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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar
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ThesisItem Restricted Detection and molecular characterization of foot and mouth disease virus from persistently infected bovine(2023-07) Ankit Pannu; Dahiya, SwatiFoot-and-mouth disease (FMD) is an OIE-listed highly contagious and economically devastating disease of cloven-hoofed animals. Routinely, FMD virus (FMDV) 3AB3 non-structural protein (NSP) based indirect ELISA test is used for population serosurveys as an indicator of exposure to virus to differentiate between infected and vaccinated animals (DIVA) in India. For this, serum samples (n=377) collected from 6-18 month old cattle and buffaloes from nine villages of district Hisar, Haryana during 2022 as per the sample plan developed by ICAR-NIVEDI were subjected to DIVA ELISA. NSP reactivity was found to be 5.3% (20/377) in both, cattle (11.3%; 12/106) and buffaloes (3.0%; 8/271). Two buffaloes from 20 NSP reactors when sampled at a gap of nine months were found to be negative for FMDV 3AB3 NSP Abs as well as for FMDV by real time RT-PCR and RT- Multiplex PCR (RT-mPCR). No FMD outbreak was reported from Haryana during 2022. Further sampling was carried out from an organized cattle farm in which bi-annual FMD+HS combined vaccination has been carried out regularly. There was no history of FMD outbreak on this farm for more than a decade. DIVA reactivity of 43.75% (7/16) was observed in cattle (6-18 months age) of this farm which could be linked to either false positive reactions or scars of past exposure and virus elimination at the time of OPF sampling. The blood parameters between NSP positive and NSP negative animals were statistically insignificant. The only animal exhibiting antibody titresThesisItem Open Access Serosurveillance, seromonitoring and kinetics of humoral immune response against foot-and-mouth disease virus in goats(LUVAS Hisar, 2023-07) Kaur, Aman deep; Lathar, AnshulSmall ruminants comprise the majority of the world's foot-and-mouth disease (FMD) susceptible population. But FMD surveillance and control strategies in the country largely ignore small ruminants, known to be critical in the epidemiology of the disease. Continued seromonitoring and serosurveillance of FMD in small ruminants is essential to extend support to FMD control decisions particularly regarding vaccination. In order to determine the vaccinal immune response of goats belonging to the different districts of Haryana, 416 pre-vaccination and 400 post-vaccination serum samples of goats were analyzed by SPCE. The percentage of serum samples having protective antibody titres (≥1.65 log10) during pre-vaccination was 43.2, 23.0 & 27.1 which increased to 57.2, 31.2 & 30.7 one month after FMD vaccination against FMDV serotypes O, A and Asia -1, respectively. The antibody titres were found highest against type O serotype. The E. coli expressed recombinant 3AB3 NSP based indirect ELISA were used on serum samples of goats collected from different districts of Haryana to detect the percent of infected/carrier animals in the state. Out of 903 serum samples, one hundred forty-eight (16.3%) goats were found to be positive for NSP (DIVA) reactivity providing a serological evidence of viral activity. One aspect of the work was to study the kinetics of humoral immune response in the FMDV vaccinated goats at an organized farm. In this study, forty goats (twenty kids and twenty adult) of beetle breed were vaccinated with oil adjuvanted trivalent (having FMDV serotypes O, A and Asia-1) inactivated FMD vaccine. Six goats were kept as control animals in which vaccine was not administered. Blood samples were collected regularly upto seven months. No adverse reaction was observed in the vaccinated goats. The FMDV vaccinated goats developed a strong humoral immune response against all the three FMDV serotypes with peak antibody titers observed at four weeks post-vaccination. The protective antibody levels (≥1.65 log10) persisted in both the vaccinated groups (kids as well as adults) for seven months. The mean antibody titres in adult goats were found superior and statistically significant in comparison to the kids during the study. Based on these findings, it is recommended that FMD vaccinations on a regular basis should be performed in small ruminants in order to effectively implement the FMD control plan in the state/country and achieve FMD-free status in future.ThesisItem Open Access Molecular detection and characterization of porcine rotavirus from piggery units in