wetinfection but with Ascaris-hookworm-Trichuris mount) and concentrated saturated sodium chloride“Triad' combination as the most ubiquitous floatation techniques according to Neva and Browncombination.(1994) Cheesbrough (1999) were used for the analysisof the faecal samples for helminth ova and larvae.Although several studies on parasitic infections ofDiagnosis was based on identification of theschool children have been carried out in some parts ofcharacteristic helminth ova/larvae usingNigeria, it is still necessary to do similar studies incharacteristics outlined by Neva and Brown (1994)different other parts of the country at different times,and Cheesbrough (1999). The egg of Trichostrangylusin view of the changing dynamics of parasiticis longer and thinner than a hookworm egg whichinfections. The present study aims at the identificationmeasures 85–115 µm in length, being more pointedof various intestinal helminths, which infect primaryon one or both ends and usually appears moreschool children, to determine the overall prevalencesegmented. On collection of faecal samples from theof infection and the pattern of infestation in relation topupils, each pupil was interviewed on some of theage and sex of the children, and reports the results offollowing points: parents' occupation, foot wearthe investigation on intestinal helminthiasis in schoolhabits, pet/domestic animals reared, regularity of dechildrenin Owerri Urban Primary Schools, Imo State,worming and availability and type of toilet facility etc.Nigeria.MATERIALS AND METHODSRESULTSStool samples from a total of 578 primary schoolFive hundred and seventy eight school children, agedchildren comprising 244 males and 334 females, aged5–16 years, in eight randomly selected primarybetween 5 and 16 years, were examined for intestinalschools in Owerri Urban, Imo State, were investigatedhelminth infections. Four intestinal helminths,for their intestinal helminthic infections betweenAscaris lumbricoides, hookworm, Trichuris trichiuraOctober 2003 and March 2004. The schools included:and Trichostrongylus sp. were identified. Of the 578pupils examined, 122 (21.1%) were infected with one

46 Opara et al.or a combination of the worms with A. lumbricoides Table II shows that out of 578 pupils examined, 264having the highest prevalence rate (65.6%), followed (45.7%) and 314 (54.3%) were males and females,by hookworm (35.2%) and T. trichiura (19.8%), respectively, of these 57 (21.6%) males and 65whereas Trichostrongylus sp. had the least rate of (20.7%) females were infected; the difference was notinfection (6.6%). These results are shown in Table significant (p > 0.05). The prevalences of infection ofI. The finding of Trichostrogylus sp. infection is A. lumbricoides and hookworm were higher insignificant, as this is the first record/report of its females than in males, whereas those of T. trichiurainfection in man in this part of the country.and Trichostrongylus sp. were higher in males, theThe prevalence of infection among the schools rangeddifferences are not significant (p > 0.05).between 5.6% (S1) and 31.3% (S7). There was no Of the 122 infected subjects, 16 (13.1%) had multiplesignificant difference in the prevalence of infection intestinal helminth infections, with 11 (9.0%) and 5between schools (p > 0.05).(4.1%) subjects having double and triple infections,respectively. Ascaris lumbricodes occurred mostlyTable II shows that the overall prevalence of infectionwith the other helminths, Ascaris + hookworms, andof the helminths was highest in pupils aged 11–13Ascaris + hookworms + Trichuris were the most(25.1%) and lowest in pupils aged between 5 and 7common occurring combinations. These results areyears old (14.4%). The prevalence of A. lumbricoidesshown in Table III.and hookworm infections were highest in pupils ofage range 5 and 7 years and 14–16 year old, DISCUSSIONrespectively, and both lowest in pupils of age bracket11–13 years. There was no significant difference inOva of four intestinal helminths, Ascarisinfection prevalence with age (p > 0.05). The Tablelumbricoides, hookworm, Trichuris trichiura andfurther shows that T. trichiura and TrichostrongylusTrichostrongylus sp. were recorded with 122 (21.1%)sp. infections were highest in 11–13 and 14–16 yearof the 578 school children positive for one or moreold pupils, respectively, while lowest infection ratestypes of helminths. The overall prevalence ofof both helminths occurred among the 5–7 and 8–10infection (21.1%), when compared with reportedyear old pupils, respectively. There was not muchresults of previous studies in other parts of the country,difference in T. trichiura and Trichostrongylus sp.(Mafiana, 1995; Ukpai and Ugwu, 2003), is low andinfection among the 14–16 year old pupils.suggestive of improved personal hygiene awarenessand environmental sanitation in the study area andTable I. The prevalence of intestinal helminths in school children in selected primary schools in Owerri UrbanschoolsSchool No. of No. Helminths identified / % prevalencecode pupils infected Ascaris Hookworm T. trichiura Trichostrongylusexamined (%) (%) (%) (%) (%)S1 54 3 (5.6) 3 (100.0) 0 (0.0) 0 (0.0) 0 (0.0)S2 70 19 (27.1) 12 (63.2) 7 (36.8) 0 (0.0) 0 0.0)S3 74 15 (20.3) 13 (86.7) 5 (33.3) 0 (0.0) 0 (0.0)S4 75 20 (26.7) 11 (55.0) 8 (40.0) 4 (20.0) 2 (9.1)S5 80 10 (12.5) 6 (60.0) 5 (50.0) 3 (30.0) 0 (0.0)S6 84 14 (16.7) 10 (71.4) 4 (28.6) 4 (28.6) 1 (7.1)S7 83 26 (31.3) 16 (61.5) 10 (38.5) 4 (15.4) 3 (11.5)S8 58 15 (25.9) 9 (60.0) 4 (26.7) 3 (20.0) 2 (13.3)Total 578 122 (21.1) 80 (65.6) 43 (35.2) 18 (14.8) 8 (6.6)% prevalence of infection in parenthesis. * p > 0.05

Intestinal helminth infections in school children in Nigeria47Table II. Prevalence of intestinal helminth infections in relation to age and sex of pupilsAge No. No. A. Hookworm T. Trichostrongylusrange examined infected lumbricoides (%) trichiura sp. (%)(yrs.) (%) (%) (%)5–7 m 29 6(20.7) 6(20.7) 3(50.0) 0(0.0) 1(16.7)f 68 8(11.8) 8(11.8) 3(37.5) 0(0.0) 0(0.0)T 97 14(14.4) 14(100.0) 6(42.9) 0(0.0) 1(7.1)8–10 m 101 20(19.8) 15(70.0) 5(25.0) 3(15.0) 0(0.0)f 64 10(15.6) 10(100.0) 6(60.0) 1(10.0) 0(0.0)T 165 30(18.2) 25(83.3) 11(36.7) 4(13.3) 0(0.0)11–13 m 69 18(26.1) 6(33.3) 5(27.8) 8(44.4) 1(5.6)f 134 33(24.6) 16(48.5) 8(24.2) 3(9.1) 1(3.0)T 203 51(25.1) 22(43.1) 13(25.5) 11(21.6) 2(3.9)14–16 m 65 13(20.0) 6(46.2) 6(46.2) 2(15.4) 2(23.0)f 48 14(29.2) 13(92.9) 7(50.0) 1(7.1) 2(14.3)T 113 27(23.9) 19(70.40) 13(48.1) 3(11.1) 5(18.5)Total m 364(45.7) 57(21.6) 33(57.9) 19(33.3) 13(22.8) 5(8.8)f 314(54.3) 65(20.7) 47(72.3) 24(36.9) 5(7.7) 3(4.6)T 578 122(21.1) 80(65.6) 43(35.2) 18(14.8) 8(6.6)% prevalence of infection in parenthesis. * p > 0.05m = male f = female T = totalTable III. Polyparasitism in school children in Owerri Urban schoolsParasite combination No. infected % infectionAscaris + Hookworm 4 3.3Acaris + Trichuris 2 1.6Ascaris + Trichostrongylus 2 1.6Ascaris + Hookworm + Trichuris 4 3.3Hookworm + Trichuris 1 0.8Hookworm + Trichostrongylus 2 1.6Ascaris + Hookworm + Trichostrongylus 1 0.8Total number infected with intestinal helminths = 122.% Prevalence of infection in parenthesis based on 122 infected.16 13.1definite communal control efforts. Previous studieshad attributed the high endemicity to poorenvironmental and personal hygiene, shortage ofgood water supply and indiscriminate defaecation.Ascaris lumbricoides, in contrast to the other geo-transmitted helminths, hookworm, T. trichiura andTrichostrongylus sp. had the highest prevalence ofinfection probably because Ascaris ova can live in soilfor years. The standard of hygiene of the pupils andtheir voracious eating habits probably predisposethem to infected faecal matters.However, the prevalence of ascariasis and hookworminfections decreased with age up to 11–13 year agegroup, but increased in the 14–16 year age group,probably indicating reduced parental personalhygiene supervision, voracious eating habit andactivities linked with soil contaminated with infected

48 Opara et al.faecal matters. In this study, though sex difference in who are fond of handling dogs and cats (Nwoke, 2001;the prevalence of intestinal helminth infections was 2004), whereas dipterans transmit the parasites tonot significant, females were more infected with food stuff.Ascaris lumbricoides and hookworm. Females aremore engaged in gardening and such related activitiesPrevention of these intestinal helminth infections islikely to bring them in contact with soil contaminatedpossible by restricting sheep, goat and cattle fromwith infected faecal matters. The finding ofstraying, avoiding ingestion of contaminated food,Trichostrongylus sp. infection in this study isavoiding use of human and animal excreta as fertilizersignificant and of public health importance. Many ofin vegetable gardens and by maintaining personalthe children infected with this helminth are fromhygiene.homes in which goats, sheep or rabbits are REFERENCESdomestically reared and their faeces used as fertilizerAdeyeba OA and Akinlabi AM. 2002. Intestinal parasiticin peridomestic vegetable gardens. Further, these infections among school children in a rural community,pets as well as domestic or stray animals often come in southwest, Nigeria. Nig J Parasitol 23:11-18.contact with children and the food (vegetables, etc.) Boreham RE, McCowan MJ, Ryan AE, Allworth AM, Robsonthey eat. In this way diseases previously found in JM. 1995. Human Trichostrongylus in Queensland.animals now emerge in man, Nwoke (2004). In Pathology 27:182-185.addition, cattle, sheep and goats, the potential hosts of Braide EI. 2004. Parasites and Politics: Parasites, poverty andTrichostrongylus sp., stray into school premises,ndpolitics, 22 Inaugural lecture of the University of Calabar,grazing lawns and defaecating indiscriminately Cross River State. 11-27.thereby contaminating the surrounding. These Bundy DAP and Guyatt HL. 1995. The health of school agefactors, probably, predisposed the children to children, report of a workshop. Parasitol Today 11:116-167.Trichostrongylus sp infection. Human infection with Cheesbrough M. 1999. District Laboratory Practice in TropicalTrichostrongylus sp. has been reported from countries countries. Part I. (Low-Price Editions) University Pressin the Middle East including Iran, Iraq and Egypt, in Cambride. 212-213.the far East (Japan, Indonesia and Korea), in Russia Etim SE and Akpan PA. 1999. Studies on geography as a riskand Armenia, in South America, in Africa (Morocco factor for geohelminthiasis in Calabar, Cross River State,and Ethiopia) and in Australia (Boreham et al., 1995). Nigeria. Nig J Parasitol 20:91-98.Although nine species of Trichostrongylus have been Etim SE, Akpan PA, Abeshi SE, Effion OE and Enyi-Deh KC.found in man, none has been reported in Nigeria. This 2002. Intestinal helminth infections in children:probably is the first recorded Trichostrongylus sp. Implications for helminth control using school-based masschemotherapy. Nig J Parasitol 23:53-59.infection in man in Nigeria. It is possible that mostinfections reported as hookworm may be Kutty VR, Soman CR and K Vijaya Kumar. 2003. Pattern ofhelminthic infestation in primary school children ofTrichostrongylus sp., since both have more or lessThiruvananthapuran district.similar morphological features. Ova of http://krpeds.org/publication/ramankutty.htmTrichostrongylus sp., which are larger with moreMafiana CF. 1995. Intestinal helminthiasis with particularpointed ends than hookworm eggs (Cheesbrough,reference to ascariasis among school children in Idewo-1999) may have been misdiagnosed or overlooked in Orile, Ogun State, Nigeria. Nig J Parasitol 16:47-53.previous studies.Mba IEK and Amadi AN. 2001. Helminth infection in schoolMixed infection due to Ascaris, hookworm andchildren in Aba, Abia State. J Med Invest Pract 2:43-45.Trichuris trichiura, often described as “ubiquitousthNeva FA and Brown HW. 1994. Basic Clinical Parasitology, 6triad”, is common. In this study, a mixed infection of ed. Prentice Hall International Inc. USA. pp 356.Ascaris and hookworm was common, which is in Nwoke BEB. 2001. Urbanization and livestock handling andconsonance with the findings of Mba and Amadi farming: The public health and parasitological(2001) and Ukpai and Ugwu (2003). The high implications. Nig J Parasitol 22:121-128.prevalence of intestinal helminths in primary school Nwoke BEB. 2004. Our Environment and Emerging and Re-children in the municipality could be due to refuse emerging Infections and Parasitic Diseases. SupremePublishers, Owerri Nigeria, pp 55.dumps around market areas, along the major roads,which attracted stray cats, dogs, goats, sheep, rodents Ukpai OM and Ugwu GD. 2003. The prevalence of gastro-and dipterans. These animals serve as potential intestinal tract parasites in primary school children inIkwuano Local Govt. Area of Abia State, Nigeria. Nig Jsources of zoonotic transfer of parasites to children,Parasitol 24:129-136.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 49-53Original paperJ P DNeuroanatomy of gill parasite Chauhanellus indicus(Monogenea: Ancyrocephalinae) from Mystus seenghala(family: Bagiridae) in Meerut (U. P.), India1 1 2P. Rastogi , D. Mishra and H. S. Singh1Department of Zoology, Meerut College, Meerut.2Department of Zoology, C. C. S. University, Meerut.Received 2 November 2006; revised 22 June 2007; accepted 2 July 2007ABSTRACT. 5-Bromoindoxyl acetate has been used for first time in India to describe the nervoussystem of an oviparous monogenean gill parasite Chauhanellus indicus. The huge arrangement ofdorsal, ventral and lateral nerve cords and its innervations have been examined. The centralnervous system consists of paired cerebral ganglia from which emanate one pair each oflongitudinal ventral, lateral and dorsal nerve cords, connected at intervals by transverseconnectives. The central nervous system better developed ventrally than dorsally or laterally.Keywords: Chauhanellus indicus, monogenea, Mystus seenghala, neuroanatomyINTRODUCTIONChauhanellus indicus (Rastogi et al., 2004;Monopisthocotylea: Anchyrocephalinae) anoviparous monogenean has been described from a newhost Mystus seenghala (family: Bagiridae) withgreater emphasis on its nervous system to indicate thepathway of acetylcholine in it. Several authors haveused the method of demonstrating cholinesteraseactivity as an indirect evidence of the presence ofacetylcholine in the nervous system of flatworms,including monogeneans, digeneans and cestodes(Reda and Arafa, 2002). Present work involved the useof 5-bromoindoxyl acetate to study the nervoussystem of C. indicus.MATERIALS AND METHODSThe gills of infected Mystus seenghala yielded 25Corresponding author: Dr. Pragati Rastogi, Department ofZoology, Meerut College, Meerut -250 001, India. E-mail:soniapr@rediffmail.comworms that were washed, fixed in cold 4% neutralformaldehyde under cover glass for 8 h, subsequentlywashed with cold distilled water and mountedpermanently by using standard method (Rastogi et al.,2004). The study of nervous system was accomplishedwith the help of histochemical localization ofesterases (Halton and Jennings, 1964) and cameralucida sketches were made. Microphotographs weretaken with the help of Motic Image Plus software.RESULTSNeuroanatomy (Fig. 1; Fig. 2a–f)The central nervous system (CNS) comprises ofpaired cerebral ganglia (cg) from which anterior andposterior neuronal pathways, interlinked by crossconnectivesand commissures, are derived. Theperipheral nervous system (PNS) includes innervationof the alimentary tract, reproductive organs,attachment organs (anterior adhesive areas andhaptor), subtegumental and haptoral muscles. Boththe CNS and PNS are bilaterally symmetrical, and

