Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes (original) (raw)
Related papers
Parasitology Research, 2008
In an attempt to develop a suitable serological test for early detection of Fasciola gigantica infection in buffaloes, a group of proteins were isolated from the somatic antigen of the parasite by immunoaffinity chromatography. The process of isolation of the proteins has been standardized and significant level of repeatability was achieved. To test the diagnostic potentiality of the antigens, two serological tests, viz., enzyme-linked immunosorbent assay (ELISA) and dot enzyme-linked immunosorbent assay, were standardized using the sera from experimentally noninfected (group A) and infected (group B) animals. Further, the sensitivity and the specificity of the tests were evaluated employing the field sera from animals of different parasitic load viz., F. gigantica positive (group C), F. gigantica and Gastrothylax crumenifer positive (group D), F. gigantica and Gigantocotyle explanatum positive (group E), a group of sera without F. gigantica but other trematode infection (group F), only G. crumenifer positive (group G), only G. explanatum positive (group H), G. crumenifer and G. explanatum positive (group I), and PM negative (group J) collected from slaughterhouses of Bareilly (Uttar Pradesh, India) and Patna (Bihar, India). In plate ELISA, the sensitivity of the antigen and the test was 75.75% while the specificity was 97%, 95%, and 98%, respectively, against G. crumenifer, G. explanatum, and mixed infection of G. crumenifer and G. explanatum, respectively. In the case of dot ELISA the sensitivity was 86.5% and specificity was 92.3%, 94.7%, and 90%, respectively, against G. crumenifer, G. explanatum, and mixed infection of G. crumenifer and G. explanatum, respectively. The potentiality of the antigen in the diagnosis of field infection is discussed.
Detection of Fasciola gigantica infection in buffaloes by enzyme-linked immunosorbent assay
Parasitology Research, 2008
The process of isolation of the 27-kDa glycoprotein from the somatic antigen of Fasciola gigantica was standardized and the diagnostic potentiality was evaluated for the detection of bubaline fasciolosis by indirect enzymelinked immunosorbent assay. Initially, the test was standardized using the sera from experimentally noninfected(n=20) and infected (n=5)animals. Further, the sensitivity and the specificity of the test were evaluated through the sera of buffaloes with different natural infections, i.e., F. gigantica (n=8 animals), F. gigantica and Gastrothylax crumenifer(n= 15), F. gigantica and Gigantocotyle explanatum (n=6), trematode infections other than F. gigantica (n=9), only G. crumenifer (n=36), only G. explanatum (n=18), G. crumenifer and G. explanatum positive (n=39), and PM negative (n=102). All animals came from the slaughterhouses of Bareilly (Uttar Pradesh, India) and Patna (Bihar, India). The level of sensitivity observed in the present study was 81.0%, while 97-98% specificity against G. crumenifer, G. explanatum, or a mixed infection with both parasites was noted. The study showed F. gigantica prevalence rate of 18-20% in the buffaloes of the study area. Enzyme-linked immunosorbent assay with a 27-kDa glycoprotein could be a feasible diagnostic method for the early detection of bovine fasciolosis.
Aim: The objective of the present study was to know the seroprevalence status of Fasciola gigantica infection in cattle and buffaloes using cysteine proteinase (CP) antigen in dot enzyme-linked immunosorbent assay (ELISA) format under field conditions. Materials and Methods: As per the standard protocol, the sera were collected from the blood of 112 cattle and 38 buffaloes of coastal areas of Navsari district, South Gujarat, India. The indirect ELISA was performed on the strip of nitrocellulose paper blotted with 1 µl of CP antigen, to detect F. gigantica seropositive animals. Results: The native CP of F. gigantica revealed a single visible band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was no any noted cross-reaction between the selected antigen and sera of Gastrothylax crumenifer-infected animals in ELISA. Out of 150 screened bovines, the sera of 47 (31.33%) were found to be reactive in dot-ELISA, with a prevalence rate of 31.25% and 31.58% in cattle and buffaloes, respectively. The seropositive bovines with heavy, moderate, and light level of infection were 44.68%, 34.04%, and 21.28%, respectively (p<0.05 between heavy and light; p>0.05 between moderate and heavy or light). The share of F. gigantica seropositive and negative animals was 31% and 69%, respectively. The optical density at 450 nm of pooled sera of seropositive bovines with heavy, moderate, and light reactivity in plate-ELISA was significantly higher with field or reference negative sera. Conclusion: The CP-based dot-ELISA can be useful for field veterinarians for quick and timely isolation of the animals requiring urgent flukicide therapy.
The Enzyme-Linked Immunosorbent Assay (ELISA) was evaluated for the diagnosis of Fasciola gigantica infection in cattle and buffaloes. The excretory-secretory (E-S Ag) antigen of Fasciola gigantica adult flukes obtained after invitro incubation was used as an antigen. The test was conducted with 276 sera collected from cattle and buffaloes which included 22 sera each from naturally infected cattle and buffaloes (known positive serum) and with similar number of samples with healthy cattle and buffaloes (known negative serum). The positive results were observed in 18 and 19 of the sera from naturally infected cattle and buffaloes with sensitivity of 81.8% and 86.3% respectively. Out of 188 serum samples which were found negative on faecal examination 32 (34%) sera of cattle and 40 (42.5%) sera of buffaloes were found positive by ELISA respectively. The sensitivity of the test was found to be 91.6% and 95.6% in cattle and buffaloes respectively.
