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Polymerase chain reaction (PCR) primers specific to exon 2 of the bovine lymphocyte antigen (BoLA)-DRB3 gene were used successfully to amplify the equivalent region in 34 Murrah and 36 Surti buffaloes selected at random. The 304 bp amplified product of the DRB3 gene was separately digested with BstγI, HaeIII and Rsal enzymes. Digestion with BstγI enzyme did not reveal any polymorphism and all animals showed a single restriction pattern, which corresponded exactly to the BstγI pattern ‘b’ previously described for cattle. Digestion with HaeIII enzyme resulted in five patterns, four of which corresponded to the Haelll patterns previously reported in cattle. The new HaeIII pattern was observed in both the breeds of buffaloes studied. The fragment analysis with RsaI revealed 13 different patterns. All of these RsaI patterns corresponded to the RsaI patterns previously described for cattle. The high degree of similarity in the restriction fragment length polymorphism (RFLP) patterns of cattle and buffalo observed in the present study provide evidence for the strong conservation amongst other bovine species, of restriction sites previously reported in cattle.

A velogenic Newcastle disease virus isolate was passaged 50 times in Vero cell culture and the virus was assessed for the molecular changes associated with the passaging. At every 10th passage, the virus was characterized conventionally by mean death time (MDT) analysis, intracerebral pathogenicity index (ICPI) and virus titration. At increasing passage levels, a gradual reduction in the virulence of the virus was observed. Molecular characterization of the virus included cloning and sequencing of a portion of the fusion gene (1349 bp) encompassing the fusion protein cleavage site (FPCS), which was previously amplified by reverse transcription-polymerase chain reaction. Sequence analysis revealed a total of 135 nucleotide substitutions which resulted in the change of 42 amino acids between the velogenic virus and the 50th passage virus. The predicted amino acid motif present at the cleavage site of the virulent virus was (109)SRRRRQRRFVG(119) and the corresponding region of the adapted adapted virus was (109)SGGRRQKRFIG(119). Pathogenicity studies conducted in 20-week-old seronegative birds revealed gross lesions such as petechial haemorrhages in the trachea, proventricular junction and intestines, and histopathological changes such as depletion and necrosis of the lymphocytes in thymus, spleen, bursa and caecal tonsils in the birds injected with the velogenic virus and absence of the lesions in birds injected with the adapted virus. The 50th-passage cell culture virus was back-passaged five times in susceptible chickens and subjected to virulence attribute analysis and sequence analysis of the FPCS region, with minor difference found between them.