50Rastogi et al.better developed ventrally than laterally and dorsally;they display strong staining for cholinesterase (ChE).The CNS and PNS (Fig. 1; Fig. 2a–f) were found to bestained extensively in blue and purple colour. TheCNS consists of a thick, curved mass of paired cerebralganglia located ventrally just anterior to pharynx. Asingle anterior ventral commissure (avc) originatesfrom the postero-lateral regions of the cerebralganglia, and runs in a semicircular manner in theanterior region of the head, just posterior to the headlobes. Two considerably thick projections (ap) extendfrom the anterior median region of the cerebralganglia, imparting to this organ a butterfly-likeappearance. Each projection gives rise to cerebralnerves (cn), which extend to the anterior to enter thehead-lobes where they innervate the anterior adhesiveareas. A pair of thin pharyngeal nerve (Pn) arises fromthe postereo- median region of the cerebral ganglia,which innervate the pharynx with four pharyngealconnectives (Pc1–Pc4).ventral nerve. It is connected to the ventral nerve cordsvia two dorsoventral connectives (dvc1–dvc2). Fourdorsal connectives (dc1–dc4) are detected connectingthe two dorsal nerves.The staining for cholinergic elements revealed thepresence of two pairs of large neurons distributedbilaterally down the main body of the worm. Thesecells are located on the lateral side of the body(L1–L2). The first pair of lateral cell bodies (L1) ispositioned at the level of the receptaculum seminis andthe second pair (L2) at the level of the ovary.The haptor is extensively innervated by a pair of outer(ohn) and two pairs of inner (ihn), relatively thickhaptoral nerves. The outer of these arise one from eachposterior prehaptoral ganglion (Phg2), whereas theinner members are derived from the ventralconnective (vc4). The outer and inner haptoral nervesrun ventrally in a posterior direction before branchinginto a plexus of numerous fine nerves in the anteriorregion of the haptor. The inner haptoral nervesinnervate the intrinsic haptoral muscles. There are twodorso-lateral haptoral nerves (dhn) arising one fromeach posterior prehaptoral ganglion (Phg2), and theserun towards posterior end and eventually branch tosupply the marginal hooklets.Two thick ventral nerve cords (vnc) arise one eachfrom lateral region of the cerebral ganglia, and runposterior, where each of them joins a prehaptoralganglion (Phg1). Another pair of prehaptoral ganglia(Phg2) is located, one on each side of the body, a shortdistance posterior to the prehaptoral ganglia (Phg1), inthe region anterior to the haptor. Both sets ofDISCUSSIONprehaptoral ganglia, on each side of the body, are This is the first attempt to describe the acetylcholineconnected via two fine ganglionic connectives. Five component of the nervous system of C. indicus. Theventral transverse connectives (vc1–vc5) were also CNS of the worm comprises mainly of a mass ofdetected, three between the two ventral nerve cords, cerebral ganglia and three pairs of ventral, lateral andone between the anterior prehaptoral ganglia (Phg1), dorsal longitudinal nerve cords connected byand one between the posterior prehaptoral ganglia transverse commissures; it is better developed(Phg2). A pair of subtegumental nerves (Stn) arises ventrally than dorsally and laterally. In these respects,from the ventral nerve cord near the postero lateral the CNS of C. indicus resembles in basic structure withside of pharynx and enters the subtegumental muscles that of all previously studied flatworms, includingof the prohaptoral region. Two subtegumental ventral monogeneans, digeneans and cestodes (Reuter, 1987;connectives (Stvc1–Stvc2) connect the subtegumental Reda and Arafa, 2002).nerve with the ventral nerve cords.The staining with 5-bromoindoxyl acetate alsoTwo considerably thin lateral nerve cords (lnc) arise revealed the basic orthogonal pattern in CNS of C.from the postero-lateral region of the cerebral ganglia indicus described for other monogeneans. Typically,and run towards posterior side, one adjacent to eachlateral margin of the body where they join the prehaptoralganglia Phg1. Two lateral connectives(lc1–lc2) are also detected in the region of testis. Atregular intervals, the ventral nerve cords communicatewith the lateral nerve cords by means of three pairs ofventrolateral connectives (vlc1–vlc3). Two thindorsal nerve cords (dnc) arise from the posterolateralregion of the cerebral ganglia and connect to thethe PNS innervates the alimentary system,reproductive organs, attachment organs and intrinsichaptoral muscles. These neural pathways and theirorganisation have previously been described in tenother monogeneans, namely Diplozoon paradoxum,Polystoma integerrimum, Gyrodactylus salaris(Reuter 1987), Pseudodactylogyrus anguillae (Redaand Arafa, 2002), Eudiplozoon nipponicum,Diclidophora merlangi, Pseudodactylogyrus bini

Neuroanatomy of gill parasite Chauhanellus indicus51CnCephalic glandapavcstvc1stnInc stvc2vncvlc1dnccirrusCgPnPcdc10.1 mmdvc1L1vc1dvc2dc2L2vc2dc3vlc2dc4lc1lc2thnIntrinsic haptoralmusclevlc 3vc 3ohn dhnVC 4VC 5 Phg 1Phg 2Fig. 1. Camera lucida drawing of schematic of the nervous system as revealed by 5-bromo indoxyl acetate staining.

52 Rastogi et al.(a)(b)(c)(d)(e)(f)(g)Fig. 2. a anterior head region showing cerebral ganglia (cg)with two anterior projections (ap) and anterior ventralcommissure (avc). Innervation of the pharynx. b middleportion of the body showing ventral nerve cord and ventralconnective near the cirrus (male copulatory complex). cmiddle portion of the body showing ventral nerve cord andventral connective near the ovary. d middle portion of thebody showing dorsal connectives and lateral connectives inthe testicular region. e eduncular region showing ventralconnective and ventrolateral connectives. f posterior regionshowing pre haptoral ganglia. g haptoral region showinghaptoral nerves.

Neuroanatomy of gill parasite Chauhanellus indicus53(Reda and Arafa, 2002), Entobdella soleae, can also be attributed the haptoral nerves andDiscocotyle sagittata (Cable et al., 1996) and neuroendocrine secretion of haptoral glands.Protopolystoma xenopodis.ACKNOWLEDGEMENTS5-bromoindoxyl acetate staining revealed numerouspairs of neuronal cell bodies associated with theThe authors are thankful to the Head, Department ofcerebral ganglia, pharynx, prehaptoral nerve plexusesZoology, Meerut College, Meerut, for the laboratoryand prehaptoral commissure of C. indicus togetherfacilities. The financial assistance from Universitywith scattered cell bodies along the ventral nerveGrants Commission, New Delhi, is thankfullycords.acknowledged.A striking feature of all monogeneans studied so farREFRENCEShas been the finding of a ring commissure around the Cable J, Harris PD and Tinsley RC. 1996. Ultrastructuralmouth (Rohde, 1968; Reuter, 1987). Rohde (1968) adaptations for viviparity in the female reproductive systemof gyrodactylid monogeneans. Tissue Cell 28:515-526.suggested an oral commissure is a character, whichdistinguishes the nervous system of the Monogenea Halton DW and Jennings JB. 1964. Demonstration of thefrom that of Digenea.nervous system of the monogenetic trematode Diplozoonparadoxum Nordmann by the indoxyl acetate method forIt is noteworthy here that some reproductive organs of esterases. Nature 202:510-511.C. indicus (Rastogi et al., 2004) are innervated by Rastogi P, Kumar K and Singh HS. 2004. Review of the genusventral commissure nerves, notably the male and Chauhanellus (Young, 1967) Bychowsky and Nagibina,female copulatory complex. It seems probable that the 1969 with a report on a new species from freshwater fishesnerves innervating the male and female copulatory of Meerut (U.P.), India. Uttar Pradesh J Zool 24:121-128.complex help in copulation. Cable et al. (1996) Reda ES and Arafa SZ. 2002. Cholinergic components of thesuggested that the neuropeptide secretions might nervous system of the monogenean gill parasites,influence movement of ova and vitelline cells, release Pseudodactylogyrus bini and P. anguillae from the eelof spermatozoa and Mehlis' gland secretions, Anguilla anguilla in Nile Delta waters. Egyptian J Zoolperistaltic contractions of the ootype which shape the 38:41-54.egg and regulate the release of egg from the ootype Reuter M. 1987. Immunocytochemical demonstration ofinto the uterus. The activity of prohaptoral head organs serotonin and neuropeptides in the nervous system ofcan be attributed to the cerebral and subtegumental Gyrodactylus salaris (Monogenea). Acta Zool 68:187-193.nerves and neuroexocrine secretions of cephalic Rohde K. 1968. Das Nervensystem der Gattung Polystomoidesglands. High degree of mobility of marginal hooklets Ward, 1977 (Monogenea). Z Morphol 62:58-76.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 54-55Short communicationJ P DPathology of nodular tapeworm in backyard poultryP. Ramesh Kumar , Reghu Ravindran , Bindu Lakshmanan , P. Senthamil Selvan , H. Subramanian1and T. Sreekumaran1 2 2 3 21Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Lakkidi.2Department of Veterinary Parasitology, College of Veterinary and Animal Sciences, Lakkidi.3Department of Veterinary Anatomy, College of Veterinary and Animal Sciences, Lakkidi.Received 20 October 2006; revised 11 December 2006; accepted 17 December 2006ABSTRACT. Intestinal samples were collected from backyard poultry brought for post-mortem atthe College of Veterinary and Animal Sciences, Pookot. Adult Railettina echinobothrida parasiteswere recovered from the intestinal contents. The specimens were subjected to gross andhistopathological examination. Grossly, nodules of varying sizes were present on the peritonealsurface of the intestine. Histopathologically, these lesions revealed degeneration and necrosis ofintestinal villi and cross-sections of parasite attached to the mucosa. Parasitic granulomas withsinus which occupy various layers of the intestinal tissue were also observed.Keywords: granuloma, intestine, nodules, Railettina echinobothrida, sinusThe tapeworm Raillietina echinobothrida is one of themost pathogenic parasites which occur in the smallintestine of chickens and turkeys in most parts of theworld. This cestode infection is still a commonproblem in poultry raised on range or in backyardflocks. However, there are only a few reports on thehistopathology of this disease (Nath and Pande, 1963;Bhoumik and Sinha, 1983; Samad et al., 1986).Therefore, an attempt has been made to find out thepathological lesions produced as a result of naturalinfections of R. echinobothrida in domestic fowls.Twelve out of 25 birds, reared by a private ownerunder backyard poultry system, died and were broughtto the College of Veterinary and Animal Sciences,Pookot, for autopsy. The parasites seen at the time ofautopsy were collected and later identified afterstaining with acetic alum carmine (Soulsby, 1982).Corresponding author: Dr. Reghu Ravindran, Department ofVeterinary Parasitology, College of Veterinary and AnimalSciences, Pookot, Lakkidi-673 576, India. E-mail:drreghuravi@yahoo.comThe intestinal segments which showed grossabnormalities were preserved in 10% buffered neutralformalin, and were processed through conventionaltechniques. Paraffin sections of 45 µ thickness wereused for staining by Grams, Zeil Neelsen's and PASstaining methods for differentiation.During autopsy, gross thickening of the intestinal walldue to connective tissue proliferation could beobserved. The nodular lesions of different sizes frommillet seed to 'pea' were seen as projections on theperitoneal surface throughout the length of theintestine. The intestinal mucosa appeared oedematousand pale with catarrhal exudates. Necrotic foci andhaemorrhagic spots were observed at the sites ofparasitic attachments. The cestodes were identified asR. echinobothrida as per the morphology described bySoulsby (1982).Histopathologically, the mucosa showed atrophy andloss of villi. The attachment of parasite to the villicaused disruption of the secretory glands,proliferation and degenerative changes leading tonecrosis, and desquamation of the epithelial cells. The

