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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
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ThesisItem Restricted Molecular diagnosis of brucellosis in sheep using polymerase chain reaction(LUVAS, 2006) Sanjiv Kumar; Puran ChandBrucellosis is a highly infectious disease which is conventionally diagnosed using culture and isolation and serological methods. PCR assay has been shown to be a promising option for the diagnosis of brucellosis. However, a few studies have been reported with field samples for evaluating the assay as a diagnostic tool. In the present study, a genus-specific PCR assay has been assessed for the detection of Brucella melitensis DNA directly from various types of clinical samples collected from apparently healthy and recently parturated ewes. The assay is based on the gene encoding Brucella cell surface protein (BCSP-31), a 31 KDa protein in Brucella chromosome, giving a product of 223bp after PCR amplification. PCR assay was standardized using DNA extracted from pure culture of Brucella melitensis biovar 1. Detection limit of this PCR assay was found to be 167.6fg of DNA per reaction which corresponds to DNA extracted from 30 Brucella organisms. A total number of 40 ewes were examined of which 35 were belonged to a sheep farm known to have endemic brucellosis for more then two decades and 5 were from a brucellosis free sheep farm. The clinical samples including uterine discharge, milk and blood were collected from each animal at the same time. The samples of milk, uterine discharge and blood were processed for PCR amplification whereas milk and uterine discharge were subjected to culture and isolation for the recovery of Brucella melietnsis. Brucella melitensis was isolated from six animals (17.14%). All the isolates were identified to be Brucella melitensis biovar 1. Of these six Brucella melitensis, three (8.57%) were from uterine discharge and three (8.57%) from milk samples collected from different animals. The Brucella specific DNA was detected by PCR in 24 (68.57%) samples of uterine discharge, 11 (31.43%) samples of milk and 5 (14.28%) samples of blood. All these samples were from 35 animals belonging to the brucellosis endemic sheep farm. None of the samples from 5 ewes belonging to the brucellosis free farm gave positive result either in PCR or in culture and isolation. Taken together 25 ewes were detected positive by PCR. From these 25 (71.43%) ewes only one sample of uterine discharge was PCR negative but the milk and blood samples of this ewe were PCR positive. In addition, serological status of these animals was also determined by RBPT, mRBPT, SAT and ELISA, that gave positive results with three (8.57%), 11 (31.43%), 11 (31.43%) and 29 (82.86%) animals, respectively. All PCR positive animals were also positive in ELISA except one ewe. The number of ELISA positive animals was higher than that of PCR positive animals, but ELISA being an antibody detection assay it can not reveal the current presence of Brucella in the animal body. The presence of Brucella could only be established by demonstrating the presence of organism or its DNA in samples from ewes is very much likely to be detected by PCR as seen in the present study. The present study leads to the conclusion that PCR is a more sensitive technique as compare to the culture and isolation, and uterine discharge appears to be a better source than milk and blood for the same, if collected within 6-12 hours of parturition. Being simple, sensitive, fast and accurate diagnostic assay, PCR could be used as a routine laboratory test for the diagnosis of brucellosis in sheep.ThesisItem Restricted Immunogenicity of oil adjuvanted polyvalent foot and mouth disease vaccine in sheep(LUVAS, 2006) Lamba, Kapil; Sharma, R.The present study was carried out to analyze the immunogenicity of Foot- and-Mouth Disease Virus (FMDV) antigens in sheep and lambs vaccinated with polyvalent oil adjuvanted FMD vaccine. Humoral immune response was evaluated by monitoring the serum antibody titres in the sera of vaccinated sheep and lambs on 0, 7, 14, 21, 28, 35, 45 and 60 days