effect of infection of the filarial parasite brugia malayi - Pondicherry ...

EFFECT OF INFECTION OF THE FILARIAL PARASITE BRUGIA 

MALAYI (NEMATODA: ONCHOCERCIDAE) ON A RODENT 

MODEL MASTOMYS NATALENSIS 

Thesis submitted 

to 

THE PONDICHERRY UNIVERSITY 

for the degree of 

DOCTOR OF PHILOSOPHY 

in 

MICROBIOLCG\ 

BY 

K. GOMATHI, MSC . 

Senior Research Fellow 

VECTOR CONTROL RESEARCH CENTRE 

PONDICHERRY-605 006 

Under the guidance of 

Dr. K. BALARAMAN, M.SC.. PL.D. 

Deputy Director (Sr. Grade) 

VECTOR CONTROL RESEARCH CENTRE 

PONDICHERRY-605 006 

INDIA. 

APRIL 2000

CTOR CONTROL RESEARCH CENTRE 

Ian Council oI Med~cd Keacarchi 

;i 

jhTmrn-k 

rd Napar. Pondicherr! - 605 (~16. India ?'R dmaff - 504 OOE. 

Telephone l.pF4 372396 372397 372797 8 372422 Fax l h 91-413-372041 

Tdegram I im MOSQUITO E-mad mosqutto@mU2 mnl netln 

CERTIFICATE 

Dated: 24 lir / 2000 

This is to cert~fi that this thesis entitled "Effect of the fJorial parasite Brugia mdayi 

(Nematoda: Onchocercidae) on a rodent model Mastomrys nardensis" incorporates results of 

original research work carried out on the subject by Mrs. & Gomathi, Vector Control Research 

Centre, Pondicherv during the study period and that the thesis has not formed as the basisfor the 

award ofany degree, diploma, associateship, fellowship or other title. 

The thesis represents orrginal workdone by the candidate under my guidance. 

WOK1,Il HEALTH ORGANIZATION 

Collrhoratmg Cenur IOI Rtsear~h & Twnmg In 

Integrrted Method\ ol Vector Control 

DEPUTY DIRECTOR (SRCRADE) & 

SUPERVISOR TO THE CANDIDATE 

wmm 

amm4imtmmaw* 

4

I wish to express my deep sense of gratitude to my esteemed guide Dr. K. 

Balaraman, Deputy Director (Sr. Grade), Vector Control Research Centre, Pondicheny for 

his meticulous guidance, invaluable suggestions and critical review of this manuscript. 

1 thank Dr. Vijai Dhanda and Dr. P.K. Das, former and present Directors of Vector 

Control Research Centre, Pondicheny, for giving me this opportunity and the necessary 

facilities to work at this centre. 

1 am grateful to Dr. K.C. Varshney, Prof. and Head, Pathology Department, Rajiv 

Gandhi College of Veterinary and Animal Sciences, Pondicheny, for his help in 

histopathological studies, constant encouragement and timely suggestions. 

I am grateful to Dr. K.P. Paily, Research officer, VCRC for his help in providing 

the parasite colony and also for his help and suggestions throughout the study. 

My thanks are due to, Dr. S. Sudha Rani (Dept. Biotechnology, Pondicherry 

University), Dr. S.P. Pani and Dr. S.L. Hoti, Deputy and Assistant directors respectively 

for their constant help and suggestions throughout the course of work. 

The help of Mrs. A. Srividya, Mr. A. Manoharan, Mr. P. Vanamail and Mr. S. 

Subramaniurn in the statistical analysis is gratefully acknowledged. 

I am grateful to Mr. B. Ravishankar, Research Scholar, Dept. of Endocrinology, 

PGIBMS, University of Madras for his help in conducting the Radio Immuno Assay and 

Dr. Kumar, Asst. Prof. RAGACOVAS, Pondicheny for his help in histopathological 

studies. 

I am fortunate in receiving help from Mrs. I. Geetha, Mrs. T. Sumathy, Mrs. M.K. 

Vijayalakshmi, Mr. V. Padmanaban, Mrs. Sundrammal, Mrs. Shantha Bheema Rao and 

Mr. P. Shakthivel. 1 acknowledge the assistance provided by Mr. A. Ramamoorthy, Mrs. 

K. Sahayamary, Mr. V. Anthonysamy and Mr. Y. Krishnan. 

I owe my special thanks to my parents Mr. M. Kapali and Mrs. Sivagami Kapali, 

my husband Mr. Rajiv Giroti and Sister, K. Nandhini for their patience, encouragement at 

the time of crisis, good wishes and appreciation which had been source of inspiration 

throughout the period of study. 

The finical assistance provided by C.S.I.R. New Delhi is gratefully acknowledged. 

Last but not the least, I thank all the staff of VCRC for their kind co-operation 

during my stay here.

Superoxide disrnutase 

Glutathione peroxidase 

LIST OF ABBREVIATIONS 

Glutathione reductase 

Glucose-6- phosphate dehydrogenase 

Reduced glutathione 

Oxidised glutathione 

Lipid peroxidation 

Gamma glutarnyl transpeptidase 

Phosphate buffered saline 

Thio barbituric acid 

Trichloroacetic acid 

Malondialdehyde 

Ethylene diamine tetmcetic acid 

Diethylene triamine penta acetic acid 

1 -chloror- 2,4 dinitrobenzene 

Adenine triphosphate 

Nicotinamide adenine dinucleotide phosphate 

Nicotinamide adenine dinucleotide phosphate (reduced) 

Magnesium chloride 

Hydrogen peroxide 

5,5'dithio-bis-2-nitrobenzoic acid 

Flavin adenine dinuceotide 

Dinitrophenyl hydrazine 

Micromoles 

Microlitre 

Microgram (s) 

Nanomoles 

Millimole 

Milligram (s) 

Picogram 

Glutathione-s-transferase 

- SOD 

- GSH 

- GSSG 

- LPO 

- y-GTP 

- PBS 

- TBA 

- TCA 

- MDA 

- EDTA 

- DETAF'AC 

- CDNB 

- ATP 

- NADP 

- NADPH 

- MgC12 

- Hz02 

- D m 

- FAD 

- DNPH 

- pmoles 

- nmoles

1. INTRODUCTlON 

2. REVIEW OF LlTERATLTRE 

3. MATERIALS AND METHODS 

CONTENTS 

COLONY MAINTENANCE. OF BRUGIA MA L4 YI 

4. EXPERIMENTS 

4.1 Changes in antioxidant enzymes and antioxidants in Mastomys mtalensrs 

Page No 

... 13 

after Bruga mlqi infection and its relation to DEC treatment. . . .I4 

4.2 Changes in the activity of membrane bound enzymes and haematological 

parameters in Mastomys natalensis infected with Bruga malayi. . . .81 

4.3 Changes in the testes ofMastomys natalensis infected with 

Brugra malayi . . .97 

4.4 Histopathologicalchanges in Mastomys natalensis infected with Brugra malayi 

and its relation to DEC treatment. .. 103 

5. SUMMARY ... 109 

6. CONCLUSION ... 113 

BIBLIOGRAPHY 114 

APPEM)IX . . ,133 

... 1 

. . .6

Filariasis is one of the most debilitating diseases of the tropics. Mortality due to 

filariasis is negligible. Howevm, there is a high d e p of morbidity due to its acute and 

chronic manifestation. Filariasis has been the subject of cons~derable attention for long, but 

it is still poorly understood. 

Over two-thirds of the lymphatic filariasis cases of the world is contributed by India, 

China and Indonesia. It is an age old disease ncorded in India as early as in 6th century 

B.C. Both bancroftian and brugian lymphatic filariasis are prevalent in India. However, 

bancroftian filariasis is the commonest, accounting for 98% of the filarial infection. India 

alone accounts for 42.8% of the global problems due to Wuchereria bancrofti and 20.2% 

due to Brugia malayi. There are about 429 million people exposed to the risk of infection 

and 3 1.3 microfilaria (mf) carriers (Appavw et al., 1999). 

Lymphatic filariasis is caused by the lymphatic dwelling filarial nematodes such as 

I.Y bancrofti (Cobbold, 1877), B. malayi (Lichtenstein, 1927) and B. timori (David and 

Edeson, 1964). Three physiological races exist in W. bancroofii and B. malayi, depending on 

the microfilarial periodicity (Sasa and Tanaka, 1972; 1974). They are the nocturnally 

periodic, nocturnally subpenodic and diurnally subperiodic forms. In the Indlan sub 

continent both the parasites exist as nocturnally periodic forms. The vector of W bancrofri 

is Culer quinquefmciatu and vector ofB. malayi is Mansonoldes species. 

Recurrent fevers, inflammation of lymph nodes and vessels, sometimes 

accompanied by transient swelling of the limbs are the earliest s~gns of the disease. Later 

symptoms include scrota1 swelling and permanent swelling of the limbs caused by 

collection of fluid in cells, tissues or body cavihes. This may lead to the grossly swollen, 

thick-skinned limbs, a condition commonly called as elephanhasis. The general approach to 

control filariasis is to reduce morbidity and transmission. Diethylcarbamatine (DEC), is still 

considered the drug of choice for treating or suppressing lymphatic filarial infections 

(Hawking, 1979, Mak et al., 1991).

As stated earlier, the host immune mechanism and its role in the suppression or 

propsion of filarial disease is very poorly understood. Therefore, experiments were carried 

out using the animal model Mastomys natalensis and the filarial parasite, B. malayi to 

understand the influence of infection on the following parameten of the host. 

1.1 Antioxidants and antioxidant enzymes 

Host immune effector cells such as phagocyks, macrophages, neutrophils and 

eosinophils release oxidants or free radicals such as superoxide dcal, hydroxyl radicals, 

singlet oxygen and hydrogen peroxide as defense mechanisms to kill the invading parasites 

(Bannister and Bannister, 1985; Callahan et al., 1988). In the host system, oxidants are also 

produced during normal cellular metabolic processes, and due to certain antiparasitic drugs. 

(Klebanoff, 1980; Klebanoff , 1982). The leucocytes can also secrete oxygen radicals from 

their outer membrane into the smundiigs (?-lathan and Root, 1977). This indiscriminate 

and self inflicting process of generation of oxygen radicals can contribute to the tissue 

damage in the host. Over recent years, there had been an increasing awareness that free 

radicals directed by the host to destroy the parasites can be an important cause of cellular 

injury and damaging membranes, proteins and nucleic acids. 

All l~ving tissues are protected from the damaging effects of free radicals by 

detoxifying them to harmless products by andoxidants and antioxidant enzymes. The process 

of generation of free radicals and antioxidant defense system may be same in the case of host 

defense against the invasion of filarial parasites. The host antioxidant defense mechanisms 

with respect to LPO and the enzymes SOD, catalase and xanthine oxidase had been reported 

in the case of animal filarial parasite, Diperalonema viteae, in Mastomys natalensis (Barn et 

a/.. 1989). However, the complete system of defense including various antioxidant enzymes 

such as SOD, catalase, GST, GPx, GR and G6PDH and antioxidants such as GSH, ascorbic 

acid and total thiol status and LPO are yet to be investigated especially in the case of a human 

filarial parasite. The mode of action of DEC, the drug of choice for the mtment of filariasis, 

is poorly understood. Cesbron et (11. (1987) had suggested that DEC activates the platelets to 

kill mf by free radicals. But so far, the role of antioxidant defense system after DEC 

treatment of filariasis has not yet been investigated. Henoe, the comfkte antioxidant system

in an animal model M. natalensis infected with human lymphatic filarial parasite, B. malayi 

and the effect of DEC tr&bnent was taken up in the present study. 

1.2 Membrane bound enzymes and haematological parameters 

In recent years, there had been reports about the possible role of the highly reactive 

oxygen radicals in the pathogenesis of malaria and other parasitic infections (Thumwood er 

al., 1989; Das et al., 1991; Mahdi and Ahmed, 1991). The production of free radicals in the 

CNS leads to LPO and degradation of brain lipids, with the loss of membrane integrity that 

causes irreversible brain cell damage (Tripathi et al., 1994). Recently, presence of mf of 

B.mallyi in the brain of Mrrrtomys was reported by Paily el al. (1995), but their effect on the 

brain cells and brain metabolism have not been studied. 

The ability of eosinophils to kill filarial parasites, Onchocerca wlvulus and Brugia 

maluyi, under in vitro condition had been demonstrated (Green el al., 1981; Sim et a!., 

1982). It was shown that serum eosinophils adhere to 0. wlvulus rnf to depulate, 

resulting in their death (Gibson et al., 1976). Similarly, Mackenzie (1980) recognized the 

presence of eosinophils associated with mf in affected lymph nodes, ocular tissues and skin. 

Eosinophil dependent killing is generally mediated through oxygen dependent and oxygen 

independent mechanisms (Spry, 1988). 

When circulating phagocytes are triggered to secrete superoxide anions during 

parasitic infection. then erythrocytes would be among the first cells to experience 

increased stress, leading to their membrane damage. The role of fke radicals during 

Plasmodia1 infections on the host tissues and the enhanced oxidative stress within 

erythrocytes had been reported by many workers (Etkin and Eaton, 1975; Garnham, 1987). 

When the membrane bound enzymes are disturbed or inactivated, the concentration of the 

ions are altered which cause cell injury. (Trump er al., 1979; 1980). Considering the 

importance of membrane compositional and the hematological changes, the effect of 

B.malayi on M. natalensis was investigated.

13 Testicular changes 

Testosterone plays an important role in maintaining secondary sexual characteristics, 

accessory sex organs, spermatogenesis and the secretory products of the sertoli cells (French 

and Ritzen, 1973). Testosterone accompanied by follicle stimulating hormone (FSH) is 

necessary for spermatogenesis in the seminiferous tubules. Initiation of spermatogenesis in 

monkeys by testosterone alone had been reported, which was associated with a 2- 4 fold 

elevation of intratesticular androgen concentration (Marshall el a[., 1984). Testosterone 

alone had been shown to maintain the spermatogenesis qualitatively in a dose dependent 

fashion in monkeys (Weinbauer et al., 1988). The initiation of spermatogenesis by 

testosterone and dehydrotestostemne had been reported in mouse (Singh el nl., 1995). 

The dependence of spermatogenesis on sertoli cell function had been reported by the 

existence of a blood-testis barrier establishment by the tight junctions between sertoli cells, 

which modulate the entry of substance into the seminiferous tubules. The nutrient and 

growth factors required for spermatogenesis have been reported to be kmsported ffom the 

extratubular environment or synthesized by the sertoli cells and delivered to the developing 

germinal cells within the tubule (Skinner, 1987). 

Exhaustive efforts have been made in the search for enzymatic parameters useful as 

markers of specific events in testicular function. Although some parasitic infections cause 

alterations In the host's reproductive processess, the effects of B. malayi infection on the 

animal models have not been evaluated. Testosterone and LDH-X are very sensitive 

indicators of reproductive function, and 1-GTP acts as an enzyme marker for sertoli cell 

division.

1.4 Histopathology 

Although traditionally regarded as lymphatic filarial parasites in their natural hosts, 

such as man, cats and monkeys, B, malayi and related species show a characteristic 

tendency to localize within the major organs like lungs, heart and testes of M. natalensis 

(section 4.1.2). in Meriones unguiculatuc (Ash, 1973b, Ash and Riley, 1974b; ElBihari 

and Ewert, 1971). Such development has been interpreted as an aborrant mode of 

development (Ash, 1973a; Vincent et al.. 1976). Vincent el a/. (1976) studied the 

chronological development of pulmonary pathology associated with B.malayi, B.pahangi 

and B. patei. Histological studies by Hawking et a1.(1950) had concluded that DEC in 

some way promotes phagocytosis of the mf. The enzyme studies in B.malayi infected 

animals in the present study has shown cellular injury and membrane damage, hence 

histopathological studies on various organs after infection and after DEC treatment was 

carried out

2. REVIEW OF LITERATURE 

Filariasis is an important disease of mankind in the tropical and subtropical regions 

of Africa, Asia and America. Filariasis was known as early as 600-250 BC (Lawrence, 

1967). It is a group of human and animal infectious disease caused by nematode parasites 

belonging to the order filariidea. Parasites known to cause human infections belong to the 

genera Wuchereria, Brugia, Onchocerca, Dipetalonema, Mansonella, Loo and Dirofilaria. 

However only two genera Wuchereria and Bnrgia are reponsible for human lymphatic 

filariasis (Sasa, 1976). 

2.1 Life cycle of lymphatic filarial parasite 

Lymphatic filarial parasite requires two hosts the definitive host-man or vertebrate 

animal (monkey, cat, rat and jirds) and the intermediate host- mosquito to complete its life 

cycle (Fig.]). The adult worms live in the lymphatic system and produce mf, which is an 

embryonic or prelarval stage produced viviparously. The microfilariae are ingested during 

the blood meal of the vector mosquito. Mf taken up by the mosquito exsheath in the gut and 

within an hour penetrate the midgut, migrate to the thoracic muscles. In the thoracic muscles 

the parasite becomes thicker and shorter as compared to the mf and is called the fust stage 

larva (LI). At about 5th day the L1 moults to become the second stage larva (L2) which is 

slightly more active than the LI. By 9th - 10th day the L2 moults to become the infective 

stage (L3). This is a very active stage, which at maturity migrates mainly to the proboscis, 

but can also be seen in other parts of the mosquito. When the infected mosquito feeds on the 

human host, L3 larvae are deposited on the skin surface, during the process of probing and 

prior to the puncture of the host skin with the proboscis. After withdrawal of the proboscis 

the L3 migrates, gets into the wound and travels to the efferent lymphatics and subcapsular 

sinus. Approximately 9-10 days after entry the L3 moults to become the fourth stage larva 

(L4). The final moult occurs at approximately 35-40 days after the entry of L3 (Edeson and 

Laing, 1959). The L4 larva attains sexual maturity after 25-50 days.

