Tropical Fasciolosis Research Articles

Development of an enzyme linked immunosorbent assay using recombinant cathepsin B5 antigen for sero-surveillance of bovine tropical fasciolosis

Bovine tropical fasciolosis, caused by Fasciola gigantica, is a major parasitic disease in tropical countries responsible for significant production losses in animal husbandry practices. The disease is transmitted by the Radix sp. snails. In the early developmental stage of the parasite, the juveniles and immature flukes cause considerable damage to the liver parenchyma of the bovine host while migrating through the liver. The cathepsin (cat) B5 is a cysteine protease that is present in the excretory-secretory product of the fluke both in immature and adult stages. The early detection of fasciolosis is very critical in effective disease management. In this study, the cathepsin B5 gene from newly excysted juveniles were cloned, sequenced and analyzed. The phylogenetic analysis revealed existence of two distinct clades. The clade I includes cat B 1 to B3 whereas clade II consist of cat B4 to B7. Further, the present study was aimed to develop an enzyme linked immuno sorbent assay (ELISA) using recombinant cat B5 antigen. The developed enzyme immuno assay showed 95.3 % sensitivity and 92.4 % specificity with a cut-off of 60 % percent positive. It revealed weighted Kappa value as 0.768 (95 % CI 0.648–0.889) when compared with ELISA using native cathepsin protein. Hence, the developed assay can be exploited as a potent tool in the diagnosis and sero-surveillance of bovine tropical fasciolosis.

Efficacy of flukicides on Fasciola gigantica, a food-borne zoonotic helminth affecting livestock in Bangladesh.

Fasciola gigantica, the causative agent of tropical fasciolosis, is a food-borne zoonotic trematode that affects around 80% livestock of Bangladesh. Triclabendazole (TCBZ), nitroxynil (NTON) and oxyclozanide (OCZN) are frequently used against fascioliasis; however, the current status of potency of these flukicides was unknown. In this study, in vitro efficacy of TCBZ, NTON and OCZN at various concentrations on F. gigantica has been evaluated by relative motility (RM), morphological distortions of apical cone through an inverted microscope, architectural and ultra-structural changes through histopathological and scanning electron microscopy (SEM). It is observed that TCBZ, NTON and OCZN at higher concentrations significantly (P < 0.05) reduced RM of the flukes compared to untreated control. NTON at 150 μg mL−1 was the most potent to reduce the motility within 4 h whereas TCBZ and OCZN were much delayed. Histopathological changes showed swollen, extensive cracking, numerous vacuoles and splitting of the tegument surrounding the spines; spine dislodged from its socket in treated flukes compared to untreated worms. Histopathological changes were more conspicuous at higher doses of TCBZ, NTON and OCZN. SEM has shown the disruption of the apical cone, apart from swelling of the tegument on the ventral surface corrugation and disruption of the ventral apical cone. All these changes indicate that NTON is the most potent in killing flukes in vitro among the tested flukicides and suggest the presence of TCBZ-resistant fluke populations in Bangladesh. It is imperative to explore the in vivo effects of these flukicides and subsequently their molecular mechanisms.

Potential of recombinant 2-Cys peroxiredoxin protein as a vaccine for Fasciola gigantica infection

Helminth 2-cys peroxiredoxin (Prx) is a major antioxidant enzyme that protects parasites against hydrogen peroxide-generating oxidative stress from the hosts' immune responses. This enzyme has been found in all stages of the tropical liver fluke, Fasciola gigantica. To investigate the potential of the recombinant F. gigantica Prx-2 (rFgPrx-2) as a vaccine candidate, vaccine trials in mice were carried out. In this study, the ICR mice were immunized with rFgPrx-2 combined with Freund's adjuvant and infected with F. gigantica metacercariae. The vaccine efficacy was estimated by quantitate fluke recovery, antibody levels and liver function. The protection by rFgPrx-2 against F. gigantica infection was achieved at 43–46% compared with adjuvant-infected and non-immunized-infected control groups, respectively. The vaccine elicited both Th1 and Th2 humoral immune responses with predominance of Th2 as indicated by the higher level of IgG1 in sera of immunized mice. However, the levels of liver damage markers, serum glutamate oxalic transaminase (SGOT) and serum glutamic pyruvate transaminase (SGPT) in rFgPrx-2 immunized group did not show significant difference in comparison with the controls. This study suggested that rFgPrx-2 may have a potential as a vaccine against tropical fasciolosis.

Immunoprophylactic Control Strategy for Tropical Fasciolosis: A Possibility

Fasciolosis is a wide spread economically important helminthosis caused by Fasciola hepatica and F. gigantica and considered as a limiting factor for domestic livestock production. The strategic control measures against fasciolosis mainly depend upon judicious use of the anti-fluke drugs.