Pathology of nodular tapeworm in backyard poultry55PgCtSadjacent intestinal secretory glands from the site ofgranuloma. The size and location of these granulomasshowed variations. Individual granulomas occupyingthe mucosa, submucosa and muscularis wereobserved. However, a single granuloma including allthese layers was also not uncommon. The granulomasin the duplicate stained sections revealed no otherorganisms. Interestingly, very few granulomasrevealed a narrow sinus (Fig.1) communicating withthe intestinal lumen. The available literature on thepathology of the disease showed no reports on theoccurrence of such sinuses in the granuloma. Theother pathological lesions were concurrent with theearlier reports (Nath and Pande, 1963; Bhoumik andSinha, 1983; Samad et al., 1986).ACKNOWLEDGEMENTSAuthors are thankful to the Associate Dean, College ofVeterinary and Animal Sciences, Pookot, for thefacilities provided for the study.GGREFERENCESBhoumik MK and Sinha PK. 1983. Studies on the pathology oftaeniasis in domestic fowl. Ind Vet J 60:6-8.Fig. 1. Granuloma communicating with intestinal lumenthrough sincus.parasitic granulomas were typical and werecharacterized by a central area of necrosis surroundedby neuterophils, eosinophils and mononuclear cellssurrounded by a thick layer of connective tissuecovering. In some areas, this connective tissueproliferation was so extensive that it replaced theNath D and Pande BP. 1963. A histological study of the lesionsin tapeworm infestations of domestic fowls. Ind J Vet Sci33:1-7.Samad MA, Aslam MM and Bari ASM. 1986. Effect ofRaillietina echinobothrida infection on blood values andintestinal tissues of domestic fowls of Bangladesh. VetParasitol 21:279-284.Soulsby ELJ. 1982. Helminths, Arthropods and Protozoa ofthdomesticated animals. (7 ed.) The English Language BookSociety and Bailliere Tindall. London. pp 448.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 56-60Short communicationJ P DIntestinal parasitic infections in children of Rajpuratown, PatialaHarpreet Kaur and SweenDepartment of Zoology, Punjabi University, Patiala.Received 23 November 2006; accepted 27 February 2007ABSTRACT. A comparative study was carried out to determine the prevalence of variousgastrointestinal parasitic infections in the children (aged up to 10 years) of Rajpura town. Faecalsamples (25 numbers) were collected from both urban (Group A) and slum (Group B) areas, andanalysed. Out of the total 50 samples analysed, 34 (68%) samples were found to harbour single ormultiple infections. The parasites detected were Entamoeba histolytica (91.17%), Ascarislumbricoides (44.11%), Hymenolepis nana (17.64%) and hookworm (17.64%). The percentageincidence of parasites was found to be higher in the children belonging to the age group of 5–10years. The percentage rate of gastrointestinal parasitic infection in children belonging to the agegroup of 6–9 months (infants) was found to be 40%. Males presented the higher prevalence for allthe parasites. Mixed infections were also reported and all of them included E. histolytica. The mostcommon mixed infection combination was that of E. histolytica and A. lumbricoides (35.29%). Thepresent study indicated that gastrointestinal parasitic infections were more frequent in slum areas(54.83%) than in urban areas (45.16%). The total percentage of symptoms like abdominal pain, lossof appetite, diarrhoea, fever and constipation was higher in cases infected with H. nana (100%) andhookworm (100%), followed by A. lumbricoides (86.66%) and E. histolytica (51.6%).Keywords: children, gastrointestinal parasitic infections, prevalenceIntestinal parasitic infections are rampant throughoutthe world, with higher prevalence rates amongst thechildren from underdeveloped and developingcountries. India, being a tropical country, withcomparatively poor sanitary conditions and lowsocioeconomic status, bears the major brunt ofintestinal parasitic infestations. A big segment ofpopulation carries one or more intestinal parasite(s).Epidemiological status has shown that protozoandiarrhoea is common among infants and youngchildren (Wolfe, 1978; Zaki et al., 1986).Amoebiasis, ascariasis, hookworm infections andCorresponding author: Dr. Harpreet Kaur, Department ofZoology, Punjabi University, Patiala-147 002, India. E-mail:harpreet_bimbra@yahoo.comtrichiuriasis are the major geohelminthic infectionsthat affect man; however, the prevalence and intensityis highest in the children of 4–15 years of age, and thusis closely associated with the age of the affectedpersons. These infections continue to be global healthproblems, particularly among the children in poorcommunities in developing countries. However, indeveloped countries, protozoans are more commonlythe cause of gastrointestinal infections as compared tohelminths (Harp, 2003). Most of the intestinalparasitic infections are faecal borne infections, andtheir transmission occurs either directly hand-to-handor indirectly through the ingestion of food or watercontaminated with human or animal faeces (Katz andTaylor, 2001). Heavy roundworm infections can leadto bowel obstructions accompanied by anaemia.

Gastrointestinal parasitic infections in children57Chronic infections in children can lead to long term salt floatation method and zinc sulphate floatationretardation of their physical and mental development. method. Detection and identification of parasites wasdone with the help of techniques and guidelinesGastrointestinal parasitic infections are important indescribed by Zeibig Elizabeth (1997) and Chatterjeeour country and particularly in Punjab, where most of(1980).the people are unaware of these infections.Defaecation in field/in open, lack of attention to Out of 50 stool samples studied, 34 samples werepersonal hygiene and lack of knowledge, all favour the found to be positive for one or the other parasite. Thespread of these infections. No survey work has been total percentage infection rate was found to be 68%.done in the past to ascertain the parasitic infestations The most common parasitic infections werein this area, therefore a preliminary study was Entamoeba histolytica (91.17%), Ascarisundertaken to study the prevalence of various lumbricoides (44.11%), Hymenolepis nana (17.64%)gastrointestinal parasitic infections in children up to and hookworm (17.64%). The percentage incidence of10 years of age, of Rajpura town, Punjab. This area is a different parasitic infections was found to be higher incomparatively low-lying area, gets flooded in rainy the children aged 5–10 years (88.23%). Out of a totalseasons, and has poor cleanliness and sanitary of 10 cases analysed in the children of age group 6–9conditions.months (infants), 40% of cases were found to beinfected with E. histolytica and 10% cases reported aA total of 50 stool samples were collected from themild infection of A. lumbricoides, along with E.children belonging to urban and slum areas of Rajpurahistolytica. The percentage incidence of varioustown, during the period of four months (January tomixed infections was found to be 52.92% positive inApril, 2006). Along with the samples, basicthe children of 5–10 years. The most common mixedinformation such as name, age, sex, social/economicinfection was that of E. histolytica and A.status and enquiries about any kind of gastrointestinallumbricoides, with a prevalence rate of 35.29%. Thesymptoms were made and recorded in a Performa.percentage incidence of parasitic infection was foundThese samples were brought to the laboratory andto be more in the children belonging to lowwere then analysed for the presence ofsocioeconomic status. According to gender, malestrophozoites/cysts/eggs of various gastrointestinal(65.37%) showed higher percentage infection rate ofparasites. For this purpose, two methods were usedTable I. Percentage incidence of various gastrointestinal parasites in young children of Rajpura town in relationto age, economic status, gender and symptomsParametersTotal Percentage incidencenumber ofsamples Entamoeba Ascaris Hymenolepis Hookwormanalysed histolytica lumbricoides nanaAge 6–9 months 10 40% 10% - -9 months – 33 56.52% 26.08% - 13.04%5 years5–10 years 17 82.35% 47.05% 35.29% 17.64%Economicstatus Low status 25 54.83% 100% 100% 100%Middle status 25 45.16% - - -Gender Males 30 54.83% 73.33% 66.66% 66.66%Females 20 45.16% 26.66% 33.33% 33.33%Symptoms Symptomatic 20 51.60% 86.66% 100% 100%Asymptomatic 30 48.38% - - -

58 Harpreet and Sweenvarious gastrointestinal parasites as compared to exhibited higher rate of infection (14.98%) asfemales (34.62%). The highest prevalence of infection compared to Giardia lamblia (7.08%). During thereported in males was that of A. lumbricoides present study, E. histolytica was found in all the mixed(73.33%) followed by H. nana and hookworm each infections. The most common mixed infectionwith percentage infection rate of 66.66%. Out of the combination was with A. lumbricoides (35.29%), H.total 34 positive cases, symptoms like abdominal pain, nana (11.76%), hookworm (17.64%), A. lumbricoidesdiarrhoea, loss of appetite and constipation was and H. nana (17.64%), A. lumbricoides andrecorded in 84.56% of cases, indicating that most of hookworm (11.76%) and A. lumbricoides and H. nanathe positive cases complained of one or the other and hookworm (5.88%). Azubike and Enekwechisymptom(s).(1994) in Nigeria also recorded mixed infections, andIn the present study, out of 50 stool samples, 34 stoolall of them included E. histolytica.samples of young children tested positive for various Children (44.11%) were found to be infected with A.gastrointestinal parasites viz. E. histolytica, A. lumbricoides. Pandey and Mishra (2000) in Bihar andlumbricoides, H. nana and hookworm. The total Mahanta et al. (1996) in Dispur reported A.percentage infection rate was found to be 68%. Ganga lumbricoides as the most common species withand Ravichandran (1995) in Tamil Nadu reported high prevalence rate of 80.0% and 28.6%, respectively.parasitic infection rate i.e. 73.39% in young children. However, Saha et al. (1995) in Dhanbad (Bihar)Saifi and Wajihullah (2001) in Budaun (Lucknow, recorded lowest percentage of A. lumbricoides duringU.P.) revealed that 38.41% of school children their study. Also, Abual-Saud et al. (1995) in Riyadhharboured one or more intestinal parasites. (Saudi Arabia) reported percentage incidence of A.lumbricoides to be as low as 0.30%. In the presentDuring the present study, the total infection rate of E.study, the percentage infection rate of A. lumbricoideshistolytica was found to be very high i.e. 91.17%.was found to be 100% in the children belonging to lowHowever, Bhattacharya and Laha (1996) insocioeconomic status. Also, Arya et al. (1998) foundMukteswar (U.P.) and Saifi and Wajihullah (2001) inA. lumbricoides as the most common parasite amongBudaun reported E. histolytica infection rate inthe children belonging to slum areas of Prabhanichildren to be 14.40% and 14.98%, respectively.(Maharashtra) with a percentage incidence of 90.66%.It was found that in the age group of 6–9 months, 40%of cases were positive for E. histolytica. E. histolyticainfections were found to be more prevalent in childrenbelonging to low socioeconomic status (54.83%).Ganga and Ravichandran (1995) in Kumbabornam(Tamil Nadu) reported E. histolytica as one of the mostcommon parasite with a percentage infection rate of20.7%. Also, Saifi and Wajihullah (2001) in UjhainiTown (Budaun, U.P.) found that E. histolyticaThe percentage incidence of A. lumbricoides infectionwas found to be 26.08% in the age group of 9months–5 years and 47.05% positive in the age groupof 5–10 years, whereas infants (6–9 months) reporteda very low rate of infection i.e. 10%. However, Saha etal. (1995) in Bengal observed lower rate of infection inchildren aged > 4 years of age as compared to olderchildren.Table II. Percentage incidence of mixed parasitic infections in children of Rajpura town belonging to different agegroupsMixed infections Total percentage Age group Age groupincidence 0–5 years 5–10 yearsE. histolytica + A. lumbricoides 35.29% 29.11% 5.88%E. histolytica + H. nana 11.76% - 11.76%A. lumbricoides + hookworm 17.64% 17.64% -E. histolytica + A. lumbricoides + hookworm 11.76% - 5.88%E. histolytica + A. lumbricoides + H. nana 17.64% - 11.76%E. histolytica + A. lumbricoides + H. nana + hookworm 5.68% - 17.64%

Gastrointestinal parasitic infections in children59The present study indicates the total percentage REFERENCESincidence of H. nana to be 17.64%. Khan et al. (1993)Abual-Saud AS, El-Zaher F, Oni GA and Osoba AO. 1995.in Pakistan found H. nana as the most common Intestinal parasitic infestation in Saudis and non-Saudis inparasite with a percentage prevalence rate of 6.79%. the Armed Forces Hospital, Riyadh. Saudi Med J 16:242-Also, Mirdha and Samantray (2002) revealed higher 247.prevalence of H. nana (9.9%) among the slumArya A, Pansambal PR and Devi R. 1998. Prevalence of wormdwellers of Delhi, as compared to other parasitic infestation and its relationship with anthropometricinfections. However, Saifi and Wajihullah (2001) in measurements of slum preschool children. J MaharashtraBudaun (U.P.) recorded percentage incidence of H. Agri Uni 23:50-52.nana to be as low as 2.72%.Azubike CN and Enekwechi LC. 1994. Survey of theThe percentage incidence of hookworm in the age prevalence of intestinal parasites in children ofprimary school age. West Afri J Med 13:227-230.group 5–10 years was found to be 17.64%. Mahanta etal. (1996) in Dispur, Assam, reported 6.5% of Bhattacharya D and Laha RG. 1996. A study on humanhookworm infections. Sharma (1991) in Bihar gastrointestinal parasitic infections in Kumaon Hills. Jobserved that hookworm infection was more prevalent Parasitol and App Animal Biol 5:29-39.in adults (12%) than in children (6%).Chatterjee KD. 1980. Parasitology in Relation to ClinicalMedicine. 12th Ed. Chatterjee Medical Publishers,The overall percentage incidence of parasitic infection Calcutta, India. pp 210-211.was higher in the age group of 5–10 years. Khan et al.(1993) in Pakistan reported that highest incidence of Dhanachand C and Anand L. 1998. Intestinal parasiticinfection was observed in children aged between 4–9infestation in children. J Parasitol and App Animal Biol7:87-91.years. However, Saha et al. (1995) in Bengal reporteda lower rate of infection in children aged less than 1 Ganga N and Ravichandran R. 1995. Intestinal parasites inyear of age (24.4%) as compared to older children children from middle income families. Ind Pediatr 32:828.(66.4%), whereas Dhanachand and Anand (1998) in Harp JA. 2003. Parasitic infections of the gastrointestinal tract.Manipur and Mirdha and Samantray (2002) in Delhi Curr opinion Gasteroenterolo19:31-36.revealed that the parasitic infections were independentof age.Katz DE and Taylor DN. 2001. Parasitic infections of thegastrointestinal tract. Gasteroenterol Clin N AmericaHigher incidence of parasitic infections was reported 30:797-815.among children belonging to low socioeconomic Khan SA, Azra, Shaikh A, Rizwana and Khan M. 1993.status. Saifi and Wajihullah (2001) in Budaun (U.P.) Incidence of intestinal parasites in residents of Hyderabadalso observed higher percentage incidence of parasitic and Latifabad, Pakistan. Proc Parasitol No. 15/16:1-4.infections among the children belonging to lowMahanta J, Narain K and Srivastava VR. 1996. Intestinalsocioeconomic status. Marcos et al. (2003) in Chile parasitic infestation in a rural population of Upper Assam. Jconcluded that intestinal parasitoses was more Parasit Dis 20:57-58.frequent in rural than in urban populations and wasMarcos L, Maco V, Terashima A, Samalvides F, Miranda Eassociated with poverty, poor environmental sanitaryand Gotuzzo E. 2003. Intestinal parasitoses in rural andconditions and lack of hygiene. However, Saha et al. urban populations in Sandia, Peru. Parasitologica(1995) in Bihar found that there was no major Latinoamericana 58:35-40.difference in parasite prevalence between rural andurban areas.Mirdha BR and Samantray JC. 2002. Hymenolepis nana: acommon cause of pediatric diarrhoea in urban slum dwellersPrado et al. (2001) in Brazil observed that males in India. J Trop Pediatr 48:331-334.presented highest prevalence for all species. But Pandey BN and Mishra SK. 2000. Prevalence of intestinalDhanachand et al. (1998) in Manipur found that parasitic infestations among the tribal populations of Purniafemales (89.1%) were more infected than males District. J Ecobiol 12:33-36.(42.9%). However, Mahanta et al. (1996) observedPrado M Das, Barreto ML, Strina A, Faria JAS, Nobre AA andthat there was no major difference in the prevalence of Jesus SR. 2001. Prevalence and intensity of infection byparasitic infections according to gender of the host.intestinal parasites in school aged children in Salvador.Revista da Sociedade Brasileira de Medicina Tropical.33:99-101.

