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The article discusses a study done to characterize the Assamese buffaloes in the Brahmaputra valley, river islands and bordering wildlife sanctuaries. Preset questionnaires were used to gather information on Assamese buffaloes and data on morphometric traits was recorded on 29 adult buffaloes from different sites of the valley. It concludes that cytogenetic screening of buffaloes from different areas of Central Asom and further comparison with other sub-populations of Assamese buffaloes can establish the genetic classification of swamp type buffaloes.

The article discusses a study on the existence of swamp buffalo population in Manipur state in India. Cytogenetic profiling and mitochondrial DNA (mtDNA) analysis were carried out to prove the existence of such a type of buffaloes. It provides information on the materials and methods used in the study including cytogenetic analysis and sequencing and sequence analysis. Several figures are also presented showing the metaphase spread and karyotype of Manipuri male and female buffaloes and the multiple alignment of different Manipuri mtDNA haplotypes.

Duck virus enteritis (DVE) also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with a chicken embryo-adapted live virus that is known to be poorly immunogenic and affords partial protection. Further, the risk of harboring other infectious agents in the embryo particularly the deadly and zoonotic avian influenza virus is also high. In this paper, we report propagation of a chicken embryo-adapted vaccine strain of duck enteritis virus in duck embryo fibroblast (DEF) cell line. Thirty serial passages were done in DEF cell that made the vaccine virus further attenuated which was tested in ducks. The growth behaviors of the virus in DEF cells were studied and at 30th passage level the virus titre was found to be 10(6.8) TCID(50)/ml. Ducks were immunized with this virus and challenged after 21 days with high dose of virulent DEV. All the immunized ducks withstood challenge with no clinical symptoms in any of the ducks while all the control ducks died. DEF cell which is free from other infectious agents appears to be a good system for cultivation of duck enteritis virus vaccine strain., (2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.)

Objective: Mutations in connexin 26 gene (GJB2) are the most common cause of hearing loss in different populations. The aim of our study was to determine the prevalence of GJB2 mutations in the population of Kerala, India., Methods: This study was conducted on the genomic DNA of 86 affected subjects and their relatives from 59 families of Kerala, India. Mutation detection was done by sequencing and PCR-RFLP., Results: 36% of the probands had mutations in the GJB2 gene. We found that 45% (15/33) of the families that had a family history of deafness had mutations in GJB2 gene. Two different mutations were identified. W24X mutation was detected in 32.5% of the affected patients. Analysis of control samples revealed a carrier frequency of 0.0357 for this mutation. The estimation of haplotype frequency revealed that there was a significant association between the W24X mutation and the haplotype in this region with respect to the markers, D13S143 and D13S175 suggesting a founder effect for this mutation in this population. A novel mutation, R32L was detected in 3.5% of the affected patients. Structural prediction revealed that this mutation alters the helical structure of the first transmembrane domain of GJB2 protein resulting in defective gap junctions., Conclusion: Mutations in connexin26 is responsible for 36% of non-syndromic sensorineural deafness in the population of Kerala, India.

Purification of bluetongue virus (BTV) group-specific VP7 protein, expressed in prokaryotic system as histidine-tagged fusion protein is described in the present study. The major antigenic portion of VP7 gene of BTV 23 was amplified from the extracted RNA by reverse transcription polymerase chain reaction and cloned. The recombinant expression construct (pET-VP7) was identified by the polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion truncated VP7 protein with a molecular mass of 36 kDa was determined by Western blot analysis using anti-His antibody. The expressed VP7 was purified to near homogeneity by chromatography on nickel-agarose column as judged by sodium dodesyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP7 protein was recognized by antibody to BTV in Western blot analysis. The capability of the recombinant VP7 protein to differentiate hyperimmune serum of rabbit to BTV from normal rabbit serum was evident in the enzyme-linked immunosorbent assay (ELISA). The purified VP7 reacted well with the 24 BTV serotype-specific sera obtained from OIE Reference Laboratory on bluetongue. Our results indicated that the expressed VP7 protein could be used as antigen for development of antibody-capture ELISA for detection BTV group-specific antibodies. This recombinant protein may also be used as antigen in competitive ELISA format.