Fig.1 LIFE CYCLE OF LYMPHATIC FILARIAL PARASFTE 

A- dult 

LlFE CYCLE OF MOSQUITO 

5 dcvdopmcni of rniamfilaria a- Adult mosquito 

C- micmlilatia , LEggraft 

D mosquito biting man and hgamg blood with miao6laria c- Lwa 

E L1 -€F(ssu~sgestege) d- F.fph) 

F- L2- bmaghgaduh 

G Fade mosquitoes Ebarticlg L3 luM

2.2 Clinical signs 

Inflammalation of lymph nodes (adenolymphangitis) and lymphatic vessels 

particularly of the extremities is the main clinical feature of brugian filariasis. Pitting odema 

and finally leading to elephantiasis are the clinical hallmarks of the chronic stage (WHO, 

1992). Other manifestations of the disease include the asymptomatic microfilaraemia, 

obstructive consequences like lymphoedema, hydrocoele, occult infection called TPE and 

acute episodes of adenolyphangitis (ADL) (Ottesen and Nutman, 1992). 

2.3 Experimental fdariasis 

Efforts to combat the widespread parasitic diseases have benefitted from the 

availabilty of the animal models. The ideal animal model for human parasitosis was aptly 

described as the one faithfully recapitulating the parasitic, immunologic and pathologic 

attributes of the disease, making available large quantities of parasitic material and leading to 

the detailed immunological analysis (Philip et al., 1988). 

The only filarial parasite naturally occurring in man and easily transmissible to 

animals is the subperiodic form of 8. malayi. The use of Brugia species in experimental 

studies are of special significance since they are the same as or closely related to those found 

naturally infecting in man (Sasa, 1976). Edeson et al. (1960) succeeded in transmitting B. 

pahangi from cats to cats and other animals. He reported that the prepatent period was 59 

days in cats and 57 to 84 days in jirds (Meriones unguiculatu). Among the rodents, male 

Meriones unguiculatus and male M, natalensis were susceptible to B. malayi infection. Zaini 

et a]. (1962) reported worms in the heart of hamsters infected with B. pahangi. 

Ramachandran and Pacheco (1965) found that in the early developmental stages B. pahangi 

worms were mainly in the skin, subcutaneous tissues and the caracass of cotton rats, whereas 

after maturity they were only in the heart and pulmonary artery. Ahmed (1967) reported B. 

malayi and B, phangi worms mainly from lymph glands and testes of spleenectomized rats 

and cotton rats. Dissanaike and Paramananthan (1961) reported that B, buckleyi inhabits the 

heart and pulmonary arteries of the Ceylon Hare. Vincent 4 al. (1976) studied the 

*

development of B. malayi, B.pahangi and B. patei in Menones unguiculatus and they 

showed a characteristid tendency to localize within the heart and pulmonary arteries. 

Ash and Riley (1970a,b) succeeded in transmitting both 5. pahangi and B. malayi in 

the Mongolian jird from cats and dogs. The adult worn were found in heart, lungs and 

testes and more numbers were present in the latter than the former. The mode of distribution 

of mature B. malayi was investigated in cats by Ewert, 1971 and In rhesus monkeys and jirds 

by ElBihari and Ewert (1971). He found that the majority of worms remained in the lymph 

vessels distal to the popliteal node for approximately 6 weeks and thereafter, they moved to 

the heart and lungs. 

McCall et 41. (1973) reported that intraperitoneal infection of Meriones unguiculatus 

with B. pahangi lead to a high percent recovery of developing larvae or adult filariae and 

about 90- 100°/o of the filariae that was recovered were located in the peritoneal cavity. 

Pabanyi et al. (1975) found that in M. natalensis infected by 5. malayi, 47% of female 

worms were found in lymphatic system and the prepatent period was 128.8 days. Whereas 

Sanger et al. (1981) had observed a prepatent period of 107 days and recovered majority of 

adult worms from heart and lungs and less number from the testes and lymphatics. Murthy et 

41. (1997) showed that the peritoneal environment of M. natalensis is not conducive to the 

development of B. malayi, which is due to its high macrophage activity in the peritoneum 

compared to that in the jirds, where large ~x.unber of worms were recovered through intra 

peritoneal infection (Mak et al., 1994). Tyagi et 01. (1998) reported that 5. malayi in 

immunosuppressed (cortisone) M, coucha showed prepatent period of 90.7 days and the 

adult worm recovery was also higher as compared to the controls. 

2.4 Changes in host physiology in relation to parasitic infection 

The last few decades have seen great advances in the parasite response and host 

defence, especially on the free radical and the antioxidant defence mechanisms. 

Mamphages, neutrophils and eosinophils nlease supemxide radicals as a host defense 

mechanism to kill the invading parasites (Bannister and Bannister, 1985). Electron 

4 

microscopic and phase contrast microscopic studies have shown th& eosinophils damaged

the tegument by the release of their granules on the surface of the parasite (Ackerman et al., 

1981). Initially it w& believed that the membrane basic proteins were the most powerful 

toxic component of eosinophils responsible for the killing of the parasite. But later, it was 

shown that the eosinophil cationic protein is more toxic than membrane basic proteins in 

vitro. The ability of eosinophils to kill the parasite 0. volvulus (Green et al., 1981) and B. 

malayi (Sim et al., 1982) in vitro had been well demonstrated. 

Batra et al. (1989) studied the status of superoxide dismutase, catalase, xanthine 

oxidase and lipid peroxidation in liver, lungs and spleen of M, natalensis during D. viteae 

infection. Xanthine oxidase and lipid peroxidatuion exhibited stimulation, while these 

antioxidant enzymes showed depression in liver and spleen. On the other band in lungs, the 

antioxidant enzymes were elevated and this lowered the lipid peroxidation. But no parallel 

study had so far been reported regarding B. malayi infection induced changes in M. 

natalensis, especially antioxidant enzymes and its relation to DEC treatment, membrane 

bound enzymes and their damage, testicular damage and the histopathological changes. 

2.5 Chemotherapy 

The microfilaricidal efficacy of DEC was detected by Hewitt et al. (1947) in L. 

carinii infected cotton rats and D. immitis infected dogs and also for the treatment of human 

bancroftian filariasis (Santiago and Stevenson, 1947). Hawking et al. (1950) demonstrated 

that the drug had no direct action rn vitro on mf and adults of L. carinii. But when 

administered to cotton rats infected with the parasite, mf rapidly decreased from circulating 

blood and were trapped in the sinusoids of liver, where they were destroyed by phagocytosis, 

such an action of DEC was interpreted as 'Opsonin-like". Subsequent studies on DEC 

showed strong activity against microftlaria of 0. wlvulus, Loa loa, W. bancrofti and B. 

malayi in man (Zahner and Schaus. 1993). Significant lethal action against adults of B. 

malayi and B. pahangi in cats (Edeson and Buckley, 1959) and against Wuchereria and 

Brugia in man (Chen, 1964) was reported. Kume et al. (1964) and Tulloch et al. (1970) 

reported that daily adminstration of DEC prevented maturation of Dirofilnria immitis in 

dogs. Aubrey and Copeman (1972) reported that Dirojlaria imm? does not mature in dogs 

when adequate doses of DEC was given for 3 successive days ev@ 2 months. Ewert and

Emerson (1975) showed that DEC treatment of cats infected with B. malayi resulted in a 

reduction of living lakae at 2-10 mgkg body weight. Ewert et al. (1983) reported that 

weekly adminstration of DEC was the most effective in B. malayi infected cats. 

DEC has direct physiological effects on eosinophils (Mackenzie, 1980) and after 

DEC treatment eosinophils can be found degranulating on the surface of microfilaria 

(Gibson et al., 1976; Kephart, 1984; Racz, 1982). The mechanism by which filarial parasites 

are killed by DEC is not clearly understood. Most of the clearance occurs in the liver, spleen 

and lungs in association with a m~xed inflammatory cell reaction with large number of 

eosinophils surrounding the dying mf (Woodraff, 1951). Ackerman et al. (1981) had 

demonstrated eosinophilia and elevated levels of membrane basic proteins in banmttian 

filariasis after treatment with DEC. Lammler et al. (1975) reported that M. natalensis 

infected with L. cannii developed anaemia, an increased sedimentation rate and leucopenia. 

After treatment with DEC and a compound HOE 258V. there was a transient change towards 

normal in the peripheral blood values. Morikov el al. (1991) reported that the antioxidant 

propertla of drugs including DEC citrate in various microsoma1 lipid peroxidation models: 

NADPH, ascorbate and CC14 dependent. The most strong antioxidant of direct action turned 

out to be DEC citrate and dipyridamole. The dose most generally for treating banmftian 

filariasis is 6 mg of DEC per kg body weight per day orally for 12 days. For brugian 

filariasis, the recommended doses range h m 3 mg to 6 mg per kg of body weight per day 

upto a total dose of 36-72 mg of DEC per kg of body weight (WHO, 1992). 

The anthelminthic agents such as macwyclic lactone ivermectin, the amoscanate 

derivative CGP 6140 and the bemhazole derivative CGP 20376 were investigated for 

their invibo modulatory effects on eosinophilic effector cells by Tischendorf et al. (1993). 

The results indicated that the reactive oxygen metabolites were produced at an increased rate 

at low doses of ivermectin and CGP 6140. The toxic potential of eosinoplls includes the 

secretion of stored granular cationic proteins and the de novo generation of oxygen 

intermediates. The effect of the macrofilaricidal agent of 2, 2'dicarbomethoxylamine -5.5' 

dibmrimrhlyl ketone on the metabolism of reactive oxygen species in A. viteae and M. 

natalensis was studied by Batra el al. (1992). The host tissues i.e., subcutaneous and the 

adjoining muscle tissues exhibited elevated levels of antioxidants aih( GSH. It is shown that

the compound kills the filarial worn by paralysing the Hlq detoxyfylng capacity without 

altering reactive oxygen species metabolism of the host. 

2.6 Histopathological studies 

The pathological changes associated with larvae and adults of B, pahangi in cat and dog 

included hyperplasia of lymph follicles and reticular cells of the nodal strorna (Schacher and 

Sahyon, 1967). Mak, (1983) had observed inflammatory reactions with granuloma 

formation with the death and disintegration of the W. bancrofti either due to treatment or 

other causes. The dead parasites were engulfed in cellular mass consisting of large number of 

eosinophils, lymphocytes and other mononuclear cells. He had also observed that, during 

heavy microfilaraemia, there was desbuction of mf in the spleen, giving rise to acute and 

chronic inflammatory reactions. Lesions consisting of granulomatous reactions to dead and 

msintegrating larvae had been observed in B, malayi infected jirds (Mak, 1983). The 

development of B. mulayi, B. pahangi and B. palei in the puhonery arteries of Meriones 

unguiculahcr and the associated pathological changes were reported by Vincent et al. (1976). 

The major pathological changes included granulomas induced by larvae and adults, 

obstructive endarteritis and chronic interstitial inflammation. Malone et al. (1976) reported 

cellular infiltration of plasma cells and eosinophlls in organs of hamsters infected with B. 

pahmgi. Live and dead worms with eosinophils and mononuclear infiltration near the area 

were observed in testis. Hamiderosis and gamt cells were observed in lungs. Crandell er 

al. (1 982) reported lesions in organs of fmts infected with B. mdayi and B. pahangi. Case 

et a/. ( 199 1 ) reported fragments of worms in kidney, spleen, liver, lungs, pulmonary blood 

vessel and lymphatics of ferrets infected with B. malayi. Eosinophilic abcesses, epitheliod 

and giant cell granulomas, penvascular cellular infiltration and pigmentation in organs were 

also noticed.

MATERIALS 

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3. MATERIALS AND METHODS 

COLONY MAINTENANCE OF BRUGIA MALAY1 

The B, malayi (in vivo) colony was maintained on the multimammate rats, Mastomys 

natalemis for carrying out various experiments and the protocol is as follows: Aeda aegypti 

(Liverpool strain) mosquitoes were maintained in the laboratory. The adult females were 

starved for 12 hrs and allowed to feed on B. malayi infected Mastomys rats. The fed females 

were maintained on glucose for 13 days until the parasite developed into infective larvae L,. 

Then the mosquitoes were slightly anaesthetmd with ether and gently crushed (with a glass 

rod) in a cavity block containing 0.9% saline. Later the crushed mosquitoes were transferred 

into a h e 1 (with a rubber tube attached to its stem and clamped), which had a layer of 

muslin cloth and 0.9% saline. The filtrate containing infective stage larvae was collected in 

the rubber tube (Lii and Sim, 1983). AAer about one hour, the LG, which have settled at the 

bottom of the rubber tube was collected in a beaker, washed several times with d ie and 

divided into lots of 200. 

Six to eight weeks old M. mtalensis males weighmg 35 to 40 gm were selected and 

inoculated subcutaneously with 200 L, placed in 1 ml of d ie. Normal healthy controls as 

well as infected M. natalemis were maintained for 120 days on pellet diet and water. 

Statistical tests used 

Student's I-test was used to test the significance of values accordingly and simple 

linear regression was used to test the correlation relationship between values.

4. EXPERIMENTS 

CHAPTER 4.1 

CHANGES IN ANTIOXIDANT ENZYMES AND ANTIOXIDANTS IN MASTOMYS 

NATALENSIS AFTER BRUGIA MALAY1 INFECTION AND ITS RELATION TO 

4.1.1. MATERIALS AND METHODS 

DEC TREATMENT 

Male animals of the age of 6-8 weeks, weighing 35-40 gms were used for the study. 

A group of 20 animals was inoculated subcutaneously with 200 infective larvae of B. malayi 

subpenodic strain, while another group of 20 animals was kept as control. The animals were 

maintained for a maximum period of 120 days. 

Four animals from each group were necropsied on 0, 30, 60,90 and 120 days post 

inoculation and assayed for antioxidants and antioxidant enzymes as per the methods given 

below. Organs such as liver, testes, bwin, heart and lungs were excised out, washed 

thoroughly with saline and blotted dry. A 10% (wlv) homogenate was prepared in Tris-HCI 

buffer (0.01 M, pH 7.4) containing 0.25 M sucrose using a potter elvehjem homogeniser 

fined with a power driven teflon pestle. For assays of mitochondnal and cytosolic enzymes, 

subcellular fractionation was done by centrifugmg the 10Ph homogenate in a Sorval (RC5C) 

refrigerated (4'C) centrifuge. The nuclear fiaction was obtained by centrifuging at 800g for 

ten minutes and the mitochondrial fiaction was obtained by centrifuging at 8000g for 20 

minutes. The post mitochondria1 supernatant was centrifuged at 100,000g (Sorval-OTD-50) 

for I hr to isolate the mimsomes. The soluble supmatant served as cytosol. 

4.1.1.1 Superoxide dismutase 

Superoxide dismutase is estimated by the method of Marklund and Marklund (1974). 

'he dtgnt of inhibition of autoxidation of pyrogallol at an alkaline pH by SOD was 

pa a measwe of the enzyme activity.

Ragenb 

1. Tris-HCI buffer . : 0.1 M, pH 8.2 containing 2 mM of diethylene hiamine penta 

acetic acid. 

2. Tris- HCI : 0.05 M, pH 7.4. 

3. Pyrogallol stock solution : 25.2 rng of pyrogallol was dissolved in 1 ml of 0.05 M Tris- 

HCI buffer, pH 7.4 in a test tube stoppered and wrapped with an aluminum foil. 

4. Pyrogallol working solution : At the time of assay 0.5 ml was diluted to 50 ml with 0.05 

M TricHCl buffer, pH 7.4 to give a 2 mM solution and shielded from exposure to light. 

5. Absolute ethanol. 

6. Chlomfonn. 

Partially purified SOD was prepared as described by McCord and Fridovich (1969). 

To 1 ml of the tissue homogenate, 0.25 ml of absolute ethanol and 0.15 ml of chloroform 

was added. After 15 minutes of shalung in a mechmcal shaker, the suspension was 

centrifuged and the supematant obtained constituted the enzyme extract. The reaction 

mixture for autoxidation consisted of 2 ml of the buffer containing DETAPAC, 0.5 ml of 2 

rnM pyrogalbl and 1.5 ml water. Irutially, the rate of autoxidation of pyrogalbl was noted at 

an interval of one minute to three minutes. The assay mixture for the enzyme contained 2 ml 

of 0.05 M Tris-HCI buffer, 0.5 ml pyrogallol, aliquots of the homogenate and water to give a 

final volume of 4 ml. The rate of inhibition of pyrogallol autoxidation after the addition of 

the enzyme was noted. Iron accelerates pyrogallol autoxidation even in trace amounts. 

DETAPAC acts as a chelator and thus prevents the interference from ~ e as ~ well ' as from 

CU" and hIn2*. 

The enzyme activity was expressed in terms of unitsimg protein in which one unit 

corn& to the amount of enzyme that mhibited the autoxidation reaction by 50%. 

The activity of catalase was assayed by the method of S~nha (1972). 

The caFakPc myme pnparation was allowed to split hydrogen peroxide for 

~ifFcmnt paiod of time. The reaction was stopped at specifibdgme intervals by adding

dichromate/acetic acid mixture. The dichromate in acetic acid is reduced to chromic acetate, 

whcn heated in the presence of hydrogen peroxide with the formation of perchloric acid as 

an unstable intermediate. The chromic acetate thus produced is measured at 610 nm. Since 

dichrornate has no absorbance in this region, the presence of this compound in the assay 

mixture does not interfere with the calorimetric determination of chromic acetate. 

Resgents 

I. Stock dichromatdacetic acid reagent : This reagent was prepared by mixing a 5% 

solution of potassium dichromate with glacial acetic acid (1 :3) by volume. 

2. Working dichromatdacetic acid reagent : The stock was diluted (1:s) with water to 

make the working dichromatdacetic acid solution. 

3. Hydrogen peroxide (0.2 M) : 1.0 ml of 30% hydrogen peroxide was made 

upto 45.0 ml with water. 