60 Harpreet and SweenSaha DR, Gupta DN, Sengupta PG, Mondal SK, Ghosh S, Wolfe MS. 1978. Current concepts in parasitology. Giardia.Saha NC, Sikder, SN and Sircar BK. 1995. Intestinal New Eng J Med 298:319-321.parasitism: a childhood problem in rural Bengal. JCommuni Dis 27:170-174.Zaki AM, Dupont HL, Alamy ELM, Arafat R, Amin K, AwodMM, Bassiouni L, Imam L, Malla ELG, Marsafie AG,Saifi MA and Wajihullah. 2001. Intestinal parasitic infestation Mohiedin MS, Naguib T, Rakha MA, Sidaros M, Wasef N,in school children of Ujhaini, Budaun. J Parasit Dis 25:26- Wright CE and Wyatt EC. 1986. The detection of29. enteropathogens in acute diarrhoea in a family cohortpopulation in rural Egypt. Am J Trop Med and Hyg.Sharma RK.1991. Strongyloides stercoralis, Trichuris35:1013-1022.trichiura and Enterobius vermicularis infection in humanpopulation of Bermo coal field area in Bihar. Bioved 1 :173- Zeibig Elizabeth A. 1997. Clinical Parasitology-A Practical176. Approach. W. B. Saunders Company, Philadelphia. pp 140-151.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 61-64Short communicationJ P DPrevalence of helminth parasites in desi fowl (Gallusgallus domesticus) at ParbhaniR. R. Hange, Y. V. Raote and A. K. JayrawDepartment of Parasitology, College of Veterinary and Animal Sciences, Parbhani.Received 4 December 2006; accepted 8 March 2007ABSTRACT. The intestines of 200 desi birds (Gallus gallus domesticus), from Parbhani region,were screened for the assessment of the prevalence of helminthic infections. Overall, 63% birdswere found infected with various helminth parasites. Different species of nematodes viz. Ascaridiagalli (30.5%), Heterakis gallinarum (13%), Subulura brumpti (6%), Dispharynx nasuta (2%),Cheilospirura hamulosa (0.5%), Gongylonema ingluvicola (1%) and Tetrameres mohtedai (0.5%)were observed. The cestode parasites which infected birds included Raillietina echinobothridia(7.5%), R. tetragona (9.5%), R. cesticillus (3%), Davainea proglottina (4%), Amoebotaeniasphenoides (6.5%), Choanotaenia infundibulum (1.5%) and Hymenolepis carioca (1%). Both single(61.91%) and mixed (38.09%) parasitic infections were observed; 25% birds were infected with acombination of A. galli and H. gallinarum. In most (63%) cases, small intestine was infected,followed by caeca (19%), proventriculus (2.5%), oesophagus (1%) and gizzard (0.5%). Highestincidence of infection occurred in winter (66.67%), followed by rainy season (63.07%) and summer(58.73%).Keywords: desi birds, helminth parasites, intestine, Parbhani, prevalencePoultry farming is a very important source ofadditional income to farmers and landless labourersbut enteric pathogens inflict colossal monetary lossesin the form of high morbidity and mortality, decreasedproduction and low meat quality. Amongst these,helminth parasites assume tremendous economicimportance as they are responsible for decreasedweight gain of the host and egg production. Matta andAhluwalia (1980) reported losses to the tune of Rs. 150million through reduced weight gains caused byAscaridia galli. Matta (1981) also estimated the lossesto the tune of 301.96 million rupees through reducedegg production as a result of ascaridiosis in birds. Toreduce the monetary losses, formulation of a suitableCorresponding author: Dr. Y.V. Raote, Department ofParasitology, College of Veterinary and Animal Sciences,Udgir-413 517, India. E-mail:jayrawant1@rediffmail.comdeworming schedule and other prophylactic measuresis of immense significance. For effective formulationof deworming schedule, an epidemiological survey ofhelminth parasites in a particular region becomesmandatory. High incidence of helminth parasites inpoultry is well documented from different parts of thecountry (Bhalerao, 1935; Bhalero and Rao, 1944;Dutt, 1950; Bhatnagar and Ruprah, 1969; Pillai andPeter, 1971; Hedge et al., 1973; Gogoi, 1975; Mattaand Ahluwalia, 1981; Kulkarni et al., 2001). However,scanty literature is available from Maharashtra(Shastri et al., 1974). Hence, the present investigationwas undertaken to study the prevalence of helminthparasites in desi fowl (Gallus gallus domesticus) fromParbhani region of Maharashtra.A total of 200 intestines of desi birds, slaughtered atabattoir in and around Parbhani, were collected over aperiod of one year. The intestines were brought to the

62Hange et al.laboratory in thermos flask containing ice, stored at gallinarum (13%) second, which is in consonance4°C without any preservative, and were examined with Nadakal et al. (1972; A. galli, 31.2% and H.within 8 h of their collection. The parasites from each gallinarum, 18.7% ) and Raote et al. (1991; A. galli,organ were collected and preserved separately in glass 43.52% and H. gallinarum, 9.63%). The incidence ofvials. These vials were labelled for the number of other nematodes viz. Subulura brumpti (6%),sample, date of collection, etc. Parasites collected Dispharynx nasuta (2%), Cheilospirura hamulosafrom each organ of the bird were counted before and (0.5%), Gongylonema ingluvicola (1%) andafter identification. For identification of parasites, Tetrameres mohtedai (0.5%) recorded was very low.staining was performed and morphological features of Comparatively higher incidence of these parasites wasthe parasites were studied under a light microscope. noted by Varghese and Peter (1973) and Shastri et al.Various morphological characters described by (1974). Out of seven species of tapeworms, RaillietinaSoulsby (1982), Reid (1992) and Ruff (1992) were echinobothridia (7.5%) and R. tetragona (9.5%)used for the identification of parasites.occurred relatively more. These two species areconsidered to be the most pathogenic as R.The present investigation revealed 63% incidence ofechinobothridia produces 'nodular taeniosis' in birdshelminth parasites, which is in line with the findings of(Soulsby, 1982). The incidences of R. cesticillus (3%),Bali and Kalra (1975), Matta and Ahluwalia (1981)Davainea proglottina (4%), Amoebotaeniaand Raote et al. (1991), who have recorded 63.42%,sphenoides (6.5%), Choanotaenia infundibulum63.70% and 71.09% incidence of helminth parasites(1.5%) and Hymenolepis carioca (1%) were relativelyfrom Punjab, Uttar Pradesh and Maharashtra,low. The present study recorded low incidence ofrespectively. On the contrary, higher incidences weretapeworms, as compared to the reports of Nadakal etrecorded by Nadakal et al. (1972) from Kerala (100%)al. (1972; 97.3%) and Hedge et al. (1973; 77.1%). Theand by Hedge et al. (1973) from Karnataka (80.50%).species of tapeworms encountered in the presentThe data pertaining to the prevalence of helminthinvestigation from Maharashtra were also evidencedparasites and species recorded are presented in Table I.by Shastri et al. (1974), Raote et al. (1991) andAmongst the incidence of nematodes recorded, A.Kulkarni et al. (2001). The present studygalli (30.5%) was ranked first and HeterakisTable I. Prevalence of gastrointestinal helminths in desi fowlParasite Birds infected (%)NematodesA. galli 30.5H. gallinarum 13S. brumpti 6D. nasuta 2C. hamulosa 0.5G. inglovicola 1T. mohtedai 0.5CestodesR. echinobothridia 7.5R. tetragona 9.5R. cesticillus 3D. proglottina 4A. sphenoides 6.5C. infundibulum 1.5H. carioca 1

Prevalence of helminth parasites in desi fowl63demonstrated an overall low incidence of nematodes detected in 25% infected birds, which is in closeas well as cestodes in comparison to earlier workers proximity with the findings of Ghosh (1986; 22.56%)from different parts of the country, which could be and Kulkarni (1999; 16.57%). The 43.75% infectionattributed to the variations in the climatic conditions, caused by a combination of A. galli and tapewormsavailability of intermediate hosts or adoption of corroborate the findings of Raote et al. (1991;managemental practices. 37.41%).The number of nematodes/bird recorded were A. galli Organ-wise, the highest percentage of infection was(5.31), H. gallinarum (6.69), S. brumpti (4.91), D. recorded in intestine (63%) and caeca (19%);nasuta (11), C. hamulosa (2), G. ingluvicola (5.5) and however, lower percentage was documented fromT. mohtedai (12). The number of trematodes/bird was oesophagus (1%), proventriculus (2.5%) and gizzardR. echinobothridia (5.86), R. tetragona (5.89), R. (0.5%). Identically, higher percentage of infection incesticillus (6.33), D. proglottina (4.37), A. sphenoides intestine was reported by Nadakal et al. (1972; 48.9%)(212.3), C. infundibulum (3.66) and H. carioca (5.5). and Kulkarni (1999; 53.20%).Similar findings were documented by Kulkarni (1999)from the Marathwada region of Maharashtra, perSeason-wise analysis of the data revealed highestcontra to the findings of Bhatnagar and Ruprahincidence during winter (66.67%), followed by rainy(1969), who encountered more number ofseason (63.07%) and summer (58.73%). Similarly,parasites/bird at Hisar, Haryana. This is attributable tohigh incidence during winter and autumn and lowdifferent climatic conditions of Haryana andduring spring and summer has been reported byMaharashtra.Bhatnagar and Ruprah (1969). Matta and Ahluwalia(1981) have documented highest incidence duringSingle infection caused by one species of helminth autumn (87.80%) and lowest during summer (53%).parasite was recorded in 61.91% birds, whereas mixed Raote et al. (1991) have stated highest incidenceinfection was noted in 38.09% birds (Table II). Mixed during winter (93.65%), followed by rainy seasoninfection caused by two or more than two species of (75.29%) and lowest during summer (39.34%).helminth parasites was reported by Raote et al. (1991;50.97%), Mpoame and Agbede (1995; 93.50%) andACKNOWLEDGEMENTSKulkarni (1999; 22.85%). Mixed infection caused by a The authors are thankful to the Associate Dean,combination of A. galli and H. gallinarum was College of Veterinary and Animal Sciences, Parbhani,Table II. Occurrence of mixed helminthic infection in desi fowlCommon parasite combinations Birds infected (%)A. galli + H. gallinarum 25A. galli + S. brumpti 12.5A. galli + D. nasuta 6.25A. galli + C. hamulosa 2.08A. galli + G. ingluvicola 4.16A. galli + R. echinobothridia 8.33A. galli + R. tetragona 12.5A. galli + R. cesticillus 4.16A. galli + D. proglottina 10.42A. galli + A. sphenoides 8.33A. galli + H. carioca + T. mohtedai 2.08A. galli + D. nasuta + S. brumpti 2.08A. galli + R. tetragona + S. brumpti 2.08