2015
Buffaloes are the important multipurpose farm animals in the Indian sub-continent, contributing significantly to meat and milk production. The humoral immune responses to Fasciola gigantica cysteine proteinase in experimentally infected buffaloes have been widely studied. However, scarcely any literature is available on serodiagnosis of bubalian fasciolosis using purified cysteine proteinase under field conditions. In the present study, cysteine proteinase dot–ELISA using dipstick (dipstick - ELISA) was developed for the detection of natural F. gigantica infection. Faecal and serum samples were collected randomly from buffaloes (n=100) slaughtered at a local abattoir. Serum samples of buffaloes revealed 54 positive cases in Dipstick–ELISA, out of which only 35 were coprologically positive. The sensitivity, specificity and accuracy of cysteine proteinase dipstick–ELISA under field conditions were 100% whereas corresponding values for coprological examination were 62%, 100% and 80%, r...
Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection
Veterinary Parasitology, 2006
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
2014
A developed ELISA was established to diagnose Fasciola gigantica infection in lactating buffaloes by detecting antibodies in both serum and milk samples. An immunogenic fraction was obtained from crude somatic antigen after fractionation on CNBr-Sepharose 4 B affinity column chromatography and used in detection of fasciolosis in lactating buffaloes. The prevalence of infection in 61 lactating buffaloes was established using serum and milk ELISAs compared with detection of Fasciola eggs in fecal samples. Serum ELISA gave the highest diagnostic value (68.9%) followed by milk ELISA (65.6%), while parasitological examination gave 36.1%. By SDS-PAGE, the isolated fraction resolved into five bands of molecular weights 97, 84, 65, 21 and 17 KDa. Immunoreactive bands of the isolated fraction were detected by immunoblot using naturally infected buffalo sera and positive defatted milk. Two bands of molecular weights 97 and 84 KDa were detected by positive sera while other two bands of molecul...
Genetika, 2021
Fasciolosis, caused by liver fluke species of the genus Fasciola, are well recognized because of its high veterinary impact. Stool examination for Fasciola eggs is not a sensitive method, and limited efforts to find a reliable and cheaper means of detection are available. The present study aimed to develop rapid diagnostic ELISA test against fasciolosis. The excretory/secretory (ES) and somatic (SA) products of Fasciola helminths were analyzed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity was evaluated by immunoblotting using hyperimmune sera raised in rabbits and seroprevalence was determined by indirect ELISA. The results of SA antigen of Fasciola species showed polypeptide bands ranged from 10kDa-100kDa, while ES antigen of Fasciola showed bands of 15kDa-55kDa. The immunoblotting results showed the most prominent bands against ES antibodies were 25, 35, 55-70, 100 and 250 kDa and SA antigens showed 10, 15-25, 35, 70, 100 and 250 kDa polypeptide bands. The sensit...
International Journal of Research -GRANTHAALAYAH
This study was undertaken to identify Fasciola giganticai on the basis of its morphology and histology to be the common cause of fasciolosis in infected buffaloes. Material & Method: Adult liver flukes were recovered from the liver of naturally infected buffaloes slaughtered in various abattoirs in Gujarat. Some adult flukes were flattened, put between two slides , pressed and stained in Borax carmine, and some flukes were sectioned in the median sagittal plane and histological slides of the flukes were prepared for detailed morphological and histological studies. Result: Microscopic pictures of the parasite used in identification defines the similarity in the morphology and histology of the F. gigantica on the basis of morphology of flukes; anterior sucker, posterior sucker (acetabulum), pharynx, uterus, ovary and type of epithelium. Conclusion: It can be concluded that the most common species found in buffaloes infected with Fasciola gigantica on the basis of its histo-morphologic...
Prepatent detection of Fasciola gigantica infection in bovine calves using metacercarial antigen
Indian journal of experimental biology
Metacercarial antigen of Fasciola gigantica was evaluated for early immunodiagnosis of experimental bovine fasciolosis using ELISA and Western blot. In ELISA, the experimental F. gigantica infection was detected as early as 2 weeks post-infection (WPI). The gradual increasing trend of antibody level was observed from 2 to 7 WPI, followed by a plateau, which was maintained up to 14 WPI. In Western blot, sera from experimentally infected calves recognized one distinct polypeptide of 21 kDa in fractionated metacercarial antigen as early as 10 th day post infection. From 2 WPI, more polypeptide bands were reacting. Recognition of these protein bands persisted till the end of the experiment (14 WPI). Cattle sera collected from the field showed 34.5% seroprevalence of fasciolosis by ELISA using MAg. Comparative immunoblot studies of metacercarial antigen with anti-Gigantocotyle explanatum and anti-Paramphistomum epiclitum sera revealed that 21 and 25 kDa polypeptides of metacercarial antigen did not cross-react with any of these sera and appear to be unique to F. gigantica and having the desirable qualities of early and specific immunodiagnosis.