Haryana(LUVAS,Hisar, 2023) Sheoran,Deepika; Bhanot,VandnaPorcine Rotavirus A (PRVA) belong to the family Sedoreoviridae and genus Rotavirus. It is non- enveloped virus and contains double stranded RNA genome of about 16-21 kbp in size with 5’ terminal cap and lacks poly A tail at 3’ end. Genome codes for 6 structural (VP1-4, VP6-8) and 5/6 non-structural proteins (NSP1-NSP5/6). PRVA has been reported from diarrheic and non-diarrheic/asymptomatic pigs from several countries worldwide including India. However, there is no report of molecular characterisation of PRVA from Haryana. So, the present study was designed to detect the presence of PRVA in Haryana by RT-PCR targeting VP6 gene and to characterize it at molecular level. In the present study, out of the total 100 rectal swab samples, 36% (36/100) samples; (38% (19/50) diarrheic and 34% (17/50) non-diarrheic) were tested positive for PRVA by RT-PCR of partial length VP6 gene. Of the positive samples, highest positivity was observed among suckling piglets (45%) followed by weaning piglets (39.13%). None of the samples from growing piglet was found positive for PRVA. Further, the phylogenetic analysis of VP6, VP4 and VP7 genes revealed that the genotype I1, P[13], P[6], G11, G4 and combinations of G4P[6], G4P[13] and G11P[13] are circulating in pig population of Haryana. The most common combination found was G4P[6] (66.66%) followed by G11P[13] (22.22%) and G4P[13] (11.11%). The genotype G11 and the combinations G4P[13] and G11P[13] have been reported for the first time from pigs in India in the present study. The results of this study indicate co-circulation of different genotypes at the same time in pig population of Haryana and reveals high genetic diversity among different PRVA genotypes. These findings are useful for the development of more accurate diagnostic tools and provides information to understand the epidemiology of PRVA in Haryana.ThesisItem Restricted Molecular detection and characterization of porcine rotavirus from piggery units in Haryana(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-04) Sheoran, Deepika; Bhanot, . VandnaPorcine Rotavirus A (PRVA) belong to the family Sedoreoviridae and genus Rotavirus. It is non- enveloped virus and contains double stranded RNA genome of about 16-21 kbp in size with 5’ terminal cap and lacks poly A tail at 3’ end. Genome codes for 6 structural (VP1-4, VP6-8) and 5/6 non-structural proteins (NSP1-NSP5/6). PRVA has been reported from diarrheic and non diarrheic/asymptomatic pigs from several countries worldwide including India. However, there is no report of molecular characterisation of PRVA from Haryana. So, the present study was designed to detect the presence of PRVA in Haryana by RT-PCR targeting VP6 gene and to characterize it at molecular level. In the present study, out of the total 100 rectal swab samples, 36% (36/100) samples; (38% (19/50) diarrheic and 34% (17/50) non-diarrheic) were tested positive for PRVA by RT-PCR of partial length VP6 gene. Of the positive samples, highest positivity was observed among suckling piglets (45%) followed by weaning piglets (39.13%). None of the samples from growing piglet was found positive for PRVA. Further, the phylogenetic analysis of VP6, VP4 and VP7 genes revealed that the genotype I1, P[13], P[6], G11, G4 and combinations of G4P[6], G4P[13] and G11P[13] are circulating in pig population of Haryana. The most common combination found was G4P[6] (66.66%) followed by G11P[13] (22.22%) and G4P[13] (11.11%). The genotype G11 and the combinations G4P[13] and G11P[13] have been reported for the first time from pigs in India in the present study. The results of this study indicate co-circulation of different genotypes at the same time in pig population of Haryana and reveals high genetic diversity among different PRVA genotypes. These findings are useful for the development of more accurate diagnostic tools and provides information to understand the epidemiology of PRVA in HaryanaThesisItem Restricted Serological and Molecular Studies on Brucellosis and Q-fever in Goats of Haryana(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-04) Priya; Chhabra), RajeshBrucellosis and Q-fever are two important zoonotic bacterial diseases that cause reproductive problems in goats, including mid to late-term abortions and has a significant impact on animal production and public health. Despite this, there have been very few studies on the prevalence of brucellosis and Q-fever in goats in Haryana. The present study was done to determine the prevalence of Q-fever and brucellosis in Haryana. A total of 400 sera sample was