Development of a recombinant NP protein based indirect ELISA for the detection of antibodies against Newcastle disease virus and differentiation of infected or recombinant vaccine immunized chickens.

Newcastle disease (ND) is a highly contagious and economically important disease in the poultry industry caused by avian avulavirus-1, historically known as Newcastle disease virus (NDV). Control of ND primarily relies on prophylactic vaccination of flocks, and many vaccines are available on the market, both conventional and more recently introduced new generation recombinant types. To assess the protection level achieved by vaccination ELISA tests are typically used, they also are to track an infection with field strains in non-vaccinated flocks. Special modifications of ELISA can be used as a screening tool to detect infection in flocks vaccinated with new generation vaccines. In this study, we have developed an ELISA test for the detection of antibodies against the nucleoprotein (NP) of NDV and for differentiation of chickens vaccinated with commercial and prototype in-house recombinant vector vaccines from those infected with field NDV strains. The NP gene of LaSota NDV strain expressed in a baculovirus vector was used as a coating antigen in the ELISA. The developed test was optimized, validated and compared to other serological tests. The sensitivity, specificity and accuracy of recombinant NP protein-based ELISA were respectively 96.1%, 96.3%, and 96.2%. Inter-rater (kappa) agreement between the NP-ELISA and the gold standard HI test was calculated to be 0.995. In our comparisons, commercially available ELISA tests revealed different specificities ranging from 95.5-100% and sensitivities at variance, ranging from 90.1 to 99.0%. A high level of maternally derived antibodies was measured in the serum of 1-day-old broilers in the NP-ELISA assay. These antibodies had disappeared and were undetected at 3, 5 and 6 weeks post-vaccination but birds became positive again at 2 weeks after control infection with a velogenic NDV strain. In SPF chickens, antibodies against NP protein were detected only after a challenge. The recombinant NP protein-based ELISA test is sensitive, specific and accurate when compared to the gold standard HI test and commercially available kits. Moreover, the method could be also used for the differentiation between vaccinated and infected birds.

Specific detection of GII-1 lineage of infectious bronchitis virus.

Infectious bronchitis virus (IBV) is a worldwide prevalent RNA virus that causes highly contagious and economically devastating disease in chicken. The virus exists in many different genetic forms which made the disease control very difficult. The present study describes the development and validation of TaqMan probe-based real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) targeting the S1 coding region of S gene characteristic for the GII-1 lineage (formerly the D1466-like variant) of IBV. These strains are quite different from other European IBV belonging to different lineages of the GI genotype. The developed method was 30-fold more sensitive than used so far for standard nested RT-PCR with detection limit of 56 RNA copies per reaction. The specificity of the assay was also evaluated with a panel of different poultry pathogens. Repeatability and reproducibility of the method was very high with coefficients of variation lower than 4%. One hundred and twenty-seven IBV-positive samples were tested by this method and GII-1 strains were detected in four of them (3·15%) which indicate a decrease in the GII-1 IBV prevalence in Poland. The assay was proven to be a valuable tool for rapid diagnosis of GII-1 lineage of IBV strains and moreover it enabled the monitoring of viral loads which can be used to assess disease progression. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a TaqMan probe-based real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for rapid and accurate identification of GII-1 lineage (formerly D1466-like variant) of infectious bronchitis virus (IBV). The assay revealed to be more sensitive than standard nested RT-PCR assay, previously used for this purpose. The developed assay has been tested on numerous field samples and revealed 3·15% prevalence of this lineage of IBV in Polish chicken population. Moreover, this new assay enables the assessment of viral load measurement which might be useful for epidemiology and pathogenesis studies.

Cross-sectional survey of selected enteric viruses in Polish turkey flocks between 2008 and 2011.

