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Both A and B grade cumulus-oocyte complexes (COCs) aspirated from cattle ovaries at slaughter were matured in vitro under normal (38.5 °C) and elevated temperatures (41 °C). Maturation competence based on cumulus expansion, COC diameter, and nuclear maturation were compared with and without antioxidant supplementation incorporating 7.5 µM retinol, 1 nM melatonin, and 1.5 µg/mL zinc chloride in an oocyte maturation medium. Heat stress significantly reduced cumulus expansion by approximately 20%, while only retinol could bring a significant (P ≤ 0.05) increase. Heat stress also decreased expansion of A (approximately 50%) and B (approximately 40%) grade COC diameter. All antioxidants significantly increased COC diameter at 38.5 °C in grade A COCs, but only retinol could significantly increase grade B COCs. At 41 °C, only retinol in grade A COCs significantly enhanced diameter. Elevated temperature also decreased the metaphase II stage of nuclear maturation by approximately 75%, and no antioxidant was protective, except retinol, which was only marginally so. Retinol (7.5 µM) was further supplemented in maturation and culture medium for in vitro embryonic development at 38.5 °C and demonstrated significantly higher (P ≤ 0.01) maturation, fertilization, and a 2-4 cell cleavage rate. Retinol supplementation not only showed better maturation results, but also was a better antioxidant in overcoming the deleterious effects of elevated temperature.

Domesticated fowls, pigeons and turkey birds were screened for avipoxvirus infection from different areas in Jammu region. Based on typical pox lesions the overall occurrence in fowl was found to be 18.52%, 17.03% in pigeons and 57.14% in turkeys. Mortality recorded in chicks was 41.96%, 45.36% in squabs, 100% in poults, and 20.00% in adult turkeys. Both cutaneous and diphtheritic forms of the disease was observed of which the latter was particularly prevalent in young birds. One sample of putative fowlpox virus (FWPV) from skin lesions of a fowl, and two samples of putative pigeonpox virus (PGPV) from skin and diphtheritic lesions each were inoculated on chorio-allantoic membrane (CAM) of 10-12 days old chicken embryonated eggs. A confirmatory diagnosis was made by PCR amplification of a highly conserved P4b gene locus detected in tissue samples from skin, diphtheritic membrane and virus inoculated CAM yielding a predicted 578 bp product. Phylogenetic analysis based on the same P4b gene locus revealed FWPV and turkeypox virus (TKPV) to be 99% related and belonging to clade 1, while PGPV was found to belong to clade 2. All three isolates illustrate considerable heterogeneity within the conserved P4b gene locus. The study indicates that the closely related FWPV and TKPV isolates may have the potential of cross infection between fowls and turkeys and therefore cross transmission studies are suggested.

Transmission electron microscopy (TEM) was employed for describing skin and scab lesions in goats affected by orf virus and to demonstrate the parapoxvirus from clinical suspensions by negative staining and ORFV confirmation by immunogold electron microscopy. All samples were confirmed as parapoxvirus by semi-nested PCR amplification of partial gene encoding for the B2L envelope protein. Skin lesions were characterized by ballooning degeneration and loss of desmosomes of the spinosum cells, cytolysis and vesicle formation. Nuclear changes included chromatin margination and increase in electron density. Cytoplasmic changes were typical of cell swelling, vacuolation and the presence of uniform, moderately electron dense viroplasm, situated in the perinuclear region. Various intracellular forms including immature virions (IV), mature virions (IMV) and wrapped virions (WV) were observed in the cytoplasm. All these forms of ORFV were observed morphologically akin to vaccinia virus (VACV). Negative staining of clinical samples and viral suspensions showed typical parapoxvirus morphology with a characteristic criss-cross tubular surface

Continuous foetal lamb testis cells OA3.Ts was used to compare the isolation of a vaccine orf virus (ORFV) strain that had been adapted to primary lamb testes cells, with scab derived wild-type/field ORFV isolates. The wild type virus showed an accelerated and exaggerated cyto-pathic effect (CPE) than the vaccine virus as has been demonstrated by immunofluorescent detection of viral antigen using ORFV monoclonal antibodies. ORFV could be successfully isolated in OA3.Ts foetal lamb testes cells and can be used for direct isolation of the virus from clinical samples.Two different methods of cell culture infection were also compared during sub-culture, one using infected supernatant as the inoculum and the other using infected cells as a modified method for infecting healthy culture cells. The present study also indicates that a higher infection can probably be achieved with inoculating infected cells together with healthy ones as a co-culture method during propagation of ORFV. This has been revealed as visibly pronounced CPE and presence of larger and multiple aggregates of cytoplasmic inclusions than cells infected with infected supernatant alone.

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Occurrence of Newcastle disease (ND) and characterization of the Avian Paramyxovirus-1 (APMV-1) amongst backyard fowls (Gallus gallus domesticus) and pigeon (Columba livia domestica) was studied. ND was suspected in two fowl flocks (100% morbidity, 86% mortality) and six pigeon flocks (21.68% morbidity, 14.16% mortality) with respiratory and/or enteric clinical signs in fowls and predominantly neurological signs in pigeons was observed. Cloacal swabs, tissue lysates or infected allantoic fluid could haemagglutinate (HA) chicken erythrocytes and pigeon convalescent serum inhibited haemagglutination (antibody titres >1/16). A 534 bp product was amplified from clinical samples and infected allantoic fluids by Reverse Transcriptase PCR (RT-PCR) targeting a partial Fusion protein gene including its cleavage site. The fowl and pigeon isolate was found to be velogenic and lentogenic respectively by virulence characterization. Fowl isolate grown in chicken embryonated eggs (CEE) showed 10-8.68 Embryo Infectious Dose (EID50) with a Mean Death Time (MDT) of 51.43 h and the Intra-Cerebral Pathogenicity Index (ICPI) in day-old chicks was 1.51. Pigeon isolate had corresponding values of 10-7.20 EID50, MDT of 92.00 h and ICPI of 0.43. Deduced amino acid sequence at Fusion protein cleavage site showed 112R-R-R-K-R*F117 velogenic motif for the fowl isolate and 112G-R-Q-G-R*L117 lentogenic motif for the pigeon isolate. Both isolates were found to be 84.1% related phylogenetically, while fowl isolate clustered with genotype XIII, the pigeon isolate clustered with genotype II and vaccine strains of APMV-1. This is the first instance of APMV-1 characterization in the region with the possibility of a vaccine spill over in pigeons.

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