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

BackgroundTropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.MethodsIn this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.ResultsThe lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test).ConclusionsThese two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

Evaluation of an in-house enzyme linked immuonosorbent assay (ELISA) for detection of anti – Fasciola gigantica antibodies in sheep

Tropical fasciolosis caused by infection with Fasciola gigantica is regarded as one of the most important single helminthes infections of ruminants in Asia and Africa. Fasciolosis diagnosis, due to low sensitivity of coprological diagnostic method has been challenging for a long period. To evaluate the usefulness of ELISA for diagnosis of anti – Fasciola gigantica antibodies in parasitized sheep, crude, as well as excretory-secretory (E/S) antigens were examined with 50 positive and 45 negative serum samples. Results were compared with liver inspection in slaughterhouse. The cut off values were determined in 0.469 and 0.516 for crude and E/S antigen respectively. We found 82% of sensitivity, 95.6 of specificity, 95.3% positive predictive value and 83 % negative predictive value using crude antigen. We have also found 92% of sensitivity, 93% specificity, 93.8% positive predictive value and 91.4 % negative predictive value using E/S antigen. We concluded that ELISA, specially using E/S antigen, is a good tool for serodiagnosis of Fasciola gigantica infection in sheep herds.

Liver histopathology and an in-house indirect ELISA method for the diagnosis of bovine fasciolosis

Tropical fasciolosis is regarded as one of the most important helminth infections of ruminants in Asia and Africa. Throughout most of its geographical range, Fasciola gigantica is of great importance as a parasite in cattle and buffalo. Fasciolosis diagnosis, due to low sensitivity of the coprological diagnostic method has been challenging for a long period. In this study, an in-house indirect enzyme-linked immunosorbent assay (iELISA) method; one of the most sensitive tests in this regard, using excretion–secretory (ES Ag) and Crude (Cr Ag) antigens and liver histopathology of F. gigantica were described for diagnosis of fasciolosis in cattle. For this purpose, the sera and liver specimens of slaughtered cattle were collected and their liver examined macroscopically and microscopically for infestation to fasciolosis. Sera from two groups of cattle, one infected with fasciolosis (n = 50) and the other non-infected with fasciolosis (n = 50), were used in the iELISA test; grouping based on histopathology results. All sera were tested by an in-house iELISA using optimum concentration of antigens. The sensitivity, specificity, accuracy, positive and negative predictive values of iELISA with ES Ag was 88%, 80%, 85%, 81%, and 87%, respectively, and with Cr Ag was 90%, 74%, 82%, 76%, and 88%, respectively. In conclusion, there was a good correlation between these two diagnostic tests of fasciolosis although iELISA using ES Ag was more closely correlated with the histopathology findings and could be regarded as a rapid diagnostic method in bovine fasciolosis.

Vaccination of buffaloes with Fasciola gigantica recombinant glutathione S-transferase and fatty acid binding protein

Fasciola gigantica, causative agent of tropical fasciolosis, inflicts substantial economic losses on the livestock industry, affecting severely buffalo productivity in the tropical countries. Very few vaccination trials with different target antigens against F. gigantica infection have been conducted in this host. Present study describes a vaccination trial in buffaloes with F. gigantica recombinant glutathione S-transferase and fatty acid binding protein. The two recombinant proteins were expressed in Escherichia coli and evaluated for their immunoprophylactic potential in buffalo calves, using montanide 70 M-VG, a mineral oil-based adjuvant, for delivering the antigens. Buffalo calves were distributed in three groups, with group I, II and III calves immunized with recombinant glutathione S-transferase, fatty acid binding protein and a cocktail of these two antigens, respectively. Immunization of the calves evoked a mixed IgG1 and IgG2 antibody response. Present vaccination trial in these animals achieved a maximum protection level of 35%, when the two antigens were used in combination. Eosinophils were measured in both immunized and non-immunized challenge control animals, which showed a steady increase in their count in response to immunization with both the antigens and infection with F. gigantica, respectively.

Epizootiology, pathogenesis and immunoprophylactic trends to control tropical bubaline fasciolosis: an overview.

On the Indian sub-continent, nearly 5,000years ago, the domestication of the riverine buffalo-the incredible Asian dairy animal was initiated. It plays a versatile role in socio-economic upliftment of its owners from the rural agricultural communities in Asian, African, South American and a few European countries. Comparatively, buffaloes are lesser evolved and susceptible to infectious diseases than cattle. However, poor body thermoregulation and wallowing nature predisposed them to snail borne infections, especially tropical fasciolosis-an incessant major constraint on buffalo production and improvement programmes. This review article is an insight into the global prevalence, varied epizootiological factors, offers possible explanation to pathophysiological clinical signs, deleterious effects of the tropical liver fluke, involving hepato-biliary system, haemopoitic system, endocrine glands and their secretions, oxidative stress, altered metabolism and significant fall in food conversion efficiency with unaffected digestibility of nutrients. Besides, the authors have briefly discussed and reviewed the developments and significance of successful immunodiagnostic approaches for detecting and forecasting the disease during early pre-patency and feasibility of developing a cost effective immunoprotection strategies against tropical fasciolosis.

