Poultry Diagnostic and Research Center

College Park, GA, United States

Poultry Diagnostic and Research Center

College Park, GA, United States

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Reis A.,Poultry Diagnostic and Research Center | Ritz C.,University of Georgia | Garcia M.,Poultry Diagnostic and Research Center
Poultry Science | Year: 2012

To determine the risk of infection associated with exposure to low-pathogenic avian influenza (AI) virus-contaminated poultry litter, the tenacity of low pathogenic A/Ck/CA/431/00(H6N2), A/Mallard/MN/355779/00(H5N2), and A/turkey/ Ohio/313053/04(H3N2) was evaluated. Viral stocks were incubated with poultry litter from commercial flocks at 25°C. Three types of poultry litter, wood shavings, shavings plus gypsum, and shavings plus peanut hulls, from commercial broiler flocks were used. The 3 low-pathogenic avian influenza viruses retained infectivity for one day in wood shavings and shavings plus peanut hulls litter types, whereas in wood shavings plus gypsum, litter viruses remained infective for up to 3 d. In contrast to the survivability in litter, all the viruses maintained infectivity in water for 4 d at titers of log104.5. The infectivity of A/Ck/CA/431/00(H6N2) shed by experimentally infected layers, broilers, and turkeys was retained for one day, independently of the type of litter. In commercial production where a high density of birds are housed, the viral load shed by an infected flock will be significantly higher than the viral load shed 3 d postinfection obtained under the experimental conditions used in this study. Therefore proper management and disposal of poultry by products, such as windrow composting of litter and the composting of carcasses during an AI outbreak should be implemented. © 2012 Poultry Science Association Inc.


Kuriakose T.,Poultry Diagnostic and Research Center | Hilt D.A.,Poultry Diagnostic and Research Center | Jackwood M.W.,Poultry Diagnostic and Research Center
Avian Diseases | Year: 2012

In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptasePCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 × 10 8 to 1 × 10 9 copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 × 10 2.5 to 1 × 10 3.2 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes. © 2012 American Association of Avian Pathologists.


PubMed | Poultry Diagnostic and Research Center
Type: Evaluation Studies | Journal: Avian diseases | Year: 2012

In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptase-PCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 x 10(5) to 1 x 10(9) copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 x 10(2.5) to 1 x 10(3.2) 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes.


PubMed | Poultry Diagnostic and Research Center
Type: Journal Article | Journal: Archives of virology | Year: 2011

For a better understanding of evolution of influenza viruses, a chicken-origin and wild-bird-origin low-pathogenic avian influenza virus (LPAI) was serially passaged in chickens. Sequences of the hemagglutinin (HA) and neuraminidase (NA) genes at each passage level were compared to those of the parental virus. Multiple mutations occurring early during passage were detected, but these were maintained during passages. Interestingly, a number of the observed mutations already existed in the parental virus, as indicated by the presence of single-nucleotide polymorphisms. The greatest numbers of mutations occurred during passage of wild-bird-origin LPAI, where a 20-amino-acid deletion in the NA gene that was observed during the first passage was maintained during subsequent passages. Subsequent experiments showed that this NA deletion was already present as a minority population in the parental virus. These results showed that a selection process favoring a viral subpopulation had occurred.

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