post vaccination (dpv) by liquid phase blocking ELISA (LPB ELISA). The FMDV specific IgG and IgM antibody titres were also estimated on different dpv by indirect double antibody sandwich ELISA. The cellular immune response was monitored by MTT based lymphoproliferation assay. Higher LPBE antibody titres were observed in sheep compared to lambs with the peak titres in both the groups obtained between 21-28 dpv. Significant LPB ELISA antibody titres were detected up to the end of study i.e. 60 dpv in sheep and lambs. Higher antibody titres were detected against FMDV serotype A followed by O and Asia1 in both the groups. The virus specific IgG antibody titres followed the same trend as observed in LPBE antibody titres. Further, IgG titres were highly correlated to LPBE antibody titres (correlation cofficient 0.86-0.90). Peak virus specific IgM titres were recorded between 7-14 dpv in both the groups and were higher in sheep compared to lambs. The lymphoproliferative responses against FMDV serotypes O, A and Asia1 in both the groups initiated as early as 7 dpv with peak response observed between 21-28 dpv followed by gradual decline in sheep but declined abruptly in lambs after attaining peak response. Significant proliferative response was maintained up to 35 dpv in sheep compared to humoral immune response, which was maintained at significant level up to 60 dpv, the day of last sampling. The lymphoproliferative responses against Con-A stimulated lymphocytes cultures in vaccinated groups were not significantly different from that of the control groups of sheep and lambs.ThesisItem Restricted Molecular detection of field strains of Marek’s disease virus in vaccinated poultry flocks(LUVAS, 2006) Kamal Deep; Verma, P.C.Marek’s disease (MD), which is a lymphoproliferative disease of chickens, remains one of the most important disease. In the present study, occurrence of Marek’s disease virus in ten vaccinated poultry flocks of Haryana (India) from different geographical locations was determined on the basis of clinical signs, gross and histopathology, precipitation test and by polymerase chain reaction (PCR). While all the samples have gross and histopathological lesions suggestive of Marek’s disease i.e. visceral tumors in liver, spleen, kidney and ovary, only five feather follicle tip samples gave a positive precipitation reaction. The feather follicle epithelium of all the samples from ten poultry flocks were positive by polymerase chain reaction. In this assay, primers derived from ICP4 gene of MDV serotype-1 and US3 region in case of HVT were used for amplification. PCR product of four samples on nucleotide sequencing revealed that they were very closely related to virulent Marek’s disease virus and their per cent similarity ranged from 99.2%-100%.ThesisItem Restricted Studies on foot and mouth disease virus type specific antibodies in milk and serum of vaccinated buffaloes(LUVAS, 2006) Yadav, Vandna; Sharma, AnshuThe present investigation was carried out on 15 randomly selected milch buffaloes divided into three groups on the basis of lactation of an organized farm, to study the foot and mouth disease virus type specific antibodies in milk and serum following FMD vaccination. Milk and serum samples collected before vaccination i.e. 0 day and on 7, 14, 28, 42 and 56 days post vaccination, were analyzed for the detection of FMD virus type specific antibodies by liquid phase blocking ELISA (LPBE) and virus specific IgG1, IgG2 and IgA antibody response by indirect double antibody sandwich ELISA. In all the cases milk samples were treated with Arkalone, centrifuged and top fat layer was removed before conducting the assay. Significant FMD virus type specific antibody titres (LPBE, IgG1, IgG2 and IgA) were detected in milk and serum of buffaloes on different days post vaccination, though the levels of antibodies were lower in milk as compared to serum. FMD virus type specific IgG1 was found to be the predominant subclass as compared to IgG2 and IgA both in milk and serum of vaccinated buffaloes. Milk and serum LPBE, IgG1, IgG2 and IgA antibody titres were positively correlated with values of regression coefficient (R) as 0.6376, 0.506, 0.434 and 0.396, respectively. Milk LPBE antibody titres against different FMD virus types were significantly different showing higher antibody titres against FMD virus type O followed by A and Asia 1. Serum LPBE antibody titres against FMD virus type O and Asia 1 were not significantly different, except for type A. Milk IgG1 and IgG2 antibody titres against FMD virus type O, A and Asia 1 were not significantly different. The serum IgG1, IgG2 and IgA antibody titres against FMD virus type O were significantly low as compared to type A and Asia 1 in all the three lactations of buffaloes. Inspite of the limitation of milk for detection of antibodies against FMD virus types that it can only be used in milch animals, we can not undermine the importance of present study. In country like India milch animals are more important and kept in large number than draught animals.ThesisItem Restricted Studies on in vitro interaction of bovine lymphocytes with foot-and-mouth disease virus(LUVAS, 2007) Joshi, Gurudutt; Sharma, RavindraThe present study was conducted to study the in-vitro interaction of bovine lymphocytes with Foot-and-Mouth Disease Virus (FMDV) types O, A and Asia-1. Peripheral blood samples collected from six unvaccinated cow calves and six vaccinated cow calves were used for separation of peripheral blood mononuclear cells (PBMCs). These PBMCs were employed to study the alteration of different T-cell subpopulations i.e. boCD4+, boCD8+ and boWC1+ T-cells, Functional competence of PBMCs using lymphocyte proliferation assay, detection of FMD virus by RT-PCR and expression of 3A-NSP antigen at different hours of post in-vitro infection (hpi) with either FMDV types O or A or Asia-1. The alterations in T-lymphocyte subpopulation were analyzed by cyto-flurometric analysis using T-cell subpopulation specific monoclonal antibodies (mAb) at various hpi. It was demonstrated that FMDV types (O, A, and Asia-1) down-regulate boCD4+ and boCD8+ T cells up to 48 hpi of peripheral blood lymphocytes from unvaccinated cattle calves whereas down regulation of boWC1+ cells was seen up to 48 hpi with FMDV type O only. However, in contrast to unvaccinated animals, lymphocytes from vaccinated animals showed significant up-regulation of boCD4+, boCD8+ and boWC1+ T-cell following exposure to FMD virus serotypes O, A and Asia-1. Lymphoproliferative test was used to characterize functional competence of PBMCs obtained from unvaccinated and vaccinated animals following in-vitro infection with either FMDV types O or A or Asia-1 using PHA or inactivated FMDV type O, A and Asia-1 antigens. The finding revealed that PHA driven LPR was significantly depressed at 24, 48 and 72 hpi in unvaccinated animals. Homotypic as well as heterotypic FMDV antigen specific LPRs were depressed in PBL cultures of unvaccinated animals which were infected with either FMDV type O or A or Asia-1 and stimulated with FMDV antigens. FMDV replication was demonstrated by Reverse transcription-PCR (RT-PCR) and 3A-NSP FMDV antigen expression in PBMCs of unvaccinated and vaccinated cattle calves infected in-vitro with FMDV serotype O, A and Asia-1 at 12, 24, 48 and 72 hpi.. Agarose gel electrophoresis analysis of amplified product, revealed 1D gene specific product of FMD virus serotype O (1301bp product), A (866bp product) and Asia-1 (914bp product) in lymphocytes of unvaccinated calves and vaccinated calves at different hpi with either FMDV type O or A or Asia-1, respectively. The 3A-NSP mAb based flowcytometry revealed significant increase in 3A-NSP antigen expression in lymphocytes of unvaccinated calves and vaccinated calves at different hpi with either FMDV type O or A or Asia-1. The interaction of macrophages with FMDV was demonstrated using MTT dye based assay on macrophages at various hpi (24, 48 and 72hrs). Macrophages obtained from unvaccinated and vaccinated calves when incubated in presence of FMD virus serotype O, A and Asia 1 showed significant depletion of macrophages at 48 and 72 hpi. FMDV cDNA in in-vitro FMDV infected macrophages was detected at 48 and 72 hpi.ThesisItem Restricted Cloning and expression of genome segment 