64Hange et al.for providing necessary facilities for the successfulcompletion of this work.REFERENCESBali HS and Kalra IS. 1975. Studies on the incidence ofhelminths in domestic and wild birds in Punjab state. J ResPAU 12:313-316.Bhalerao GD. 1935. Helminth Parasites of DomesticatedAnimals in India. ICAR Monogram. 6:365.Bhalerao GD and Rao NSK. 1944. Some helminth parasites ofpoultry. Proc Ind Acad Sci 20:30-39.Bhatnagar PK and Ruprah NS. 1969. Incidence of helminths infowl at Hisar. J Res PAU 6:411-414.Dutt SC. 1950. Effects of light infections of fowl tapewormRaillietina cesticillus (Molin) on young chickens. Proc IndSci Cong pp 243.Ghosh SS. 1986. A short note on incidence of helminths inpoultry in Nagaland. Ind J Anim Hlth 25:83-84.Gogoi AR. 1975. Occurrence of nematodes and trematodes inlocal fowls in Assam. Kerala J Vet Sci 5:131-134.Matta SC and Ahluwalia SS. 1980. Studies on effect ofAscaridia galli infection on growth rates of chicks. Ind JPoult Sci 15:1-4.Matta SC and Ahluwalia SS. 1981. Note on the survey ofgastrointestinal helminths of domestic fowls in UttarPradesh. Ind J Anim Sci 51:1013-1015.Mpoame M and Agbede G. 1995. The gastrointestinal helminthinfections of domestic fowls in Dschang, WesternCameroon. Revue d' Elevfage et de medecine Veterinairedes pays Tropicaux 48:147-151.Nadakal AM, John KO and Mohandas A. 1972. A quantitativeanalysis of gastrointestinal helminths of domestic fowls inSouthern Kerala. Ind J Anim Hlth 11:5-9.Pillai K and Peter CI. 1971. Studies on tapeworms commonlyencountered in fowls. Ind Vet J 48:430-431.Raote YV, Joshi MV, Bhandarkar AG and Bhagwat SS. 1991.Incidence of helminth parasites in desi fowls. Ind J Poult Sci26:188.Reid WM. 1992. Cestodes. In: Diseases of Poultry. HofstudthMS and others (Eds.), 8 Edition, Prima Educational BookAgency, New Delhi. pp 649-666.Hedge KS, Abdul Rahaman S, Rajasekariah GR, Ananth M andRuff MD. 1992. Nematodes and Acanthocephalans. In:Bhagiratha J. 1973. Comparative studies on the incidence ofthDiseases of Poultry. Hofstud MS and others (Eds.), 8intestinal helminths in desi birds reared on free range systemEdition, Prima Educational Book Agency, New Delhi. ppand farm birds under hygienic conditions. Mysore J Agri Sci614-648.7:102-105.Shastri UV, Kamath GR and Ghafoor MA. 1974. Occurrence ofKulkarni GM. 1999. Incidence of parasites of digestive tract ininternal parasites of desi fowl in Marathwada region ofpoultry birds. M. V. Sc. Thesis. Marathwada AgriculturalMaharashtra state. Ind Vet J 51:233.University, Parbhani.Soulsby EJL. 1982. Helminths, Arthropods and Protozoa ofKulkarni GM, Narladkar BW and Deshpande PD. 2001.thDomesticated Animals. 7 Edition, ELBS and BailliereHelminthic infections in desi fowl (Gallus gallusTindall, London. pp 102-103.domesticus) in Marathwada region. J Vet Parasitol 15: 137-139. Varghese CG and Peter CT. 1973. Studies on the incidence ofnematodes of domestic fowls in Kerala state. Ind Vet JMatta SC. 1981. Effect of Ascaridia galli infection on the egg50:1219-1220.production of poultry. Ind J Poult Sci 16:283-284.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 65-67Short communicationOccurrence of Trichomonas gallinae in pigeons*J P DH. R. Parsani, R. R. Momin, N. M. Shah and B. S. ChandelDepartment of Parasitology, S. D. Agricultural University, Sardarkrushinagar.Received 2 November 2006; revised 12 March 2007; accepted 13 April 2007ABSTRACT. Trichomonas gallinae is a pathogenic protozoan often affecting themouth and crop of domestic pigeons. Fifty pigeons were examined for infection bycollection of oesophageal swabs and crop contents under phase-contrast microscope.Out of 50 oesophageal swabs, 35 (70%) were found positive for trichomonads by wetand stained smear examination. On the other hand, from 50 crop content samples, 24(48%) and 19 (38%) were found to be positive by wet and stained smear examination,respectively.Keywords: osmic acid vapour, pigeon, Trichomonas gallinaePigeons act as reservoir host or carrier and animportant source of infection for other avian host,which share the common parasitic fauna (Kumar,1998). There are quite a large number of problemsrelated to management of parasitic infestation in birds.Clinical or sub-clinical parasitoses leads to anaemia,as a result of continuous sucking of blood by parasites.Clinically, birds show partial or complete anorexia,diarrhoea, dehydration, loss of weight and alterationin body microelements, which results inimmunosuppression in birds. Trichomonas gallinae ispathogenic protozoan often affecting the mouth andcrop of domestic pigeons, wild columbids and raptors.Morbidity and mortality in pigeon squabs may be high(Soulsby, 1982) due to this infection. The incidence ofthis infection has been studied in Australia (Grimesand Elder 1971; Mckeon et al., 1997), Greece(Githkopoulos and Liakos 1987), Spain (Kaminjolo et*Part of M.V.Sc. thesis submitted to S. D. AgriculturalUniversity, Sardarkrushinagar.Corresponding author: Dr. H. R. Parsani, Department ofParasitology, College of Veterinary Science and AnimalHusbandry, S. D. Agricultural University, Sardarkrushinagar-385 506, India.al., 1988, Martinez - Moreno et al., 1989) andYugoslavia (Kullisic et al., 1996). In India, theinfection was reported by Bhattacharya et al. (1997).The present study elucidated the method for collectionof samples, techniques for identification andoccurrence of T. gallinae in columbid birds.Collection of samples for trichomonad: Fiftypigeons (20 from zoo and 30 from wild) wereexamined for infection.Collection of oesophageal swab samples: Mouths ofpigeons were examined for the presence ofdiphtheritic lesions, if any. Two throat swabs werecollected form each pigeon. The swabs were keptimmediately in tubes containing sterile normal salineand examined immediately.Collection of crop content: Crop contents wereaseptically collected from pigeons by flushing thecrop with 1.5 ml of sterile saline using a blunt ended18G rubber tube. Tubes and syringes were flushedwith 70% ethanol after sampling each pigeon.Suspended crop contents were transferred in to asterile 5 ml tube, till examination.

66 Parsani et al.Techniques for identification of trichomonadWet smear examination: Oesophageal swabs andcrop fluid samples were examined as wet preparationwithin 2 h of sampling. A drop of swab sample or cropfluid was placed on a clean glass slide covered withcover-slip and examined for motile Trichomonasspecies at 200x and 400x magnifications, under aphase-contrast microscope.flagellum attached to an undulating membrane. Aprominent axostyle was also present at the posteriorend of the organism (Fig. 2). Fixing of smears withosmic acid vapour before staining with Field's orGiemsa stain as described by Mandel et al. (1963) wasfound to be essential for the detection of the flagella.Staining method: A smear on clean glass slide wasprepared from oesophageal swab and crop fluidsamples. It was dried and fixed with osmic acid vapourand stained with Giemsa (Mandel el al., 1963). Theexamination was done under 400x and 1000magnification for trichomonad.Out of 50-esophageal swabs, 35 (70%) were positivefor trichomonads by wet and stained smearexamination. Out of 50 crop contents, 24 (48%) and 19(38%) were positive by wet and stained smearFig. 2. Staining of flagella after fixing with osmic acid vapour.examination, respectively. Direct wet-mountexamination of crop contents and oesophageal swabsThe clinical signs of dullness and diphtheritic lesion inrevealed 48% and 70% infection, respectively. Motilemouth have been reported by several authors (Grimespyriform trophozoites were readily detected in wetandElder, l971; Panigrahi et al., 1982). In the presentmount preparations under phase-contrast microscopystudy, diphtheritic lesions were detected in 70% offor up to 6 h at room temperature (Fig. 1). The presentpigeons, which showed the presence of T. gallinae inobservations are in close agreement with McKeon etoesophageal swabs; however the pigeons did not showal. (1997) who have reported 46% prevalence of T.any clinical signs. The high prevalence of T. gallinaegullinae in crop content in wild Senegal dove and 71%infection in adult pigeons was not surprising as thisin racing pigeons in Perth, Australia; Martinezspeciesof columbiformes is considered the naturalMoreno et al. (1989) have reported 79.2% prevalencehost of this organism (Stabler, l954). In nature, thefrom Spain. Stained smear of crop content anddisease is transmitted from the adult to the squabs inoesophageal swab have shown 38% and 70%the pigeon milk, which is produced in the crop andprevalence, respectively, for T. gallinae. Thesquabs are infected in minutes after hatching.organisms were pyriform in shape and measured 619 x29 µm, which have four 813 µm long anterior flagella ACKNOWLEDGEMENTSarising from a blepheroplast and one posteriorThe authors are highly grateful to Dr. D. K. Pethkar,former Professor of Parasitology, College ofVeterinary Science and Animal Husbandry, S. D.Agricultural University, Sardarkrushinagar, forencouragement.REFERENCESBhattacharya HM, Laha R and Bhattacharya D.1997.Trichomoniasis in pigeons. Ind Vet J 74:341.Githkopoulos PR and Liakos VD. 1987. Parasites of thealimentary tract of pigeons in Greece. Deltiontes EllenikesKteniatrikes Etaireias 38:79-83.Fig.1. Motile pyriform trophozoites in wet mount preparationunder phase-contrast microscope.Grimes TA and Elder JK. 1971. The isolation of trichomonadsfrom pigeons. Aust Vet J 47:160-161.

Occurrence of Trichomonas gallinae in pigeons67Kaminjolo IS, Tika Singh ES and Ferdianand GAA.1988. Mckeon T, Dunsmore J and Radial SR.1997. TrichomonasParasites of common pigeon (Columba livia) from the gallinae in budgerigars and columbid birds in Perth,environs of Port of Spain, Trinidad. Bull Ani Health Prod Western Australia. Aust Vet J 75:652-655.Africa 36:194-195.Panigrahi B, Grimes JE, Glass SE, Nagi SA and Hall CF. 1982.Kulisic Z, Pavlovic I and Milutinovic M.1996. Contribution to Disease of pigeon and doves in Texas Clinical findings andknowledge of the parasite fauna of pigeons in the Belgrade recommendations for control. JAVMA.181: 384-386.area in the period 1990-1994. Veterinarski Glasnik 50:785-790.Stabler RM.1954. Trichomonas gallinae,A review. Exp Parasit3:368-402.Mandel LN, Prasad H and Prasad AK.1963. Observations onthe staining techniques of Trichomonas foetus. Ind Vet JSoulsby ELJ. 1982. Helminths, Arthropods and Protozoa ofth40:263-265.Domesticated Animals. 7 Ed. Elbs and Bailliere Tindall,London.Martinez-Moreno FJ, Martinez-Moreno A, Becerra-Martell Cand Martinez-Cruz MS. 1989. Parasite fauna of pigeons inCordoba province; Spain. Revista Iberica de Parasitologia49:279-281.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 68-71Short communicationAnaemia in experimental caprine schistosomosisJ P D1 2Samidha Gupta and M. C. Agrawal1Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur.2Department of Parasitology, College of Veterinary Science and Animal Husbandry, Jabalpur.Received 23 November 2006; revised 11 April 2007; re-revised 8 June 2007; accepted 24 June 2007ABSTRACT. Haemoglobin (Hb) concentration was estimated at 15 day intervals in ten Barberigoats aged 23 months, of which six goats were infected each with 2000 cercariae of Schistosomaincognitum and 2000 cercariae of S. spindale, one goat was infected with 3000 cercariae of S.incognitum and 1000 cercariae of S. spindale, and the remaining three goats served as non-infectedcontrols. The infected goats had 5.8–12.8 g/dl Hb, whereas the controls had 11–13.8 g/dl Hb duringthe experimental period i.e. 315 days post-infection. The three animals which died due toschistosomosis (124, 135 and 233 days post-infection) showed Hb concentration as low as 5.89 g/dl,the values which were never observed in the non-infected goats. There was a decrease of17.02–48.2% Hb in infected animals as compared to the controls. The analysis of variance showed asignificant variation (p < 0.05) in Hb levels between infected and non-infected goats.Keywords: anaemia, Barberi goats, experiment, haemoglobin, schistosomosisTill recently, the pathological studies in H, Jabalpur. Three goats were kept as non-infectedschistosomosis have been done by conventional controls, whereas six goats were infected each withmethods of following post-mortem and 2000 cercariae of Schistosoma incognitum and 2000histopathology. Though such studies were important, cercariae of S. spindale, and one goat was infectedyet these were of limited use in treating the animals or with 3000 cercariae of S. incognitum and 1000 of S.forecasting prognosis of the case. Therefore, in recent spindale, with details given elsewhere (Gupta et al.,years, attention has been drawn towards biochemical 2006). Clinical observations were made on these goatsparameters (Das et al., 1988; Rajkhowa et al., 1997; (Gupta et al., 2006) associated with studies onAgrawal and Singh, 2000 a and b), which not only excretion of schistosome eggs/miracidia in the faecesreflect pathogenesis of schistosomosis but also help in (Gupta and Agrawal, 2005). For Hb estimation, 2 mlits treatment and prognosis. Studies on blood blood was drawn from jugular or ear vein in thehaemoglobin (Hb) assume greater significance as they morning at 15 days intervals, starting from the pre-directly affect animal production (Srivastava et al., infective day (0 day) to 315 day post-infection and1964; Ahluwalia and Dutt 1972, Rajkhowa et al., processed by routine procedure for Hb estimation1992; Jain et al., 2000) and severe anaemia may prove using Sahlis haemoglobinometer.fatal to the infected animals.All infected goats developed clinical schistosomosisTen Barberi goats, 23 months old, were procured from characterized by diarrhoea or dysentery, anorexia andLivestock farm, College of Veterinary Science and A loss of condition starting from eight to nine weekspost-infection. The marked fall in Hb value was firstCorresponding author: Dr. M. C. Agrawal, College of observed on 75 days post-infection, when two goatsVeterinary Science and Animal Husbandry, Jabalpur-482 001,viz. GI 4 and GI 8 revealed 8.5 g/dl and 9 g/dl Hb, inIndia. E-mail:mcagrawal@yahoo.comcontrast to 12.8–13.20 g/dl Hb in non-infected goats

Anaemia in experimental caprine schistosomosis69(Table 1). These and other infected goats continued to disease nor their faeces turned positive for fluke eggsshow lower Hb values up to 135 days post-infection or miracidia, whereas the other animals, mainlyand even more (Fig. 1) . Interestingly, three infected domestic ones, exhibited symptoms such as anorexia,goats (GI 2, GI 4 and GI 7) died on 124, 135 and 233 loss of body weight or diarrhoea, and anaemia wasdays post-infection; the Hb value of these animals observed as an associated symptom. Thus Srivastva etwere recorded to be 5.8 g/dl, 9 g/dl and 7.6 g/dl, al. (1964) demonstrated 27% fall in Hb in sheeprespectively, prior to their death. The infected goats infected with 7000–15000 S. indicum cercariae,which survived acuteness of the infection retained Hb whereas Ahluwalia and Dutt (1972) and Jain et al.concentration of 9 g/dl and above, except in GI 5 (2000 ) recorded 8.2 g/dl and 10.4 g/dl in pigs infectedwhich experienced 8.4 and 8.8 g/dl Hb on two with 20,000 or 1500 S. incognitum cercariae,occasions (Table 1). The Hb values below 8 g/dl respectively. Rajkhowa et al. (1992) recorded as lowproved crucial and led to the death of the animals. At as 4.83 g/dl Hb in bull calves having 112 day oldthe end of the experiment (315 days post-infection), experimental S. indicum infection, as against 10 g/dlHb values of infected goats ranged between 5.8–12.8 on 0 day of the infection. Saad et al. (1984) andg/dl and those of non-infected goats ranged between Kassuku et al. (1986) also recorded anaemia in the11–13.8 g/dl ( Table I, Fig. 1), which yielded a goats infected with S. bovis though no such work hasdecrease of 17.02–48.2% Hb concentration in been reported on goats from India.infected goats. The analysis of variance alsoconfirmed significant variation in Hb concentrationAnaemia also co-related with the excretion of flukebetween infected and non-infected goats (p < 0.05).eggs in the faeces, which was responsible forintestinal haemorrhage and enteritis. Sucking of theThe Hb values in schistosome infections varied blood by the blood flukes also contributes towardsdepending on host compatibility, dose of the cercariae decrease in Hb concentration but this alone might beand onset of the clinical form of the infection. This compensated in animals not suffering from clinicalmight be the reason as to why Das et al. (1988) failed infection. Therefore, development of anaemia, into record any appreciable decrease in Hb values in present studies, between 75–225 days post-infectionRhesus monkeys infected with S. incognitum, as when egg excretion is noticeable, further supports theneither monkeys showed any clinical features of the previous view that fall in Hb is associated with onset1614Infected groupControl group12Haemoglobin level10864200 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315Days (post-infection)Fig. 1. Mean haemoglobin concentration in experimentally infected and non-infected goats.