Serum enzyme and hematological profile of Fasciola gigantica immunized and experimentally infected riverine buffaloes

Sixteen male, yearling Murrah buffaloes were randomly assigned to four groups of four buffaloes each. Each animal in Gr-I and II were immunized, respectively, with 4.8 mg of excretory secretory antigen and 1,300 microg Infection Specific Antigen, in three divided doses. Subsequently, each animal in Gr-I, II, and III was per os infected with 800 viable Fasciola gigantica metacercariae (bubaline origin) on week-6 after the start of the experiment, while animals in Gr-IV served as healthy controls. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations progressively increased during the prepatency and respectively attained the highest levels during week-6 and 8 post-infection (PI). The alkaline phosphatase (AP) exhibited elevated trends from eighth week PI onward and continued to be higher until the end (p < 0.05-0.01). With the cessation of traumatic activities of the diastomes, the AST and ALT levels declined yet were throughout significantly higher than the healthy controls (p < 0.05-0.01). In the immunized animals (Gr-I and II), the fluctuation patterns were similar but the values were significantly lower than the non-immunized (Gr-III) animals (p < 0.05-0.01). The elevated levels of the enzymes had positive correlation with depressed erythrocytic indices, leucocytosis, eosinophilia, necropsy worm recovery, and hepatic lesion score in the respective groups. The increased concentrations of the enzymes revealed two clearly demarcated stages: (a) remarkably elevated AST (40.8%) and ALT (140.0%) levels during the prepatency, signifying traumatic lesions inflicted by the F. gigantica adolescercariae and (b) the significant increase in AP (107.9%), suggestive of bile duct hyperplasia, cholangitis, periportal fibrosis, and biliary obstruction etc. from sixth week PI onward. None of healthy controls developed clinical signs and had normal hematological and serum enzyme profiles. Diagnostic significance of these marker enzymes in the disease forecasting and in time application of control strategies to combat tropical fasciolosis in buffaloes in the endemic areas has been discussed.

Kinetics of serum immunoglobulin isotype response in experimental bovine tropical fasciolosis

The present communication reports on the kinetics of immunoglobulin isotype response in Fasciola gigantica infected bovine calves. Fifteen Holstein–Friesian cross-bred male calves were assigned to 3 groups (Gr) of 5 calves each and infected with 4-month (Gr-A) and 16-month-old (Gr-B) F. gigantica metacercariae ( n = 400), respectively, while Gr-C calves served as uninfected control. Infection was terminated by treating the animals with triclabendazole on 28 weeks post-infection (WPI). Sera were collected on 0, 4, 10 and 14 days post-infection (DPI) and subsequently at weekly interval up to 32 WPI. The immunoglobulin isotype response was analyzed by ELISA, using anion exchange purified antigen fraction. An IgG response against F. gigantica infection was evoked by 3 and 2 WPI in animals of Gr-A and Gr-B, respectively with peak antibody response at 13 WPI. Elicitation of an early IgG1 response by 10 and 14 DPI but a delayed IgG2 response at 6 and 4 WPI, in animals of Gr-A and Gr-B, respectively was recorded. An early IgM response was evoked by 10 and 14 DPI and the level peaked at 13 and 12 WPI, with no detectable level at 21 and 15 WPI in animals of Gr-A and Gr-B, respectively. IgA response was elicited at 4 WPI in both the groups and showed the highest titre at 25 and 27 WPI in animals of Gr-A and Gr-B, respectively. Present study indicated an early and predominant response of IgG1, with concurrent expression of delayed and weak IgG2 in calves experimentally infected with F. gigantica.

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.

Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle

Vaccine trials were conducted in Brahman cross cattle evaluating the efficacy of 4 native antigens purified from adult Fasciola gigantica flukes, and 1 recombinant F. gigantica antigen, as vaccines against tropical fasciolosis. The antigens tested were native glutathione S-transferase, cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli, and were formulated in 1 or more of several adjuvants (Quil A, Squalene Montanide 80 ®, MF59-100, Auspharm ®, NAGO, polylactoglycolide microspheres, Algammulin, DEAE, Freund's). Vaccination induced low, moderate or high antibody titres to the various antigens which were dependent on the adjuvant. Low but significant reductions in fluke burdens (31%, P < 0.026) and fluke wet weight (36%, P < 0.041) were only observed in cattle vaccinated with the native FABP in Freund's adjuvant. There was no correlation between total antibody titres to FABP and protection. The protection observed in cattle vaccinated with native FABP of F. gigantica supports the notion that this class of proteins is a useful target for protection of animals against Fasciola and extends the efficacy of FABPs to the tropical liver fluke. This is the first report of vaccination of cattle against F. gigantica with a purified protein.