4. Phosphate buffer : 0.01 M, pH 7.0 

The reaction mixture contained 0.5 ml of hydrogen peroxide, 1.0 rnl of buffer, 0.4 ml 

of water and 0.1 ml of diluted homogenate (1: 10). After 15, 30 and 60 seconds of incubation, 

2.0 ml of dichromateJacetic acid reagent was added. To the control tube, the enzyme was 

added after the addition of acid reagent The tubes were then heated and the colour 

developed was read at 610 nm.The activity of catalase was amved at from the amount of 

hydrogen peroxide consumed and was expressed as poles of hydrogen peroxide 

consumed/rninutdmg protein. 

'Ibis was assayed by the method of Beutler (1984). 

GST catalyses the reaction of I-chloro, 2,4-dinitrobenzene with the -SH group of 

glutahone. The conjugate formed is measured at 340 nrn. 

Reagents 

1. Potassium phosphate buffer : 0.5 M, pH 6.5 

2. I-chlm -2,4 dinitrobemne (CDNB) : 30 mM in 95% ethandl

3. Reduced glutathione : 30 mM 

To I ml of buffer and 0.1 ml of CDNB, 1.7 ml of water was added and incubated at 

37'C for 5 minutes. After incubation, 0.1 ml of reduced glutathione was added and fwther 

incubated for 5 minutes. The reaction was initiated by addmg 0.1 ml of the homogenate. 

The increase in absorbance was measured at 340 nrn at one minute interval for 5 

minutes. The values are expressed as unitslmg protein. 

4.1.1.4 Glutathione Peroxidase 

Glutathione peroxidase was assayed by the method of Rotruck et al. (1973) with 

some modifications. 

Ln this procedure, the rate of oxidation of glutathione by Hz& is used as a measure of 

peroxidase activity. Glutathione remaining in the solution at a given time is determined by its 

reaction with DTNB. EDTA is used in the incubation medium to reduce the non-enzymatic 

reaction rate at a low level. 

Reagents 

1. Sodium phosphate buffer : 0.3 M, pH 7.0 

2. Sodium azide : 1Omhl 

3. Reduced glutathione :4mM 

4. Hydrogen peroxide :2.5 mM 

5. TCA :lPh 

6. Phosphate solution : 0.3 M disodium hydrogen phosphate. 

7. DTNB : 40 mg/100 ml of 1% sod~um cihate 

8. EDTA : 0.8 mM 

9. Standard : 20 mg of reduced glutathione in 100 rnl distilled water. 

This solution contained 20 pg of glutathione/O. 1 ml. 

A known volume of the homogenate was added to the incubation medium which 

containal0.4 ml of buffer, 0.2 rnl of sodium azide, 0.2'ml of ED%, 0.2 ml of Hydrogen

paoxide and 0.2 ml of reduced glutathione. The incubation medium was made upto a final 

volume of 2.0 ml with water. The tubes were incubated at 37'C for 90 and 180 minutes. The 

d o n was terminated by the addition of 1.0 ml of the precipitating agent. The reaction 

mixture was centrifuged and to the supernatant, 6.0 ml of disodium hydrogen phosphate was 

added. One ml of DTNB reagent was added just prior to the calorimetric analysis. The 

absorbance was read at 412 nm against a blank, which contained only 6.0 rnl of disodium 

phosphate and 1.0 ml of DTNB reagent. Suitable aliquot5 of the standards were taken and 

treated in a similar manner. The activity was expressed in terms of pgm of glutathione 

utilizedlrninutelmg protein. 

4.1.1.5 Glutathione reductase 

Glutathione reductase was assayed according to the method of Beutler (1984). 

Glutahone reductase catalyses the reduction of oxiW glutathione (GSSG) by 

NADPH or NADH to reduced glutathione. The activity of the enzyme is measured at 340 

mn following the oxidation of NADPH. GR is a flavin enzyme and it has been found that it 

is not fully activated by FAD in normal samples. Complete activation of apoenzyme requires 

the preincubation of enzyme with FAD. This is done prior to the addition of GSSG or 

NADF'H to the reaction system. 

Reagents 

1. Tris-HC1 : 1 M with EDTA 5 mM, pH 8.0 

2. FAD : l o p 

3. Oxidised glutathione (GSSG) : 0.033 M 

4. NADPH :2mM 

To 100 p1 of Tris-HCI buffer, 20 tr] of the sample, 1.58 ml of water and 200 pl of 

FAD is added and incubated at 3PC for 10 minutes. To the test sample, 200 tr] of GSSG is 

lddcd and again incubated at 3PC for 10 minutes. Later 100 tr] of NADPH is added to both 

the blank and the test samples and the decrease in optical density was measured against the 

at 340 tun (3PC). The values are expressad as uniWgm proth

4.1.1.6 Glucose-6-phosphate dehydrogenase 

modifications. 

This enzyme was assayed by the method of Z i et a/. (1958) with some 

Glucose-&phosphate dehydrogenase is assayed by measuring the increase in 

absorbance, which occurred at 340 nm when NADP is reduced to NADPH. The reduction 

takes place when two electrons are transferred fkom glucose-6-phosphate to NADP in the 

reaction catalysed by glucose-&phosphate dehydrogenase. 

Reagents 

I. NADP : 0.2 mM 

2. Tris-HCI buffer : 0. l M, pH 8.0 

3. Magnesium chloride : 0.1 M 

4. Glucose-6-phosphate : 6 mM 

To 0.1 ml of NADP, buffer and MgC12, 0.5 ml of water and 0.1 ml of the 

homogenate was added. After 10 minutes, the reaction was initiated by the addition of 0.1 ml 

of glucose-6-phosphate solution. The increase in optical density was measured at 340 nm at 

2S°C. The activity of the enzyme was expressed in terms of units/mg/protein where one unit 

cornponds to the amount of the enzyme required to bring about a change in optical density 

of 0.01 /minute. 

4.1.1.7 Reduced glutathione 

Reduced glutathione was estimated by the method of Moron eta/. (1979) 

This method is based on the development of yellow colour when 5,5'dithio-bis-2- 

n~trobic acid (DTNB) is added to the compounds containing sulphydryl pups.

Reagent6 

1. DTNB : 0.6 mM in 0.2 M phosphate buffer, pH 8.0. 

2. Phosphate buffer : 0.2 M, pH 8.0. 

3. TCA : 5% 

4. Standard : 10 mg of reduced glutathione in I00 ml water. This contained 

10pgm of glutathione in 0. I ml solution. 

To 0.1 mi of the homogenate, 1.0 ml of water and 2.0 ml of precipitating agent were 

added, mixed thoroughly and centrifuged after 5 minutes. To an aliquot of the supernatant, 

2.0 ml of DTNB reagent was added and made upto a final volume of 3.0 ml with phosphate. 

buffer. The absorbance was read at 412 nm against a reagent blank The amount of 

glutathione was exprrssed as pgm of glutathionel mg protein. 

4.1.1.8 Total thiol groups 

(1 %8). 

Total thlol groups were estimated acconhg to the method of Sedlack and Lindsay 

The sulphydryl pups in tissues were determined by using the Ellman's reagent. In 

this method, DTNB is reduced by -SH groups to form 1 mole of 2-nitro, 5-mercaptobenzoic 

acid per mole of -SH. 

Reagents 

1.0.01 MDTNEI : 99 mg DTNEI dissolved in 25 ml of absolute methanol. 

2. Tris-HCl buffer : 0.2 M, pH 8.2 containing 0.02 M EDTA. 

The sampk was pnpartd by homogenising 100 mg of tissue in 4.0 ml of 0.02 M 

EDTA. To an aliquot of the tissue homogenate, 1.5 ml of Tris-HCI buffer @H 8.2) and 0.1 

ml of DTNB was added, mixed and made upto 10.0 ml with absolute methanol. A reagent 

blank without the sample and sample blank without DTNEI were prepared in the same 

manna. The test tubes were stoppered and allowed to stand with occasional hkmg for 15 

minutes. The reaction mixture was cenhifuged at 3000g at R.* for 15 minutes. The

absorbance of the clear supernatant was read at 412 nm. Calibration curves were obtained 

with reduced glutathione as standard. Values were expressed as pgm of glutathiondmg 

protein. 

4.1.1.9 Ascorbic acid 

Ascorbic acid was estimated by the method of Omaye ef al. (1979). 

Ascorbic acid is oxidzed by copper to form &hydroascorbic acid and 

diketogluconic acid. When treated with 2.4, dmirophenyl hydrazie, it reacts to form the 

derivative bis 2,4, dinitrophenyl hydrame. This compound in strong sulphuric acid 

undergoes a rearrangement to form a product with an absorption band that is measured at 

520 nm. The reaction is run in the presence of thlourea to provide a mildly reducing medium, 

which helps to prevent the ~nterference from non-ascorbic chromogens. 

Reagents 

1. TCA : 5% 

2. DTC reagent : 3 gms DNPH (ditropbenyl hydrazine), 0.4 gms thiourea and 0.05 gms of 

copper sulphate were dissolved in 10 ml of 9N sulphuric acid and made upto 100 ml with the 

same solution. 

An aliquot of the homogate was precipitated with ice-cold TCA, cenhihged for 20 

minutes at 3,500g and 1.0 ml of the supernatant was made upto 3.0 ml with 5% TCA and 

then, treated with 0.2 ml of DTC and incubated for 3 hrs at 37'C. Then 1.5 ml of icecold 

65% sulphuric acid was added, mixed and kept at RT. for an additional 30 minutes. Blank 

contained only 3.0 ml of TCA. Standards in the range of 10-50pgms were treated in the 

same way. The intensity of the color was measured at 520 nm. Results were expressed as 

pgm of ascorbic acid! mg protein.

4.1.1.10 Lipid peroxidation 

Lipid peroxidation in the tissue was estimated by the method of Stoch and 

Dormandy (197 1 ). 

Malondialdehyde (MDA), produced during peroxidation of lipids, served as an index 

of lipid pemxidation. In this method MDA reacts with thiobarbihuic acid to generate a 

coloured product, which is read at 532 nm. 

Reagents 

I. Isotonic phosphate buffered saline (PBS), pH 7.4. 

2. TBA : 1% in 0.05 mMit sodium hydroxide. 

3. TCA : 10°/o solution in 0.1 moVlit dmn arsenite. 

4. Standard MDA : stock solution of MDA (400 nmol/ml) was prepared using 1,1,3,3 

tetraethoxy propane. This was stored at 4OC and diluted with distilled water to make 

working standard of 50 dm1 concentration. 

5. EDTA : 0.1 momit. 

One rnl of the homogenate was suspended in 0.1 ml of PBS and 0.5 ml of TCA, 

followed by 2.0 ml TBA and 0.075 ml EDTA. Tubes were mixed and kept in a boiling water 

bath for I5 minutes. Tubes were cooled at R.T. and centrifuged. Absorbance was read at 532 

nm. Each test sample had its own blank tube, which was not boiled. Subtraction of 

absorbance of unboiled sample from boiled sample eliminated absorbance increase due to 

any non-TBA reactive material in the sample. EDTA was added to chelate any ironlother 

metal in the ahact which, otherwise can initiate lipid peroxidation during boiling and may 

result in falsely elevated TBA reactivity. Results were expressed as nmoles of MDA 

fonndmg protein! 20 minutes. 

4.1.1.11 Protein 

Protein was estimated by the method of Lowry et al. (I%])

-eats 

I. Lowry's reagent :Solution A: 2% sodium carbonate in 0.1 N sod~um hydroxide solution. 

prior to use. 

: Solution B: 0.5% copper sulphate in 1% sodium potassium tartarate. 

The reagent was prepared by mixing 50 ml of solution A with 1.0 ml of solution B 

2. Folins Ciocalteau w ent 

To 0.1 ml of (1:lO) diluted homogenate, 0.9 ml of water and 5.0 ml of Lowry's 

reagent was added, mixed well and kept at RT. for 10 minutes. To this, 0.5 ml of Folin's 

reagent was added and mixed. 

Standard Bovine serum albumin solution containing 20-100 micrograms of protein 

and a blank were treated in a similar manner. The intensity of the colour developed was 

measured after 20 minutes at 660 nrn. The values were expressed as mg/p tissue. 

4.1.2 RESULTS 

The experimental animals w m necropsied on 0 day and on 30,60,90 and 120 days 

post-inoculation with infective larvae of B. malayi for biochemical studies. On 120th day 

post-inoculation, various organs were examined for the presence of adult worms. The worm 

recovery was 25.1 5linfected animal and they were distributed in lungs, testes and heart and 

liver and brain did not have adult worms. Lungs contained the maximum number of worms 

followed by testes and heart (F1g.2). 

Biochemical analyses were done to determine whether the B.malayi infection caused 

any change in the production of oxygen €ree radicals, antioxidants and antioxidant enzymes 

in different organs of the host animal, M. natalemis.

Fig 2:B.malayi distribution in Lungs 

testes & heart of M-natalensis 

Testes (37.07) 

Lungs (44.38) 

Figures in parenthesis are %age of adult 

worms recovered

4.1.2.1 Changes in antioxidant enzymes and antioxidants in control and 

B.m&yi infected animals 

4.1.2.1.1 Superoxide dismutase 

The changes in the activity of SOD in the organs of infected and control animals are 

presented in Figs. 3-5. In control animals, all the organs (viz., the liver, testes, brain, heart 

and lungs) showed a continuous increase in the activity of this enzyme from 0 to 120 days. 

Contrary to this, in the testes, heart and brain of the infected animals, the activity of this 

enzyme showed an increasing trend upto 30 days and thereafter the enzyme activity declined 

s~gn~ficantly w0.05), while in the liver, the activity increased sigmficantly upto 60 days and 

thereafkr declined. And in the lungs the enzyme activity showed increasing trend throughout 

and the activity was higher than that of control . 

4.1.2.1.2 Catalase 

The changes in the catalase activity are shown in Figs. 6-8. All the organs of the 

control animals showed increase in catalase activity from 0 to 120 days. Compared to this, 

the tesw, heart and brain of the infected animals showed decline in the enzyme activity from 

30th day while liver showed significant m.05) decline from 60th day. However, the lungs 

showed a steady increase in catalase activity from 30 days (significant at F0.05) and the 

increase was much higher than that observed in control. 

4.1.2.13 Clutathione-s-transferase 

The activity of GST in various organs of the infected and control animals are shown 

in F~gs. 9-1 1. The GST activity was found to increase in control animals from day 0 to 

day 120.

LIST OF ABBREVIATIONS USED IN THE GRAPHS 

Liver - Li 

Testes - T 

Heart - H 

Brain - B 

Lugs - Lu 

Normal -N 

Infected - 1 

Na' K' ATPase - Na 

ca2' ATPase - Ca 

Mg 2' ATPase - Mg 

Haemoglobin - Hb 

Acetylcholinesterase - Ach est 

y-glutarnyl transpeptidase - G P 

Alkaline phosphatase - AP 

Catale -CAT

Fig.3: Effect of B.malayi on SOD in 

in Liver and Testes of M.natalensis 

0 30 w so 320 

Days of lnfecuon 

4 - 

30- 

25 - 

LI-N + LI-l - T-N - T-1 


Brain and Heart of M.natalensis 

0 30 60 90 120 

Days of Infection 

+ B-N --t 8-1 - H-N e H-l 

Fig.5: Effect of B.malayi on Lungs 

of M. natalensis 

0 30 W 90 120 

Days of Infection

:iq 

Fig.6: Effect of B.malayi on Catalase 

in Liver and Testis of M.natalensis 

; - 9 16 

5 12 

10 

e 

"- 

34 - 

1 32- 

- 3 

9 30- 

= 21). 

8 

I 20 

24 - 

22 

30 BO UO i 20 


-- ti-N + La-l - T-N * T-1 

Fig.7: Effect of B.malayi on Catalase 

in Brain and Heart of M.natalensis 

0 30 60 90 120 


c B-N -+- 6-1 - H-N - H-l 

Fig.8:Effect of B.malayi on Catalase 

in Lungs of M. natalensis 

-4 

30 60 90 120 

Days of Infection

Fig.9: Effect of B.rnalayi on GST in 

Liver and Testis of h4.natalensrs 

24. . 

2 2- 

1 - 

= 0. 

0 2 

0 

0 0. 

0 55- 

Days of lntect~on 

+ b-N -+- LI-1 - T-N -++ T-l 

Fig.10: Effect of B.malayi on GST in 


30 80 SO 120 


t 8.N --c 6-1 - H-N - H-1 


Lungs of M.natalensis 

Days ot \ntection 

I

Whereas in the infected animals, organs such as testes, brain and heart showed significant 

0.05) decline in GST activity from 60 to 120th day while the liver showed a decreasing 

bend from 90 to 120 days. However, in the lungs, the enzyme activity showed a significantly 

wO.05) increasing bmd from60 to 120 days, but was lower than that of the control. 

4.1.2.1.4 Glutathione peroxidase 

The organs such as testes, brain and heart of the infected animals showed an increase 

in GPx activity upto 30 days (Figs. 12-14) and thereafter it declined gradually to significant 

(pcO.05) level as against an increase in the comls. The liver showed significant decline 

w.05) from 0 to120 days while the lungs showed significant increase w0.05) from 30 

days as in the case of control. However, the increase in enzyme activity in infected lungs was 

much lower than that observed in the control 

4.1.2.1 .S Glutathione reductase 

The activity of GR in Liver, testes, brain and heart of the mfected animals was found 

to decrease significantly @

Fig.12: Effect of 6.rnalayi on GPx in 

Liver and Testis of M.natalensis 

22. 

21- . 

14- 

13- 

12, 

111, 

10- 

g 14. 

- 3 # 

2 12- 

= 10t% 

El 

z 8- 

11- 

'. 

30 W 90 120 


-) LI-N C LI-l - T-N - T-l 

Fig.13: Effect of 6.malayi on GPx in 


- 

4. 0 30 M 90 120 

10- 

0 s- 

Days of lntoct~on 

Fig.14: Effect of B.malayi on GPx in 

Lungs of M. natalensis 

0 30 80 90 120 


.