70 Ravindran and WilliamsTable I. Change in haemoglobin concentration in experimentally infected goatsInfected group Control groupDP1 GI-2 GI-3 GI-4 GI-5 GI-6 GI-7 GI-8 Avg. Range GC-1 GC-2 GC-3 Avg. Range0 11.10 11.20 11.20 11.00 12.00 11.10 12.10 11.38 11.00-12.10 12.80 11.80 11.60 12.06 11.60-12.8015 11.20 11.00 11.20 10.40 11.40 10.40 11.20 10.97 10.40-11.40 11.00 11.40 11.40 11.27 11.00-11.4030 11.40 11.20 11.00 11.20 10.80 11.20 11.20 11.14 10.80-11.40 11.20 11.40 11.00 11.20 11.00-11.4045 11.00 11.60 11.80 10.80 11.20 10.20 11.20 11.11 10.20-11.80 12.00 11.40 11.00 11.47 11.00-12.0060 11.10 11.60 10.15 10.90 11.60 10.20 11.10 10.95 10.15-11.60 11.20 11.30 11.20 11.23 11.20-11.3075 11.20 11.60 8.50 11.00 12.00 10.20 9.00 10.50 8.50-12.00 12.80 13.20 13.00 13.00 12.80-13.2090 11.60 9.00 9.00 10.00 11.20 9.00 9.30 9.87 9.00-11.60 12.20 12.50 12.40 12.37 12.20-12.50105 9.60 9.00 9.00 10.40 11.90 9.40 9.60 9.84 9.00-11.90 12.00 12.80 12.60 12.47 12.00-12.80120 9.60 9.00 5.80 10.20 10.40 9.20 9.40 9.09 5.80-10.40 11.80 12.80 12.70 12.43 11.80-12.80135 9.80 10.00 Died 9.00 9.20 9.00 9.20 9.37 9.00-10.00 12.20 12.00 12.40 12.20 12.00-12.40150 7.70 10.80 10.00 10.00 Died 12.20 10.14 7.70-12.20 11.20 11.80 11.20 11.40 11.20-11.80165 8.40 10.60 10.00 10.20 12.00 10.24 8.40-12.00 11.80 11.80 12.00 11.87 11.80-12.00180 7.20 10.20 9.80 11.80 11.60 10.12 7.20-11.80 12.00 13.00 12.20 12.40 12.00-13.00195 8.00 10.80 9.80 11.20 10.80 10.12 8.00-11.20 12.20 13.00 12.80 12.67 12.20-13.00210 8.40 10.00 8.80 11.20 11.00 9.88 8.40-11.20 12.00 13.00 12.60 12.53 12.00-13.00225 7.60 10.00 9.00 11.00 10.60 9.64 7.60-11.00 11.80 13.00 12.80 12.53 11.80-13.00240 Died 11.20 8.40 11.80 10.60 10.50 8.40-11.80 11.60 12.60 Died 12.10 11.60-12.60255 11.50 10.40 12.00 12.50 11.60 10.40-12.50 11.80 12.00 11.90 11.80-12.00270 11.50 10.40 12.00 12.50 11.60 10.40-12.50 11.00 12.00 11.50 11.00-12.00285 11.80 9.50 12.40 12.20 11.48 9.50-12.40 11.60 13.80 12.70 11.60-13.80300 12.00 9.80 13.00 12.00 11.70 9.80-13.00 12.40 13.20 12.80 12.40-13.20315 12.60 9.40 12.20 12.80 11.75 9.40-12.80 13.00 13.80 13.40 13.00-13.80Avg. 9.70 10.82 9.73 10.00 11.40 9.90 11.10 5.80–12.80 11.90 12.43 12.05 11.00–13.80-(35.1) -(8.92) -(40.02) -(10.9) -(1.66) -(18.9) -(3.38) – – -(1.53) +(14.4) +(9.52)Note: Parenthesis indicates variation of haemoglobin on percentage basis.Data are gm/dl, fortnightly.

Anaemia in experimental caprine schistosomosis71of egg production (Humphries et al., 1994). It may benoted that marked anaemia was limited only up to 225days post-infection, though goats continued toharbour infection for much longer period. Thisobservation is in contrast to that of previous workers(Srivastava et al., 1964; Ahluwalia and Dutt, 1972).The reason for recovery at later stage, in the presentstudy, might be due to erratic egg excretion with muchlower number of flukes afterwards. The death ofinfected goats was always associated with severeanaemia and dysentery, thereby providing clue for thetreatment of animals with supportive medicines (fordehydration, haemoglobin and glucose loss etc.),besides specific treatment.Gupta S and Agrawal MC. 2005 Excretion of eggs andmiracidia of schistosomes in experimentally infected goats.J Parasit Dis 29:23-28.Gupta S, Agrawal MC and Vohra S. 2006. Clinical observationsin experimental caprine schistosomosis. J Vet Parasitol20:29-31.Humphries D, Vella AT and Pearce EJ. 1994 Increased CD4 + Tcell-dependent anti-erythrocyte antibody levels followingthe onset of parasite egg production in Schistosomamansoni infected mice. Parasit Immunol 16:469-477.Jain J, Agrawal MC and Rao KNP. 2000. Effect of praziquanteland closantel treatment on haematology and faecal eggcount in experimental porcine schistosomosis. Ind J Ani Sci70:23-825.ACKNOWLEDGEMENTSKassuku A, Christensen NO, Nansen P and Monrad J. 1986.Clinical pathology of Schistosoma bovis infection in goats.The authors are thankful to the Dean, College ofVet Parasitol 62:83-91.Veterinary Science and Animal Husbandry, Jabalpur, Rajkhowa C, Gogoi AR, Borkakoty R and Sarma P. 1992. Somefor providing necessary facilities.observations on haematological changes in experimentalSchistosoma indicum infection in cattle. Ind J Ani SciREFERENCES 62:125-126.Agrawal MC and Singh KP. 2000a. Effect of Schistosoma Rajkhowa C, Gogoi AR, Borkakoty MR and Sarma P. 1997.incognitum infection on serum biochemistry of rabbits. J Some biochemical changes in the blood of bull calvesVet Parasitol 14:31-34.experimentally infected with Schistosoma indicum. J VetAgrawal MC and Singh KP. 2000b. Trials of oxamaniquine onParasitol 11:169-173.Schistosoma incognitum infected rabbits. J Vet Parasitol Saad AM, Hussein MF, Dargie JD and Taylor MG. 1984.The14:141-143. pathogenesis of experimental Schistosoma bovis inAhluwalia SS and Dutt SC. 1972. Clinical study onSudanese sheep and goats. J Comp Pathol 94:371-385.Schistosoma incognitum in domestic pigs: Srivastava HD, Muralidharam SRG and Dutt SC. 1964.symptomatology, haematology and effect on growth rate. Pathogenicity of experimental infection of SchistosomaInd Vet J 49:863-867.indicum Montgomery (1906) of young sheep. Ind J Vet SciDas M, Kushwah A, Agrawal MC, Joshi SC and Shah HL. 1988.34:35-40.Biochemical determination of pathogenesis of Schistosomaincognitum infection in Macaca mulatta. J Vet Parasitol2:133-135.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 72-73Short communicationJ P DOccurrence of Liponyssus sp. (Feather mite) in IndianturkeysHarkirat Singh, A. K. Mishra, S. Ghosh and J. R. RaoDivision of Parasitology, Indian Veterinary Research Institute, Izatnagar.Received 4 January 2007; revised 8 June 2007; accepted 30 June 2007ABSTRACT. An attempt was made to know the occurrence of mite infestations in turkeys atCentral Avian Research Institute, Izatnagar. Pooled faecal samples from 102 turkeys weresubjected to floatation technique, and a mite of the Liponyssus species was found along with eggs.Keywords: India, Liponyssus sp., turkeysLiponyssus bursa (Hirst, 1916), the tropical fowl mite,is a non-burrowing mite of poultry, and is also knownas Ornithonyssus bursa (Sambon, 1928) andLeiognathus bursa (Berlese, 1888). L. bursa is knownto infest a variety of poultry birds including chickens,turkeys, ducks, pigeons, sparrows, starlings and mynabirds. Nesting birds are the worst affected andabandon their nests. This mite feeds on blood andtissue fluids of birds at night and hides in nearbycracks and crevices during day time. Sometimes, themites appear as red dots on skin of birds when they arefeeding, and may also be visible in the environment. Itfeeds on mammals, if birds are not available.Infestation with L. bursa (O. bursa), along with O.sylviarum, results in “dirty-looking” blackenedfeathers, and may cause cracks and scabs around thecloaca and decreased productivity. It can even carrythe Western Equine Encephalitis virus. It is mainly aproblem on small rural poultry farms and in breederflocks, and has become a pest to man in areas of highbird populations or where birds are allowed to roost onroofs, around the eaves of homes, and office buildings.They are uncommon in commercial poultry layeroperations where the birds are raised in cages(Soulsby, 1982; Denmark and Cromroy, 2003). It isCorresponding author: Dr. A. K. Mishra, Division ofParasitology, Indian Veterinary Research Institute, Izatnagar-243 122, India. E-mail: drashokkumarmishra@gmail.commostly restricted to warm and tropical regions ofAfrica (Egypt, Nigeria, Malawi and Republic of SouthAfrica), Asia (China, India, Thailand), Indonesia(Java, Mauritius), Australia (New South Wales,Queensland, South Australia), Central America(Canal Zone), Islands of the Indian Ocean (ComoroIslands, Zanzibar), Islands of the Pacific (Hawaii,New Guinea), North America (Canada, United States),South America (Argentina, Colombia) and WestIndies (Bahamas Islands).In the present study, an attempt was made to check theprevalence of Liponyssus sp. in turkeys and guineafowls, reared at Central Avian Research Institute(CARI), Izatnagar. Turkeys were reared on deep littersystem in CARI, Izatnagar. Six male turkeys, 69months of age, were kept in one shed. Seventeenpooled samples of turkeys were collected, three-times,during March to June 2004. Samples were brought onice to the laboratory, and were then subjected tofloatation by centrifugation (1500 rpm, 25 min) insaturated salt solution. Micrometry was performed todetermine the size of eggs and adult mite.The mite was identified based on the descriptionprovided by Baker et al. (1956). The adult mite wasdark brown in color and 108 µm x 57 µm in size (Fig.1), and the eggs were elliptical in shape, doublewalledand 90 µm x 72 µm in diameter (Fig. 2). The

Occurrence of Liponyssus sp. (Feather mite) in Indian turkeys73Fig. 1. Adult Liponyssus sp. (400x) DP-dorsal plate; H-hair.species of mite possesses a number of characterssimilar to that of L. bursa, the distinct shape ofcapitulum (Fig. 1) of the present specimendifferentiates it from L. bursa and also from otherpoultry mites. As per the nature's law, to be successfulin different hosts as well as in different environmentalconditions, parasites have changed and are changingsome of their characters essential to survive in newhabitats. L. bursa is a well established non-burrowingmite and has been reported in poultry birds. In thepresent study, the mite specimen with a distinct type ofcapitulum was collected from turkeys reared in deeplitter system. However, the distinct capitulumcharacter is not sufficient to declare a specimen as anew species because it will add further confusion inspeciation. In the present day knowledge, the time hascome to resist from designating new species status to aspecimen before it has been thoroughly studiedgenetically. Species identification on the basis ofmorphological and bio-metrical parameters haveadded a number of confusions because there is a strongpossibility of human error in the explanation ofmorphological characters and in the analysis ofmorphometrical parameters by different authors. Toavoid confusion, we thought it is reasonable todesignate the mite specimen as Liponyssus sp.ACKNOWLEDGEMENTSThe authors are grateful to Dr. Deepak Sharma, SeniorScientist, CARI, Izatnagar, for rendering his valuablehelp in the course of the present work.REFERENCESBaker EW, Evans TM, Gould DJ and Hull WB. 1956. In: AManual of Parasitic Mites of Medical or EconomicImportance. A Technical Publication of the National PestControl Association Inc. N. I. pp 22.Berlese A.1888. Acari, Myriapoda et Scorpiones hucusque inItalia reperta. Mesostigmata Fusc 52:143.Fig. 2. Egg of Liponyssus sp. (400x) W-double wall.dorsal plate of mite was tapered and blunt posteriorly,and not extended up to the posterior extremities. Longhairs were present at the posterior extremity. Thepossession of the above characters placed the miteunder the genus Liponyssus Kolenatis, 1858. Theshape of the dorsal plate differentiates the presentspecies from O. (Liponyssus) sylviarum. Although theDenmark HA and Cromroy HL. 2003. DPI EntomologyCircular 299. Entomology and Nematology Department,Florida Cooperative Extension Service, Institute of Foodand Agricultural Sciences, University of Florida.Hirst S. 1916. On some new acarine parasites of rats. BullEntomol Res 6:183-190.Sambon LW. 1928. The parasitic acarions of animals and thepart they play in the causation of the eruptive fevers andother diseases of man. Preliminary considerations basedupon an ecological study of typhus fever. Annals Trop MedParasitol 22:67-132.Soulsby ELJ. 1982. Arthropods. In: Helminths, arthropods andprotozoa of domesticated animals. Belliere Tindall. pp 448-449.

