Antigenic characterization of 52-55kDa protein isolated from Trypanosoma evansi and its application in detection of equine trypanosomosis.

Trypanosoma evansi is a haemo-protozoan parasite responsible for the disease surra, an economically important disease of wide range of domestic and wild animals. The present diagnostic methods using soluble antigens have inherent problems like lack of standardized and reproducible antigens, as well as ethical issues. This entails further efforts for search of defined antigenic molecules with satisfying sensitivity and specificity for sero-epidemiology of trypanosomosis. In present investigation, we have identified and purified 52-55kDa immuno-dominant protein cluster in molecular mass ranges by preparatory SDS-PAGE methods from T. evansi proteome. The purified protein was further characterized by hyper immune serum raised in rabbits and also further evaluated for its immunodiagnostic potential using experimentally infected horse serum samples by different immunological tests. The immunoblot, ELISA and dot blot assay using purified cluster in infected pooled serum samples showed detection of infection early as 10th days post infection till termination of experiment. The observations revealed that purified cluster is expressed not only at early stage but also persisted and detected throughout course of infection. Further, whole cell lysate antigen separated out and detected 141 spots by 2-D gel electrophoresis. The isoelectric focussing (PI) of 52-55kDa was determined in pH range between 6.9 and 7.5 along with two other cluster of proteins recognised by immune sera of ponies infected with T. evansi. MS/MS analysis of the purified protein cluster identified five proteins i.e. pyruvate kinase 1, beta tubulin, paraflagellar rod protein, alanine aminotransferase and variable surface glycoprotein showing homology to protein present in Trypanosome database. These identified proteins may be useful for development of vaccines and diagnostic targets against animal trypanosomosis.

Comparative evaluation of recombinant HSP70 (N & C-terminal) fragments in the detection of equine trypanosomosis.

Trypanosomosis (Surra) is an economically important disease caused by Trypanosoma evansi which is an extracellular parasite present in the plasma, tissues and other body fluids of a wide range of hosts including domesticated animals. Currently, serological reports are based on detection of antibodies by ELISA using whole cell lysate (WCL) antigen, which has a limitation of persistence of anti-trypanosomal antibodies after successful treatment of the disease. Moreover, it has some ethical issues also like requirement of mice for in vivo maintenance of parasite for preparing the antigen. Therefore, in the present study, an attempt was made to evaluate the in vitro production of recombinant heat shock protein 70 (HSP70) for detection of antibodies in experimentally infected ponies. The amino acid sequence analysis of HSP70 revealed that N-terminal region of the protein was highly conserved while the C-terminal region was most divergent. The four different regions of HSP70 protein viz. HSP-1, HSP-2, HSP-3 and HSP-4 were cloned and expressed, among which HSP-1 (N-terminal region) & HSP-2 (C-terminal region) were truncated while HSP-3 & HSP-4 were complete C-terminal proteins. The recombinant fragments were probed with sequentially pooled experimental serum samples where antibodies were detected in these fragments from 10(th) day post infection till the termination of the experiment. Further, these recombinant fragments were also comparatively evaluated with WCL antigen in ELISA using experimental as well as field serum samples. It was observed that after successful treatment of infected ponies, there was a sharp fall in antibodies (within 90 days) when tested with recombinant HSP's fragments, while antibodies persisted even after 469 days when tested against WCL antigen. The sensitivity and specificity of all HSP70 fragments were also estimated from field serum samples with reference to WCL antigen ELISA. The HSP-1 showed minimum sensitivity (41.03%) among all the recombinant fragments. Among the C-terminal fragments, maximum sensitivity was observed with the HSP-2 (61.54%) while minimum was observed with HSP-4 (48.72%). The specificity increases for recombinant fragments from N-terminal to C-terminal region of protein and maximum specificity was observed with HSP-4 fragment (91.3%).

Comparative efficacy of different in vitro cultivation media for Trypanosoma evansi isolated from different mammalian hosts inhabiting different geographical areas of India.

