Experimental inoculation of a crow derived influenza A (H5N1) virus in chickens and its pathological and genetic characterization

We report the infectivity of a crow derived influenza A (H5N1) virus (A/crow/India/01TR01/2012) in chickens and its pathological and genetic characterization. Histopathological changes and immunohistochemistry staining of internal organs and skeletal muscle were consistent with influenza A virus infection. Real time RT-PCR and virus isolation results demonstrated the systemic spread of the virus in chickens with 100% mortality. Comparatively higher level of virus shedding was detected in oropharyngeal swab (7.63×106 viral RNA copy) than in cloacal swab (6.66 × 106 viral RNA copy). Concentrations of viral antigen in kidney, lungs, brain, spleen and large intestine were higher compared to pancreas and skeletal muscle. No genetic change was observed on interspecies transmission of the virus. The study revealed that the crow derived H5N1 virus is able to kill the poultry, underlining the need for close monitoring of presence of virus in poultry near crow roosting areas so that further transmission to other avian and mammalian hosts can be prevented

Dietary Supplementation with Soluble Plantain Non-Starch Polysaccharides Inhibits Intestinal Invasion of Salmonella Typhimurium in the Chicken

Soluble fibres (non-starch polysaccharides, NSP) from edible plants but particularly plantain banana (Musa spp.), have been shown in vitro and ex vivo to prevent various enteric pathogens from adhering to, or translocating across, the human intestinal epithelium, a property that we have termed contrabiotic. Here we report that dietary plantain fibre prevents invasion of the chicken intestinal mucosa by Salmonella. In vivo experiments were performed with chicks fed from hatch on a pellet diet containing soluble plantain NSP (0 to 200 mg/d) and orally infected with S.Typhimurium 4/74 at 8 d of age. Birds were sacrificed 3, 6 and 10 d post-infection. Bacteria were enumerated from liver, spleen and caecal contents. In vitro studies were performed using chicken caecal crypts and porcine intestinal epithelial cells infected with Salmonella enterica serovars following pre-treatment separately with soluble plantain NSP and acidic or neutral polysaccharide fractions of plantain NSP, each compared with saline vehicle. Bacterial adherence and invasion were assessed by gentamicin protection assay. In vivo dietary supplementation with plantain NSP 50 mg/d reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue, by 98.9% (95% CI, 98.1–99.7; P<0.0001). In vitro studies confirmed that plantain NSP (5–10 mg/ml) inhibited adhesion of S.Typhimurium 4/74 to a porcine epithelial cell-line (73% mean inhibition (95% CI, 64–81); P<0.001) and to primary chick caecal crypts (82% mean inhibition (95% CI, 75–90); P<0.001). Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was associated with increased short circuit current but no change in electrical resistance. The inhibitory activity of plantain NSP lay mainly within the acidic/pectic (homogalacturonan-rich) component. Supplementation of chick feed with plantain NSP was well tolerated and shows promise as a simple approach for reducing invasive salmonellosis

Occurrence of <i>sop</i>E<i> </i>gene and its phenotypic expression among different serovars of <i>Salmonella enterica </i>isolated from man and animals

631-634Salmonella pathogenesis is a complex phenomenon and a Type III secretion system plays a central role in the development of Salmonella-induced enteritis. One such Type III secretion protein is Salmonella outer protein E (SopE). Prevalence of sopE gene and its phenotypic expression (SopE protein) among different serovars of Salmonella enteric isolated from man and animals were investigated. Of 305 strains of S. enterica belonging to 11 serovars tested for the presence of sopE, 130 strains belonging to three serovars viz., Enteritidis, Gallinarum and Virchow were found to carry sopE gene irrespective of their source of isolation when tested by PCR amplification technique using its specific primers. Of these 130 strains, 112 strains were found to express SopE protein phenotypically as detected by Dot-ELISA using SopE antibody. Among the different serovars tested only serovars Gallinarum, Enteritidis and Virchow expressed SopE protein phenotypically in vitro. Role of SopE protein in pathogenesis of salmonellosis has been discussed

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Not AvailableHighly pathogenic avian Influenza (HPAI) is an important zoonotic disease and is becoming a great threat to poultry industry. India has experienced continual outbreaks of H5N1 HPAI virus since February, 2006 especially in Eastern India. Survivability in poultry faeces is an important determinant in evaluating the persistence of the virus in the poultry sheds and their vicinity. In this paper, survivability of Indian H5N1 HPAI virus in dry and wet poultry faeces at 42, 37, 24 and 4 °C, respectively is reported. The effect of different temperatures was determined by linear regression model and defined in terms of linear equation. The virus survived up to 18 h at 42 °C, 24 h at 37 °C, 5 days at 24 °C and 8 weeks at 4 °C in dry and wet faeces, respectively. The coefficients of determination (R2) values for dry and wet faeces revealed that the difference in viral persistence in dry and wet faeces at all temperatures was not very marked. Results of the present study indicated that H5N1 HPAI virus may remain viable for extended periods of time in faeces at low temperatures and may act as a long term source of influenza virus in the environment.Not Availabl

