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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
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ThesisItem Restricted Identification and differentiation of meat of cattle, sheep, goat, pig and chicken by polymerase chain reaction assay(LUVAS, 2006) Satish; Ashwani KumarPolymerase chain reaction (PCR) assay was standardized to identify and differentiate the meat of cattle, sheep, goat, chicken and pig. The study included raw, cooked, adulterated and unknown meat samples of these species of animals. DNAs from meat samples were extracted by following the standard protocol. DNAs free from proteins and RNA and having an OD ratio of 260/280 ~1.7 to 1.9 were included for identification and differentiation of species of meat origin. The yield of DNA from raw and cooked meat of various species of animals varied from 100 g to 500 g and 20g to 100g per 500 mg of tissues respectively. Conventional and multiplexplex PCRs were standardized by using common forward primer (SIM GACCTCCCAGCTCCATCAAACAT- CTCATCTTGATGAAA) and species-specific reverse primers. Six PCRs ,one of each species was formulated and were run on respective six species of DNAs. All the PCRs amplified the target mitochondrial cytochrome b gene sequences successfully and produce a single band of size specific to each species except cattle and buffalo. PCR products from goat, chicken, sheep and pig produced DNA fragment of 157, 227, 331 and 398 bp respectively whereas cattle and buffalo meat amplified a fragment of similar size of 274 bp. Having confirmed species specificity of each primer independently, a multplex PCR was designed by mixing all the primers in a single reaction but targeting DNA of a single species of animal. The primer concentrations when mixed in ratio of 1:0.2:3:0.6:0.6:3:0.6 for SIM:G;Ch:C:C:S:P gave species specific amplification and characteristic band pattern on agarose gel electrophoresis was obtained. Amplified PCR products from six species ranged from 157-398 bp. Multplex PCR profiles of cooked meat samples of each species of animal revealed species-specific bands for all species except cattle and buffalo where band of same size was obtained. The primer pair specific to cattle also amplified buffalo target cytochrome b gene sequence and the band size in both the species was of 274 bp. To differentiate these two species, restriction enzyme analysis with six different restriction enzymes viz. AluI, BamHI, HaeIII, HinfI, PstI, and RsaI was carried out. However, it did not differentiate the meat of cattle and buffalo. A 20% adulteration of meat of one species of animal into the meat of other species was detected specifically by multiplex PCR in five binary mixtures of cattle and pig, cattle and sheep, cattle and goat, sheep and goat and chicken and goat. All the twelve unknown meat samples were identified for their species origin of meat.ThesisItem Restricted “Prrevallence off Escherriichiia collii serrottypes iin poullttrry and effffiicacy off a kiilllled bactterriin iin iitts prreventtiion””(LUVAS, 2005) Rajesh Kumar; Mahajan, N. K.Escherichia coli infections remain one of the major causes of economic losses in poultry industry worldwide, because of greater intensification of poultry husbandry and ubiquity of pathogenic Escherichia coli in litter, dust, water and as a commensal organism in the host body. Present study was carried to find out the prevalence of E. coli serotypes associated with various disease conditions, to know their antibiogram pattern and to evaluate the efficacy of a formalin killed bacterin prepared from most predominate serotype along with adjuvant in protection from challenge. Heart blood of birds from 115 flocks was plated on MLA which were suffering from colibacillosis, omphalitis, Chronic respiratory disease complex and Swollen head syndrome (SHS). E. coli was isolated and identified on the basis of standard cultural and biochemical tests. E. coli isolates were sent to CRI Kasauli (H.P.) for serotyping. Serotype O78 was most predominant (40.8%), isolated from 47 out of 115 samples followed by O2, O8, O101, O9, O147, O50, O88, O53 and O89. Serotype O78 was isolated from all the disease conditions and from all the age groups of the birds. Antibiogram pattern showed E. coli isolates were most sensitive to chloramphenicol, gentamicin, cephalexin, ciprofloxacin