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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

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2007 - 200912020 - 20242
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Subject: Veterinary Parasitology × Thesis Type: Ph.D ×
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    Detection of anthelmintic resistance against gastrointestinal nematodes in sheep and goats
    (Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-04) Santosh; Santosh; Vohra, Sukhdeep; Vohra, Sukhdeep
    e study was conducted to evaluate the status of anthelmintic resistance in gastro intestinal nematodes (GINs) in sheep and goats at two organised farms (CSBF and UCBF) located in Hisar, Haryana. Sheep and goats reared at these farms exhibited reduced efficacy for multiple anthelmintics following treatments with Fenbendazole (FBZ), Closantel (CLS), Morantel (MOR), and Ivermectin (IVM) in the Faecal Egg Count Reduction Test (FECRT). The results suggested that the overall efficacy was highest for CLS at 89.41% and 77.23% in sheep and goats, respectively, on the 14th day post-treatment, and least for FBZ at 58.73% and 64.55% in sheep and goats, respectively. The pre-treatment faecal culture revealed Haemonchus contortus, Trichostrongylus spp. Strongyloides spp., and Oesophagostomum spp.; however, in post-treatment samples, H. contortus was predominantly observed. ED50 values of EHA demonstrated the presence of thiabendazole resistance in one sheep farm (UCBF) and two goat farms (CSBF and UCBF). Further, the 180 infective larvae of H. contortus from both sheep and goat farms were subjected to allele-specific PCR (As-PCR) for accurate diagnosis of BZ resistance. The As-PCR revealed 40% homozygous resistant (rr), 46.11% heterozygous (rS), and 13.88% homozygous susceptible (SS), with the overall frequency of the resistant (r) allele being 63.5% and for the susceptible allele (s) being 36.5%. The survey indicated that the GINs of sheep and goats on the farms have developed multiple anthelmintic resistance to FBZ, CLS, MOR, and IVM, and the condition is alarming on the farm.
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    Screening, identification and evaluation of some novel target specific therapeutic compounds against Trypanosoma evans
    (Lala lajpat rai university Hisar, 2022-06) Gupta, Snehil; Vohra, Sukhdeep; Rajender Kumar, Rajender Kumar
    The Trypanosoma evansi is the most pathogenic animal trypanosome, which leads to annual economic loss of US $ 671.1 million in India. Limited drug availability, high toxicity, severe side effects and emerging drug resistance necessitate the investigation of novel and safer chemotherapeutic agent. Therefore, in the present study, a total of 36 drug molecules were evaluated for their anti trypanosomal activity against T. evansi and cytotoxicity against mammalian cells. Drugs were divided into 4 categories, viz. novel target specific drugs (11), re-purposing based drugs (7), drugs effective on other kinetoplastids (6) and antibiotics (12) and they were investigated for their mode of action by studying the mRNA expression of 13 drug target genes using qPCR. Drugs with high anti trypanosomal activity and low cytotoxicity were evaluated for therapeutic efficacy in mice model. In the novel target specific drugs, etoposide and IC-261 increased the longevity of T. evansi infected mice by 48 h in comparison to control mice. Among the drugs effective on other kinetoplastids, amphotericin B raised the survival period of mice by 120 h. Nifurtimox and buparvaquone checked the level of parasitaemia on regular injection. However, imidocarb raised the survival period of mice by 20 days without any significant organotoxicity. Data generated showed that qPCR profile of highly efficient anti-trypanosomal drug was similar to standard drugs and imidocarb can be suggested as an alternate therapeutic compound for treatment of Surra infection. The data generated will be of immense use in future drug development strategies for curtailment of Surra in animals.
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    Characterization of surface antigens of Trypanosoma evansi by monoclonal antibodies and development of suitable assays
    (LUVAS, 2007) Rayulu, V. Chengalva; Chaudhri, S.S.
    The present investigation has been carried out on an economically important vector-borne disease, known as ‘surra’, caused by a hemoprotozoan Trypanosoma evansi in domestic animals, with objectives to characterize the surface antigens of T. evansi Indian isolate by monoclonal and polyclonal antibodies, and to develop and validate sensitive, specific, simple, cost-effective and field-adaptable diagnostic tests. A field isolate of T. evansi collected from infected cattle was propagated in rats and preserved by freezing in liquid nitrogen. Whole cell lysate (WCL), flagellar (FL) and cell membrane (CM) antigens were prepared from DEAE- cellulose column chromatography purified trypanosomes. In SDS–PAGE, 16 polypeptide bands from 11.93 to 91.31 kDa in WCL, 14 bandsfrom 11.23 to 116.42 kDa in FL and 4 bands from 11.93 to 59.68 kDa in CM preparation were resolved. Eight hybridoma clones were produced in two attempts by fusion of B lymphocytes from T. evansi surface antigen immunized BALB/c mice with Sp2/0 Ag14 myeloma cells. One clone, identified as 1D7, showed good titre against surface antigen of T. evansi and therefore amplified both in culture flasks and BALB/c mice. Monoclonal antibody (mAb) from the mice ascites fluid was partially purified by precipitation with 50% saturation of ammonium sulphate and found to be of IgA isotype by isotype-specific indirect ELISA, protein G-agarose chromatography, SDS-PAGE and immunoblotting. HIS recognized 13 antigen bands ranging from 13.89 to 77.51 kDa, while mAbs were strongly reactive with a 54.7 kDa polypeptide in immunoblotting. MAb-based latex agglutination test (LAT) and Ag-ELISA were developed to detect circulating antigens of T. evansi in the sera of domestic animals. The shelf–life of the latex reagent was found to be about eight months. Analytical sensitivity of LAT and Ag-ELISA was 1.0 µg/ml and 100 ng/ml antigen respectively. The diagnostic sensitivity and diagnostic specificity were recorded as 95.38% and 59.74% for LAT and 83.08% and 67.14% for Ag-ELISA respectively using microhematocrit technique (MHCT) as reference test, and 90.33% and 88.30% for LAT using Ag-ELISA as reference test. Monoclonal antibody- based CIC-ELISA was also performed for detection of T. evansi antigens in circulating immune complexes (CIC) in the MHCT-positive, Ag-ELISA-negative bovine sera samples. LAT and Ag-ELISA were found to be more sensitive than wet blood film (WBF) and MHCT. A total of 1538 blood and sera samples from cattle (n=826), buffaloes (n=285), equines (n=395) and camels (n=32) in different places of Haryana during summer, rainy and post-rainy seasons of the years 2005 and 2006 were screened for T. evansi infections by WBF, MHCT, LAT and Ag-ELISA. The overall prevalence of T. evansi infection was 2.47, 4.23, 42.59 and 34.98 per cent by WBF, MHCT, LAT and Ag-ELISA, respectively. Prevalence of T. evansi was 3.03, 6.05, 50.73 and 40.92% in cattle; 0.70, 1.05, 37.19 and 30.18% in buffaloes; 2.78, 3.04, 31.90 and 27.85% in equines, and 0, 0, 12.5 and 12.5% in camels by WBF, MHCT, LAT and Ag-ELISA, respectively. Variations in the prevalence of T. evansi infection in different animal species, seasons and regions of Haryana state were observed and discussed.