- a 

g 25- 

to- 

Fig.15: Effect of B.malayi on GR in 

Liver and testes of M. natalensis 

0 30 M) 80 120 


Fig. 16: Effect of B.malayi on GR in 


0 30 60 90 120 

Days of infection 

+ 6.N --C B-I - H-N - H-l 

Fig.17: Effect of 6.malayi on GR in 


0 30 90 120 

Days of infection

Fig. 18: Effect of B.malayi on G6PDH in 

Liver and Testes of M. natalensis 

4 - 

3 5- 

3 -I I 

30 M 90 120 


-) LI-N 4 b-l - T-N - T-l 

Fig.19: Effect of B.malayi on G6PDH in 


5 i 

30 M So 110 


-, 8.N - 

8.1 - 

H-N - H-l 

Fig.20: Effect of B.malayi on GGPDH in 

Lungs of M. natalensis 

16- 

1s. 

g 14. 

% 13, / 


Lu-N -+- Lu-l 

/ 

./," 1 

i 

120

4.1.2.1.7 Reduced glutathione 

The GSH levels in the control animals showed an increasing trend throughout the 

study period (Figs. 21-23). Whereas, in the infected animals, the liver, testes and heat 

showed significant w0.05) decrease in the activity from day 30 to day 120 and in the brain 

from day 0 to day 120. However, the lungs showed increasing trend from 30 days to 120 

days as in control, but the activity was lower than that of the control. 

4.1.2.1.8 Total thiol group 

The total hi01 status in the organs of the control animals showed a steady increase 

from 0 to 120 days (Figs. 24-26), whereas, the liver, testes and brain of the infected animals 

showed a significant decrease w.05). The heart also showed similar trend, but from 30th 

to 120thday only. In the lungs, however, the thiol level declined from 0 to 30 days and then 

increased significantly wO.05) as in the control. But the values were lower than the control. 

4.1.2.1.9 Ascorbic acid 

Ascorbic acid content in the organs of mfected and contml animals are pmted in 

Figs. 27-29. In the organs of the control animals it increased from 0 to 120 days whereas, in 

the infected animals, significant decrease w0.05) was observed throughout the study pencd 

in testes and heart and it decreased in liver and brain from 30th day to 120th day. However, 

in the lungs significant increase (~~0.05) was noticed from 30th day to 120th day and the 

values were lower than that observed in control. 

4.1.23 Lipid peroxidation 

The level of lipid peroxidation was measured in terms of malondialdehyde 

released and is presented in Figs. 30-32. The LPO was significantly higher w0.05) in 

liver, testes, brain and heart of both control and B.m~layc infected animals. On the 

contrary, in the lungs of infected animals there was significant wO.05) reduction in LPO 

levels as compartd to the controls.

12- 

3 m- 

15- 

lor, 

Fig.21: Effect of B.malayi on GSH in 

Liver and Testis of M.natalensis 

30 w 120 

Days of tnfectlon 



7 -. 

54 

0 30 0 90 120 


+ 8-N + 8-1 -C H-N ---+ H-1 




5 12g 

10- 

= 

Fig.24: Effect of B.malayi on SH Group 

in Liver and Testis of Mnatalensis 

6- 

Days of ~nfecbon 

+ LI-N + b-l - T-N C- T-1 

Fig.25: Effect of B.rnalayi on SH Group 

in Brain and Heart of M.natalensis 

- 

4- 0 30 W 00 120 


Fig.26: Effect of B.malayi on SH Group 

in Lungs of M.natalensis 


L,,.N . . Lu-l 

. 

sf!

Fig.27: Effect of B.malayi on Ascorbic 

acid in Liver & testes of M.natalensis 

2 0 ~ I 

64 1 

30 00 90 1 zc 

Days of Infecbon 

Fig.28: Effect of 6.malayi on Ascorbic 

acid in Brain and Heart of M.natalensis 

301 I 

04 

30 6a 80 

I 

12C 


B-N - 0.1 --- W.N + n.1 

9 57 

Fig.29: Effect of B.malayi on Ascorbic 

acid in Lungs of M.natalensis 

0- 

Days of inteetlon

137 

12- 

11 - 

a- 

? 5. 

s 6," 

-9 6. 

5 58- 

= ,- 

4 5. 

Fig.30: Effect of B.malayi on LPO in 

Liver and Testis of Mmatalensis 




Days of lntecbon 



44 

0 90 120 

Days of cnfsction

4.1.2.3 Change in the antioxidant enzymes, antioxidants and lipid 

peroxidation in infected animals after Diethylcarbamazine @EC) 

administration 

A group of 16 animals were selected of which, half of them were infected with B. 

malayi and the other served as control. Four animals each from the control and infected 

group were adrmnistered with DEC (6 rng kg body weight orally for 5 days) and grouped as 

(I) control (uninfected, Group I), (2) control-DEC treated (Group 2), (3) infected (B.malayi 

infected, Group 3) and (4) infected-DEC treated (Group 4). The animals were necropsied 

and the ~nfluence of the drug, DEC on the antioxidant enzymes, antioxidants and LPO in 

various organs were studied. 

4.1.23.1 Superoxide dismutase and catalase 

The activity of SOD and catalase were significantly low @

Fig.33:Effect of 6.malayi on SOD in 

Liver & Testis after DEC treatment 

, 

9 

8 

7 

- 6 

g 5 

S 4 

s 2 

1 

0 

Testis 

Organ 

1 Control Control+D Infected Infected+[) 1 


Brain,Heart & Lungs after DEC treatment 

Organ 

Lungs 

Control Coml+D Infected Infected+[)

Fig.35: Effect of B.malayi on CAT in 


T&ie 

Organ 

I Control Control+D Infected lnfected+D I 

Fig.36: Effect of B.malayi on CAT in 


Organ 

( Control Control+ D Infected Infected+ D 1



Liver Testis 

Organ 

I Control = ControlcD infected f--/ Infected+ D / 

1.4 

g 1.2 

I1 

5 0.8 

K 0.6 



0.4 

0.2 

0 

Organ 

Lungs

m 20 

0) - 15 

9 

g 10 

il! 5 

0 



Liver Testis 

Organ 

Control Control+D Infected Infected+[) 1 



". 

Organ 

Lungs 

Control Coml+D Infected Infected+[)

The GR and G6PDH activities in liver, brain, heart and lungs of B.mlayi infected 

animals (Group 3), which were decreased, were restored to nonnal level (significant at 

pc0.05) after DEC treatment (&up 4) (Figs. 41-44). However, in testes, despite an increase 

in activity, these enzyme levels did not reach the levels found in the testes of the control 

animals. In control (Group 2), the administration of DEC, did not bring about any sh~ft in the 

activity of these enzymes. 

4.1.233 Antioxidants and lipid peroxidation 

The decteased levels of GSH, total thiol and ascorbic acid in liver, brain, heart and 

lungs of the infected animals (Group 3) were restored to normal levels (significant at F0.05) 

after DEC trtatment (Group 4) (Figs. 45-50), however, in lungs, GSH levels did not show 

any significant difference. In testes, although there was significant @

30 

25 

8 - a 20 

2 15 

C 

I 10 

I 

5 

0 

35 

3 30 

2 25 

9 20 

C 

I 15 

f 10 

5 

0 

Fig.41: Effect of B.malayi on GR in 


Tor* 

Organ 

Control Control+D Infected Infected+D 

Fig.42: Effect of B.rnalayi on GR in 


Organ 

Lungs 

Control Control+D lntected Infected+D

Fig.43: Effect of B-rnalayi on GGPDH in 


Teatis 

Organ 

Control Control + D Infected I niected + D 

Fig.44: Effect of B-malayi on GGPDH in 

Brain.Heart & Lungs after DEC treatment 

16 

UJ 14 

5 12 

3 10 

5 

6 

2 4 

2 

0 

Organ 

Lungs 

Control Control+D Infected Infected+ D

Fig.45: Effect of B.maiayi on GSH in 


[ Control 

Organ 

~ontrol+ D Infected Infected + D I 

35 

8 30 

25 

P 20 

3 15 

Z 10 


brain,Heart & Lungs after DEC treatment 

5 

0 

Lungs 

I Control 

Organ 

Control+D Infected Infected + D I

Fig.47: Effect of B.malayi on SH in 


Organ 

I Control Control+D Infected Infected+ D I 

18 

16 

$ 14 

- 3 12 

g 10 

C 8 

a 6 

8 4 

2 

0 

fig.48: Effect of B.malayi on SH in 


Hurt Lungs 

Organ 

Control Control+D Infected Infected + D

Fig.49: Effect of B.malayi on Vit-C in 


Liver Testis 

Organ 

Control Control+D Infected Infected + D 

Fig.50: Effect of B.malayi on Vit-C in 


25 

3 20 

15 

5 

z" lo 

5 

0 

Organ 

Lungs 

Control Control+D Infected Infected+ D



Organ 

I Control Control+D Infected Infected+ D 1 

12 

g 10 

- 3 

m 8 

> 

5 

2 

2 


Brain,Heart & Lungs after DEC treament 

0 

Organ 

Lungs

4.1.3 DISCUSSION 

4.1 3.1.1 Toxic oxygen radicals 

The last few decades have seen great advances in understanding the parasite induced 

host physiology in t m of host defense mechanism especially the role of oxidants (free 

radicals) and antioxidant enzymes (Callahan et al., 1988). A free radical is any species 

capable of independent existence that contains one or more unpaired electrons. They are 

highly reactive and unstable and can injure some biological targets such as proteins, lipids, 

carbohydrates and key molecules in membrane and nucleic acids. Moreover, free radicals 

initiate autocatalytic reaction whereby, molecules with which they react are themselves 

converted into free radical and thus propagate the chain of damage (Halliwell and 

Guneridge, 1989). 

The free radicals may be initiated within cells during the normal cellular metabolic 

processes (Klebanoff, 1974; 1980), by ionizing radiation and certain anti-parasitic drugs 

(Docampo and Moreno, 1984) and the host response is also a potent source of oxidants. 

Molecular oxygen is a buadical and a relatively unreactive compound. It undergoes a four- 

electron reduction to form water. However, it can be metabolized inviw to form highly 

reactive derivative oxidants. A series of sequential one-electron transfer yields three reactive 

intermediates superoxide anions, hydrogen peroxide and hydroxyl mhcal. When two free 

radicals meet, they can join their unpaired electrons to form a covalent bond. When radicals 

react with non-radicals. new radicals are generated invivo, which is likely to set off free 

radical chain reaction (Halliwell et al., 1995; Thomas, 1995) (Fig. 53). The best studied 

biologically relevant fra radical chain reaction is lipid peroxidation. 

4.13.1.2 Lipid peroddation 

Lipid peroxidation is an important consequence of oxidative cellular damage 

(Plea and Witschi, 1976) involved in a variety of diseases and stress (Slater, 1984; 

Halliwcll and Guttnidge, 1989). LPO involves the direct reaction of oxygen and ply 

unsaturated fatty acid (PUFA) to form fm radicals and semistable -ides (Tappel,

FIG. 53 FORMATION AND DETOXIFICATION OF REACTIVE 

OXYGEN SPECIES IN BIOLOGICAL SYSTEMS. ( Clark et al., 1986) 

0; L-- .v,,%!Gq 

O? ' - 

,Fez' GSH GSSG 

- h-+* 

t 

616 'qbi2 

-Fe3' i hot4 

CELL 

TOXICITY 

Superoxide GSH-PX - glutathione peroxidase 

SOD - Superoxide dismutase GR - Qlutahone reductase 

GSH - reduced glutathione .OH - hydroxyl radical 

GSSG - oidised glutathtone S - secondary radical

1973). The two major systems of LPO are non-enzymatic ascorbate induced system 

(Ottolenghi, 1959) and enzymatic NADPH induced system (Hochstein and Enister, 1963) 

mediated by NADPH cytochrome-c-reductase. Substances such as ascorbate and ferrous 

ions, which induce ~e" to ~e", and peroxides enhance LPO (Halliwell and Guaeridge, 

1989). LPO decreases membrane fluidity, increases leakiness of the membrane (Jacob 

and Lux, 1968) to substances that are normally impermeable, inactivates membrane 

bound enzymes (Kesner et a/., 1979) along with the fatty acid and antioxidant depletion. 

Damage to the membrane may be subtle and involve only small changes in the 

composition of fatty acids, yet often sufficient to greatly increase the susceptibility of the 

membrane to oxidative damage (Halliwell and Guneridge, 1985). 

Oxygen radicals are generated inside leucocytes enabling them to kill phagocytosed 

microorganisms (Babior el al., 1973). Unfottunately for the host, these leucocytes can also 

secrete superoxide radicals along with other mediators from their outer membrane into the 

surroundings (Nathan and Root, 1977). This indiscriminate and self-inflicting process 

contributes to the tissue damage or inflammation (Fig. 54) (Fantone and Ward, 1982; Weiss 

and LoBuglio, 1982; Fteeman and Crapo, 1982) and has the capacity to cause tissue damage 

in parasite induced disease. Johnson and Ward (1982) reponed that these events occur much 

more vigorously when certain receptors on the surface of leucocytes, such as those 

responsible to antigen-antibody complexes (Fc receptors) and to complement components 

are activated. Thus endothelial damage in he rat, ~nitiated by immune complexes or C5 

activation, can be prevented by depleting animals of neutrophils (Till er al., 1982), infusing 

SOD or catalase (Johnson and Ward, 1981; McCormick er a!.. 1981). radical scavengers 

(Ward er al., 1983% Fligiel era/.. 1984; Fox 1984) and iron chelators (Ward ef al., 1983b). 

Baba ef al. (1989) reported that, in M. nalalensis infected with D. vileae, there was 

increase in LPO in the organs such as liver and spleen and decrease in lungs. Red blood cells 

infected with P. bwghei have been observed to show five-fold increase in LPO than the 

normal (Etkin and Eaton, 1975). Mahdi et a/. (1992) reported increased levels of lipid 

Wxid~~ in brain of M. natalemis infected with P. berghei. Elevated levels of LPO 

products during malarial infection had been reported (DesCamps er a1.,,1987; Nalr ef al., 

3

FIG. 54 THE SEQUENCE OF EVENTS IN FREE RADICAL MEDIATED 

LIPID PEROXIDATION (FANTONE AND WARD, 1982) 

R'+ PUFA -+ PIJFA'. R initialion phase 

puFi.oi -> PROTEIN (- propagation 

l'l'i A scavenger' WFA.OH. H,O henc conjugates 

malonyld~aldchydr 

crhanc penlanc 

phase 

Free & ~ Q~ndude S reduced glutahon+ other hols (for mple, protan hols). 

vitunin $ d ud flat em^ Scavepyg auyim include supmade htase, 

-dBkuat~av=

1981), and their increase in human patients with P.falciparum infection are associated with 

complications and death. P. vinckei infected RBCs have also been reported to generate 

significant levels of malondialdehyde (Clark et al., 1984a, b; Stocker et al., 1985) and it has 

been suggested that LPO can be set into motion whenever conditions of increased oxidative 

stress or decreased antioxidant defences occur in the cell. 

Production of reactive oxygen species in mice infected with P. berghei and P. yeolii 

had been reported (Li and Li, 1987; Dockrell and Playfair, 1984). It has been suggested that 

HI@ and the reactive oxygen radicals from the respiratory burst have the potential to initiate 

LPO, resulting in the formation of toxic aldehydes. Clark ei al. (1987) reported that products 

of LPO are toxic and inhibitory against malarial parasite P. falcipam. The products of 

macrophage secretions such as oxygenderived free radicals and tumour necrosis factor are 

shown to kill the human malarial parasite P. falciparum invitro (Worncraft er al., 1984) and 

also initial stages of P. berghei in rodents (Wozencrafl et al., 1985). Hydrogen peroxide 

injected intravenously caused a drop in parasitaemia in mice with P. yeolii or P. chaubadi 

and can kill P. yeolii and P, berghei maintained invifro (Dockrell and Playfair, 1983). Gharib 

et al. (1999) reported a two fold increase in LPO and decreased antioxidants at the site of 

pulomatous inflammation in liver infected with Schistosoma mansoni. 

ln the present study, it has been found that following infection of B.malayi, LPO 

was sigruficantly high in liver, testes, brain and heart of M.natalemis, whereas in lungs it 

was low. A negative correlation was observed between the LPO and activity of SOD1 

catalase in liver, testes, brain and heart (Figs.55-58, 60, 62-64) and the correlation was 

significant 0 .05) in liver (SOD I= -0.96). testes (SOD r= -0.97), Brain (catalase -0.89) 

and heart (catalase r--0.92) only. The LPO levels, on the contmy, had a positive correlation 

with catalase in testes and with SOD in lungs, however it was significant in the lungs (SOD 

r= -0.88, p4.05) only (Figs. 59 & 61). This increase in LPO is due to the decrease in the 

antioxidant caqmcs and GSH production in these organs. But in lungs LPO operates below 

the normal level, although superoxide anions may be produced at a greater rate than in 

control, it however, appears to be removed by elevated SOD and catalase (sec. 4.1.2.1.1 

Br4.1.2.1.2).