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Not AvailableIn the present study, 27 field materials from different sources were tested in SYBR Green I dye based Real Time PCR optimized using WHO recommended conventional PCR primers targeting Bacillus anthracis protective antigen (PA) gene of pX01 plasmid. The assay had detection limit of 0.000002 pg in terms of target DNA quantity and up to 4 copies of pX01 plasmid per reaction, in terms of copy number. Melt curve analysis of PCR products of field samples showed deviation up to 3.2°C in melting temperature from standard anthrax strain suggestive of possible mutation/ mutations in PA gene. Targeted region of PA (596 bp) encompasses domain 2, involved in its binding with lethal factor (LF) and/or edema factor (EF) and mutation in this region may alter the efficacy of PA to traffic toxins inside the cells and results in differed level of virulence of pathogen. The test is useful not only for detection but may be suggestive of mutation also. Evaluation of a protective antigen gene based SYBR Green I real time PCR for detection of Bacillus anthracis in field samples (PDF Download Available). Available from: https://www.researchgate.net/publication/265013123_Evaluation_of_a_protective_antigen_gene_based_SYBR_Green_I_real_time_PCR_for_detection_of_Bacillus_anthracis_in_field_samples [accessed Jun 3, 2017].Not Availabl

PCR-RFLP analysis of HA and NA genes of a H5N1 isolate being a possible escape mutant.

Not AvailableNew AIV strain continue to emerge because of genetic instability of influenza virus and lack of proof reading and repair mechanism makes AIV genome easily prone to error that occur during replication. This study provide an update of H5N1 being an escape mutant which escaped from a immunized bird in aimmunization experiment conducted on birds with virus strain A/Duck/Tripura/103597/2008(H5N1). This article provide an update of H5N1 virus as it was evolving, creating the potential for new strain that is efficiently transmitted from birds to birds, birds to human or human to human remains highly lethal. The emergence of escape mutant may be either because of antigenic drift or genetic shift which results in H5N1 variants.Not Availabl

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Not AvailableElucidation of the molecular pathogenesis underlying virus-host interactions is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) virus infection in chickens. However, the pathogenesis of HPAI virus in chickens is not completely understood. To identify the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we analyzed the lung proteome of a chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). Mass spectrometry data sets were searched against the chicken UniProt reference database. At the local false discovery rate level of 5%, a total of 3313 proteins with the presence of at least one unique peptide were identified in the chicken lung proteome datasets. Differential expression analysis of these proteins showed that 247 and 1754 proteins were downregulated at 12 h and 48 h postinfection, respectively. We observed expression of proteins of the predominant signaling pathways, including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and JAK-STAT signaling. Activation of these pathways is associated with the cytokine storm effect and thus may be the cause of the severity of HPAI H5N1 infection in chickens. We also observed the expression of myeloid differentiation primary response protein (MyD88), inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), interleukin 1 receptor associated kinase 4 (IRAK4), RELA proto-oncogene NF-κB subunit (RELA), and mitochondrial antiviral signaling protein (MAVS), which are involved in critical signaling pathways, as well as other, less-commonly identified proteins such as hepatocyte nuclear factor 4 alpha (HNF4A), ELAV-like RNA binding protein 1 (ELAVL1), fibronectin 1 (FN1), COP9 signalosome subunit 5 (COPS5), cullin 1 (CUL1), breast cancer type 1 susceptibility protein (BRCA1), and the FYN proto-oncogene Src family tyrosine kinase (FYN) as main hub proteins that might play important roles in influenza pathogenesis in chickens. In summary, we identified the signaling pathways and the proteomic determinants associated with disease pathogenesis in chickens infected with HPAI H5N1 virus.Not Availabl

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Not AvailableDucks are the “Trojan Horses” for Asian H5N1 avian influenza viruses (AIV) and attain carrier status without displaying overt infection. These birds help in the spread of the virus among the poultry and human population through direct or indirect contact. Preen oil is the secretion of preen gland of water birds such as ducks. In a process called preening, the water birds spread preen oil across their feather and body. Preen oil has been known to play a significant role in the accumulation of various pathogens including Highly Pathogenic Avian Influenza (HPAI) from water onto feathers. However, the studies are scarce on the role of preen oil in the survivability of HPAIV. We conducted a simulative study to analyse the effect of preen oil on the survivability of the HPAI virus (H5N1) on duck feathers. Duck feather samples along with relevant controls were spiked with the H5N1 virus at two different initial concentrations (104 EID50 and 106 EID50), stored at 37°C, 25°C and 10°C temperatures and tested at regular intervals for percent infectivity by egg culture method and qRTPCR. The infectivity and viral load were significantly higher in naturally preened duck feathers in comparison to the three preen oil deficit controls at both low and high initial concentrations of virus (104 EID50 and 106 EID50). Maximum persistence was seen at 10°C in naturally preened duck feathers spiked with 106 EID50 concentration of viruses. It was also seen that depletion of preen oil from duck feathers reduced the persistence of the virus. These results demonstrate that preen oil plays a significant role in survivability and protection of HPAIV on duck feathers. This study herein will present new avenues in understanding one of the epidemiological niches of HPAIV.ICA

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Not AvailableHighly pathogenic avian influenza (H5N8) viruses were detected in waterfowl at 2 zoos in India in October 2016. Both viruses were different 7:1 reassortants of H5N8 viruses isolated in May 2016 from wild birds in the Russian Federation and China, suggesting virus spread during southward winter migration of birds.Not Availabl