The present study was undertaken to establish an optimal medium for primary culture initiation and maintenance of T. evansi isolated from different mammalian hosts of diverse geographical regions of India viz. donkey/1 (Hardoi, Uttar Pradesh), donkey/2 (Junagarh, Gujarat), pony/1 (Hisar, Haryana), camel/1 (Bikaner, Rajasthan) which represented isolates 1, 2, 3 and 4, respectively. Primary cultures were initiated with all four isolates in five different in vitro cultivation media with seeding density of 1 × 10(6) trypanosomes/ml. The parasites of all four isolates could remain viable only for 48 h in medium E (Alsever's solution) and for 72 h in medium A, C and D. Parasites reached to a maximum density (2.5-3.75 × 10(6)/ml) within 24 h and thereafter, a sharp decline (0.5-0.75 × 10(6)/ml) in the next 72 h was observed in 1, 2 and 3 isolates cultured in medium B. In isolate 4, parasite counts got more than doubled in 24 h and then decreased gradually up to sixth day post initiation of cultivation which thereafter increased gradually up to 34 days and a constant parasite number of 10(5)/ml could be achieved for 90 days in medium B. During this prolonged culture the trypanosomes retained their long slender morphology and infectivity to mice.

Early detection of Trypanosoma evansi infection and monitoring of antibody levels by ELISA following treatment.

In present communication, we report an outbreak of Trypanosoma evansi in equine herd n = 30 (horse and mules) which, were reared in fly proof stables as well as in open paddock maintained under semi-intensive system of management, and its effective control using trypanocidal drug. The infection was monitored by antibody ELISA up to 180 days post-treatment (PT). A total of 8 out of 14 equines (57.14 %) which were maintained only in open paddocks were found positive with T. evansi infection parasitologically. The infected animals were treated with quinapyramine methyl sulphate and chloride combination administered at the prescribed dose rate on 3rd day of screening. The parasite could not be detected from any treated animals from day-3 PT up to 6 month. Further, we also could not observe relapse of infection, neither in treated group nor in equine herd maintained at the farm. Sero-conversion was observed in all eight animals by 10th day of screening, indicating that immune response was due to recent infection as the animals became chronologically positive. The antibody titre reached at the peak by 10-14th day in all infected animals, and started declining by 17th day of screening, further reached to near cut off level by 180 days. Since, antibodies persisted up to 6 month PT and antibody detection assays are not able to differentiate between current and past infections in treated cases. The detection of circulating antigen assay and parasitological techniques in combination may be performed for effective diagnosis and management of T. evansi infection.

Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines.

Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immuno blot. Three immuno dominant clusters of proteins i.e. 62-66 kDa, 52-55 kDa and 41-43 kDa were identified based on their consistent reactivity with donkey sequential serum experimentally infected T. evansi up to 280 days post infection (dpi). The protein cluster of 62-66 kDa was purified in bulk in native form and comparatively evaluated with whole cell lysate antigen (WCL). ELISA and immuno blot showed that polypeptide of this cluster is 100% sensitive in detection of early and chronic infection. Further, this protein cluster was also found immuno reactive against hyper immune serum raised against predominantly 66 kDa exo antigen, revealed that this is a common immunodominant moieties in proteome and secretome of T. evansi.

Development of an antibody-ELISA for seroprevalence of Trypanosoma evansi in equids of North and North-western regions of India.

The importance of Trypanosoma evansi as the etiological agent for surra is often overlooked due to difficulty in accurate diagnosis of the disease. In the present study, an antibody-ELISA was developed using whole cell lysate antigen prepared from purified trypanosomes and used for seroprevalence study of T. evansi in equids. A total of 3695 equids were surveyed and blood samples were collected from each animal during September 2009 to August 2011. Out of these, 420 serum samples were found positive for presence of antibodies against T. evansi collected from equids of six agro-climatic zones of North and North-western regions of India comprising eight states viz., Gujarat (36/479), Haryana (11/275), Himachal Pradesh (14/83), Jammu and Kashmir (32/221), Punjab (1/38), Rajasthan (90/1148), Uttarakhand (141/753), and Uttar Pradesh (65/330). The maximum seroprevalence (19.69%) for T. evansi infection was observed in equids of Uttar Pradesh state with an overall seroprevalence of 11.36% in North and North-western regions of India. The results indicated that surra is endemic in equids of North and North-western parts of India.

Identification and characterization of cysteine proteinases of Trypanosoma evansi.

Trypanosoma evansi is a causative agent of 'surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.

A randomized double blind placebo controlled clinical evaluation of extract of Andrographis paniculata (KalmCold) in patients with uncomplicated upper respiratory tract infection.