Tuberculosis (TB) caused by Mycobacterial organisms has emerged as one of the major diseases in captive elephants. In vitro Interferon-gamma (IFN-gamma) assay is being used as an ancillary test for early detection of TB in domestic and captive wild animals. In the present study, basic sequence information and immunological cross-reactivity of this major cytokine of Asian elephants were explored. At predicted amino acid level, IFN-gamma of Asian elephant showed maximum identity to that of horse (73%). Other IFN-gamma amino acid sequences that showed high level identity were that of giant panda (72%), dog (71%), nine-banded armadillo (69%), cattle (63%) and human (62%). IFN-gamma promoter sequences of Asian elephant, human, cattle and mouse showed high level conservation of the putative transcription factor binding sites, TATA box and transcriptional start site. The functionally important human IFN-gamma promoter elements, such as AP-2IRE-BE, YY1-gammaIFN-BED, ATFCS and AP-1gammaINF binding sites, were absolutely conserved in the corresponding elephant sequence. There was only a single nucleotide variation in the other two important elements, NFAT-gammaINF and IFN-gammaPE, indicating the highly conserved regulation of IFN-gamma expression across different species. Phylogenetic analysis based on IFN-gamma protein sequences revealed a closer relation of Asian elephants and nine-banded armadillo. This shows a closer evolution of these members of Afrotheria and Xenarthra, respectively; and supports the previous reports based on mitochondrial DNA studies. In Western blot analysis, IFN-gamma of Asian elephant expressed in Escherichia coli was detected using an anti-bovine IFN-gamma monoclonal antibody, indicating immunological cross-reactivity.

Interferon-gamma, a major immunomodulatory cytokine, of Indian water buffalo (Bubalus bubalis) was characterized at molecular level. Complementary DNA and essential promoter region were cloned and sequenced, and functional recombinant protein was expressed in bacterial system. The cDNA has 97.8% nucleotide identity with 11-nucleotide and four-amino acid variations, and the essential promoter region has 98.4% identity with five-nucleotide variations and a four-nucleotide deletion in comparison with the corresponding bovine sequences. All the major promoter elements such as NF IL-2 like motif, cyclosporin sensitive binding element and GATA motif are strictly conserved. Recombinant buffalo-IFN-gamma expressed in bacterial system reacted with an anti-bovine-IFN-gamma monoclonal antibody in Western blot and showed antiviral activity against buffalo pox virus in cultured Madin-Darby bovine kidney (MDBK) cells by inhibiting virus induced cytopathic effect. The study shows high level sequence similarity of IFN-gamma among ruminants. In view of the immunomodulatory and antiviral activities of IFN-gamma, this molecule will be useful in better understanding of the immune system of water buffaloes.

RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. It is projected as a potential tool to inhibit viral replication. In the present paper, we demonstrate the suppression of replication of an avian herpes virus (Anatid Herpes Virus-1, AHV-1) by siRNA mediated gene silencing in avian cells. The UL-6 gene of AHV-1 that codes for a protein involved in viral packaging was targeted. Both cocktail and unique siRNAs were attempted to evaluate the inhibitory potential of AHV-1 replication in duck embryo fibroblast (DEF) cell line. DEF cells were chemically transfected with different siRNAs in separate experiments followed by viral infection. The observed reduction in virus replication was evaluated by cytopathic effect, viral titration and quantitative real time PCR (QRT-PCR). Among the three siRNA targets used the unique siRNA UL-B sequence was found to be more potent in antiviral activity than the cocktail and UL6-A-siRNA sequences.

Phytohemagglutinin (PHA)-induced delayed-type hypersensitivity is an immunocompetent trait considered an indicator of cell-mediated immune or T-cell responses. Divergent selection was performed to generate high and low lines for response to PHA-P. Extreme-responder birds of the F2 generation in each line were used to study possible differences in macrophage activity and the associated functional genes. To evaluate macrophage activity, nitric oxide (NO) was estimated both systemically in serum and in in vitro monocyte culture. Semi-quantitative RT-PCR was used to detect the differential mRNA expression patterns of iNOS and MIP-1beta in monocyte culture, whereas T(H)1 cytokines (IL-2 and IFN-gamma) were studied in peripheral blood mononuclear cells (PBMC) at different time intervals after lipopolysaccharide (LPS) induction. The high line showed strong systemic, as well as in vitro NO production, compared to the low line, upon stimulation with NDV and LPS, similar to early and high iNOS mRNA expression. Following the pattern of iNOS gene expression, an early strong expression of cytokines with powerful iNOS-inducing action, such as IFN-gamma and the chemokine MIP-1beta, was observed in the high line. In contrast, for response to PHA-P, low expression of IL-2 was observed in the high compared to the low line. In conclusion, the study revealed that divergent selection for response to PHA-P resulted in a divergent effect on T(H)1 cell activity, resulting in altered macrophage function in chickens. Selection, based on response to PHA-P, could lead to more resistant birds or birds with an enhanced immune response.