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 74-75Short communicationJ P DImmune response analysis in Toxoplasma gondiiinfectedchicksY. V. Raote, A. V. Gaikwad, G. P. Bharkad and A. K. JayrawDepartment of Veterinary Parasitology, Maharashtra Animal and Fishery Sciences University, Udgir.Received 6 May 2007; accepted 2 July 2007ABSTRACT. A trial was conducted for the assessment of immune responses in Toxoplasma gondiiinfectedchicks. Eighteen chicks were equally divided into three groups (Gr): viz. Gr-I served asuninfected control group, chicks of Gr-II were infected with 1000+56 sporulated oocysts of T. gondiiand the birds of Gr-III were infected with 1000+56 sporulated oocysts of T. gondii and injected withlevamisole hydrochloride @ 2mg/kg b. wt., intramuscularly. On day 38 post-infection (PI),cell-mediated immune (CMI) responses were analysed using hapten 1-chloro 2-4di-nitrobenzene (DNCB). On day 28 PI, immunoglobulin concentration in serum was estimated byemploying immunoglobulin flocculation test. Twenty-four h after application of DNCB, skin ofbirds was found to be warm, hyperaemic, oedematous and thickened, which confirmed CMIresponse. However, maximum skin thickness (3.6 mm) was recorded in chicks of Gr-I, whereas theskin of chicks in Gr-III and Gr-II was observed to be 3 mm and 2.9 mm thick, respectively.Similarly, maximum diameter of indurative skin reaction was observed in chicks of Gr-I (35.3 mm),compared to the birds of Gr-III (31.6 mm) and Gr-II (26.6 mm). Maximum (16 mg/100 ml serum)level of immunoglobulins was observed in the birds of Gr-III, intermediate in Gr-II (15 mg/100 mlserum) and minimum in Gr-I (13.3 mg/100 ml serum). Reduced weights of bursa were noticed in thebirds of Gr-II (297.3 mg%) compared to the birds of Gr-III (451.2 mg%) and Gr-I (547.3 mg%).Spleen revealed severe congestion throughout the parenchyma. Relatively underdeveloped follicleswere thinly populated by lymphoblasts. In thymus, there were focal areas of necrosis in lymphoidfollicles.Keywords: chicks, immune response, Toxoplasma gondiiToxoplasma gondii is an obligate intracellular generally asymptomatic in immunocompetent adults,coccidian parasite of the felids. Felines serve as whereas > 95% of Toxoplasma encephalitis in AIDSdefinitive host, whereas non-felines serve as patients is due to recrudescence of latent infection, as aintermediate hosts with disseminated tissue result of progressive loss of cellular immuneinfections. T. gondii infection is widely prevalent in surveillance (Scott et al., 1997). Hence, an attemptman and animals that significantly impact animal was made to analyse the impact of T. gondii infectionproduction and public health throughout the world on immune responses of chicks.(Dubey and Beattie, 1988). Toxoplasmosis isCorresponding author: Dr. Y. V. Raote, Department of VeterinaryParasitology, College of Veterinary and Animal Sciences,Udgir-413 517, India. E-mail:jayrawant1@rediffmail.comEighteen chicks were equally divided into threegroups (Gr): viz. Gr-I served as uninfected controlgroup, chicks of Gr-II were infected with 1000+56spourlated oocysts of T. gondii, whereas birds of Gr-IIIwere infected with 1000+56 sporulated oocysts of

Immune response analysis75T. gondii and injected with levamisole hydrochloride produced by Toxoplasma organisms in the bursa of@ 2mg/kg b. wt., intramuscularly. On day 38 postthfabricius. On 14 day PI, Gautam et al. (1982)infection (PI), cell-mediated immune (CMI) documented the occurrence of Toxoplasma organismsresponses were analysed, using hapten 1-chloro 2-4 in bursa of chicks.di-nitrobenzene (DNCB). On day 28 PI,immunoglobulin concentration in serum was Microscopic examination of the sections of spleenestimated by employing immunoglobulin flocculation revealed severe congestion throughout thetest.parenchyma, which is in consonance with the findingsof Biancifiori et al. (1986), who recorded enlargementTwenty-four h after application of DNCB, the skin of of spleen with focal necrosis. Gautam et al (1982)birds was found to be warm, hyperaemic, oedematous reported depletion of lymphocytes in white pulp andand thickened which confirms the CMI response. reticular hyperplasia in red pulp. HistologicalHowever, maximum thickness was recorded in chicks examination of sections of thymus showed focalof Gr-I (3.6 mm), compared to the chicks of Gr-III (3 necrotic changes in lymphoid follicles, atrophy ofmm) and Gr-II (2.9 mm). Similarly, maximum bursa and underdeveloped bursal follicles. Indiameter of indurative skin reaction was documented mammalian toxoplasmosis, Beverley (1974) reportedin chicks of Gr-I (35.3 mm), as compared to Gr-II (26.6 depletion of cortical lymphocytes of spleen andmm) and Gr-III (31.6 mm). Suppression of CMI thymus. Further he stated, this depletion might be toresponses in chicks of Gr-II was evident, as T. gondii cope with cellular immunity required to counterinfection has a direct bearing on immune system. infection.Whereas CMI responses in chicks of Gr-III were notseverely affected, as their CMI response was enhancedREFERENCESby treating them with levamisole hydrochloride. Beverley JKA. 1974. Some aspects of toxoplasmosis, a worldMandakhalikar (1992) recorded haemorrhages and wide zoonosis. “Parasitic Zoonoses, Clinical andExperimental Studies”. Soulsby, ELJ (Ed.), Academiccoagulative necrosis in mesenteric lymph nodes ofPress, INC. New York, San Fransisco, London.dog. Beverly (1974) reported reactive changes in thelymph nodes, spleen and thymus of man. Suppression Biancifiori F, Rondini C, Grelloni V and Frescula T.of CMI responses could be attributed to damage 1986.Avian toxoplasmosis: experimental infection ofchicken and pigeon. Comp Immun Microbiol Infect Disproduced by T. gondii infection in thymus of chicks.9:337-346.Maximum (16 mg/100ml serum) level of Chhabra MB, Mahajan RC and Ganguly NK. 1980. Antibodyimmunoglobulins was observed in birds of Gr-III, response and serum protein alterations in experimentalintermediate in Gr-II (15 mg/100ml serum) and toxoplasmosis. Ind Vet J 57:627-631.minimum in Gr-I (13.3 mg/100 ml serum). Variation inDubey JP and Beattie CP. 1988. Toxoplasmosis of animals andimmunoglobulin level was not evident between the man. CRC Pressm Boca Raton, Florida.groups, and values of Gr-III and II were comparableGautam OP, Chhabra, MB, Gupta SL and Mahajan SK. 1982.with those of control group. T cells are responsible forIncidence, diagnosis and control of toxoplasmosis inthe activation of B cells for the secretion of antibodies animals. Final report of ICAR research scheme (Nov. 1978-against infective agent. T. gondii infection damaged Nov. 1982). Department of Veterinary Medicine, College ofthymus lobes and caused atrophy of bursal follicles in Veterinary Sciences, Hariyana Agricultural University,infected birds, which might have resulted in low Hisar.production of antibodies. However, in birds of Gr-III, Mandakhalikar MV. 1992. Studies on characteristics ofestablishment of infection was low due to Parbhani isolate of Toxoplasma from cat. M.V.Sc. Thesisenhancement of CMI responses. Remington and submitted to Marathwada Agricultural University,Hackman (1966) and Chhabra et al. (1980) recorded Parbhani.the elevated levels of α, β and γ globulins in Remington JS and Hackman R.1966. J Immunol 95:1023.Toxoplasma-infected mice.(cited by Chhabra et al., 1980).Reduced bursal weights were noticed in birds of Gr-II Scott J Arnold et al. 1997. Disseminated toxoplasmosis. ArchPathol Lab Med 121:531-537.(297.3 mg%) as compared to the birds of Gr-III (451.2mg%) and Gr-I (547.3 mg%). Reduction in size ofbursal follicles as well as % weights of bursa recordedin infected birds is attributable to the damage

Journal of Parasitic Diseases: June 2007, Vol. 31, No. 1, 76Book reviewJ P DVector BiologyVinod P. Sharma and Jagbir S. Kirti (Eds.)Published byThe National Academy of Sciences, India, Allahabad (2006)Pages: 256B. N. DhawanChief EditorProceedings of the National Academy of SciencesSection B: Biological SciencesThe National Academy of Sciences, India organized a malaria, the mosquitoes, both Anopheles and Culexsymposium on Vector Biology jointly with the groups. Three articles are devoted to dengue vectors,National Academy of Vectors and Vector-borne their epidemiology and control. Papers are includeddiseases at the Punjabi University, Patiala from on sandflies that cause Kala-Azar, also on ScrubFebruary 18–20, 2006. The proceedings of this Typhus, bovine tropical Theileriosis, control of bitinginternational symposium were published by the midge and the control of vectors of veterinaryBiological Sciences Section of the NASI, India at importance. The topic of the symposium was veryAllahabad in 2006. Prof. V. P. Sharma, the well-known general; hence the various presentations are of veryworld renowned Vector Biologist in the area of malaria varied and of general nature. Nevertheless, the variousand Prof. Jagbir Singh Kirti have edited these papers in the book have adequately underlined thepresentations, numbering 20, in the form of a coherent importance of understanding the biology of vectorsvolume entitled “Vector Biology”.without which it would not be possible to successfullyThe vectors play a vital role in the transmission ofdraw up strategies for control of parasites.parasitic diseases and hence their study is of This publication should be greatly welcome by allparamount importance. However, it so happens that shades of parasitologists as a useful addition to the listdue to the scanty knowledge of the vectors, the of books on a rather difficult topic for which themajority of which are insects, we fail by and large in editors, Professors Vinod P. Sharma and Jagbir S. Kirtiour efforts to eradicate the parasitic diseases. The deserve praise. We should also be thankful to Prof. B.weakest point in the life cycle of parasites mostly N. Dhawan, the Chief Editor of publications oncomes when they are outside their definitive hosts and Biological Sciences and the National Academy ofin the process of being transmitted from one host to the Sciences, India at Allahabad for its timely publication.other. So far our major efforts and energies weredirected towards preventing the diseases orProf. M. Shamim Jairajpuri, FNAcontrolling the parasites after they have reached theINSA Senior Scientisthosts and have got well settled in their bodies. This isDepartment of Zoologynot always the easiest way to tackle them. The majorAligarh Muslim Universitypart of the book is obviously devoted to vectors ofAligarh-202 002

JOURNAL OF PARASITIC DISEASESInstructions to AuthorsJ P DGeneralThe Journal of Parasitic Diseases is the official organof the Indian Society for Parasitology (ISP).Manuscripts are accepted from international authorsirrespective of whether they are members of the ISP ornot. The scientific standard as related to quality,originality, clarity, brevity and the potential to attract awide range of readership is the sole criterion foraccepting a manuscript for publication. Each volumeof the Journal of Parasitic Diseases consists of twoissues released in June and December. “Instructions toAuthors” are published in the June issue of the journaleach year. Authors are requested to read theseinstructions very carefully and follow them strictly toensure efficient and quick editorial processing andpublication of their manuscripts. Manuscripts that areincomplete or are not prepared strictly according to theinstructions and style of the journal will be returnedwithout editorial processing.Aims and scopeThe primary constituency of the Journal of ParasiticDiseases is parasitology. It publishes original papers(pure, applied and clinical), which contributesignificantly to any area of parasitology. Researchpapers on various aspects of computational, cellularand molecular parasitology are especially welcome.Submission of manuscriptsAll manuscripts, in triplicate (three hard copies + 1disk copy) along with a duly complete “Undertakingby Authors”, and correspondence should be addressedto:Professor Prati Pal Singh, Ph. D., F. N. A. Sc.Editor-in-ChiefJournal of Parasitic DiseasesRoom No. 113, Block-FNational Institute of PharmaceuticalEducation and ResearchSector-67, Phase-XS. A. S. Nagar-160 062, Punjab, INDIAE-mail: drppsinghjpd@yahoo.co.inAlternatively, the manuscripts (Word format or PDFfiles; text, references, tables, legends and figures) andundertaking in electronic form can be submitted as ane-mail attachment. However, simultaneously, theymust also be submitted as three hard copies + 1 diskcopy along with the hard copy of the “Undertaking byAuthors”.Manuscripts are received with the understanding thatthe research reported therein was conducted withproper animal care as per the approved guidelines ofthe Institutional Animal Ethics Committee. Similarly,manuscripts which report experiments on humansubjects and materials, must have obtained necessaryapprovals from the relevant institutional ethicscommittees. Submission of a paper to the Journal ofParasitic Diseases implies that all the authors haveapproved submission of the paper, that the paper is notunder consideration for publication elsewhere, andthat if the paper is accepted for publication orpublished, the article or portions thereof will not bepublished elsewhere without written permission fromthe Editor-in-Chief. It is the responsibility of theauthor(s) to obtain all the necessary permission toinclude items like figures, tables and other materials.In the selection of papers for publication, the decisionof the Editor-in-Chief is final and no query regardingthe decision will be entertained.Statements or opinions expressed in the Journal ofParasitic Diseases reflect the view(s) of the author(s)and do not represent the official position of the ISP.Neither the Society nor the Editorial Board acceptsany responsibility for the statements made by thecontributors.Forms of publicationThe Journal of Parasitic Diseases publishescontributed articles falling into the followingcategories.• Original papers: that report original research.i