A randomized, double blind placebo controlled clinical study was conducted to evaluate the efficacy of KalmCold, an extract of Andrographis paniculata, in patients with uncomplicated upper respiratory tract infection (URTI). The assessment involved quantification of symptom scores by Visual Analogue Scale. Nine self evaluated symptoms of cough, expectoration, nasal discharge, headache, fever, sore throat, earache, malaise/fatigue and sleep disturbance were scored. A total of 223 patients of both sexes were randomized in two groups which received either KalmCold (200 mg/day) or placebo in a double blind manner. In both the treatments, mean scores of all symptoms showed a decreasing trend from day 1 to day 3 but from day 3 to day 5 most of the symptoms in placebo treated group either remained unchanged (cough, headache and earache) or got aggravated (sore throat and sleep disturbance) whereas in KalmCold treated group all symptoms showed a decreasing trend. Within groups, mean scores of symptoms in both the groups decreased significantly (p < or = 0.05) from day 1 to day 3 and day 5 while from day 3 to day 5 all symptoms except expectoration in placebo group did not improve significantly whereas in KalmCold treated group all symptoms improved significantly (p < or = 0.05) except earache. Comparing mean between both groups, all symptoms at day 1 and day 3 were found to be the same while at day 5 all symptoms except earache in KalmCold treated group improved significantly (p < or = 0.05) than placebo group. Similarly, within groups, overall scores of all symptoms in both the groups decreased significantly (p < or = 0.05) from day 1 to day 3 and day 5 while from day 3 to day 5 placebo group did not improve significantly whereas KalmCold treated group showed significant improvement (p < or = 0.05). On between groups analysis, KalmCold group showed significant reduction (p < or = 0.05) in overall symptom scores as compared to placebo group. In both placebo and KalmCold treated groups, there were only a few minor adverse effects with no significant difference in occurrence (Z = 0.63; p > 0.05). The comparison of overall efficacy of KalmCold over placebo was found to be significant (p < or = 0.05) and it was 2.1 times (52.7%) higher than placebo. The findings of this study revealed that KalmCold was effective in reducing symptoms of upper respiratory tract infection.

Immune responses to polyethylenimine delivered plasmid DNA encoding a Fasciola gigantica fatty acid binding protein in mice and rabbits.

Fasciola gigantica fatty acid binding protein (FABP) was evaluated for evoking an effective immune response in mice and rabbits, when delivered as a DNA vaccine in muscle cells. Polyethylenimine (PEI), 25 kDa, branched cationic polymer was used as a delivery vehicle for this DNA in the muscle cells of mice and rabbits. Naked DNA evoked mixed Th1 and Th2 responses in mice. PEI condensed DNA, at amine nitrogen over DNA phosphate (N/P) ratios of 4, 6 and 8 and with various DNA concentrations, failed to evoke a significantly higher antibody response compared to naked DNA in mice. Similarly, the humoral immune response to naked DNA administration in rabbit thigh muscles was poor and no boosting of this antibody response on administration of DNA complexed to PEI was observed. On metacercarial challenge, rabbits failed to show any significant protective immune response in both the naked DNA and PEI-DNA immunized groups. Administration of PEI alone (12.5 mug) in mouse thigh muscles caused significant muscle cytotoxicity but condensation of DNA with PEI had less of a toxic effect on muscle cells, which was inversely related to the N/P ratio. Delivery of plasmid DNA encoding F. gigantica FABP by high molecular weight polyethylenimine (branched, 25 kDa) did not boost the effective immune response in both the animal species, which could either be attributed to cytotoxicity associated with this cationic polymer or muscle cells being unsuitable target cells for PEI condensed DNA delivery.

Nonoxynol-9 vaginal pessary: a preliminary Indian experience.

BACKGROUND: Barrier methods of contraception do not have systemic effects and allow the user complete control over their use. For women, the ease of use of a contraceptive is often more important than its efficacy. Hence, barrier methods could be offered as a useful alternative method of contraception. Nonoxynol-9 (a spermicide) is a locally acting, non-hormonal method free from systemic side-efforts. It is a woman-controlled, reversible method which is to be used before intercourse. There are little data available on its efficacy, side-effects and acceptability among Indian women. METHODS: The vaginal pessary nonoxynol-9 was offered as a contraceptive option to 3200 women attending the Family Planning clinics at 31 Human Reproduction Research Centres (HRRCs) of the Indian Council of Medical Research. The other contraceptives offered included an intrauterine device, oral pills, condoms, Norplant, tubal sterilization and vasectomy using the cafeteria approach. Those who accepted nonoxynol-9 were followed up to assess the rates of continuation, failure and side-effects. RESULTS: The nonoxynol-9 pessary was accepted by 541 women who were followed up for 3470 woman-months of use. The reasons given for acceptance were that it was user-controlled and/or they did not wish to use other methods because of the side-effects or contraindications of these methods. The overall continuation rates were 41.2% and 33% at 9 and 12 months of use, respectively. Most women (31.3%) discontinued its use due to personal reasons such as husband dissatisfaction, desire for further pregnancy, irregular use of pessary and difficulty in insertion. Twenty-nine women became pregnant during the study period (15 due to method failure and 14 due to user failure) giving a use-effectiveness of 8.8% at 12 months. The method failure rate was 4.3% at 12 months of use. The failure rates were lower compared with the reported failure rates of barrier contraceptives (1%-30% at 1 year of use) and the side-effects were minimal. CONCLUSION: Nonoxynol-9 had low acceptability (16.9%) and overall continuation rates--41.2% and 33% at 9 and 12 months of use. It could be offered to women looking for a short term, user-controlled contraceptive.