The cDNAs encoding the interleukin-10 and interleukin-18 of Indian water buffalo (Bubalus bubalis) were cloned and sequenced. A 537 bp IL-10 cDNA fragment and a 623 bp IL-18 cDNA fragment were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from concanavalin A stimulated splenocytes. Sequence analysis of these cytokines revealed high level conservation at nucleic acid and protein level. Both these cytokines also showed strict conservation in the predicted secondary structure and critical amino acid residues compared to the ruminant homologues. Basal level expression of both IL-10 and IL-18 was observed in liver, lung and spleen. The expression level of IL-10 was not affected by mitogenic stimulation, whereas IL-18 was up regulated upon stimulation. The availability of these cytokine molecules will aid in the study of their role in the immunology and pathogenesis of infections in water buffalo.

An Indian isolate of Goatpox virus (GTPV) was adapted and propagated in Vero cells for development of an attenuated virus. The virus was initially passaged in primary lamb testes cells and subsequently in Vero cells. At the 55th passage, the virus showed evidence of attenuation when tested for safety in seronegative goats. At this stage, the virus was found to be completely non-pathogenic. The virus was passaged further and the 60th passage was used for testing its immunogenicity in goats. The latter were inoculated with 10, 100 and 1000 TCID50 of the attenuated virus by intradermal (i.d.) route and challenged after 28 days with virulent GTPV. The attenuated virus produced no adverse reaction even at the highest dose and conferred complete protection even at the lowest dose against challenge with a high dose (2 x 10(6) of 50% skin-reactive dose SRD50) of virulent virus. Increased levels of virus-specific serum antibodies could be demonstrated by both indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test in all the immunized goats. No horizontal transmission of the virus from the immunized to in-contact animals took place. Our results suggest that this attenuated virus could be a safe, immunogenic and potent candidate for developing a vaccine against goatpox.

Antigenic determinant analysis was carried out on VP3, one of major immunogenic proteins of Infectious bursal disease virus (IBDV) using computer algorithms. Altogether 17 peptides were synthesized for predicted putative regions and were tested for their reactivity with IBDV-positive polyclonal sera as well as with antisera to other common avian viruses to confirm specificity and to rule out cross reactivity. Of 17 peptides tested, three were selected and synthesized in multiple antigenic peptide (MAP) format. The immunization of rabbits with the three MAPs resulted in high humoral immune response. The purified antipeptide antibodies were screened against native IBDV antigen and the respective titers were determined. Out of the three antisera to MAPs that raised against the MAP3, spanning the amino acids (aa) 974-995 region on the VP3 protein had a very high titer (2048) and reacted specifically with IBDV. Thus, the antiserum to MAP3 detected native virus in enzyme-linked immunosorbent assay (ELISA), revealing the presence of a potential antigenic determinant on the C-terminus of the protein. This study proved that an antipeptide antibody could be used as a safe and specific tool for the diagnosis of IBD in chickens.

Bovine herpesvirus 1 (BHV-1) has been reported from the Indian subcontinent in late 70's. In order to identify the origin of an Indian isolate of BHV-1 (IBR/H 167 VS) and its molecular relationship to known strains of BHV-1, a 680 bp region of the glycoprotein gene gIV was amplified by polymerase chain reaction (PCR), cloned and sequenced. Comparison of this sequences with the corresponding one of an European strain of BHV-1 (Cooper) revealed more than 99% nucleotide (nt) homology. We conclude that the Indian isolate under study does not differ from the Cooper strain regarding the gIV gene.

The Indian cattle is known to be more tolerant to tropical infections than the European cattle. In order to verify the genetic basis of this variation, the DR B exon-2 of the major histocompatibility locus, known for coding the antigen recognition site, from the Hallikar breed of Indian cattle was amplified by PCR, cloned and sequenced. Comparison of this sequence with the information available on taurus cattle brought out six unique nucleotide changes and three amino acid changes. The amino acid positions were at 17, 72 and 87. A major variable region was observed at amino acid position 85 to 87 from all the alleles so far reported for the bovine locus.

A pair of oligomers of 20 and 23 bp were designed for amplifying a 381 bp sequence from glycoprotein IV gene of bovine herpesvirus 1. The primer pairs were used for amplifying genomic DNA of BHV-1 directly from cell culture fluids under different experimental conditions such as, untreated cell culture fluid, thermal denaturation and proteinase K treatment in presence of detergent. The results reveal that direct thermal denaturation of cell culture fluid is sufficient to detect the virus by polymerase chain reaction.

The genomic variation of three isolates of bovine herpes virus 1 (BHV-1) originating from different geographical regions and displaying different clinical symptoms were studied by restriction analysis using four different restriction endonucleases. EcoRI displayed an uniform restriction pattern for all the isolates and HindIII showed marginal variation among the isolates. BstEII and PstI displayed unique restriction patterns, based on which the isolates could be grouped into two subgroups of BHV-1.1. BstEII appears to be the enzyme of choice for differentiating BHV-1.1.