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DEVELOPMENT OF DOT-ELISA FOR THE DIAGNOSIS OF BOVINE VISCERAL SCHISTOSOMOSIS

KrishiKosh

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Title DEVELOPMENT OF DOT-ELISA FOR THE DIAGNOSIS OF BOVINE VISCERAL SCHISTOSOMOSIS
 
Creator SUDHAKAR, KOMMU
 
Contributor Dr. G. S. SREENIVASA MURTHY
 
Subject organic acid salts, biological phenomena, diseases, research methods, organic compounds, toxicity, amino acids, body weight, enzymes, proteins
 
Description Schistosomosis has been recognized as one of the major parasitic diseases of livestock and human beings. Schistosoma spindale is the major cause of visceral schistosomosis among bovines. A laboratory standardized Dot-Enzyme Linked Immmunosorbent Assay (Dot-ELISA) was used to diagnose the field cases of visceral schistosomosis in bovines and the sensitivity and specificity of the same was compared with CCIEP. The morphological characters of S. spindale were also studied by light microscopy and scanning electron microscopy (SEM) in the present investigation.
The mesenteries collected from bovines slaughtered at slaughter house, Chengicherla, Hyderabad, Telangana state (Erstwhile Andhra Pradesh) and found positive for tiny, white, cream coloured flukes in the blood vessels were brought in ice within 1 hr of collection to the laboratory. The flukes in the blood vessels were harvested by perfusion
technique. The abattoir study indicated an overall prevalence of 31.42 per cent (33/105) of schistosomosis in slaughtered bovines.
The harvested schistosomes were identified by light microscopy based on their morphological characters viz., smooth cuticle along with number of testis (3-5) and 15-20 spindle shaped eggs containing spine in male and female worms of S. spindale, respectively and spiny or tuberculated cuticle along with oval shaped eggs with a terminal spine in case of S. indicum male and female worms respectively.
Scanning electron microscopy (SEM) confirmed the male and female worms of Schistosoma spindale in copula stage with well defined gynaecophoric canal, originating from just below the ventral sucker up to the posterior end of the body. The posterior part of the male worm ended in a wide conical projection. The oral sucker of the male was hollow, triangular and sub terminal in position with thick muscular rim without spines. The ventral surface of the oral sucker was completely covered with numerous spines measuring about 3μm in length. Irrespective of the position on the oral sucker, all spines were directed downwards in to the aperture of oral cavity.
The ventral sucker was pedunculated, round, thick rimmed. The rim of the ventral sucker was 22μm in width and the inner side of which had numerous pointed spines measuring around 2.5μm in length and were directed towards the center of the ventral sucker. An aspinulated region measuring nearly 5-6μm was noticed beneath the lower border of rim of ventral sucker which was followed by a spinulated region with numerous spines blunter than the spines on oral sucker. The tegument surface of S. spindale showed ridged layers with large uniciliated and pit like papillae measuring approximately 3μm in diameter which were recorded more in posterior end. There was no spination in between
uniciliated and (or) pit like papillae. The cuticular ridges on the tegument of female S. spindale were smoothly perforated, compact, coarsely placed when compared to male worm. The number of epidermal papillae was more in female than in the male worm.
The worms identified as S. spindale by light microscopy were collected and washed thrice in PBS (pH. 7.2) to remove host contamination. Excretory and Secretary (ESA) and Whole Worm (WWA) antigens were extracted from S. spindale. ES antigens were extracted from 500 live worms which were incubated at 37˚C for overnight in 5 per cent carbon dioxide incubator followed by centrifugation at 13,000 rpm for 30 min at 4˚C and the protein content of supernatant was quantified as 2.01mg/ml.
The WWA was extracted from 500 worms by homogenizing in a glass tissue homogenizer followed by sonication at 20 kHz for 10 cycles for 90 seconds each with a gap of one minute interval. Then the contents were centrifuged at 9,500×g for 15 min at 4˚C in a high speed centrifuge. The supernatant containing a protein content of 3.88 mg/ml was used as whole worm antigen.
Hyper immune sera was raised against ESA and WWA of S. spindale in New Zealand white rabbits and the antibody development was checked by DID and CCIEP techniques using healthy rabbit sera as control.
Dot-ELISA was standardized in the laboratory for the diagnosis of visceral schistosomosis caused by S. spindale infection in bovines. The different dilutions of antigens (WWA & ESA) were titrated against different dilutions of respective hyper immune sera and goat anti rabbit IgG HRP conjugate. Irrespective of intensity, the appearance of brown color dot reaction was considered as positive.
The nitro cellulose strips dotted with 1.5μl each of either WWA (91ng) or ESA (93ng) were incubated at 37˚C for 1 hr followed by blocking with 5 per cent BSA for 1hr and again incubation at 37˚C for 1hr in 1:100 dilutions of hyper immune sera of respective antigens. The strips were washed in 0.005 per cent Phosphate Buffered Saline Tween-20 (PBST) for 6 times (each for 10 min) and incubated in goat anti rabbit IgG HRP conjugate (1:2000) for 1hr at 37˚C followed by washing in 0.005 per cent PBST for 6 times (each for 10 min). The strips were treated with DAB for 2-3 min and the positive reactions were recorded as brown dot formation and reaction was stopped by adding double distilled water.
Two out of three sera samples collected from cattle, positive for amphistome ova by faecal examination showed cross reaction with only WWA but not with ESA.
Specificity studies were conducted on 11 sera samples collected from cattle reared in an organized farm found negative for helminth ova and with no history or symptoms of schistosomosis. Sensitivity studies were conducted on 33 sera samples collected from cattle, positive for schistosomes by examining the mesenteries.
The sensitivity and specificity studies on counter current immuno electrophoresis (CCIEP) using WWA and ESA with known positive bovine sera samples were recorded as 82.5% (26/33) & 78.57% (8/11) and 86.84% (28/33) & 84.61% (9/11), respectively.
Similarly, the sensitivity and specificity of Dot-ELISA with WWA and ESA with kwon positive sera were 76.74% (23/33) and 73.3% (7/11) and 80.48% (25/33) and 78.57% (8/11), respectively.
The laboratory standardsized dot-ELISA was tested for its efficacy on sera samples cattle and buffaloes collected from the field. Out of 125 sera samples screened in cattle, 41 were positive indicating 32.80 per cent infection with WWA and 51 were positive
indicating 40.80 per cent infection with ESA. Whereas screening of 163 sera samples in buffaloes, 53 were positive indicating 32.51 per cent infection with WWA and 66 were positive indicating 40.49 per cent infection with ESA.
The dot-ELISA detected S. spindale specific antibodies against whole worm antigen in 94 out of 288 bovine sera samples indicating 32.63 per cent of bovine schistosomosis. Similarly, the test using excretory and secretary antigen could detect 117 positive cases of schistosomosis out of 288 serum samples screened indicating 40.62 per cent of infection.
Hence, the dot-ELISA standardized with ESA of S. spindale in our laboratory may be recommended for the diagnosis of schistosomosis in the field.
 
Date 2017-01-04T16:06:49Z
2017-01-04T16:06:49Z
2014-12-16
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/94545
 
Relation D;408
 
Format application/pdf
 
Publisher PVNR TVU