7 of Indian isolates of blue tongue virus(LUVAS, 2005) Punia, Sangeeta; Maan, SushilaBluetongue (BT) is an arthropod borne, infectious, non-contagious, economically important viral disease of ruminants. The causative agent bluetongue virus belongs to Orbivirus genus of the family Reoviridae. It is biologically transmitted by Culicoides midges to susceptible vertebrate hosts. BTV infection occurs throughout most of the tropical, subtropical and temperate regions of the world. The diagnosis of the BT is complicated owing to presence of 24 serotypes and cross reactivity with other related orbiviruses. The disease is endemic in India and anchor profound economic losses to the country’s exchequer. There is no specific information available on evolutionary origin of the Indian isolates as there were only a few characterized on the basis of genome sequences so far. Though there is a good repertoire of 21 serotypes reported to be present in India but there is no effective vaccine in the country. Furthermore, the prevalence of number of serotypes demonstrates the ability of these viruses to undergo rapid evolutionary change through gene reassortments and mutations. Keeping these problems in mind, the present study was aimed at cloning and expression of genome segment 7 of BTV. RT-PCR assays were conducted using genome segments 7 specific primers to amplify two distinct BTV isolates, i.e. BTV-1 Avikanagar and BTV-15 Hyderabad. The expected PCR products of 1154 and 1137 bp were obtained for BTV-15 Hyderabad and BTV-1 Avikanagar, respectively. The primer pairs were also evaluated for their serogroup specificity using other serotypes of BTV. The PCR amplicons of 1137bp (BTV-1Avikanagar) and 1154 bp (BTV-15 Hyderabad) were cloned into two different vectors, namely pPCR-Script-Amp SK (+) plasmid vector and pQE30 UA vector, respectively. The clones were screened for the presence of insert using restriction endonuclease digestion (Xho1) and PCR assays with VP7 gene specific primers. The orientation of clones was confirmed by digestion with Pst1. The clones were then subjected to DNA sequencing. The nucleotide sequence analysis revealed that the BTV isolates showed a 79% - 99.9% nucleotide identity, they segregated into different clades and did not display an unequivocal geographic, temporal or serotype relationship. The Indian isolates had shown comparatively less sequence similarity with American, Chinese and French isolates but has high similarity with South African and Australian isolates. Phylogenetically, the Indian isolates could not be segregated to a monophyletic group based on VP7 gene sequence. Further restriction enzyme analysis has led to the conclusion that the two Indian isolates of BTV-1 and 15 can be differentiated from each other on the basis their restriction profile analysis using EcoR1, Pst1 and Taq1. The BTV-1 Avikanagar recombinant clone in right orientation was selected for expression study. The IPTG induced expressed protein of 48 kDa was detected as a 39 kDa band (due to aberrant mobility of this hydrophobic protein) in SDS-PAGE. In immunoblot study, no distinct band was observed may be due poor level of expression of protein or due to low titer of the antiserum used. Hence, on the basis of this study it can be concluded that the nucleotide sequence analysis of genome segment 7 represents a useful basis for the identification of BTV that can be strengthened by addition of data for segment 7 of more Indian isolates of BTV. For expression of recombinant VP7 protein further investigations needs to be carried out for the development of cost effective, more consistent and non-infectious high level expression system with capability to detect all serotypes of BTV.ThesisItem Restricted Etio-pathological studies on gastrointestinal tract disorders of buffalo calves(LUVAS, 2005) Kuldeep Singh; Mishra, S. K.The present investigation was undertaken to study the clinico-hematology and pathology of gastrointestinal tract disorders in buffalo calves. Buffalo calves suffering from diarrhoea at CIRB, CCS HAU, Hisar and village Arya Nagar, Hisar were examined. The clinical examination revealed a significant