To reduce the need for antibiotics in animal production, alternative approaches are needed to control infection. We hypothesized that overexpression of native defensin genes will provide food animals with enhanced resistance to bacterial infections. In this study, recombinant porcine beta-defensin 2 (PBD-2) was overexpressed in stably transfected PK-15 porcine kidney cells. PBD-2 antibacterial activities against Actinobacillus pleuropneumoniae, an important respiratory pathogen causing porcine contagious pleuropneumonia, were evaluated on agar plates. Transgenic pigs constitutively overexpressing PBD-2 were produced by a somatic cell cloning method, and their resistance to bacterial infection was evaluated by direct or cohabitation infection with A. pleuropneumoniae. Recombinant PBD-2 peptide that was overexpressed in the PK-15 cells showed antibacterial activity against A. pleuropneumoniae. PBD-2 was overexpressed in the heart, liver, spleen, lungs, kidneys, and jejunum of the transgenic pigs, which showed significantly lower bacterial loads in the lungs and reduced lung lesions after direct or cohabitation infection with A. pleuropneumoniae. The results demonstrate that transgenic overexpression of PBD-2 in pigs confers enhanced resistance against A. pleuropneumoniae infection.
SYBR Green I based real-time RT-PCR assay was developed for the detection and quantification of duck reovirus (DRV) using ABI PRISM 7500 sequence detection system. The assay was carried out using a set of primer designed to amplify highly conserved sequences of S2 gene of DRV. A 10-fold dilution series assay using a plasmid containing the cDNA of DRV S2 gene demonstrated the high sensitivity of the assay with a lowest detection limit of ≤1.48 copies/μL Standard deviation and coefficient of variation were low for both intra-assay and inter-assay variability. The assay performance was evaluated on 80 samples obtained from artificially infected Cherry Valley ducklings and 10 field specimens compared with the conventional RT-PCR assay. It was shown that 10 artificially infected samples tested negative in gel-based assay were positive for the real-time RT-PCR. DRV could be detected in all eight different tissues collected from the ducklings infected artificially. In contrast, the higher detection rate was obtained in the bursa of fabricius (90%), lung (90%), spleen (80%), and thymus (70%) than that in the liver (30%) as well as in the pancreas (10%). This method was rapid, specific, and sensitive for the detection of DRV and will be useful in veterinary diagnostic applications.
Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne zoonotic diseases, thereby posing a serious threat to human health. Although JEV is potentially neurotropic, its pathogenesis and distribution in the host have not been fully elucidated. In this study, an infected mouse model was established using a highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, combined with anatomical imaging of the mouse brain, were used to dynamically localize the virus and construct three-dimensional (3D) images. Consequently, onset of mild clinical signs occurred in some mice at 3.5 d post JEV infection, while most mice displayed typical neurological signs at 6 d post-infection (dpi). Moreover, brain pathology revealed typical changes associated with non-suppurative encephalitis, which lasted up to 8 d. The earliest detection of viral antigen was achieved at 3 dpi in the thalamus and medulla oblongata. At 6 dpi, the positive viral antigen signals were mainly distributed in the cerebral cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 8 dpi, the antigen signals gradually decreased, and the localization of JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was detected in the brain at 21 dpi. In this model, the viral antigen was first expressed in the reticular thalamic nucleus (Rt), and the virus content is relatively stable. The expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images showed that viral signals were mostly concentrated in the parietal cortex, occipital lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in mice, a large number of viral antigens were detected in denatured and necrotic neurons, suggesting that JEV directly causes neuronal damage. From the time of its entry, JEV is widely distributed in the central nervous system thereby causing extensive damage.
9-day-old ducklings were inoculated with new type duck hepatitis virus.And the NO in the serum,liver and brain,the TNF,IL-2 in the serum were determined at 12,24,48,72,96,168 h and 14 d post inoculation(PI),and at the same time,the histopathological changes of liver and brain of inoculated duckling were observed.The result showed that the levels of serum NO increased significantly at 48,72 h and 96 h PI.The activities of liver NO began to increase significantly at 24 h PI.There were no changes in the content of brain NO.The concentration of serum TNF and IL-2 increased significantly at 24 h after inoculation,then dropped significantly at 96 h PI.Histopathological examination revealed the hemorrhagic and necrotic inflammation of the liver at 24 h PI.At 48 h to 96 h PI,proliferative inflammation was observed.All the time,the nonsuppurative encephalitis was observed in the brain.The study implied that the duckling infected with new type duck hepatitis virus resulted in the changes of the concentration of NO,TNF and IL-2,and these changes were related to the hurt of the liver and the pathogenesis of the disease.
In 2002,a new serotype of duck hepatitis virus(DHAV-3) was first reported in China.Then the infection has been found in many duck-growing areas and caused great economic losses due to the high mortality.To develop a live vaccine against DHAV-3 infection,an isolate was attenuated by serial passage in duck embryos.After 53 passages,the virus did not cause any clinical signs and microscopic lesion in tissues for high dose inoculation,and no virulence reversion was found after repeated passage in 3-day-old ducklings.Ducklings vaccinated with the 54th virus(5×105.9 ELD50) on 1 or 3-day-old of age were well protected.The protection index was 81.4% and 100% on day 4 and 6 respectively against the challenge with virulent strain.The virus neutralizing antibody reached 10-3.29 at day 7 after vaccination of 1-day-old ducklings and peaked at 4th week.The level of immunity was sufficient to protect ducklings through the susceptible period.These results demonstrate that the attenuated virus is a promising vaccine candidate for the prevention of DHAV-3 infection.
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