0 

Fig.55:Relationship between change in 

SOD and LPO levels in liver 

SOD 

1 

Observed values + Expected values 


1 

r=007 p

1 

0 


SOD and LPO levels in heart 

i l l - 

-5 1 

a 

-7 

-6 -1 0 5 0 0.5 1 1 . 5 2 2.5 3 35 4 

SOD 

( 0 ObSe~0-d values - Expected values I 


SOD and LPO levels in lungs 

= Obwed values - 

SOD 

Expected values

Fig.6O:Relationship between change in 

catalase and LPO levels in liver 

Q 

1 0 

I 

1 2 3 4 5 

, 

6 

y-- 

7 

catalase 

/ Obse~ed values + Expected values I 

Fig.61 :Relationship between change in 

catalase and LPO levels in testes 

30 I5 10 5 0 5 0 

catalase 

- - 

8 !5 

2 5 

r Observed values '-- Expected values 


catalase and LPO levels in brain 

r = 0 8 0 p

1 - 

0- ' 1 

5 - 

6 - 

7 - 


catalase and LPO levels in heart 

r=-0 92 pcO05 

y= 1 3 7 0 93.x 

B i 

1 0 1 2 3 

catalase 

4 5 6 7 

Observed values - Expected values 

1:1* 

1 

,z 1 

0 5 - 


catalase and LPO levels in lungs 

0 

43 -6 4 -2 0 2 4 6 

catalase 

I Observed vdues + Expected values I

The LPO altered during infection of B.malayi was brought back to normal levels after DEC 

treatment and this has happened due to the restoration of the activity of antioxidant enzymes 

and GSH to normal levels as observed in a further experiment. In testes, the LPO was 

remarkably high compared to controls and even after DEC treatment, did not reach normal 

level. This may be due to the irreversible tissue damage as evidenced in histopathological 

studies (sec. 4.4.2.2). 

4.1.3.2 Superoxide dismutase and catalase 

The cells are rich in the antioxidants such as GSH and an antioxidant enzyme like 

catalase, SOD. GR and GPx and contains a proteolytic system that can hydrolyze oxidatively 

modified protein (Halliwell and Gutteridge, 1989). Disrnutation of superoxide anion is 

catalysed by SOD, which yields Hz@, and it is Wer decomposed by the enzyme catalase 

and glutahone peroxidase. This reaction plays a major role in protecting the cell membrane 

from H2q (Jacob er a/., 1%5; Panicker and Zyer, 1969), but a decrease in the activities of 

SOD, catalase. GPx, GR and GSH level can exacerbate LPO. 

Studies conducted on D.viteae infected animals (Baba et a/., 1989), indicated that the 

act~vity of SOD decreased in liver and spleen, but increased in lungs. Red blood cells 

parasitized by P.krghei (Fairfield et a/.. 1983; 1986) and P. vinckei (Stocker et al., 1985) 

contained less SOD and catalase than the RBC's of uninfected. Altered levels of SOD have 

also been nponed in patients with P. falcipanun mfection and in mice with P. berghei and 

P. vinckei infection (Areekul and Boonme, 1985, 1987; Suthipak el nl., 1982; Stocker et al., 

1985; Fairfield el a/., 1988). Areckul and Boom (1987) had shown that higher 

parasitaemia of RBC's, due to Pjalcipnun is associated with higher SOD activity 

compared to the uninfected ones. Fairfield et a/. (1983) reported that P. berghei devoid of 

endogenous SOD adopts e yhqte SOD for protection against the deleterious effect of 

superoxide radicals and consequently reduces the host SOD level. SOD and catalase were 

decnesed significantly in P, v im malaria in patients (Mathews and Selvam, 1991). SOD 

showed restoration of enzyme activity while. catalase activity was increased significantly 

after primaquine trratmcnt.

Catalase had been reported to be responsible for the detoxification of Hz@ (Brenner 

and Alison, 1953; ~icholis, 1965). Small quantity of erythrocyte catalase was observed to 

exist in inactive form viz., catalase complex I1 (Leibowitz and Cohen, 1968). The conversion 

of complex I1 to active form is dependent on the reducing agent NADPH (Eaton el al., 

1972). Under conditions of increased oxidative stress, it is likely that more NADP rather 

than NADPH is formed, which is required for the activation of catalase. The decreased 

NADPH and G6PDH leads to reduced catalase activity (Eaton er al., 1972). The reactive 

oxygen radicals can also inhibit the activity of catalase and GPx as had been observed by 

Hodgson and Fridovich (1975) and Searle and Wilson (1980). 

In M nalulensis infected with D. viteae, decreased activities of catalase and 

xanthine oxidase and increased LPO were observed in liver and spleen (Batra el al.. 1989). 

And on the other hand, in lungs the elevated activity of xanthine oxidase and catalase 

prevented the LPO. Catalase activity was also found to be low in P, vivm (Mathews and 

Selvam, 1991) and P. ful~ipanun (Areekul and Boonme, 1987) infected human erythrocytes, 

and in mrce erythrocytes infected with P. berghei (Nair e! al., 1984) and P. Vinckei mckard- 

Maureau el al.. 1975). 

In the pment study, in the liver, brain, testes and heart of the infected animals, the 

actlvrty of catalase increased initially and declined later. However, in lungs, its activity 

rncreased wrth the propion of the parasite development. Fielden et al. (1974) and Bray 

(1974) observed that SOD activity never decreased in the presence of catalase, which 

suggested that the reaction product H 2q inactivated SOD (Yamakura and Suzuki, 1986). 

Kono and Fridovich (1982) demonsmted that catalase was inhibited by superoxide anion 

and concluded that SOD and catalse are mutually acting protective set of enzymes. In the 

present study, a positive correlation was obsewed between the activity of SOD and catalase 

In liver, brain. heat and lungs (Figs. 65,67-69), and the relationship was significant w0.05) 

In brain (14.94) and heart (~0.97) only. Compared to this, in the testes, a negative 

cornlation was observed Ween the activity of SOD and catalase, and it was not 

significant. The deed SOD and catalase activity in the organs of B. malayi infected 

animals may be due to the inhibition by reactive oxygen radicals released and non- 

availability of NADPH. This is &m~ed between 60 and 120 days post rnfection, which is 

&

3 

a- 

7. 

6- 


SOD and catalase levels in liver 

9 1: +- 

10- 

m 

I Observed values -+- Expected values I 


SOD and catalase levels in testes 

r= 047p>OOS 

re0 27 D>O M 

y-54-67.X 

1, 1 0 1 2 3 4 5 e 

SOD 

154 

I, 

1 4 5 0 05 1 

SOD 

1 5 2 25 3 

I I Ohewod values --c Expbcted values 


SOD and catalase levels in brain 

I 

1-091 p

- 


SOD and catalase levels in heart 

6- 

r=OS? p

the period by which, the parasite attains full development and gets lodged in the organs. In 

lungs, conhary to other'organs, the levels of SOD and catalase showed increasing trend 

throughout the study period indicating that more amount of NADPH is formed by the 

restoration of G6PDH (sa. 4.1.2.1.6), even though the parasite burden is higher than that 

observed in other organs. 

The activity of SOD and catalaw which were altered in all the organs of B.ma1ayi 

infected animals were restored to normal levels after DEC treatment. And this may be 

associated with concornittarit generation of NADPH by the increase in G6PDH activity after 

DEC treatment as observed (sec. 4.1.2.3.2). 

4.133 Glutathione and glutathione related enzymes 

GSH plays an important role in protection of cells against oxidants in most 

organisms (Bryant and Behm, 1989). Depletion of GSH leads to lethal conditions and 

accumulation of oxidii glutathione (GSSG), which is also harmful to cells, because it 

forms mixed disulphides with proteins and other molecules containing thiol groups. The 

concentration of GSH in tissues is regulated by its generating enzyme GR from oxidised 

glutathione. The utilising enzymes GPx and GST convert the reduced state (GSH) to 

oxidized form (GSSG) during the detoxification of the radicals. The NADPH reqd for 

GR activity is regenerated by the enzyme G6PDH (Halliwell and Gutteridge, 1990). 

Jakoby et al. (1976) reported that GSH is capable of protecting cells from oxidant 

stms owing to its antioxidant and nucleophilic charactenstics. It helps in maintaining the 

integrity of nd cells (Fegler. 1952) particularly when exposed to oxidant stress due to drug 

or malarial infection (Allen and Jandl, ]%I) by protecting hemoglobin and other thiol 

containing proteins from denaturation. 

GPx plays an important role in the detoxificat~on of Hz&, organic hydroperoxides or 

lipid peroxides (Cohen and Hochstein, 1963: Chnstopherson, 1%8; 1969; Flohe, 1982). 

GSH and NADPH are required in awuate amount for GPx activity @*their depletion.

esults in its reduced activity (Condell and Tappell, 1983). Also that GPx is inactivated under 

severe oxidative sees^. . 

In P. knowlesi infected monkey, the activity of GSH had been reported to be 

decreased (Fulton and Grant, 1956; Fletcher and Maepith. 1970). Bhattacharya and 

Swarupmithra (1987) reported a decline in the etythrocyte GSH In P.vivlu infected patients. 

P. knowlesi infection resulted in elevation of hepatic stress in monkeys, besides affecting the 

antioxidant enzymes of the host. Gpx activity was found to be low in P. vinckei infected 

mice erythrocytes by Stocker et al. (1985) and in P. viva by Mathews and Selvam (1993) 

and Sarin (1 993). In studies with P. falcipanrm and P. viva infected RBC's GR activity was 

also found to be reduced (Stocker et al., 1985; Kamchogwongpaisan el al., 1989). Litlov el 

al. (1981) reported that in the absence of GSH, loss of GR activity is seen. GST is one of the 

GSH utilizing enzymes and reduction in GSH leads to the decreased activity of GST 

(Srivastava el al., 1995). 

Picard- Maureau er al. (1975) had observed decrease in the activity of G6PDH of 

P.vinckei parasitized mice erythrocytes and a similar trend was reported in P. bergei infected 

mice (Nair et 01.. 1984; Grindberg and Soprunov, 1983) and in the RBC's of patients 

afflicted w~th malaria (Mathews er al., 1991). 

Srivastava er al. (1991; 1992) and Clark et al. (1986; 1989) reported an increased 

oxidative damage during malaria infection and attributed this to decreased GST level. 

Srivastava er al. (1995) had shown that P berghei infection in M.naraletzsis reduced the 

activity of GST in the hepatic mitochondria and microsome and the GST level was brought 

back to normal aAer chloroquine treatment. Mathews and Selvam (1993) reported decreased 

GST activity in P. vivor infected human erythrocytes. 

In the present study, GSH level decreased in liver, brain, testes and heart of the 

B,mafayi infected animals, but in lungs it showed an increasing trend. A negative correlation 

was observed between the activity of GSH and LPO in all the organs (Figs. 70-74) and it was 

significant w0.05) in liver (I=-0.88), testes (I=-0.95) and heari (r--0.88) only. Similarly a 

positive cornlation was observed between the activity of GST and GSI~ ip all the organs

(Figs. 75-79) and it was significant wO.05) in liver (~0.93, testes (I= 0.94) and brain 

(~0.98) only. The activity of GRI G6PDH had a positive correlation with GSH activity in all 

the organs (Figs. 80-89) and it was significant (~4.05) in testes (~0.96, 0.93), brain 

(~0.98, 0.98) and heart (~0.98, 0.88) only. There was positive and significant (~~0.05) 

correlation between the activity of GPx and GSH in all the organs (Figs. 90-94), such as liver 

(r-0.9), testes (r=0.98), brain (r= 0.97), heart (~0.94) and lungs (F0.9). In the present study, 

the level of NADPH regenerating enzyme, G6PDH was found to be low and this condition 

might have resulted in the non-availability of NADPH. The decrease in GR (GSH generating 

enzyme) activity is due to the non-availability of the reducing equivalent NADPH required 

for the conversion of oxidized glutathione to reduced glutathione which is the substrate for 

this enzyme (Halliwell and Gutteridge, 1990). The decrease in GPx and GST (GSH utilising 

enzymes) is related to the reduction of GSH levels. Thus the decrease in the GSH generating 

and utilising enzymes reduced the GSH level in the B.malayi infected animals. However, in 

lungs the availability of G6PDH in plenty had increased the GSH level. 

Following DEC administration the GSH level in all the organs of the B.malayi 

infected an~mals significantly ~ncreased and thls is due to the restoration of the activity of the 

enzymes G6PDH. GR GST and GPx as stated above. 

4.1 3.4 Total thiols 

Loss of membrane suphydryl groups renders the membrane increasingly susceptible 

to LPO with increase in membrane permeability, leading to colloid osmotic hemolysis and 

reticuloendothelial entrapment (Jacob and Jandl, 1962). The depletion of membrane 

sulphydryl attached to fatty ac~d double bonds renders them increasingly vulnerable to 

pemxidative cleavage, which in turn leads to membrane dissolution (Jacob and Lw, 1968).

g 20- 

:I-. 

F1g.70:Relationship between change in 

GSH and LPO levels in liver 

- 

,=pen pom I 

I 

yr2 703 38'r 

4 

2 1 6 (I 10 l? 14 

GSH 

I


GSH and LPO levels in heart 

. I 

-7 

1 0 1 2 3 4 5 

GSH 

I Observed values + Expected values 1 


GSH and LPO levels in lungs 

GSH 

I Observed valuer + Expected values I

12, - 

I 0 

U) 

Q 4 

2 

0 - + - 

Fig.75:Relationship 

x-, 

between change in 

GST and GSH levels in liver 

y= 089+802-x 

- - 

GST 

I Observed values + Expected values : 


GST and GSH levels in testes 

r=004 ~ ~ 0 0 5 

y=-06447 1-x 

-- 

202 0 02 04 06 08 1 12 I d 

14 

2 0 02 04 06 08 1 1 2 14 

GST 

I Observed values + Expected values 


GST and GSH levels in brain 

12 r-096, p


GST and GSH levels in heart 

-2 4 005 0 1 0 15 0.2 

GST 

0 25 0 3 C 

[ Observed values - Expected values / 


GST and GSH levels in lungs 

2 5 I - 

0 4 

-0 05 0 0.05 

GST 

0 1 0 

I 9 Observed values - Expected values 1

$ 

Fig.80:Relationship between change In 

GR and GSH levels in liver 

0 4 

2 

0 . 

2 

2 0 2 4 6 8 

G R 

10 12 14 16 18 

10- 

14. 

12- 

10' 

5 0. 

a 6. 

4- 

2- 

0, 

I Observed values --t Expected values I 


GR and GSH levels in testes 



GR and GSH levels in brain 

r=O98. pcO M 

y= 1 2+ 1 Max 

2 0 2 4 0 E 10 12 1.4 

G R 

I Oboowad values + Expected values


GR and GSH levels in heart 

- Observed 

values + Expected values 


GR and GSH levels in lungs 

- Obsefved 

values + Expected values


G6PDH and GSH levels in liver 

I r Observed values + Expected values ( 


GGPDH and GSH levels in testes 

( I Observed values -+- Expected values I 


G6PDH and GSH levels in brain


' 

G6PDH and GSH levels in heart 

I - Obsewed 

vdues + 

Expected values / 


GGPDH and GSH levels in lungs 

I = Observed values + Expected values /

V) 

(3 :; 

- 

Fig.9O:Relationship between change in 

GPx and GSH levels in liver 

GPx 



GPx and GSH levels in testes 

Observed values I - Expected values 

m 

Fig.92.Relat1onship between change In 

GPx and GSH levels In bra~n 

r-007 p

Fig.93:Relationship between change In 

GPx and GSH levels in heart 

61 1 

5- 

4- 

1 3- 

(I) 

0 2- 

1 - 

0- 

1 - 

0 

y=-0 41 +O 79.x 

1 2 3 4 5 6 

GPx 

I Observed values + Expected values I 


GPx and GSH levels in lungs 

, --A 

-0 5 0 0 5 1 15 2 2 5 3 

GPx 

I Obse~ed values - 

Expected values 

I

A reduction in the total thiol contents was observed in the present study in different 

organs of B.malayi infected animals. A significant w.05) positive comlation was 

observed between thiol status and GSH levels in all the organs (Figs. 95-99) such as liver 

(d.96), testes (~0.9). brain (~0.98). heart (r=0.96) and lungs (I4.89). (Kosower et al. 

(1982) had shown a direct link between the thiol status of the membrane and cellular GSH. 

This shows that the function of GSH is to serve as a reducer of membrane protein 

disulphides and to avert membrane thiol oxidation. The decreased GSH concentration 

observed in the present study had contributed to decreased thiol status, while in lungs the 

availability of GSH had increased the thiol level. The decreased total thiol levels in the 

B.malayi infected animals were restored to the normal levels after DEC therapy, which is 

due to the restoration ofGSH levels (sec. 4.1.2.3.2). 

4.13.5 Ascorbic acid 

The antioxidant property of ascorbic acid is often associated with its ability to 

regenerate vitamin E h m vitamin E radical (Niki eta/., 1984). Nishlkimi (1975) had shown 

that ascorbate reacts with superoxide and converts it to Hz@. Ascorbic acid levels in plasma 

were found to be decreased due to P.vivar infection (Irwin and Hutchins, 1976; Frei er a/., 

1988). Tappel (1969) and Grimble and Hughes (1967) reported that GSH is required for the 

conversion of dehydroascorbate to ascorbate. In the present study ascorbic acid levels were 

found to be low in liver, brain, testes and heart except for lungs of the B.malayi infected 

animals, which is due to the alteration in GSH level during infection. The ascorbic acid level 

and GSH had positive comlation in all the organs (Figs. 100-104) and it was significant 

w0.05) in liver (r=0.99), testes (~0.89)~ brain (r= 0.88) and heart (14.97) only. And the 

restoration of the ascorbic acid levels observed in the infected animals after DEC treatment 

is due to the restored GSH levels (sec. 4.1.2.3.2).