increase in respiration and pulse rate in diarrhoeic buffalo calves. Hematological examination showed a significant increase in hemoglobin, packed cell volume, total erythrocyte count and total leucocyte count. Differential leucocyte count showed significant neutrophilia accompanied by lymphocytopenia, whereas absolute leucocyte count revealed a significant neutrophilia. Serum biochemical examination revealed a significant increase in total serum protein and albumin and a decrease in total globulin concentration. A significant hypoglycemia, hyponatraemia and hyperkalemia were the prominent features in diarrhoeic buffalo calves. Examination of faecal samples revealed ascariasis and coccidiosis in buffalo calves. Bacteriologically, E. coli Salmonella spp., Klebsiella spp., Proteus spp. and Pseudomonas spp. were isolated from faeces of diarrhoeic buffalo calves. Among serotypes of E. coli, O9, O101, 0172, O128, O42, O44, O86, O117 and O153 were isolated. Salmonella typhimurium 4, 5:i:1, 2 and S. bareilly 6, 7:y:1,2 were the serotypes of Salmonella spp. isolated from faeces of diarrhoeic calves. On pathological study, 43 buffalo calves revealed changes in gastrointestinal tract during postmortem examination. The gross lesions in oesophagus, intestine and abomasum were petechial and diffuse haemorrhages and congestion along with ulcer in abomasum. Liver evidenced petechial haemorrhages and congestion, yellow and pale discolouration with necrotic foci. Pancreas and mesenteric lymph nodes showed congestion along with enlargement. Gross lesions in other visceral organs along with gastrointestinal tract revealed congestion, consolidation and abscess formation in lungs and haemorrhages and congestion in other organs. Histopathologically, oesophagus showed erosions and ulceration in mucosal epithelium and squamous metaplasia of glandular tissue in some cases, infiltration of lymphocytes was there. Lesions in intestine and abomasum were desquamation of epithelium, ulcer formation due to complete denudation of epithelium, lymphoid depletion in Peyer’s patches, oedema and infiltration of mononuclear cells. Liver exhibited degenerative changes viz. fatty changes, hydropic degeneration and centrilobular necrosis along with infiltration of neutrophils, lymphocytes, plasma cells and macrohages. Micro-granuloma formation and bile duct hyperplasia were also seen. Mesenteric lymph nodes were reactive and exhibited depletion of lymphoid tissue and reticular cell proliferation. Pancreas showed degenerative changes in acini, congestion and haemorrhages. Spleen also showed depletion of lymphoid cells in white pulp and reticular cell hyperplasia. Congestion and hemosiderosis were the prominent feature. Lungs exhibited serofibrinous pneumonia, micro-granuloma, thickened pleura due to serofibrinous exudate and leucocytes, desquamation of bronchial epithelium, emphysema and atelectasis of alveoli. Heart showed hyaline degeneration, sacrocysts and focal necrosis of muscle fiber. Kidneys showed congestion and mild degenerative changes. Bacteriological study of different organs from carcases of buffalo calves revealed isolation of E. coli followed by Proteus spp., Klebsiella spp. Salmonella spp. and Pseudomonas spp. E. coli strains belonged to 21 serotypes. Others were rough and untypable. E. coli ‘O25’ was most prevalent followed by ‘O9’. Serotype. Salmonella typhimurium 4, 5:i:1,2 was most prevalent salmonella spp. serotype followed by S. newport 6,8:e,h:1,2 and Salmonella muenchen 6, 8:d:1,2. Antibiogram pattern of different bacterial organisms revealed a varying degree of sensitivity to different chemotherapeutic drugs. Neoascaris vitulorum and Eimeria spp. were isolated from intestinal lumen of dead calves. Two cases revealed enteritis of obscure etiology. Mortality in buffalo calves for period of six year (1999-2004) based on postmortem records revealed affection of gastrointestinal tract (enteritis, gastroenteritis and hepatitis) as leading cause of death followed by diseases of respiratory system. Mortality was highest in buffalo calves less than one month of age. Mortality in male calves was slightly higher than females and it