Immuno-diagnosis of bubaline fasciolosis with Fasciola gigantica cathepsin-L and recombinant cathepsin L 1-D proteases.

Fasciola gigantica cathepsin-L cysteine proteinase and recombinant cathepsin L 1-D were assessed for their potential in the immuno-diagnosis of F. gigantica infection in buffaloes. A diagnostic ELISA, based on these two antigens, was developed to detect antibodies against F. gigantica in water buffaloes. Sensitivity of the ELISA was assessed using sera from buffaloes experimentally or naturally infected with F. gigantica from F. gigantica endemic areas and its specificity by probing the sera of the host from F. gigantica non-endemic area. Our earlier studies under experimental setting showed 100% sensitivity of cathepsin-L ELISA in the diagnosis of fasciolosis in buffaloes, with the earliest detection of infection at 4 weeks post-infection. However, under field situation of natural F. gigantica infection, this sensitivity declined to 97.1% but specificity of the test remained 100%. Cross-reactivity of the antigen was checked with Schistosoma indicum, S. spindale, Paramphistomum epiclitum, Gastrothylax spp., Gigantocotyle explanatum, hydatid and Strongyloides papilossus in the bubaline host, naturally infected with these helminths. F. gigantica cathepsin-L and the recombinant cathepsin L-1D does not cross-react with these helminth parasites in natural mono or mixed infection of the host. The present ELISA contributes a relatively sensitive and reliable tool for the early serodiagnosis of bubaline fasciolosis.

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection.

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.

Fasciola gigantica cathepsin-L cysteine proteinase in the detection of early experimental fasciolosis in ruminants.

Cathepsin-L cysteine proteinase was purified from Fasciola gigantica regurgitant by two-step alcoholic fractionation, followed by ion-exchange chromatography. The purification strategy was evolved to eliminate other contaminating proteins co-precipitating with the purified proteinase during alcoholic fractionation. The enzyme was stable on long-term storage at -20 degrees C rendering it more suitable for field diagnostic use. The purified cathepsin-L cysteine proteinase was assayed for detection of F. gigantica experimental infection in sheep and buffaloes and could detect infection, as early as 4 weeks post-infection by ELISA, Western blotting and Dipstick ELISA. The 28-kDa cathepsin-L cysteine proteinase seems a promising antigen for the diagnosis of tropical fasciolosis in domestic animals.

Vaccination of buffaloes with Fasciola gigantica recombinant fatty acid binding protein.

Recombinant fatty acid binding protein (rFABP) of Fasciola gigantica was expressed in Escherichia coli and used as vaccine in Freund's adjuvant to evaluate the level of protection induced in buffalo (Bubalus bubalis) calves. Fifteen buffalo calves were distributed to three groups of five calves each. An antigen dose of 400 mug for each of the three immunizations at 3-week intervals, and a challenge dose of 600 metacercariae was administered per calf. Levels of anti-FABP antibodies increased rapidly by 2 weeks after the first immunization and were always significantly higher in the immunized-challenged group than in the infected control group. Immunization with FABP induced both humoral and cell-mediated immune response in these animals. Vaccination showed a moderate level of protection in terms of reduced fluke burden (35.8%) and liver damage as assayed by aspartate aminotransferase and sulfhydryl group levels as well as anti-fecundity effect of the vaccine.

Humoral immune response against Fasciola gigantica fatty acid binding protein.