and resistant against oxytetracycline, doxycycline, ampicilin and erythromycin. Most predominant serotype O78 was inactivated with 0.3% formalin and emulsified with Freund’s incomplete adjuvant. A dose of 3.8X108 CFU in 0.5 ml was infected S/C in neck. 114 day-old broiler chicken were divided into 4 groups and were maintained on broiler mash alone. Group 1 was given vaccination on 7th and 21st day. Group II was given vaccination on 14th and 28th day. Group I and Group III were challenged on 28th day and Group II and Group IV was challenged on 35th day. Three birds from each group were euthanized at 6, 12, 18, 24, 48 and 72 hours post challenge. Gross lesion scoring, count of CFU and histopathological lesions were studied. There was no effect of vaccination on the weight gain. CFU (Log10) in blood of vaccinated groups were significantly lower (P 0.05) than unvaccinated group at all the different hours post challenge. The pathological changes in unvaccinated and challenged birds comprised of fibrinous pericarditis, perihepatitis and airsacculitis, oedema, congestion and mononuclear cell infiltration in the alveoli of lungs. Lesion score in liver, heart and lungs was significantly different from vaccinated groups. The Indirect Haemagglutination test carried out for quantification of humoral immune response following vaccination with O78 killed bacterin revealed high titres till 45 days of age. On the basis of macroscopic and microscopic lesion score, protective efficacy of bacterin using schedule II (vaccinated on 14th and 28th day of age) was 67-68%, which was slightly better than schedule I (vaccinated on 7th and 14th day of age) having 62-65% efficacy, so can be used for prevention of E. coli infections in broilers, however; further study trials using multiple serotypes may be carried outThesisItem Restricted Studies on immunosuppressive effect of commonly used Gumboro vaccines in broiler chickens(LUVAS, 2005) Yadav, Rupesh; Mahajan, N. K.The study was divided in to three experiments. In experiment-I 215 day- old broiler chicks were divided in to 5 groups A, B, C, D and E. Group A was kept as unvaccinated control. Groups B, C, D were divided into three subgroups each. Ist subgroups were given IBD vaccine; IInd subgroups were given IBD vaccines with NDV vaccine. Third subgroups were given IBD vaccine, NDV vaccine and supplemented with Levamisole @ 7.5 mg/ kg body weight & vitamin E @ 25mg / bird /day from day 13 to day 17 of age. Group E was subdivided into two subgroups EI&EII. EI served as NDV control and EII group was primed with live B 1 NDV strain and boosted with killed NDV on day 8 of age. In other subgroups live NDV vaccination was done with Lasota strain on day 5 of age followed by booster vaccination on day 21 of age. Three commercial IBD vaccines (vac1, vac2 & vac3) were given on day 14 of age in groups B, C&D, respectively. Various parameters like clinical signs, gross lesions, HI titers due to NDV, IBDV ELISA titers, body weight, bursa and spleen index, bursa spleen and thymus lesion scoring was done to assess the role of three IBD vaccines in causing immunosuppression. Clinical signs were not produced by any of vaccines. Some haemorrhagic spots were observed in thigh muscles in IBD vaccine3 inoculated groups on day 21 and 28. Body weights were significantly reduced in nonsupplemented subgroups inoculated with IBD vaccine1 & IBD vaccine3. Vaccine 2 did not cause significant decrease in HI titers whereas the HI titers against NDV were significantly lower in vac1&3 inoculated subgroups, which were not supplemented by immunomodulators, on the other hand highest ELISA titers against IBDV were produced by IBD vaccine3 followed by IBD vaccine1&2. All the three vaccinal strains caused significant decrease in bursal index to a varying degree. Supplementation of immunomodulators caused significant increase in bursal indices. There was no effect on spleen indices by different vaccines. Vaccines 1 and 3produced thymic lesions but these were absent in vac2-inoculated groups. Maximum bursal and spleen lesions were produced by vac3 followed by vac1 and 2. Thymic lesions were absent in vac2 inoculated groups but present in vac1 and 3 inoculated groups. Supplementation of immunomodulators decreased the severity of lesions in all the subgroups. Twenty chicks from 3 hatcheries were bled on day 1 of age to know the