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20243
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Subject: Veterinary Microbiology × End date: 2024 × Start date: 2024 ×
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    ThesisItemOpen Access
    ASSESSMENT OF IMMUNOGENICITY OF LIVE ATTENUATED GOATPOX VACCINE AGAINST LUMPY SKIN DISEASE IN CATTLE
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-03) SAI SINDHU MUMMADISETTY; DEEPTHI .B (MAJOR); SRIVANI .M; SUBHASHINI .N
    Lumpy skin disease (LSD) is a rapidly emerging, transboundary and notifiable disease listed by the World Organization for Animal Health. LSD is being reported regularly with escalated incidence in India ever since its first appearance in 2019. Severe systemic disease is reported in the affected cattle, resulting in devastating economic losses. Mass vaccination of susceptible animals is the foremost approach in tackling infectious diseases. However, several heterologous and homologous vaccines are being used as a prophylactic measure across the globe to control LSD outbreaks. Although the efficacy and immunogenicity of homologous LSD vaccines is known to be excellent, cost of vaccine production along with the Neethling responses observed in the vaccinated animals urged the need for alternative vaccine candidates. Owing to its high degree of co linearity and amino acid identity of LSDV with other Capripoxviruses (CaPVs), heterologous vaccines employing Goatpox vaccines (GPV) and Sheeppox vaccines (SPV) can be safely used to protect against LSDV. The present study deals with an objective to determine the optimum dose of Goatpox vaccine against LSD infections in cattle. A total of 31 samples were collected from clinical cases suspected for LSD, in different areas of Andhra Pradesh during September to December, 2022. Of the 31 samples, 26 were found positive for LSDV by PCR using primers targeting CaPV- specific P32 gene and LSDV- specific RPO30 gene. Clinical samples (n=10) confirmed by PCR were considered for isolation of LSDV in Madin-Darby Bovine Kidney (MDBK) cell line. LSDV (LSDV/Cattle/VJA PNGR/SVVU/2022) could be isolated from two samples (nasal swab and skin scab, both collected from a single clinical case) in the fifth passage showing characteristic cytopathic changes. MDBK amplified virus was adapted to Vero cell line and obtained infectivity titer of 10-7.595 per 100 μL is further employed for use in serum neutralization test. Phylogenetic analysis of the LSDV isolate based on full length ORF036 gene (RPO30) showed 100 % identity and formed a distinct clade with Middle-east Asia and African isolates. Amino acid specific signatures to differentiate CaPVs were noticed and estimates of evolutionary divergence over sequence pairs between clusters was determined that showed a distance of 9.52 % between vaccine strains and the field isolate in the present study. Vaccination trial was conducted in randomly selected heifers placed into four groups (A, B, C and D) of eight animals each. Group A served as control group, while groups B, C and D were vaccinated with 1mL, 2mL and 3mL of 1 X 103.0 TCID50/dose of Goatpox vaccine respectively. Group D vaccinated with 3 times the dose used in goats produced the best humoral immunity that was persistent till the end of the trial i.e., 35 days post vaccination(p<0.05). Thus, this study shows thrice the dose of Goatpox vaccine used in goats is considered as the optimum dose in cattle against LSD.
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    ThesisItemOpen Access
    DETECTION OF GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE AND VIRULENCE FACTORS OF Mannheimia ISOLATES FROM BRONCHOPNEUMONIA CASES OF SHEEP AND GOAT, AND EXPLORATION OF ANTIMICROBIAL PROPERTIES OF PHYTOCHEMICALS
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-02) MARUTHI SWAROOP THATAVARTHI .N.S.R.K; SIVARAMA KRISHNA .G (MAJOR); ANAND KUMAR .P; ANNIE SUPRIYA .R
    The respiratory infections of small ruminants are a burning problem for the communities who depend on sheep and goat rearing as major income source in India. The Mannheimia species is one of the major pathogens implicated in pneumonia of small ruminants and responsible for huge economic loses to livestock sector worldwide. The Mannheimia is a Gram negative, non-motile, non-spore forming, oxidase positive, catalase positive coccobacillary/bipolar organism. In the present study a total of 92 nasal swab samples were collected form clinical cases of pneumonic sheep and goats, out of which the Mannheimia was isolated from 30 samples (25 from sheep and 5 from goats). Morphologically, all the isolates characterized as bipolar & cocco-bacillary in Gram’s staining. Culturally, the Mannheimia isolates developed small pin-point pink coloured colonies on MacConkey agar, and small greyish haemolytic colonies on Blood agar. The phenotypically confirmed Mannheimia isolates were further genotyped by using genus specific primers targeting the gene coding for 16S rRNA. The genotypic primers specifically reacted and typed the isolates as belongs to the genera Mannheimia with an amplified product size of 304 bp. Overall the prevalence of Mannheimia was found to be 32.6%. The phylogenetic analysis revealed that the Mannheimia isolates from Andhra Pradesh formed a separate clade with close relation to M. hemolytica, Canada and M. caviae, Denmark strains. The biofilm forming ability of the Mannheimia isolates was evaluated by three methods such as Standard Tube method (ST), Congo red agar method (CRA) and Microtiter plate assay (MTP). Among, MTP assay was considered as best method which detected and quantified the biofilm production by 29 (96.6%) isolates. The antibiogram pattern of Mannheimia isolates revealed that high resistance to Ampicillin (80%) followed by Gentamicin (50%), Co-trimoxazole (43%), Tetracycline (40%), Amoxicillin-Clavulanic acid (40%), Ceftriaxone (36.6%) and Enrofloxacin (26.6%). On analyzing the multiple antibiotic resistance index (MAR), none of the samples revealed a MAR index of less than 0.2, which indicates a potential threat of rising multi-drug resistant (MDR) bacteria. The genes coding for antibiotic resistance of streptomycin and extended spectrum β-lactamases (ESBL) were identified using gene specific primers in multiplex PCR with a prevalence rate of 70% for strA gene, followed by 16% for blaOXA, 13.3% for blaTEM and 10% for blaSHV genes. The genes coding for virulence factor leukotoxin (Lkt) was found in only two isolates. The antimicrobial properties of selected phytochemicals were evaluated which can be tested as alternative approach to combat the threat of rising MDR strains of Mannheimia. The Eugenol, an extract of Syzygium aeromaticum, was found to effective with MIC ranging between 32-2052 µg/ml against the Mannheimia isolates whereas the MIC of Cinnamic acid was found to be ranging between 1.25 to 2.50 mg/ml. In conclusion, our study provides the detailed information on prevalence of Mannheimia in clinical cases of Pneumonia of sheep and Goats, its phylogenetic relations, pathogenic potential, danger of rising MDR strains and phytochemicals, Eugenol and Cinnamic acid, as alternative therapies to combat the MDR strains.
