Effects of Pasteurella multocida lipopolysaccharides on bovine leukocytes.

Lipopolysaccharide (LPS) is a major virulence factor of Gram-negative bacteria playing a major role in stimulating protective immune response in mammalian host. However, in many gram-negative bacterial infections, LPS also elicits immunopathology by inducing excessive inflammatory changes. P. multocida (Pm), a gram-negative bacterium, causes acute lung inflammation and fatal septicemic disease in animals. However, the effects of Pm LPS on host cells are little known. In this study, LPS isolated from three different serotypes (B:2, A:1 and A:3) of Pm were individually tested in vitro to assess the response of bovine leukocytes. Pm LPS induced cell proliferation and cell death of leukocytes, in a dose- and time-dependent manner. In these cells, mitochondrial dysfunction and caspase activation mediate cell death.

Design and evaluation of reverse transcription nested PCR primers for the detection of betanodavirus in finfish.

Viral encephalopathy and retinopathy otherwise known as viral nervous necrosis (VNN) is a neuropathological condition affecting more than 50 fish species worldwide, mostly marine. Different PCR protocols with specific primers were reported from many countries for confirmation of VNN in fishes. In the present study, two pairs of primers were designed and evaluated for the diagnosis of clinical and subclinical cases of infections from field. These primers designated as BARL-F1/BARL-R1 amplified a 902 bp product in the variable region (T4) of the coat protein gene by first step PCR. Nested PCR primers BARL-F2/BARL-R2 amplified a fragment of 313 bp. The results were comparable with other commonly used primer sets such as F2/R3 and RG668f/RG919r primers. These new primers could detect betanodavirus in standard reference samples containing low, moderate and high viral load. Known positive and negative control samples of fish also revealed a predictive value of 100 % by RT-PCR diagnosis.

White spot syndrome virus (WSSV) genome stability maintained over six passages through three different penaeid shrimp species.

White spot syndrome virus (WSSV) replicates rapidly, can be extremely pathogenic and is a common cause of mass mortality in cultured shrimp. Variable number tandem repeat (VNTR) sequences present in the open reading frame (ORF)94, ORF125 and ORF75 regions of the WSSV genome have been used widely as genetic markers in epidemiological studies. However, reports that VNTRs might evolve rapidly following even a single transmission through penaeid shrimp or other crustacean hosts have created confusion as to how VNTR data is interpreted. To examine VNTR stability again, 2 WSSV strains (PmTN4RU and LvAP11RU) with differing ORF94 tandem repeat numbers and slight differences in apparent virulence were passaged sequentially 6 times through black tiger shrimp Penaeus monodon, Indian white shrimp Feneropenaeus indicus or Pacific white leg shrimp Litopenaeus vannamei. PCR analyses to genotype the ORF94, ORF125 and ORF75 VNTRs did not identify any differences from either of the 2 parental WSSV strains after multiple passages through any of the shrimp species. These data were confirmed by sequence analysis and indicate that the stability of the genome regions containing these VNTRs is quite high at least for the WSSV strains, hosts and number of passages examined and that the VNTR sequences thus represent useful genetic markers for studying WSSV epidemiology.

Cytokine profiles, apoptosis and pathology of experimental Pasteurella multocida serotype A1 infection in mice.

Mice were experimentally infected with Pasteurella multocida serotype A1 to study the cytokine profiles, host cell apoptosis and sequential pathology at different hours of post-infection. Infected mice were dull, anorectic and depressed. A transient leukocytopenia followed by progressive leukocytosis was observed in the course of infection. Serum cytokine profiles showed significantly (P<0.01) higher amount of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6 and mouse KC) in the infected mice when compared to control mice. The circulating lymphocytes were apoptotic on annexin V staining. Apoptotic nuclei were detected in splenocytes, hepatocytes and infiltrating leukocytes of the lungs on TUNEL staining. The lungs were grossly congested and hemorrhagic, and showed infiltration with polymorphonuclear cells at early and mononuclear cells in the late hours of infection. Alveolar epithelia, inter-alveolar septa and capillary endothelium of the lungs showed ultrastructural changes. Liver had degenerative changes in histological and ultrathin sections.

Detection of rabies virus genes by in-situ polymerase chain reaction.

Rabies is diagnosed by FAT in the impression smears of brain tissues. In this study, an attempt was made to diagnose rabies using in situ polymerase chain reaction (ISPCR). A digoxigenin-labelled double-stranded probe specific for a portion of the 'N' gene of rabies virus was used. Positive signals were identified as blue dots in the intraneuronal and neuropil areas.