0 4 

0 - 

lo' 

6- 

8 

WJ 4- 

0 

2 - 

2 5- 


total thiol and GSH levels in heart 

14 

-2 1 0 1 2 3 4 5 

Total thiol 

I - Observed values + Expected values I 

2- 

I 15' 

% I- 

0 5- 


total thiol and GSH levels in lungs 

r=oes PC005 

y-035+037'x 

1 2 3 

Total thiol 

4 5 

- Observed values - Expected values

12 

6Y 

0 4 

2 

0 

Fig. 100:Relationship between change in 

ascorbic acid and GSH levels in liver 

y=035+1 50.r 

2 2 1 0 1 2 3 4 5 6 ' 

10 - 

Ascorblc acld 

1 9 Observed values -+- Expected values I 

Fig.101 :Relationship between change In 

ascorbic acid and GSH levels in testes 

I 

8- ,=Om pcODJ 

y= 2 11+0m.x 

6- 

I 

6Y 4- 

0 

I 

, 

0 2 4 6 6 10 12 '4 

/ 

Ascorb~c ac~d 

- Observed values -+- Expected values ( 

Fig.lO2:Relationship between change In 

ascorbic acid and GSH levels in brain 

Ascorbic acld 

1 - Ota.wed values - Expected ~~~~~~~1

Fig. 103:Relationship between change in 

ascorbic acid and GSH levels in heart 

Ascorbic acid 

9 Observed values +- Expected values 

Fig. 104: Relationship between change in 

ascorbic acid and GSH levels in lungs 

Observed values - 

Expected values

CHAPTER 4.2 

CHANGES IN THE ACTMTY OF MEMBRANE BOUND ENZYMES AND 

HAEMATOLOGICAL PARAMETERS IN MASTOMYS NATALENSIS INFECTED 

4.2.1 MATERIALS AND METHODS 

WITH BRUGlA MALAY/ 

M. natalensis of the same age group (6-8 weeks) and weight (35-40 grn) were 

divided into two groups, one served as the control while the other was infected with B. 

rnalayi infective larvae as described earlier. At 0, 30, 60, 90 and 120 days of post- 

inoculation, brain homogenate of the animals were prepared as described earlier and used for 

the determination of the activity of different enzymes. Blood samples were collected at 0,30, 

60, 90 & 120 days post infection and used for haematological and biochemical 

investigations. 

The erythrocyte membrane was isolated according to the procedure of Dodge et al. 

(1%3). Blood plasma was removed and the remaining packed cells were washed three times 

with isotonic phosphate buffer, pH 7.4. The washed RBC suspension was haemolysed with 

hypotonic buffer (20 milliosmolar, pH 7.2) in the ratio of buffer: cells of 14:l. The ghost 

cells were sedimented in a high speed refi-igerated centrifuge at 20,Wg for 40 minutes. The 

supernatant was decanted carefully and the ghost button was resuspended by swirling. 

Sufficient buffer of the same strength was added to reconstitute the original volume. The 

mtio of cells to washing solution was approximately 1:3 by volume. The ghosts were washed 

three times subsequent to haemolysis. The supernatant after the last wash was either pale 

pink or colourless. The pellet of erythrocyte membrane was suspended in 10 ml of 0.32 M 

sucrose solution and hornogenised. Aliquots of this homogenate were used for enzyme 

assays and estimation of protein.

4.2.1.1 N ay ATPase 

Reagents 

Nay ATPase activity was determined as per the method of Bonting (1970). 

1. Tris-HCI buffer : 184 mM, pH 7.5 

2. Magnesium sulphate : SO mM 

3. Potassium chloride : SO mM 

4. Sodium chloride :600mM 

5. EDTA :ImM 

6. ATP : 40 rnM 

One ml of Tris buffer, 0.2 ml each of all the other reagents listed above were mixed 

together such that the final volume of 2.0 ml contained 92 mM Tris buffer, 5 mM 

Magnesium sulphate, 60 mM sodium chloride, 5 mM potassium chloride, 0.1 mM EDTA 

and 4 rnM ATP. After 10 minutes equilibrium at 3PC in an incubator, reaction was started 

by the addition of 0.1 ml homogenate. The assay medium was incubated for 15 minutes and 

then the reaction was stopped by the addit~on of 1.0 ml of 10% TCA. 

The amount of phosphorous liberated by the enzyme from ATP was estimated by the 

method of Fiske and Subbarow (1925). And the enzyme activity was expressed as pmoles of 

phosphorous liberated/min/mg protein. 

4.2.1.2 ca2+ ATPase 

Pan (1983). 

The activity of ca2' ATPase was estimated according to the method of Hjerten and 

&IrgentS 

I. Tris-HCI buff^ : 125 mM. pH 8.0 

2. Calcium chloride : 5OmM 

3. ATP : 1OmM

To the reaction mixture containing 0.1 ml each of calcium chloride, ATP and buffer, 

0.1 rnl of 1:20 diluted homogenate was added and incubated at 37C for 15 minutes. Then 

the reaction was arrested by the addition of 0.5 ml of 10 % TCA. The amount of 

phosphorous liberated From the substrate was estimated by the method of Fiske and 

Subbarow (1925) and the enzyme activity was expressed as poles of phosphorous 

liberatediminlmg protein. 

4.2.13 M~'' ATPase 

al. (1982). 

Reagents 

The activity of M$ ATPase the enzyme was estimated by the method of Ohnishi et 

1 .Tris-HC1 buffer : 375 mM, pH 7.6 

2. Magnesium chloride : 25 mM 

3. ATP : IOmM 

The assay was initiated by the addition of 0.1 ml of 1:20 diluted homogenate to the 

reaction mixture containing 0.1 ml of water and 0.1 rnl of each of the above reagents. 

Incubation was carried out at 37C for 15 minutes and then the reaction was terminated by 

the addition of 0.5 ml of 10% TCA. The amount of phosphorous liberated from the substlate 

was estimated by the method of Fiske and Subbarow (1925) and the enzyme activity was 

expressed as poles of phosphorous liberatedimidmg protein. 

4.2.1.4 Acetylcholinesterase 

Acetylcholinesterase was estimated by the method of Hestrin (1949)

Reagents 

1. Acetylcholine bromide : 0.1 M 

2. Sodium chloride :1M 

3. Magnesium chloride :IM 

4. Tris-HCI buffer : 0.5 M, pH 7.5 

5. EDTA : 0.2 M 

6. Cocktail : Mix 13 ml of 1.0 M sodium chloride, 2 rnl of 1 M 

magnesium chloride, 10 ml of 0.5 M Tris-HC1 and I0 rnl of 0.2 M EDTA. 

7. Hydroxylamine hydrochloride : 2 M 

8. Sodium chloride : 3.5 M 

Mix reagents 7 and 8 before use in 1: 1 ratio viv (prepare freshly) 

9. Hydrochloric acid :6N 

10. Fenic chloride : 0.37 M in 0.1 M HCI 

1 1. Reaction &urn : Mix 17.5 rnl of cocktail with 2.0 ml of 0.1 M 

acetylcholine chloride and 5.5 ml of distilled water. Tlus reaction medium gives the final 

concentration of 130 mM sodium chloride, 20 mM magnesium chloride, 50 mM Tris-HCI, 

0.2 mM EDTA and 4 mM acetylcholine chloride. 

To the reaction mehum of 0.5 ml, 0.1 rnl of tissue homogenate and 0.4 ml of 

distilled water were added and the reaction mixture was incubated at 3% for 30 minutes. 

Then the reaction was terminated by addmg 2.0 ml of hydroxylamine hydrochloride and 

sodium chloride mixture. The contents were mixed by vortex mixer and after one minute, 1 

ml of HCI was added and mixed. Then 1 ml of femc chloride was added and absorbance was 

read at 540 nm. The activity of the enzyme was expressed as pmoles of acetyl choline 

bromide utiliscd/min/mg protein. 

4.2.1.5 7-glutamyl transpeptidase 

yGTP was assayed according to the method of Orlowski and Meister (1965) and 

modifid by Rosaki and Rau (1972).

Reagents 

1. L-y-glutamyl pnitmkilide : 30.3 mg dissolved in 10 ml of distilled water. 

2. Tris-HCI buffer : 0.1 ml, pH 8.2 

3. Glycyl glycine : 13.2 mg dissolved in 10 ml of distilled water. 

4. Acetic acid : 10% 

5. Standard : 13.8 mg of p-nitroaniline (recrystallised) (ImM) in 

100 ml of distilled water. 

The tissue homogenate (0.5 ml) was added to the reaction mixture containing 0.5 ml 

y-glutamyl pnitroanilide, 2.2 ml of glycyl glycine and 1.0 ml of buffer. AAer incubation for 

30 minutes at 3?C, the reaction was terminated by the addition of 1.0 ml of 10% acetic acid. 

The amount of pnitroaniline liberated in the supematant was measwed, as the difference In 

the optical density at 410 nm between samples, with and without substrate. The substrate 

incubated in the absence of enzyme under the same conditions was used as a reference blank. 

Optical density of solution of p-nitroaniline in the range 0.005-0.02 ples served as 

standard cwve for aniving at the amount of product formed. The activity of the enzyme was 

expressed as poles of pnitroaniline liberated/midmg protein. 

4.2.1.6 Alkaline phosphatase 

Alkaline phosphatase was assayed by the method of ffing (1965) using disodium 

phenyl phosphate as substrate. 

Reagents 

1. Carbonate buffer : 0.1 M, pH 10.0 

2. Disodium phenyl phosphate : 0.01 M 

3. Magnesium chloride :0.1 M 

4. Sodium carbonate : 15% 

5. Standard : 100 mg of redistilled phenol in 100 ml water.

To 1.5 ml of carbonate buffer, 1.5 ml of disodium phenyl phosphate, 0.1 ml of 

magnesium chlorideand 0.1 ml of 1:20 diluted tissue homogenate was added and incubated 

for 15 minutes. The reaction was stopped by adding 1.0 ml of 10 % TCA. The contents were 

then centrifuged and the residue was discarded. To the supernatant, 1.0 ml of sodium 

carbonate solution and 0.5 ml of Folin's reagent was added and the absorbance was read at 

640 nm after 10 minutes. The activity was expressed in terms of pmoles of phenol 

liberatedimg protein4 5 minutes. 

4.2.1.7 Haemoglobin 

Austin (1932). 

Reagents 

Haemoglobin was estimated by the cyanmethaemoglobin method of Drabkin and 

1. Fenicyanide-cyanide reagent: This was prepared by dissolving 200 mg potassium 

fenicyanide, 50 mg potassium cyanide and 140 mg potassium dihydrogen phosphate in a 

litre of water. 

2. Cyanmethaemoglobin standard: Purchased from Span Diagnostics Pvt. Ltd., Surat, India. 

This was kept in the dark at 4'C. It had an equivalent hemoglobin concentration of 60 mg%. 

Twenty p1 of blood was added to 4.0 ml of the fenicyanide-cyanide reagent. This 

was allowed to stand for 15 minutes and read against a reagent blank at 540 nm. The 

standards were diluted in fenicyanide-cyanide solution to obtain a range of concentrations 

and read in the same manner. 

Blood hemoglobin values were expressed as &dl. 

4.2.1.8 Total RBC counts 

Lewis, 1975). 

Total RBC counts was determined according to the standard procedure (Dacie and

The method involves an accurate dilution of a measured quantity of blood with a 

fluid, which is isotonic with the blood, which will prevent coagulation. 

The RBC pipette was filled with blood to the 0.5 mark and the diluent was filled upto 

101 mark to make it to 1 :200 dilution. The pipette was shaken for three minutes and the first 

two drops were discarded. The cover slip was placed on the haemocytometer counting 

chamber and a few drops of diluted blood were filled and left to settle for one minute and 

then counted under a light microscope. The total RBC was calculated using the following 

formula: 

RBC/cu.mm = cells counted x 5 (115 cm2 counted) x 10 (depth) x 200 (dilution factor). 

4.2.1.9 Total leucocyte count 

Total leucocyte count was estimated by the haemocytometer method (Miale, 1972). 

Blood was diluted with a fluid that lyses the non-nucleated erythrocyte precursors. If 

the blood smear showed nucleated erythrocytes, the cell count was corrected to the true 

leucocyte count according to the following formula: 

Corrected count = Observed count x 100 

100 + % nucleated erythrocytes 

The WBC pipette was filled to the 0.5 mark with blood and diluted to the 11 mark 

with I .Ph HCl. This made a 1:20 dilution. The pipette was shaken for three minutes and the 

lint two drops were discarded. The haemocytometer chamber was filled with diluted blood 

and left to settle for one minute. 

Lcucaytes present in the four large c ow squares (1 sq.mm each) were counted and 

calculated by tbe following formula: 

WBC/cu.mm = Cells counted x IOfdeotb) x 2Mdilution factor) 

4(sq.mm. counted)

4.2.1.10 Differential leucocyte count 

(John, 1972). 

Reagents 

The leucocyte differential count was detenined by Leishman's staining method 

1. Leishrnan's stain : 0.2 g of Leishman dye was dissolved in 100 ml of acetone free 

methanol at 50°C for 15 minutes with occasional swng, then cooled and filtered. 

2. Buffered water : 0.066 M Sotenson's phosphate buffer was made upto 50 ml, pH 7.0 

and mixed with 3.89 ml of KH2P04 (9.1 gm4) and 61.1 ml NazHPO4 (9.5 gm4) to I litre 

water 

A drop of blood was placed on a clean slide and a spreading slide was placed at 

an angle on the slide and moved forward to make a smear of 3-4 cm in length and air-dried. 

The slide was covered with stain for 2-3 minutes and the stain was diluted by adding drops 

of buffered water and left for 5-7 minutes. The slide was washed with water, dried and 

examined under a microscope. 

4.2.2 RESULTS 

4.2.2.1 Membrane bound enzymes of brain. 

The changes in the activity of the membrane bound e v e s of the brain are 

presented in the Figs. 105-107. The activ~ty of all the enzymes studied ( Nay ATPase, 

M ~ ATPase, ~ ' Ca2' ATPase, yGTP, acetylcholinesterase and alkaline phosphatase), 

increased continuously from day 0 to day 120 in the control animals, whereas in the 

B.malayi infected ones there was a significant (pC0.05) decrease from 60 days to 120 days. 

43.2.2 Membrane bound enzymes of RBC 

The activity N~'K' ATPase, Ca2' ATPase and h4g" ATPase in the infected animals 

1- slightly from 0 day to 30 days and d e d the~fter as aglost the continous

Fig. 105: Effect of 6.malayi on N~+K+ 

and ~ d%~~ase in brain of M. natalensis 

0 10, 

0 14- 

0 12- 

- ! 01- 

g ooe- 

= ow. 

OM- 

0-3 

0 30 W 90 120 

Days of ~nfect~on 

Fig.lO6:Effect of 6.malayi on M&P~S~ 

& Ach-est in brain of M. natalensis 

0 14- 

0 12- 

01- 

ow. 

0 M- 

3 5- 

0 30 W PO 120 

Days of lnfecbon 

Fig.107: Effect of B.malayi on GTP 

and AP in brain of M. natalensis 

Days of tnfectton 

I - GTP.N -C OW-I - AP-N * AP-I ]

increase in the control animals (Figs. I08 & 109). And the enzyme, acetylcholinesterase was 

found to decrease s ~~ficantl~ (p4.05) 6om 0 day to 120 days. 

4.2.23 Haematological parameters. 

The effect of parasitic development on some haematological parameters was studied 

(Figs. 110-1 13). The hemoglobin levels and RBC counts in the infected animals were fokd 

to fall within the normal physiological range (Appendix). The total WBC count in the 

infected animals was significantly w0.05) lower than the normal values. 

A significant decrease w0.05) was observed in the absolute count of lymphocytes 

after 80 days post infection, while the eosinophil and neutrophil count increased significantly 

(F0.0 I ). 

4.23 DISCUSSION 

4.23.1 Membrane bound enzymes of brain. 

Membrane bound enzymes such as Na'K' ATPase, M ~ ATPase, ~ * ca2* ATPase, 

plays an important role in the maintenance of the ionic gradient between the ~nbacellular and 

exhacellular compartments of the cell (Trump et al., 1979). Acetylcholinesterase is involved 

in the banmission of nerve impulses. N ay ATPase plays an important role in the active 

eansport of ~ aand ' K* ions (Mahaboob Basha and Nayeemunnisha, 1993), conduction of 

nerve impulses and synaptic function in the brain (McIlwah 1969; Sweadner, 1979). M~~ 

ATPase and ca2' ATPase have been found to regulate ionic pumps in the CNS (Farber, 

1982). 

In the present study, the activity of NaX' ATPase, M ~" ATPase and ca2' ATPase 

of the CNS daeaxd to significant levels. ATPases depend on lipids and thiol groups to 

maintain their structure and function (Gamer et a/., 1983; h hh, 1980; Shalev et al., 1981). 

Oxygen fne radical production during B.mahyi infection leads to oxidation of thiol groups 

(SCC 4.1.2.1.8). This decrease in Na'K' ATPase may be aseociated to l$t decrease in the thiol 

level as stated earlier. Thc reduced activity of N~'K' ATPase leads to decreasg: in sodiwn 

9 0

Fig.! 08:Effect of B.malayi on ~ a' K' 

and caMATPase in RBC of M.natalensis 

0.024 

0 30 60 90 

I 

120 


- Na-N - Na-t - Ca-N -- ca-l 

Fig.lO9:Effect of B.malayi on M ~%TP~S~ 

and Ach est in RBC of M.natalensis 


- -Mg-N -+- Mg-l - Ach est-N - Ach est-l I

- 

Fig.110: Effect of 6. malayi on Hb and 

RBCofM.natalensis 


I-H~J-N -HW - RBC-N - RBC-I 1 

6000 -, 

5000 - 

Q, 

3 4000- 

2 

C Q 3000g 

2000- 

Fig.111: Effect of B. malayi on Total 

WBC of M.natalensis 

I, - 

- 

+ 

0 

0 

7 

30 

T 

60 


90 

I 

120 

r- - TOW WBC-N Total WBC-I 1 

il i

801 

70 - 

0, 

- 3 60m 

> 50- 

40- 

Fig. 1 1 2: Effect of 8. malayi on 

Lymphocytes of M.natalensis 

90 -, 1 

30- 

20 - 

10- E 

0 0 30 60 90 120 


Fig.113: Effect of B. malayi on 

Neutrophils & Eosinophils in Mastomys 

7" 

40 - 

35- 

0, 

- 3 30m 

> 25- 

20" 4B 

5 

15- 

10- 

5- 

- -. 

