RAMANI PUSHPA .R.N (MAJOR)ANAND KUMAR .PANNIE SUPRIYASAIKUMAR GANGITLA2024-07-072024-07-072024-02https://krishikosh.egranth.ac.in/handle/1/5810211557Ever 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.EnglishThesis