collected from serum bank of Department of Veterinary Microbiology, LUVAS, Hisar and were analyzed using RBPT and iELISA for brucellosis and iELISA for Q-Fever. Moreover, 10 clinical samples with serum were collected from mid-term to late-term aborted goats in Haryana and subjected to PCR using bcsp31 and IS711 primers for Brucella Spp., and IS1111 for Coxiella burnetii. The overall seroprevalence was estimated as 13.75 percent by RBPT and 16.5 percent by iELISA for brucellosis and 18.25 percent by iELISA for Q-fever. It was observed that 4.5 percent samples were serologically positive for both Brucellosis and Q-fever by iELISA. The seropositivity of brucellosis and Q-fever in mid to late-term aborted goats of Haryana was observed as 20 percent by RBPT and iELISA for brucellosis and none of the samples were found positiveby iELISA for Q-fever. By using conventional PCR, 20% (2/10) clinical samples were found positive for B. melitensis and 10% (1/10) were positive for C. burnetii. Subsequently, it suggests that serological and molecular tests are suitable for diagnosing brucellosis and Q-fever in goats. Furthermore, vaccination programmes, public awareness and campaign programmes should be done to teach farmers regarding hygiene, sanitation and to stop spreading of disease.ThesisItem Restricted Serological and Molecular Studies on Brucellosis and Q-fever in Goats of Haryana(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-05) Priya; Chhabra, RajeshBrucellosis and Q-fever are two important zoonotic bacterial diseases that cause reproductive problems in goats, including mid to late-term abortions and has a significant impact on animal production and public health. Despite this, there have been very few studies on the prevalence of brucellosis and Q-fever in goats in Haryana. The present study was done to determine the prevalence of Q-fever and brucellosis in Haryana. A total of 400 sera sample was collected from serum bank of Department of Veterinary Microbiology, LUVAS, Hisar and were analyzed using RBPT and iELISA for brucellosis and iELISA for Q-Fever. Moreover, 10 clinical samples with serum were collected from mid-term to late-term aborted goats in Haryana and subjected to PCR using bcsp31 and IS711 primers for Brucella Spp., and IS1111 for Coxiella burnetii. The overall seroprevalence was estimated as 13.75 percent by RBPT and 16.5 percent by iELISA for brucellosis and 18.25 percent by iELISA for Q-fever. It was observed that 4.5 percent samples were serologically positive for both Brucellosis and Q-fever by iELISA. The seropositivity of brucellosis and Q-fever in mid to late-term aborted goats of Haryana was observed as 20 percent by RBPT and iELISA for brucellosis and none of the samples were found positiveby iELISA for Q-fever. By using conventional PCR, 20% (2/10) clinical samples were found positive for B. melitensis and 10% (1/10) were positive for C. burnetii. Subsequently, it suggests that serological and molecular tests are suitable for diagnosing brucellosis and Q-fever in goats. Furthermore, vaccination programmes, public awareness and campaign programmes should be done to teach farmers regarding hygiene, sanitation and to stop spreading of disease.ThesisItem Restricted Serological and Molecular Studies on Brucellosis and Q-fever in Goats of Haryana(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-04) Priya; Chhabra, RajeshBrucellosis and Q-fever are two important zoonotic bacterial diseases that cause reproductive problems in goats, including mid to late-term abortions and has a significant impact on animal production and public health. Despite this, there have been very few studies on the prevalence of brucellosis and Q-fever in goats in Haryana. The present study was done to determine the prevalence of Q-fever and brucellosis in Haryana. A total of 400 sera sample was collected from serum bank of Department of Veterinary Microbiology, LUVAS, Hisar and were analyzed using RBPT and iELISA for brucellosis and iELISA for Q-Fever. Moreover, 10 clinical samples with serum were collected from mid-term to late-term aborted goats in Haryana and subjected to PCR using bcsp31 and IS711 primers for Brucella Spp., and IS1111 for Coxiella burnetii. The overall seroprevalence was estimated as 13.75 percent by RBPT and 16.5 percent by iELISA for brucellosis and 18.25 percent by iELISA for Q-fever. It was observed that 4.5 percent samples were serologically positive for both Brucellosis and Q-fever by iELISA. The seropositivity of brucellosis and Q-fever in mid to late-term aborted goats of Haryana was observed as 20 percent by RBPT and