OF 

0 30 60 90 120 


-

efflux and thereby alter the membrane permeability, and also likely to affect the transport of 

Na' ions as well as nerve impulses and synaptic functions of the brain as reported above. 

The decrease in ca2' ATPase and Mg2' ATPase activities is due to peroxidation of the 

membrane lipids (sec 4.1.2.2). The lowered activity of Mg2' ATPase and ca2' ATPase 

observed in the B, molayi infected animals is bound to alter the permeability of the CNS 

membrane, and cause disturbances in the concen!mtion of ~ g and " ca2'. The decreased 

acetylcholinesterase activity in the present study may be due to altered lipid fluidity of the 

CNS membrane because of peroxidation (Howard and Sawyer, 1980). 

yGTP is an extrinsic membrane glycoprotein (Tate and Meister, 1976) widely 

distributed in kidney, brain and liver. Brain endothelial cells are also rich yGTP and alkaline 

phosphatase, which are responsible for transendothelial hansport and vascular permeability 

(Vorbrodt el al', 1986). Tripathi er a/. (1994) reported a decrease in the yGTP and atkalie 

phosphatase activity in the cerebral microcapillaries of mice infected with P. yoelii. In the 

present study, the decrease in y-GTP and alkaline phosphatase due to B.mnlnyi infection 

indicates membrane damage in the brain. The increased LPO (section 4.1.2.2) may be 

responsible for the membrane damage of the brain cells. Moreover, the decrease in ahkine 

phosphatase and y-GTP and acetylcholinesterase may increase disruption in blood brain 

barrier permeability and lead to abnormal cerebral metabolism. 

4.23.2 Membrane bound enzymes of RBCs. 

Oxygen radicals cause more damage to red cells than to other cells, since they are 

exposed to high oxygen concentrations in the lungs. The red cell membrane contains 

unusually high concentration of vulnerable unsaturated fatty acids and lacks the cytochrome 

respiratory pathway, which allows most of the oxygen in other cells to avoid sequential 

acquisition of electrons (Suhail and Rizvi, 1990). Thus, reactive forms of oxygen are 

generated at a higher rate in red cells and the balance between this stress and their 

antioxidant defmces determines their life span (Carrel et al., 1975). 

Mice red cells infected with P. vinckei had been reported to d9)age the membrane 

calcium hansporl system due to the accumulation of rnalondialdehyde (Kayen ef al., 1983),

thereby increasing intracellular calcium. Tanabe et al. (1983) have shown reduced ca2' 

ATPase and M~Z' ATPase in P, chaubadi cells. They had also reported that the Nay 

ATPase activity of the host cell membrane was 85% less than the normal. So the 

permeability changed due to decreased ATPase, which would have resulted from the 

membrane lipid changes in the RBC 

The observed decrease in N~-K' ATPase, ca2' ATPase and M~~ ATPase and 

acetylcholinesterase activity in RBC in the present study indicates membrane damage. The 

reduction in the activity of ATPases can be explained from the fact that they are lipid and 

thiol dependent enzymes. and their decreased activity can be attributed to membrane damage 

as reported above. Decrease in ca2* ATPase and M ~ ATPase ~ ' activity may be due to 

peroxidation of the membrane lip~ds. The erythrocyte membrane is normally less permeable 

to extracellular Ca2'. Enhanced host membrane permeability to Ca2+ is combined with 

reduced activity of Ca2' ATPase, leading to an increase In intracellular ca2' (Sarkadi, 1980). 

Acetylcholinesterase is an erythrocyte specific enzyme (Mitchell el al., 1965). 

Elevated cholinesterase in mouse erythrocytes infected with P. chaubadi (Konigk and Mitsh, 

1977), and P. berghei (Areekul and Cheerarnakara, 1987) had been reported. The decrease in 

acetylcholinesterase in 5. malayi infected animals may be due to altered lipid fluidity of the 

cell membrane (Howard and Sawyer. 1980). 

4.233 Haematological parameters 

Butteworth (1977) and Ogilvie et al. (1978) observed changes in the absolute count 

of neutmphils. lymphocytes. monocytes and granulocytes in helminthic mfection. Choong 

and Mak (1991) reported an increase in total count, differential counts and blood eosinophils 

to k times at 3 weeks post infection upto 13 weeks in Presbytis cristata infected with 5. 

Malayi. It further increased to five tims upto 20 weeks, but no change in total leucocyte 

count was observed. An increase in neutrophils was reported in Toxoplasma mfection 

(Frcnkel. 1973). Rahmah el 01. (1994) reported that Toxoplarma gondi infection resulted in 

the increase in neutrophils and cosinophils and a decrease in lymphocytes. Larsh (1967) and 

Cypas ( 1972) reported infection of mice by Trichinella spimlir and Nematospiroides dubia

with i n d lymphocytes and decreased neutrophils. In P. viva and P. falcipamm 

infection, decreased lymphocytes, increased monocytes and eosinophils had been observed 

without significant changesin total leucocyte, neutrophil and platelet counts (Kurnaresan and 

Selvam, 1991). Eosinophils and neutrophils play a significant role in host defense against the 

parasites. In Onchocerciasis infections, eosinophils were shown to be the major participants 

in adherence to 0. wlvulus (William er al., 1987). The cationic proteins of the eosinophil 

damage target cells through membrane interaction, since the major basic protein as well as 

eosinophil cationic proteins can disrupt cell membrane. Mackenzie (1980) had shown that 

eosinophils eventually target mf and Greene er 01. (1981) had shown that neutrophils are 

equally efficient in the same system. 

In the present study the haernoglobin levels and RBC counts were not altered due to 

the B. mnloyi mfection, while total WBC counts showed a decrease below nonnal values. 

The significant increase in neutmphil and eosinophil counts and decrease in lymphocytes 

confirm the above findings.

CHAPTER 4.3 

CHANGES IN THE TESTES OF M. NATALENSIS INFECTED WITH 

43.1 MATERIALS AND METHODS 

4.3.1.1 Testosterone 

B. MALAY1 

Testosterone Radioimmunoassay was done using Testosterone Double Antibody kit 

obtained from Diagnostics Products Corporation, Los Angeles, USA. 

The basic principle of FUA involves competition between a radioactive and non- 

radioactive antigen for a fixed number of antibody binding sites in a fixed time. The amount 

of labeled analyte bound to the antibody (goat anti-rabbit gamma globulin) is inversely 

proportional to the concentration of the analyte present in the sample. The antiserum is very 

specific for testosterone with an extremely low cross-reactivity to other steroids. 

Martomys rats were infected wth B. rnalayi as desnibed earlier. The testes was 

excised after 0, 30, 60, 90, 120 days post inoculation, washed with phosphate buffered 

saline. pH 7.4. homogenized and centrifuged at 2000 rpm for 20 minutes. The pellet was 

discarded and the supernatant was used as sample for RIA. 

Reagents 

1. Testosterone antiserum 

2. ('"1) Testosterone 

3. Calibrators (conc. 5, 10,20,50, 100 & 200 pgtube) 

4. Goat anti-rabbit gamma globulin 

5. Polyethylene glycol 

6. Borate buffer for testosterone

Tubes were labeled for calibrators, blank, maximum binding tube, total counts and 

samples (tissue homogenate) in duplicate. Two hundred pl of the buffer was taken into the 

blank tube and 100 pl ihto the maximum binding tube. Similarly 100 pi of the calibrators 

and the sample was taken in the calibrator tubes and sample tubes. Then 100 p1 of the [I2' I] 

testosterone was added into all the tubes, followed by 100 p1 of testosterone antiserum to all 

the tubes except the blank tubes. The tubes were vortexed and incubated for a minimum of 

60 minutes at R.T., which was followed by the addition of 100 pi of goat anti-rabbit gamma 

globulin to all the tubes. The contents of the tubes were vortexed and incubated for I hr at 

R.T. then 2.0 rnl of cold 4% PEG-saline solution was added and centrifuged at 2,000g for 20 

minutes. The supernatant was decanted, the rim of each tube was blotted dry and counted in 

a Gamma Counter for 1 minute. Calibration curve was prepared as indicated by the kit 

procedure. The testosterone levels were expressed in nanornoles/mg protein. 

43.1.2 7-GTP 

1-GTP was assayed according to the method of Orlowski and Meister (1965) and 

modified by Rosalki and Rau (1972) as described earlier. 

43.13 LDH-X 

The testes of M.naralemis infected with B.rnalayi were excised on different days of 

post inoculation (0,30, 60, 90. 120). washed in chilled potassium phosphate buffer (0.1 M, 

pH 8.3) and homogenised in the same buffer. The homogenate was centrifuged at 10,000g 

for 30 minutes at 4°C and the supernatant was used for LDH-X isoenzyrne assay. 

LDH-X isorymes were visualized on agar gel by following the method of Hanis and 

Hopkinson ( 1978). 

Reagents for staining 

1. 0.05 M Tris-HCl buffer. pH 8.0 : 20 ml 

2. Calcium l,actate (pentahydrate) 

mixture). 

3. NAD 

: 100 mg (8 mM final concentration in reaction

4. MTT : 5 mg in 1 ml water 

5. Phenazine metho suiphate (PMS) : 2.5 rng in 0.5 ml water 

6. Agar (approx., 2%) :20ml . 

Electrophoresis conditions 

1. Bridge buffer: 0.2 M phosphate buffer, pH 7.0 

Gel buffer : 0.01 M phosphate buffer. pH 7.0 

2. I2 Vlcm for 5 b, with cooling. 

One percent agarose gel was prepared in 0.01 M phosphate buffer and fvted on a 

horizontal electrophoretic system. About 10 p1 of each sample were added into individual 

well along with the control sample. Whatman filter paper was used as the wick between the 

bridge buffer and electrophoresis was carried out at constant current (I2 V) for 5 hrs. The 

isoenzymes were visualised by activity staining as follows. The unfixed gel was rinsed with 

cold his buffer (0.1 M, pH 8.3) and incubated for 10 minutes at 37C in dark in a staining 

solution containing 200 p1 calcium lactate, 10 mg NAD, 5 mglml m, 2.5 md0.5 ml PMS 

in 2% agar made upto 25 ml. The isoenzymes LDH 1-4 migrated anodally, while LDH-5 

migrated cathodally. The LDH-X isoenzyme migrates between LDH-3 and LDH 4. 

43.2. RESULTS 

The effect of B. ma lay^ on testosterone, y -GTP and LDH-X of testes was studied and 

the data are presented in Figs. 114 8~115. The testosterone levels were observed to increase 

initially (upto 60 days) and decreased significantly w0.05) thereafter. And althrough the 

study period. the activity of this hormone was always lower than the control. 

y-GTP level increased significantly WO.05) in the infected group during the 

parasite development than in control throughout the study period. 

The LDH-X mzyme was visualized on agarose gel by activity staining (Platel). The 

LDH-X bands in the infected animals were clearly visible on 30th day and faintly visible on 

60th day but absenthot visible on 90th and 120th day, whereas. in control the enzyme bands 

were clearly visible throughout the study period. 

9 9

Fig.114: Effect of B, malayi on GTP 

testes of M. natalensis 


- - gamma GTP-N gamma GTP-I 

Fig.115: Effect of 8. malayi on 

Testosterone in testes of M. natalensis 

104 

0 30 60 90 1 


Plate 1: Electrophoresis of LDH -X isoenzymes 

from testes of M. natalensis infected with B. malayi 

LDH-I . 

LDH - 2 

LDH - 3 

LDH - X 

LDH - 4 

Lane 1 - Control (matured adult) 

Lane 2 - 30 days post infection 




Lane 6 - Control (young adult)

433 DISCUSSION 

Testosterone is a sensitive indicator of the reproductive function (Rikihisa et al., 

1984). The present study demonstrates that the testosterone production in the host was 

decreased in the infected animals. Reduction in testosterone level may be due to degadation 

or decreased testosterone output from the testes due to parasitic infection (Rikihisa et al., 

1985). Decreased number of leydig cells or their topological changes in distribution with 

reference to blood and lymphatic vessels might cause decreased out put of testosterone in 

testes. Rikihisa el al. (1985) reported a decrease in rat testosterone due to Taenia 

taeniafonnis infection. A similar kind of inhibition is reported in Toxoplasma gondii 

infection (Dias and Stahl, 1984). 

y-GTP in the testes is primarily found in the sertolt cells of mammals and had been 

used as a sertoli cell marker in rats (Hodgen and Sherins, 1973; Lu and Steinberger, 1977; 

Fukuota et a/., 1990)). Hodgen and Sherins (1973) found an abrupt increase in y-GTP 

activity in prepubertal rat testis which coincided with the cessation of sertoli cell division and 

their maturation. Krueger er al. (1974) measured 1-GTP activity in testis of vitamin-A 

deficient rats. The level of this enzyme ~ncreased during germinal cell depletion. The 

observed increase in y-GTP In B.malayi infected animals suggests that the sertoli cell has 

been damaged and germinal cells depleted. These adverse effects due to B. malayi infection 

have been confirmed by the hstopathological studies (sec 4.4.2.1.3). 

Among vertebrate species, somat~c cells often contain lactic dehydrogenase (LDH) 

in an array of five isoenzymes. The unique LDH-X isoenzyme occurs in the mature testis of 

mammals (Blackshaw, 1973; Linda and Mayeda, 1974; Taylor and Gutteridge, 1986). It is a 

unique marker of the germinal cell rnaturatlon and the production of the sperms (Lalwani et 

al., 19%). Hodgen (1983) show4 the coincidence of LDH-X with the histological 

appearance of pachytene spematocytes. ltoh and Ozasa (1985) and Aganval el al. (1997) 

reported impcurment of testicular function due to cadmium chloride administration to rats, 

i.e., a marked duction in testicular LDH-X. The occurrence of LDH-X either in traces or

its absence in B. malayi infected animals observed in the present study may be due to the 

reduced production of spermatocytes and sperms and this has been confirmed by the 

histopathological studies'(section 4.4.2.1.3). And it may also be due to the decrease in the 

testosterone levels and damage of sertoli cells.

CHAPTER 4.4 

HISTOPATHOLOGICAL CHANGES IN MASTOMYS NATALENSIS INFECTED 

WITH BRUGIA MALAY1 AND ITS RELATION TO DEC TREATMENT 

4.4.1 MATERIALS AND METHODS 

A group of 16 animals were maintained for 120 days, of wh~ch half sewed as 

control and the other half were infected with B. malayi. Four animals each from the 

control and infected group were administered with DEC (as described earlier) and 

maintained for comparison purpose. Animals were euthanized using diethyl ether and 

necropsied. Gross pathology was studied after systematic dissection. Representative 

tissues from lungs, testes, heart, brain, liver, kidney and spleen were taken and fixed in 

10% buffered formalin. Tissues were processed by routine paraffin embedding technique 

and 5 micron thick sections were prepared and stained with hematoxylin and eosin and 

examined under light microscope. 

4.4.2 RESULTS 

4.4.2.1 Studies after B. malayi infection 

4.4.2.1.1 Lungs 

The lungs of the control animals did not have any pathological change except for 

the presence of few RBC's in the alveoli. In the infected animals, the lesions Included 

mild exfoliative changes (Plate 2) in bronchiolar epithelium and moderate to severe 

haemorrhages (Plate 3). ln such areas macrophages laden with haemosederin pigment 

were also observed (Plate 4). Either sections or complete mf were seen in lumen of blood 

vessels (Plates 5 & 6). perivascular space within the extravasited blood vessel, in the 

connective tissue (Plate 6) and beneath the pluera. In some of the areas, mf was 

surrounded by mononuclear infiltratory cells. Presence of giant cells suggests the 

chronicity of the lesions (Plate 8). In a few infected animals, adult parasites were seen in 

pockets (Plate 9) and out of them a few wen ncognised as adult female due to the 

presence of mf in them. (Plate 10).

Plate2 Section of Lung of infected group showing exfoliation of bronchiola 

epithelial cells. (Haematoxylin and Eosin 400X) 

Plates: Section of Lung of infected group showing severe hemorrhages. (Haematoxylin 

and Eosin 400X) 

Plate Q: Section of Lung of infected group showing haemosiderosis in the areas of 

hemodge (Haematoxylin and Eosin 400X) 

Plates Section of Lung of infected group showing microfilana in lumen of blood 

vessel (Haematoxylin and Eosin I OOX). 

Plate 6 Section of Lung of infected group showing microfilaria across the blood vascular 

wall (Haematoxylin and Eosin 400X). 

Plate? Section of Lung of infected group showing a complete coiled microfilaria 

extravascularly (Haematoxylin and Eosin IOOOX).

Platc 2 Plate 3 

Plate 4 Plate 5 


.

Plate$: Section of Lung of infected group showing foreign body giant cells indicating 

the chronicity of lesions (Haematoxylin and Eosin 1000X). 

Plateg: Section of Lung of infected group showing adult parasite (Haematoxylin and 

bin IOOX). 

Platew Section of Lung of mfected group showing cross sections of adult female parasite 

with microfilaria (Haematoxylin and Eosin IOOOX). 

Plate fi : Secnon of Heart of infected pup showing moderate infiltration of 

mononuclear cells in epicardium. (Haematoxylin and bin 400X). 

Plate 13 Section of Hem of u L f d group showing microfilaria in myocardium 

(Haanatoxylin and b in 1000X). 

Plate \k Sectron of Heart of infected group showing microtilaria and hemorrhage in 

myoca&rn (Haematoxylin and Eosin 1000X).

Platc X Plate 9 


Plate 12 Plate 13

4.4.2.1.2 Heart 

The heart of the control animals did not show any other significant change. 

However, in the infected animals, a few mononuclear inflammatory cells were seen in the 

connective tissue and in the myocardium around the parasite. In the epicardium, 

moderate to severe infiltration of mononuclear cells was observed (Plate I I). Some of the 

muscle fibres in the myocardium had a few fat vacuoles. Microfilariae were seen in the 

myocardium (Plate 12), in the capillaries and in interstitial connective tissues. Presence 

of some erythrocytes in the vicinity of the mf indicated haemorrhage (Plate 13). 