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION OF LUMPY SKIN DISEASE VIRUS FROM CATTLE OF ANDHRA PRADESH
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-02) SAIKUMAR GANGITLA; RAMANI PUSHPA .R.N (MAJOR); ANAND KUMAR .P; ANNIE SUPRIYA
    Ever since 2019, there have been reports of outbreaks of Lumpy skin disease (LSD) in cattle from several regions of India, with Andhra Pradesh as one of the main impacted states where animals were extremely suffered resulting in severe economic loss to farmers. Hence, a modest attempt has been made to study the LSD with regard to detection, isolation and characterization of LSDV isolates from various districts of Andhra Pradesh and its phylogenetic analysis. The clinical signs noticed in the ailing animals include papular and nodular lesions across the body ranging from 0.5 to 5 cm in diameter, enlargement of superficial lymph nodes, a rise in body temperature, oculo-nasal discharges, lameness owing to edematous swelling in their limbs. These signs were more severe in the crossbred cattle. In the current investigation, a total of 81 clinical samples (38 skin scabs, 21 nasal swabs, 18 blood samples and 4 faecal samples) from 54 clinically infected animals from different regions of Andhra Pradesh were collected. Preliminary screening of the samples was done by capripox generic PCR. Thirty four out of 38 skin scabs (89.48%), 7 out of 21 nasal samples (33.33%) and 1 out of 4 faecal samples (25%) were found positive and produced the amplification with product size of 192bp. The blood samples did not react with any primers. All the 81 samples were further screened using A33R gene primer pair. The positive percentage obtained for skin scabs, nasal samples and faecal samples by A33R gene-based PCR assay was same as that of p32 gene-based PCR assay and produced the amplification product of 588bp. Hence, the new technique of amplifying LSDV A33R genome for the detection of LSDV in clinical samples is equivalent to the OIE-recommended PCR technique that amplifies the p32 gene. The LSDV specific fusion (F) gene was detected in 29 skin scabs(76.3%), 3 nasal swabs (14.29%) and 1 faecal sample (25%) and produced the amplification with product size of 472bp. None of the blood samples reacted with the primers. The coding region of A33R gene from three field isolates was sequenced and the blast analysis showed 100% similarity with isolates from Turkey, Russia, and Kazakhstan. A phylogenetic tree was constructed with the sequences of field isolates and circulating strains available in GenBank. It was revealed that the field isolates were clustered with LSDV isolates from Turkey, Serbia, Russia, and Kazakhstan. However, vaccine strains clustered separately. Similarly, the genomic region of partial F gene from six field isolates was sequenced and blast analysis showed 100% genomic similarity with strains from Kenya, China, Russia, Bangladesh, and India. The similarity with other LSDV vaccine strains was 98% to 99%. The phylogenetic tree generated showed that the isolates from Vizianagaram region were clustered with Kenya isolate and KSGP 0240 strain. The isolate from Srikakulam was in cluster with isolates from the field outbreaks in Tamil Nadu and Odisha during 2019. Isolates from Krishna and Machilipatnam regions were in one node and clustered separately as well as Palamaner isolate which also clustered separately. All the vaccine strains were grouped together in one node The skin scab from clinically infected cattle which tested positive in PCR assay was propagated in primary lamb testicular cells (PLT). No CPE was observed until 5 days post infection (PI). The supernatant from the infected cells was used to infect fresh PLT cells (first blind passage). The PLT cells began to exhibit CPE in the form of cell rounding, cell aggregation, shrinking of cells after 72 hrs PI and prominent CPE was evident after 96 hrs PI. The virus was also propagated through chorio allantoic membrane (CAM) route in 10-day-old embryonated chicken eggs (ECE) up to 5th passage using skin scabs which were tested positive by PCR. The mortality of chicken embryos was observed after 96 h at each passage level. The isolates produced the characteristic pock lesions on CAM after 4th passage and became clear after 5th passage. The virus in both PLT cells and CAM was further confirmed by PCR using LSDV specific primers (Fusion gene). The CAM of LSDV infected chicken embryo was subjected to histopathological examination. The Haematoxylin and Eosin-stained tissue sections revealed thickening of the membrane over inoculation site with congested blood vessels and haemorrhages over the membrane. The epithelial cells showed vacuolar degeneration and often containing eosinophilic, intra-cytoplasmic inclusions which is the characteristic of pox virus. The histopathological findings of infected skin scabs revealed hyperkeratosis in the epidermis, infiltration of mononuclear cells, macrophages, lymphocytes and fibroblasts in the dermis. Eosinophilic intracytoplasmic inclusion bodies in the epithelial cells were also noticed.