iELISA for brucellosis and none of the samples were found positiveby iELISA for Q-fever. By using conventional PCR, 20% (2/10) clinical samples were found positive for B. melitensis and 10% (1/10) were positive for C. burnetii. Subsequently, it suggests that serological and molecular tests are suitable for diagnosing brucellosis and Q-fever in goats. Furthermore, vaccination programmes, public awareness and campaign programmes should be done to teach farmers regarding hygiene, sanitation and to stop spreading of disease.ThesisItem Restricted Serological and Molecular Studies on Brucellosis and Q-fever in Goats of Haryana(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-04) Priya; Chhabra, RajeshBrucellosis and Q-fever are two important zoonotic bacterial diseases that cause reproductive problems in goats, including mid to late-term abortions and has a significant impact on animal production and public health. Despite this, there have been very few studies on the prevalence of brucellosis and Q-fever in goats in Haryana. The present study was done to determine the prevalence of Q-fever and brucellosis in Haryana. A total of 400 sera sample was collected from serum bank of Department of Veterinary Microbiology, LUVAS, Hisar and were analyzed using RBPT and iELISA for brucellosis and iELISA for Q-Fever. Moreover, 10 clinical samples with serum were collected from mid-term to late-term aborted goats in Haryana and subjected to PCR using bcsp31 and IS711 primers for Brucella Spp., and IS1111 for Coxiella burnetii. The overall seroprevalence was estimated as 13.75 percent by RBPT and 16.5 percent by iELISA for brucellosis and 18.25 percent by iELISA for Q-fever. It was observed that 4.5 percent samples were serologically positive for both Brucellosis and Q-fever by iELISA. The seropositivity of brucellosis and Q-fever in mid to late-term aborted goats of Haryana was observed as 20 percent by RBPT and iELISA for brucellosis and none of the samples were found positiveby iELISA for Q-fever. By using conventional PCR, 20% (2/10) clinical samples were found positive for B. melitensis and 10% (1/10) were positive for C. burnetii. Subsequently, it suggests that serological and molecular tests are suitable for diagnosing brucellosis and Q-fever in goats. Furthermore, vaccination programmes, public awareness and campaign programmes should be done to teach farmers regarding hygiene, sanitation and to stop spreading of disease.ThesisItem Restricted Expression and characterisation of foot-and-mouth disease virus serotype O specific single domain antibodies(Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 2024-04) Vijay; Dahiya, SwatiFoot-and-mouth disease (FMD) is a highly contagious livestock disease caused by FMD virus. It is globally endemic and causes huge economic losses directly or indirectly and therefore its prevention and control during outbreaks is necessary by employing a combination of diagnostics, epidemiological surveillance, and mass vaccination. Conventional antibodies raised in animals have been predominantly used for FMD diagnosis with some disadvantages, including high production costs and poor stability. Camelid derived single domain antibodies (sdAbs) or nanobodies, offer several advantages over such polyclonal or monoclonal Abs, including their small size, high physicochemical stability, easy genetic manipulation, lower production cost and animal welfare. In this study, an existing sdAb sequence (VHH-C1) from open-source database was used to bypass the time-consuming steps in sdAb generation and constructed two different sdAb sequences through rational designing specific for FMDV serotype O based on the homology with the ‘in-house phage display library (PDL)’ sequence reads archive. These three sdAb genes were got synthesized and cloned into pET303/CT-His expression system. Two sdAbs namely VHH-C1 and VHH-SLU1were successfully expressed in E. coli BL21 (D3). The conc. of purified sdAbs was found to be 200 and 30 µg/ml for VHH-C1 and VHH-SLU1, respectively. Upon further characterization using FMDV typing DAS ELISA, VHH-C1 was found to be specific for FMDV serotype O with high affinity, while VHH-SLU1 detected all the three serotypes prevalent in India. Both the sdAbs were resistant to thermal degradation upto 85°C. In conclusion, this study demonstrated the potential of using open-source synthetic biology and bioengineering approaches to develop rapid and specific sdAbs which could be used in diagnosis for FMDV detection and sero-monitoring. Further research needs to be taken up to express sdAb clones specific for other FMDV serotypes also.