4.4.2.1.3 Testes 

The testes of the control group had seminiferous tubules with normal 

spermatogenesis. The lumina of the tubules were filled with many sperms (Plate 14). The 

interstitial tissue had normal number of leydig cells. In this group, no circulatory or 

degenerative change was noticed. 

In the infected animals, though some of the tubules appeared normal, many of 

them did not have multilayered spermatogenic cells. The lumina contained only a few 

developed sperms (Plate IS). When compared to the control, the testicular damage was 

marked. A few tubules showed initial stages of calcification. In some areas, the 

interstitial cell of leydig was normal, but in the areas of tubular damage, these cells also 

became degenerated and atrophied. 

4.4.2.1.4 Liver 

The liver of the control animals did not have any significant change. In the 

infected group, vacuolation of hepatocytes was more pronounced and in addition, some 

of the hcpatocytes showed fatty changes and necrosis (Plate 16). Portal veins showed 

hyperaemia. Nuclear hyperchromacia and presence of twin nuclei in some hepatocytes 

suggested an ongoing regenerative process (Plate 17). A few small foci of mononuclear 

cellular infiltration particularly in the areas of hepatic degeneration were also recorded 

(Platel8). Microfilaria was mainly in the sinusoids (Plate 19).

Plate w: Section of Testes of normal group showing seminifmus tubules with 

normal spermatogenesis (Haematoxylin and Eosin 400X). 

Plate 19 Section of Testes of infected group showing moderate reduction in 

spermatogenesis indicating testicular degeneration (Haematoxy tin 

and Eosin 400x1. 

Plate Y: Won of Liver of infected group showing hydropic degeneration of fatty 

changes in the perilobular areas (Hamatoxylin and Eosin 100X). 

Plate IT Section of Liver of infected group showing hyperchromatic binucleated 

hepatocytes indicating regenerative prwm (Haematoxylin and 

=n 400X). 

Plate 18: Section of Liver of infected group showing hepatic degeneration with 

moderate mononuclear cellular infiltration (Haematoxylin and Eosin 2SOX). 

Plate 19: Section of Liver of infected group showing microfilaria in hepatic sinusoids 

(Hacmatoxylin and Eosin 400X).

Platc 14 Plate 15 

Platc I6 Plate 17 

Plate 18 Plate 19

4.4.2.1.5 Brain 

The control animals did not have any significant pathological changes.Mild 

congestion of the meningeal blood vessel was noticed in the infected brain (Plate 20). 

Mild neuronal degeneration and a few areas showing satellites and neurophagia were also 

seen. Around some of the blood vessels of brain tissue of infected animals, widening of 

Virchow- Robin's perivascular space was a feature (Plate 2 1. Numerous sections of mf 

were seen in the lumen of blood vessels and in the brain tissue (Plates 22 & 23). Around 

a few mf, the infiltration by glia cells was also recorded (Plate 24). 

4.4.2.1.6. Kidney 

In the control group, kidneys did not show any other significant histological 

change. However, in the infected animals, the renal tubules showed moderate to severe 

vacuolar degeneration of the tubular epithelium (Plate 25). In a few tubules, tubular 

necrosis leading to formation of hyaline casts was noticed. The hyaline casts were more 

in the tubules of the medullary region (Plate 26). In the renal parenchyma, some areas 

showed mild haemorrhage. The subcapsular space was widened in some places and 

showed mf (Plate 27). Sections of mf were also noticed in the glomerular tuft (Plate 28), 

interstitium (Plate 29) and in some large renal blood vessels. 

4.4.2.1.7 Spleen 

In the control animals, spleen did not show any significant pathological change. 

The amount of hemosiderin pigment was in normal quantity. However, the infected group 

showed lymphoid hyperplasia (Plate 30) and presence of giant cells (Plate 31). No mf 

was observed in the section of spleenic tissue.

Plate a Section of Brain of infected p up showing congestion of meningeal blood 

vessel (Haernatoxylin and Eosin IOOX). 

Plate 3: Section of Brain of infected pup showing widening of Varsho-Robins 

perivascular space (Haernatoxylin and Eosin 400X). 

Plate a Section of Brain of infected group showing microfilaria in the blood vessel 

(Haematoxylin and Eosin 400X). 

Plate fa: Section of Brain of infected group showing microfilaria (Haematoxylin and 

Eosin 400X). 

Plate a: Section of Brain of infected pup showing infilhation of glial cells around 

microfilaria (Haematoxylin and Eosin 400X). 

Plate 15 Won of hdncy of ~nfected group showing severe vacuolar degeneration 

in the tubular epithelial cells (Haernatoxylin and Eosin 250X).

Plate ICt Section of Kidney of infected group showing hyaline casts in the lumen of 

nedu1lary tubules (Haematoxylin and Eosin ZSOX). 

Plate 17 Section of Kidney of infected group showing nicrofilaria in subscapsular 

tissue (Haematoxylin and E&i I WX). 

Plate a Section of Kidney of infected group showing microfilaria in glonemlar tufts 

(Haematoxylin and Eosin 1000X). 

Plate P W on of Kidney of infected group showing nicrofilaria in renal interstitial 

tissue (Harmatoxylin and Eosin IOOOX). 

Plates )Om of Spleen of infected p up showing lymphoid hypcrplacia 

(Haanatoxylin and Win 250X). 

Plate31: Sea~on of Spleen of infected group showing giant cells with Langhan's type 

of appearme (Haematoxylin and Eosin 400X).

I'iatc 26 Plate 2:

4.4.2.2 Histopathological changes in relation to DEC treatment 

All the organs of the infected DEC treated animals showed similar histological 

changes like that of the infected untreated animals. However, the testicular tissue showed 

severe degeneration and the seminiferous tubules were completely devoid of 

spermatogenic cells and spermatozoa (Plate 32). They contained only fibrous material. In 

some areas, moderate to severe infiltration of the lymphocytes, plasma cells and 

macrophages were recorded. Adult female parasite sections were present in pockets 

(Plate 33). In some sections calcified adult parasites were observed. Mild connective 

tissue proliferation was present around the parasite in some sections. 

4.4.3. DISCUSSION 

Vincent et al. (1976) studied the chronological development of pulmonary 

pathology associated with B. malayi, B. pahangi and B. patei in Meriones unguiculatus, 

which caused inflammatory reactions. Pulmonary granulomas were observed during the 

final molt, followed by involution and formation of residual vascular lesions and some 

were seen during sexual maturity of the worms. Obstructive endarteritis and chronic 

interstitial inflammation with degenerating mf were also observed. Malone et al., (1976) 

studied the histopathological lesions in the lymphatic system and other major organs of 

hamsters infected with B. pahangi. Cellular infiltration of plasma cells and eosinophil, 

obstruction of pulmonary arteries and obstructive granulomatous lymphangitis were 

observed. Live and dead worms were found in testicular parenchyma. Accumulation of 

eosinophils, large mononuclear cells and plasma cells were seen in interstitial tissues 

between superfacial seminiferous tubules. Heavy accumulation of hemosiderin and giant 

cells were also observed in the lung. Degenerative or necrotic hepatocytes occurred In the 

liver. Schacher and Sahyoun (1967) reported pathologic changes due to B. pahangi in 

experimentally infected cats and dogs. Hyperplasia of lymph follicles and reticular cells 

of the nodal stroma had been reported (Schacher and Sahyoun, 1967; Mak, 1983). 

Destruction of mf had-been observed in the spleen, which caused acute and chronic 

inflammatory reaction in patients infected with B. malayi (Mak, 1983). Large number of 

lesions were observed in liver, lungs and spleen of ferret infected with B, malayi and B.

Plate 31: Section of Testes of infected DEC treated group showing severe testicular 

degeneration and C.S. of adult parasite (Haernatoxylin and Eosin IOOX). 

' Plate Section of Testes of infected DEC treated group showing sections of adult 

parasite (Haematoxylin and Eosin IOOX).

Plate 32

pahangi and no lesions in the kidneys (Crandell et al., 1982). Case et al. (1991) reported 

eosinophilic abcesses and epithelioid and giant cell granulomas with fragmented worms 

in various stages of disintegration in kidney, spleen, liver, lung, pulmonary blood vessel 

and lymphatics of ferrets infected with B. malayi. Endothelial hyperplasia, blood vessel 

obliteration with marked perivascular infiltration of lymphocytes, plasma cells, 

eosinophils and numerous large macrophages laden with a coarse golden brown pigment 

was also reported. 

The pathological changes observed in the present study in various organs were of 

inflammatory and degenerative nature suggesting tissue injury and body's response. 

Since the mf circulated mainly in the blood vascular system and acted as parasitic emboli, 

they might have got lost in smaller blood vessels, such as arterioles and capillaries of 

various organs. This might have resulted in ischemia, tissue hypoxia and subsequent cell 

injury seen in the form of various retrogressive changes. Toxic metabolic products of the 

mf and adult parasites and hepatic damage leading to decreased detoxification capacity 

can be considered as added factors in causation of degenerative changes in all the organs. 

The inflammatory changes in various organs might be in response to the mf and adult 

parasites as well as the necrosed tissues, which acted as foreign body. This is in 

agreement with earlier reports by Schacher and Sahyoun (1967) and Malone el al. 

(1976). 

The adult parasite in the testes caused significant inflammatory changes, which 

led to severe damage to spermatogenesis. In an earlier study adult worms had been 

reported in testes (Malone ef al., 1976), but no significant pathological changes were 

seen. In the present study the testicular damage after DEC treatment was more 

pronounced. Since the DEC treatment was given after the development of parasites, they 

got killed, calcified and elicited a foreign body reaction. Hence the changes became more 

pronounced and testicular damage could not be reverted back. Further studies will be 

required to understand the action of DEC in relation to stage of infection and possible 

pathological outcome.

The mf could not be seen in spleen in B.rnalayi infected animals as reported 

earlier. This may be because it is an important organ of immune system of the body. The 

fact is substantiated by mild! lymphoid hyperplacia and presence of giant cells observed 

in spleen of infected animals. This is in agreement with earlier reports by Duke (1960) 

and Mak et al. (1984), where spleen was unaffected by 5, malayi infection. Thus the 

morphological changes seen in the spleen would be dependent on the state of the host's 

patho-immunological responses. To find the specific cells of the immune system 

responsible for killing of mf and the irreversible testicular damage may be another 

interesting field for further investigation.

5. SUMMARY 

The effect of B.mala)ti infection on the host, M. natalensis on antioxidant defence 

mechanism and its relation to DEC treatment, membrane bound enzymes, testicular enzyme 

markers and histopathological changes were studied. The adult worms were found in lungs, 

testes and heart and absent in liver and brain. Lungs contained the maximum number of 

worms followed by testes and heart. 

The antioxidant enzymes, antioxidants and LPO were determined in liver, testes, 

brain, heart and lungs of the control and infected animals at various time intervals. 

Following B. malayi infection, LPO increased significantly from 0 to 120 days in 

liver, testes, brain and heart and decreased in lungs to significant levels. A negative 

correlation was observed between LPO and the activity of SODIcatalase in liver, testes, brain 

and heart and it was significant in liver and testes between SOD and LPO and in brain and 

heart between catalase and LPO. On the contrary, a positive and significant correlation was 

observed in lungs for SOD. 

In infected animals the antioxidant enzymes SOD and catalase increased initially in 

testes, brain and heart upto 30 days and declined thereafter, while in liver the activity 

increased significantly upto 60 days and declined later. In lungs, the enzyme activity showed 

significantly increasing trend throughout. A positive correlation was observed between the 

activity of SOD and catalase in liver, brain, heart and lungs and it was significant in brain 

and heart only. 

The GST activity decreased significantly from 60 days in testes, brain and heart, 

while the liver showed decreasing trend from 90 days only. And in lungs, it increased 

significantly from 60 to 120 days. A positive correlation was seen between the activity of 

GST and GSH in all the organs and it was significant in liver, testes and brain only. 

GR activity decreased significantly in liver, testes, brain and heart of the infected 

animals throughout the study period, while in lungs it showed an increasing trend from 30 

days. G6PDH decreased significantly in liver, testes and heart h m 30 to 12$days, while in

ain, it decreased from 0 day itself. However, in lungs its activity increased significantly 

throughout. The activity of GRlG6PDH had a positive correlation with GSH in all the organs 

and it was significant in testes, brain and heart alone. 

GPx activity increased in testes, brain and heart upto 30 days and declined 

significantly thereafter, but liver showed a significant decline from 0 day itself. And in lungs, 

the activity increased from 30 days. There was a positive and significant correlation between 

the activity of GPx and GSH in all the organs. 

The reduced glutathione in liver, testes and heart of the infected animals showed a 

significant decrease in their activity from 30 to 120 days and in brain from 0 to 120 days. 

But, the lungs showed an increasing trend from 30 days to 120 days. A negative correlation 

was observed between the activity of GSH and LPO in all the organs and it was significant 

in liver, testes and heart. 

The total thiol level also showed significant decline in liver, testes and brain of the 

infected animals from 0 to 120 days and heart showed similar trend from 30 days only. 

However, in lungs significant increase was noticed. A significant and positive correlation 

was observed between the thiol status and GSH levels in all the organs. 

In the infected animals, ascorbic acid level decreased significantly throughout in 

testes and heart and in liver and brain, it decreased from 30 days only. However, in lungs 

significant increase was noticed from 30 days of infection. The ascorbic acid level and GSH 

activity had positive correlation in all the organs and it was significant in liver, testes, brain 

and heart alone. 

The enzymes SOD and catalase, which were sipficantly low in all the organs of the 

infected animals increased after DEC treatment except in testes. And in lungs, DEC 

treatment reduced the increased level of these enzymes to almost normal level. 

The glutathione related enzymes and antioxidants which were reduced during B. 

rnalayi infection were almost restored back to normal level after DEC treatment in liver,

ain, heart and lungs. However, in testes, despite an innzase in activity, these enzymes and 

antioxidants level did not reach the level found in the control. 

The increased LPO observed in B. malayi infected animals in liver, brain and heart 

declined after DEC treatment. In testes, though LPO declined after DEC treatment, the levels 

were still higher than that of the control. In lungs, the LPO, which was decreased, showed an 

increase after DEC treatment. The levels of these antioxidant enzymes and LPO in the 

organs of control an~mals were found unaffected by DEC treatment. 

The activity of membrane bound enzymes of brain, such as Na' Ka ATPase, ca2+ 

ATPase, M~"ATP~S~, y-GTP, acetylcholinesterase and alkaline phosphatase of the infected 

animals showed a significant decrease from 60 days. 

Nat K' ATPase, ca2' ATPase, M~~~ ATPase in RBC of infected animals showed a 

declining trend from 30 to 120 days and acetylcholinesterase decreased significantly 

throughoul the study period. 

The haemoglobin levels and RBC count in the infected animals were found to fall 

within the normal physiological range. Total WBC counts was significantly lower 

throughout the parasite development. The absolute count of lymphocytes decreased 

significantly from 30 days and the eosinophil and neutrophil count increased significantly 

throughout. 

The testosterone levels increased initially upto 60 days and declined thereafter. y- 

GTP increased significantly in the infected animals throughout the study period. 

LDH-X enzyme bands in the infected animals were visible clearly on 30th day and 

faintly on 60th day and completely absent on 90th and 120th day, while the control was 

clearly visible in all the cases. 

Histopathological studies were done in organs such as lungs, testes, heart, liver, 

brain, kidney and spleen. Either complete or sections of mf were observed in all these organs 

except in spleen. The pathological changes in these organs were of degenerative and

inflammatory nature indicative of cell injury and body's response. The adult parasite in the 

testes caused significant inflammatory changes, which led to severe damage to 

spermatogenesis. Afler DEC treatment, these changes in the testes became more chronic and 

testicular damage became irreversible.

6. CONCLUSION 

B. malayi infection of M. natalensis has brought about the following changes in different 

organs compared to healthy (control) animals: 

Decrease in the level of antioxidant enzymes and antioxidants in liver, testes, brain 

and heart, compared to an increase in lungs. Increase in the LPO of liver, testes, 

brain and heart, compared to a decrease in the lungs. All these shifts get restored to 

normal level (as in control animals) following the administration of DEC in all the 

organs except in testes. 

Haematological parameters and the level of membrane bound enzymes of the brain 

and RBC's get affected. Testicular damage occurs as evidenced by altered marker 

enzymes. Histopathological changes take place in liver, brain, testes, heart, lungs, 

kidney and spleen which do not get rectified even after DEC treatment, and in the 

case of testes the histopathological changes became more pronounced. 

Thus, it can be said that the host, M. natalensis responded to B. malayi infection, by 

damaging its own antioxidant defence system and consequently causing tissue 

injury in all the organs studied. However, in lungs the antioxidant defence system 

seems tc be less affected leading to reduced peroxidation of lipids. DEC treatment to 

infected animals restores the activity of antioxidant enzymes, but does not rectify 

the histopathological changes in all the organs and in fact, in testes, the tissue 

damage becomes more severe i.e., degeneration of seminiferous tubules leading to 

absence of spermatogenesis. 

Histopathological changes in various organs include mechanical injury caused by 

the parasite circulation and ischemia and hypoxia due to parasitic embolism. The 

degree of the pathological changes varied from organ to organ.

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APPENDIX 

Haematological values of Mastomys natalensis (Central Drug Research Centre, National 

Laboratory Animal Centre, News Letter No. 5, Jan 1990) 

Erythrocytes (I 0I2fl) : 6.3-.7.9 

Haemoglobin (nmold) : 8.0-8.9 

Packed cell volume (%) 

Reticulocytes (%) 

WBC (I 09fl) 

Neutrophils (%WBC) 

Lymphocytes (%WBC) 

Monocytes (%WBC) 

Eosinophils (%WBC) 

Basophils (%WBC)

effect of infection of the filarial parasite brugia malayi - Pondicherry ...

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