status of maternal antibodies against IBDV and NDV. Chicks from hatchery I had significantly lower HI titers against NDV but higher IBDV antibody titer as compared to hatchery II and II. Maternal antibody titers against NDV were 5.57±1.38, 13.14±1.94 and 12.00±2.6 in three hatcheries, respectively. Against IBDV maternal antibody titers were 5161.00±197.84, 3307.00±400.79 and 3645.21±242.21 in hatchery I, II and III, respectively. Five blood samples were collected from 15 farms each to know the IBD and NDV vaccinal response under field conditions. There was lot of variation in NDV and IBDV antibody titers. Some IBDV strains produced higher IBDV ELISA titers but at the same time caused decrease in HI titers against NDV. On the basis of these studies it can be concluded that the practice of vaccinating against IBD at day13-14 of age in the field still holds good and farmers should be advised to supplement the chicks with immunomodulators from 13 to 17 day of age to ameliorate immunosuppressive effect of intermediate and intermediate plus vaccines.ThesisItem Restricted Seroepidemiological studies on Japanese encephalitis among animals in Haryana(LUVAS, 2007) Nagaleelavathi, S.P.; Garg, S.R.Haemagglutination inhibition test (HI) and serum neutralization test (SNT) were standardized and used to study the seroprevalence of Japanese encephalitis (JE) antibodies in animals in Haryana. Parallel testing of 213 serum samples by both the tests showed that the sensitivity and specificity of HI in comparison to SNT was 96.29% (92.18%-100%, P0.05) and 100%, respectively. Seventyeight (9.8%) of 793 animals were tested positive for JE antibodies. The seropositivity was the highest in buffaloes (12.6%, n=182), followed by pigs (11%, n=163), horses (9.7%, n=185) and cattle (7.2%, n=263). The prevalence was higher in the animals in the rice cultivating areas (10.6%, n= 601) as compared to those in the non-rice cultivating areas (7.3%, n=192). The corresponding respective values for the two areas were 12.5% and 3.7% in pigs, 10.6% and 5.7% in horses, 7.6% and 6% in cattle, and 13.6% and 10.9% in buffaloes. JE antibody positive samples when tested for West Nile virus (WNV) antibody by HI revealed that 53 of 78 samples were positive for WNV too suggesting that both the flaviviruses are co-circulating in the region. Seroprevalence of JE in animals suggested their role in the transmission of infection which can be correlated to the outbreaks of human disease in the state.ThesisItem Restricted Studies on serodiagnosis of glanders using various antigens(LUVAS, 2008) Jinu Mary John; Kapoor, P. K.Glanders is an important reemerging zoonotic disease in India. Since there is no proper treatment or vaccine available for glanders, its timely diagnosis, prevention and control are utmost important. In view to have a quick and effective diagnostic tool, the present study on serodiagnosis of glanders was planned. The standardization of Indirect Haemagglutination (IHA) test and Dot Enzyme-linked immunosorbent assay (Dot ELISA) were performed with Complement Fixation Test (CFT) antigen, mallein PPD and recombinant antigen for the diagnosis of glanders and applied on 248 field equine sera collected from various States of the country. A significant IHA titre of 320 and above was found as positive diagnostic titre for glanders. The tanned formalinised SRBCs were found better when used on the same day of coating antigens. The albumin bovine serum- phosphate buffered saline (ABS-PBS) was found better diluent than normal rabbit serum-phosphate buffered saline (NRS-PBS). Analysis of diagnostic test characteristics for the comparative efficacy of IHA test and Dot ELISA with various antigens was done. The specificity of IHA test with all the three antigens ranged from 99.16% to 99.47%, but the sensitivity of IHA was found maximum (63.64%) with CFT antigen. The accuracy of prediction of the IHA test with all the three antigens was ranged from 95.56% to 97.58%. The specificity of Dot ELISA with all three antigens was found to be 97.47% and the sensitivity was 100% with CFT and recombinant antigens and 90.91% with mallein PPD. The accuracy of prediction of Dot ELISA varied from 97.18% to 97.58% with the various antigens. The present study revealed that the Dot ELISA with recombinant antigen can be used for field screening of equine sera for the diagnosis of glanders.