• Short communications: those report some Manuscripts and presentationpreliminary but important findings that do notwarrant publication as full paper.Manuscripts, in English, must be printed (Times NewRoman, 12 size; preferably black laser print) on one• Reviews: both invited and contributed ones on side of a white, good quality standard size bond paper.topics of current importance.All manuscripts must be typed double spaced and thepagination should be done on the top left corner. Only• Book reviewsone set of original illustrations and two first-classcopies are adequate. The order of presentation should• New and viewsbe: title Page with title, authors, affiliation,corresponding author (with full address, telephone• Letters to the Editor-in-Chief and fax number and e-mail ID), Abstract,Review of manuscriptsIntroduction, Materials and Methods, Results,Discussion, Acknowledgments and References.Abstract, tables and legends for figures should beSince June 2006 issue, the journal has been followingtyped on separate pages and not in continuation of the“Tutorial Editorial Policy”. Manuscripts received bymain text. At least one inch margin must be availablethe Editorial office, after checking their completenesson all four sides of the paper, to provide space forand suitability as per the scope of the journal, shall beeditorial markings/comments.sent to appropriate Members of the Editorial Board,who in turn shall send them to two suitable Reviewers Title: Title of paper should be short and yet sufficiently/Referees/ Consultants. They will read, evaluate and descriptive and informative.return the manuscripts along with theirreport/comments. The Editorial Board Member, on the Authors: The names of authors should include initialsbasis of the report/comments received and on the and surname and their institutional addresses.scientific quality of manuscript, shall send his or herrecommendations (regarding acceptance, noncharactersincluding spaces should be provided.Running title: A short running title not exceeding 60acceptance or modification) to the Editor-in-Chief.Finally, the Editor-in-Chief, based on all the reportsAbstract: The abstract should be limited toand recommendations and on the overall merit of theapproximately 250 words and should contain a line ofmanuscript, shall decide and communicate theintroduction, important results and theircorresponding author if the manuscript has to besignificance. In no case it should be just a statement ofaccepted, not to be accepted or has to be accepted afterresults only. References are not allowed in themajor/minor modifications. If a revised manuscript isabstract.not received within three months, and is received later,it will be treated as a new manuscript. Manuscripts not Keywords: 3-5 keywords arranged in alphabeticalaccepted for publication shall not be returned.order should be provided below the abstract.Correction of proofsProofs of the articles will be sent to the correspondingauthor for checking its correctness and completeness.No major or any editorial change in the text or data orin the authors shall be allowed at this stage. Theauthors are responsible for the final accuracy of theproofs of their manuscripts. The corrected proofs mustbe returned promptly (maximum within five days).The fax or scanned version (as e-mail attachment) ofthe corrected proofs is also acceptable. A delay inreturning corrected proofs will result in their delayedpublication or their publication as they stand. Thereference section of a manuscript is usually prone tomaximum errors, and hence authors are advised tocheck this section very carefully.Introduction: Introduction should provide theessential background, must briefly review the relevantliterature, identify the question(s) to be answered andshould state the reasons for undertaking the study.Further, to avoid suspense, it should end with a briefstatement of results.Material and Methods: The source of the materialused should be clearly mentioned. The proceduresadopted should be explicitly stated to enable otherworkers to reproduce the results. New methods may bedescribed in sufficient details, established methodscan be just described in brief with appropriatereference(s). Deviations from previously reportedmethods, if any, should be explained giving thereasons thereof.ii

Results: The results should be as clear as possible and • Cheever AW, Macedonia JG, Mosimann JE andmust state only the findings of the study reported. They Cheever EA. 1994. Kinetics of egg productionshould be comprehendible to the readers and and egg excreted by Schistosoma mansoni and S.sequenced in such a way that a logical progression is japonicum in mice infected with a single pair ofdeveloped. The data presented in figures and tables worms. American Journal of Tropical Medicineshould not be repeatedly summarized. Results should and Hygiene. 50:281-285.not be intermixed with interpretations or conclusions.The name of the journal should either be given in fullDiscussion: In this section the results should bediscussed in a focused way and not just recapitulated.or abbreviated as per the Index Medicus or Medline.Authors should try to present the principles, Edited booksgeneralizations and relationships. They shouldspecifically show how the results and interpretationsSchaniz PM and Kagan IG. 1980. Echinococcosis. In:reported in the manuscript agree or disagree withImmunological Investigations of Tropical Parasiticalready published information. The significance andDiseases. Houba. V (Ed.), Churchill Living stone,importance of the work must also be discussedNew York. pp 104-129.sufficiently. Conclusions must be stated clearly andevidence for each conclusion should be summarized.Conference proceedingsLong, rambling discussion lacking logic and flow Singh, Prati Pal and Mortensen RF. 1985. Serumshould be avoided. Speculations must be avoided to amyloid P-component (SAP) response in inbredthe extent possible.mouse strains during Listeria monocytogenesinfection. In: Proceedings of II InternationalAcknowledgements: Acknowledgements should beSymposium on Genetic Control of Host Resistance tobrief and made for specific scientific, technologicalInfection and Malignancy, Montreal (Ed. E.and monetary (grant) assistance.Skamene), Allan R. Liss, Inc., New York, pp 339-344.References: List of references should include onlypublished papers and papers in press. AbstractsReportspresented in meetings will not be considered as World Health Organization. 1984. The Leishmaniasis.references. Citations of unpublished work, where Technical Report Series No. 701. Geneva,absolutely necessary, should be incorporated in the Switzerland.text itself (examples: our unpublished observations orSharma et al. unpublished data or Sharma et al. Electronic format paperspersonal communications). Personal communicationsmust be supported by an authorized letter of Ponce LV. 2005. Adoptive transfer of dendritic cellspermission. References in the text should be cited modulates immunogenesis and tolerogenesis in aaccording to the name and year system (also known as neonatal model of murine cutaneous leishmaniasis.Harvard system) in chronological order, using 'and' for Kinetoplastid Biology and Disease. 4:2.articles with two authors and et al. for those with more http://www.kinetoplastids.com/content/4/1/2 .than two authors and semi colon between referencesPapers published only with Digital Object Identifier(Trager and Jensen, 1976; Sharma et al., 1994).(DOI) numbersReferences should be listed at the end in alphabeticalorder and it must be ensured that all references cited in Jabakhanji R. Inflammatory and neuropathic painthe text are included in the list and vice versa. The animals exhibit distinct responses to innocuous andfollowing style should be followed:motoric challenges. Molecular Pain. DOI:10.1186/Journal1744-8069-2-1.In the list of references at the end of the paper, all thenames of authors should be given. However, if thereare more than six authors, list first six names followedby et al.• Trager W and Jensen JB. 1976. Human malariaparasite in continuous culture. Science.193:673-675.iiiTables: Tables should be typed separately andnumbered consecutively with Roman numerals (I, II,III etc.). Each table should bear a brief title. Columnheadings should also be brief. Units of measurementsshould also be abbreviated and placed below theheadings. Statistical variations such as SD and SEshould be identified. Numbers less than one shouldhave a zero set before the decimal point (e.g. 0.1). The

journal does not follow floating number principle, so Interleukins: Designations recommended by IUISwrite 1 and not 1.0.committee on interleukins have to be used (EuropeanCytokine Network. 1991. vol. 2 (5). pp 309-310).Illustrations: Three sets (one original and two firstclasscopies) of illustrations should be submitted, Abbreviations: Only standard abbreviations are to benumbered consecutively in Arabic numerals (1, 2, 3 used. This should conform to the International Systemetc.). Line drawings should be made on good quality of Unit(s), the SI for the Health Professional, WHO,art paper. Letters, numbers and symbols should be Geneva, 1977.clear in figures and of sufficient size so that whenreduced they can be accommodated in a single column Short communications: These should be prepared in a(8.5 cm) or double column (17.5 cm) without loss of manner similar to research papers but should not beclarity. Title and explanation of symbols should be categorized with the subheadings as in the full paper.typed on separate pages. Data for tables, graphs etc. However, abstract and keywords should be provided.should be carefully verified. All statisticalLetters: This is the quickest way of ensuringevaluations, percentage and other calculations shouldpublications. These should not exceed 500 words.be checked thoroughly before submission of a paper.Once a paper has been accepted for publication, no Submission of manuscripts on diskalterations will be allowed.The general style of presentation as detailed abovePhotographs: Monochrome photographs should be should be followed. Please label your disk with (1)submitted in triplicate (all originals; photocopies are short version of the title of the manuscript, (2) name ofnot accepted) and should be un-mounted and the first author, (3) phone and fax number, postal andnumbered consecutively. The title of the paper and e-mail address, (4) hardware and software used,author(s), and orientation should be indicated with an including version, (5) and the list of all the file namesarrow should be penciled on the back of each (eight characters identifying the file before the dot andphotograph. The photographs should be good quality three characters after the dot for the identification ofprints on glossy paper, sharply focused showing good application). Use only one file for text, references,contrast to ensure high quality production. Their size tables and legends to illustrations. Tables should beshould conform to a single (8.5 cm) or double (17.5 placed at the end of text. Use boldface, italics and subcm)column size. Symbols, arrows and letters should and superscript where necessary. Illustrations have tocontrast with the background. Colour photographs are be stored in separate files. Digital figures should be inalso accepted; however, authors will be charged for the Word or preferably JPEG files. Preferably, please usesame.MS-Word word-processing package. A compact diskis preferred over a floppy disk. The original and twoLegends for illustrations and photographs: Legendshard copies with illustrations, containing the exactfor illustrations and captions for photographs shouldversion on the disk, and the duly completebe typed double spaced on a separate page.“Undertaking by Authors” must be submitted alongEnzyme nomenclature: The trivial names with the disk.recommended by the IUPAC-IUB Commissionsshould be used. At the first citation in the text of thepaper, its code number and systematic name should beindicated.iv

JOURNAL OF PARASITIC DISEASES(Official organ of the Indian Society for Parasitology)Undertaking by AuthorsJ P DWe, the undersigned, give an undertaking to the following effect with regard to our manuscript titled:_______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________submitted for consideration to possible publication in the Journal of Parasitic Diseases.1. Research reported was conducted within the proper animal care and according to the applicable National Laws.2. The manuscript mentioned above has not been submitted for publication in any form to any other journal.3. The paper, if published in the Journal of Parasitic Diseases, will become the sole copyright of the Indian Society forParasitology and the article(s) thereof will not be published elsewhere unless written consent is obtained from the Editor-inchief.4. We also agree to the authorship of the manuscript in the following sequence:Authors' names (in sequence) Signature of authors1. _________________________________________ _______________________________________2. _________________________________________ _______________________________________3. _________________________________________ _______________________________________4. _________________________________________ _______________________________________5. _________________________________________ _______________________________________6. _________________________________________ _______________________________________7. _________________________________________ _______________________________________8. _________________________________________ _______________________________________9. _________________________________________ _______________________________________10. _________________________________________ _______________________________________Please note:1. All authors are required to sign independently in the form and in the sequence given above. A photocopy of this form mayalso be used.2. No addition/deletion/or any change in the sequence of the authorship will be permissible at a later stage, without validreasons. If change is valid , then all authors involved should attest to the change. The decision, however, rests wth theEditor-in-chief.3. If the authorship is contested at any stage, the manuscript will be either returned or will not be processed for publication.ImportantIn order to facilitate speedy processing of the submitted manuscript, please ensure the following:1. Manuscripts are prepared as per the format of JPD.2. Three copies of the manuscript and a brief covering letter.3. Running title of not more than 60 characters including spaces.4. Keywords (3-5 words).5. "Undertaking by Authors", duly complete and signed.6. Three sets of photographs (if any) with legends and captions on a separate page.7. A CD containing exactly the hard copy version.v

The Indian Society for ParasitologyMembership ApplicationName : ____________________________________________________________________________________Educational Qualification:______________________________________________________________________Field of Specialization :________________________________________________________________________Work Address:________________________________________________________________________________________________________________________________________________________________________Phone.:_________________________Fax:____________________________E-mail:______________________Mailing Address:_______________________________________________________________________________________________________________________________________________________________________Phone.:_________________________Fax:____________________________E-mail:______________________Signature of the Applicant:_____________________________________________________________________Proposed by:______________________________ Seconded by:_____________________________________(Member, ISP)(Member, ISP)__________________________________________________________________________________________Membership FeeIndia : Life Membership : Rs. 2000.00 Other Countries : US$ 400.00(Demand draft should be drawn in favour of Secretary, The Indian Society for Parasitology, Payable at Lucknow)Membership of the Society includes subscription to the Journal of Parasitic Diseases, a biannual publication of theISP. Members are eligible to attend annual conferences of the ISP and avail discounts in registration fee.Membership is governed by the Constitution and Bye Laws of the Indian Society for Parasitology.-----------------------------------------------------------------------------------------------------------------------------------------(For Office use Only)Life Membership No. :____________________Receipt No.:___________________Signature of Secretary Signature of Treasurer----------------------------------------------------------------------------------------------------------------------------------------Note: Duly completed form along with draft should be mailed to Dr. J. K. Saxena Secretary, ISP, Division ofbiochemistry, Central Drug Research Institute, Post Box-173, Chattar Manzil Palace, Lucknow-226001, India.Personal cheques/cash are not accepted. There is no annual or ordinary membership. This form may be xeroxed.vi

THE INDIAN SOCIETY FOR PARASITOLOGYExecutive CommitteePresidentProf. Veena TandonDepartment of ZoologyNorth Hill UniversityShillongVice-PresidentDr. J. MahantaRegional Medical Research CentreDibrugarhVice-PresidentProf. P. D. JuyalDepartment of Veterinary ParasitologyGuru Angad Dev Vet. & Ani. Sci. Univ.LudhianaJoint SecretaryDr. Vasu DevMalaria Research CenterSonapurVice-PresidentProf. R. MadhviDepartment of ZoologyAndhra UniversityVisakhapatnamSecretaryDr. J. K. SaxenaDivision of BiochemistryCentral Drug Research InstituteLucknowTreasurerDr. L. M. TripathiDivision of ParasitologyLucknowMembersDr. Wasim Ahmed, AligarhDr. P. Prakash Babu, HyderabadDr. S. C. Datta, KolkataDr. S. K. Ghosh, BangaloreDr. Neena Goyal, LucknowProf. Neelima Gupta, BareilleyDr. B. V. Jadhav, AurangabadDr. 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 the Journal of Parasitic Diseases or in thepresentations during the regular meetings of the Indian Society for Parasitology are thoseof the author(s), and do not necessarily reflect the official position of the Society. TheEditorial Board, the publisher and the Society disclaim any responsibility for the accuracyof the statements made by the contributors.Copyright © 2007 The Indian Society for Parasitology

Designed & Printed at :- Azad Offset Printers (P) Ltd. 144-Press Site, I.A.-1, Chandigarh Tel. : 0172-2654411, 2652349, 2651316