Recombinant fatty acid binding protein of Fasciola gigantica was expressed in Escherichia coli and purified by nickel chelating affinity chromatography. The recombinant protein along with native fatty acid binding protein (FABP) isolated from the parasite were evaluated for their potential in the diagnosis of F. gigantica infection in sheep, cattle and buffaloes, both by ELISA and western blotting. Results of this study indicate that there is no humoral immune response generated against this protein in the experimental infection of these ruminants with F. gigantica, thereby limiting the usefulness of this antigen in the early diagnosis of fasciolosis in these animals. Also, the paper discusses the probable reasons for the failure of this protein in detecting humoral response in these animals by ELISA and immunoblotting.

28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis.

Coprological confirmation of ovine fasciolosis in the field, prior to out breaks of the disease and/or strategic antifluke medication, seem to be of little consequence. Efforts are, therefore, being made to evolve a putative antigen specific to serodiagnostic test for early diagnosis during prepatency. In the present investigation, 28 kDa cysteine proteinase was used in ELI SA and Western blot to detect Fasciola gigantica antibodies and further Dipstick-ELISA was developed for field application, using known positive monospecific sera from experimentally infected sheep with 100 F. gigantica metacercariae. Isolation of 28 kDa cysteine proteinase was achieved from bubalian origin flukes. The specific antigen, recognised homologous antifluke antibodies by Western blot as early as 2nd week post-infection (wpi) with 100% sensitivity, in sera samples of sheep harbouring 38 flukes and by 10th wpi in sheep harbouring 3-8 flukes. All sheep were found positive for the infection when ELISA and/or Dipstick-ELISA was applied from 4th wpi. In pooled sera of infected sheep, these were positive during 4th wpi.

Evaluating contraceptive choice through the method-mix approach. An Indian Council of Medical Research (ICMR) task force study.

The method-mix approach was used to evaluate informed contraceptive choices in the present study. A total of 8,077 potential clients were given a balanced presentation of all available contraceptive methods in the national program, ie, the CuT 200 intrauterine device (IUD), low-dose combined oral pills (OC), condom, and sterilization (female/male) along with a new method, Norplant(R).(1) The majority of women opted for spacing methods; among them, the IUD was preferred by about 60% of clients, followed by condoms (9%), OC (6%), and Norplant (5%). Sterilization, mainly female, was accepted by about 17% of the women making an informed choice. The economic status of couples did not influence the contraceptive choices, as all the methods were offered free of cost in the present study, which is the current practice in the national program. Illiterate women more often accepted sterilization (about 25%) than did literate women (15%). This is because illiterate women had more children; about 30% of illiterate women had three or more children, as opposed to 16.2% of literate women. However, literacy status did not influence the choice of any specific spacing method. The study also revealed that, by encouraging potential clients to make an informed choice, they could override the provider's bias while accepting a particular type of spacing method. This is evident from the observation that Norplant was the first choice of the provider for 35% of the women, whereas only 5% of women preferred and accepted Norplant. The present study stresses an urgent need to promote the practice of informed choices in the national program with a variety of contraceptive options-especially, spacing methods for improving contraceptive prevalence and reproductive health in the country.

Primary experimental infection of riverine buffaloes with Fasciola gigantica.

The clinical course of the primary experimental Fasciola gigantica infection was investigated in riverine buffalo calves of the Murrah breed. Nine male calves aged 12-15 months were randomly assigned to two groups of five (Group I) and four (Group II) animals. Each animal in Group I, was orally infected with 1000 metacercariae (mc) of F. gigantica, whereas Group II animals did not receive any infection dose and served as uninfected controls. No clinical signs of fasciolosis were observed until the sixth week post-infection (PI). Group I animals, however, developed recognised symptoms of acute fasciolosis, comprising apyrexic inappetance, anemia, poor weight gain, diarrhoea and sub-mandibular and facial oedema, respectively, from 5, 6, 8, 16 and 17 weeks PI. The signs were intermittent in nature and of variable duration. The prepatent period was of 92-97 days (mean 95.2 +/- 3.1). One of the five infected animals died on Day 147 PI. At necropsy, 36.8 +/- 11.0% of the infection dose was recovered as adult fluke population. The gross lesions were primarily biliary in nature. Group II, the uninfected controls, throughout the study period of 165 days PI, did not show any symptom and were negative for F. gigantica. The study demonstrated that the onset of adverse effects of F. gigantica on the growth and health of the infected host was mainly noted during late prepatency much before coprological prediction and diagnosis. The significance of preventive therapy against fasciolosis during prepatency has been stressed in endemic areas.