was maximum in winter season followed by rainy and summer seasons.ThesisItem Restricted Modulation of tracheal mucus related clearance of E. coli and Newcastle Disease virus in chickens on treatment with Mulhatti (Glycyrrhiza glabra)(LUVAS, 2005) Seghal, Nitin; Kalra, S. K.The present study was conducted in chicken model to assess the Kafa dosa aspect of the Tridosa Theory of Ayurveda by establishing a system design for evaluating the effect of Mulhatti (Glycyrrhiza glabra) on tracheal mucus related clearance of Escherichia coli (E. coli) and Newcastle Disease virus (NDV). An instrument was designed and standardized for determining the tracheal mucus clearance rates in chickens. Dosages for E. coli and NDV infections were standardized. Day old broiler chicks were divided into two groups, one group was fed Mulhatti root powder @ 2g/kg body weight supplemented in feed. At two weeks of age these birds were further divided into 4 subgroups each and infected with E. coli, NDV and E. coli + NDV. Uninfected controls were also kept. The birds were sacrificed at different sampling intervals and the tracheae thus collected were subjected to mucosal transport measurements. The study revealed that the infection with E. coli and NDV casued a drop in tracheal mucus clearance rates and feeding of Mulhatti root powder did not provide any added advantage to the chicks in recovery rates from the depression in tracheal mucus clearance. The study being first of its kind, it is suggested that the experiment be repeated on a larger population.ThesisItem Restricted G and P genotyping of group ‘A’ rotavirus using RT-PCR and DNA probes(LUVAS, 2005) Siwatch, Deepti; Gaya PrasadGroup ‘A’ rotavirus is the major aetiologic agent associated with diarrheal diseases of human infants and young ones of many farm animals. It is the prototype virus of the genus Rotavirus in the family Reoviridae. Due to segmented nature of the RNA genome and wide host range, vast genetic and antigenic diversity exists amongst different isolates of rotavirus. Keeping this point in view, the present study was undertaken to characterize human group ‘A’ rotavirus circulating in Haryana. Of 110 human diarrheic stool samples collected, 75 (68.18%) were found positive for rotavirus by RNA-PAGE. Of the 75 RNA-PAGE positive samples, 58 (77.33%) were of long electropherotype and 17 (22.67%) were of short electropherotype. Fifty four of the seventy five RNA-PAGE rotavirus positive samples were subjected to characterization into G and P genotypes by RT-PCR. Fourty eight APPENDIX -viisamples (88.89%) were typed for various G types and 40 samples (74.07%) for various P types. G2 and G1 were the most prevalent G types followed by G4, G9 and G3. In case of P types, P4 was the most prevalent followed by P8, P6 and P10. There was high prevalence (24.07%) of mixed G and P genotypes in the strains circulating in this geographic region. G1P8 and G2P4 were found to be associated with 27.78% of rotavirus infection in the present study population. G2P4 being the most prevalent genotypic combination. Apart from the usual combinations, several unusual combinations such as G1P4, G1P6, G3P4, G4P4, G4P6, G9P4 and G9P6 were observed which constituted 22.22% of the locally prevalent rotavirus strains. Several unusual electropherotypes and genotype combinations were also seen. Nucleic acid hybridization assays using biotinlayted full length (1062 bp) type specific probes for G1, G2 and partial length (876 bp) DNA probe P4 type yielded reasonably good results for G and P genotyping of rotavirus positive field samples. Further characterization of the strains in terms of intratypic variation with in the prevalent G i.e. G1, G2 and P types i.e. P4 and P8 using XmnI, EcoRI, VspI, AluI , PstI and StyI restriction enzymes yielded results similar to the ones inferred from in silico analysis of the reference strains of the respective types. However, G2 strains remained undigested suggesting possible absence of restriction site for the enzymes used for the digestion of PCR product or possible mutation in a single base of restriction site which needs to be further validated using sequence studies.