BACKGROUND: Enteric diseases are an important health problem for the intensive poultry industry, resulting in considerable economic losses. Apart from such microbiological agents associated with enteritis as bacteria and parasites, a lot of research has been recently conducted on viral origin of enteric diseases. However, enteric viruses have been identified in intestinal tract of not only diseased but also healthy poultry, so their role in enteritis is still unclear. The present study aimed at determination of the prevalence of four enteric viruses, namely astrovirus, coronavirus, parvovirus and rotavirus in meat-type turkey flocks in Poland as well as at statistical evaluation of the occurrence of the studied viruses and their relationships with the health status and the age of birds. Two hundred and seven flocks of birds aged 1-20 weeks originating from different regions of the country were investigated between 2008 and 2011. Clinical samples (10 individual faecal swabs/flock) were duly processed and examined using molecular methods targeting the conservative regions of viral genomes: RNA-dependent RNA polymerase gene of astrovirus, non-structural 1 gene of parvovirus, non-structural protein 4 gene of rotavirus, and 5' untranslated region fragment of turkey coronavirus. Different statistical methods (i.e. the independence chi-square test, the correspondence analysis and the logistic regression model) were used to establish any relationships between the analyzed data. RESULTS: Overall, 137 (66.2%, 95% CI: 59.3-72.6) of the 207 turkey flocks sampled were infected with one or more enteric viruses. Among the 137 flocks, 74 (54%, 95% CI: 45.3-62.6) were positive for one virus, whereas 54 (39.4%, 9 5% CI: 31.2-48.1) and 9 (6.6%, 95% CI: 3.1-12.1) were co-infected with two or three different enteric viruses, respectively. No flock was simultaneously infected with all four viruses studied. The prevalence of astrovirus infection was 44.9% (95% CI: 38.0-52.0), parvovirus 27.5% (95% CI: 21.6-34.2), rotavirus 18.8% (95% CI: 13.8-24.8), and coronavirus 9.7% (95% CI: 6.0-14.5). Young turkeys aged 1-4 weeks old had the highest (82.1%, 95% CI:71.7-89.8) prevalence of viral infection. Applied statistical methods have indicated the dependence of rotavirus infection as well as the co-infection with multiple viruses and the health status of turkeys. Furthermore, our results statistically confirm that especially young birds are susceptible to infection with rotavirus and astrovirus. CONCLUSIONS: The study demonstrated the presence of astrovirus, coronavirus, parvovirus and rotavirus infections in Polish turkey farms. These viruses were detected in both healthy and diseased birds. However, the presented results provide valuable feedback which could help to evaluate the role of some enteric viruses in the etiology of enteritis in turkey.

Molecular characterization of fowl adenoviruses isolated from chickens with gizzard erosions.

Broiler chickens with clinical signs of uneven growth, depression, and dull feathers were submitted to our laboratory and, at necropsy, lesions in proventriculus, gizzard, and intestines were detected. Fowl adenovirus serotype 1 (FAdV-1) was isolated from digestive tissues. The virus, assigned as FAdV-PL/G068/08, showed 99.5% nucleotide homology and 99.2% amino acid homology in hexon gene with chicken embryo lethal orphan (CELO) strain classified as the European reference of FAdV-1. One-day-old and 21-d-old SPF chickens were inoculated with FAdV-PL/068/08 by both nasal and ocular routes and then observed daily and examined by necropsy at 6, 10, and 14 d postinoculation. Experimental infection with isolated virus was fatal for younger chickens and major lesions occurred in the gizzards. No clinical or pathological changes were observed in chickens infected at 21 d of age, but the presence of intranuclear inclusion bodies in gizzard epithelial cells was detected. Molecular characterization was based on the long and short fibers genes sequencing and comparison of obtained sequences with other FAdV-1 strains. The homology between FAdV-PL/G068/08 and other sequences available in GenBank was between 98.9 and 99.8% (short fiber region) and 99.0 and 99.7% (long fiber region) at nucleotide level and between 98.4 and 100% (short fiber region) and 99.3 and 99.9% (long fiber region) at amino acid level. No correlation between identified amino acid changes in short and long fiber proteins and pathogenicity of studied FAdV-1 strains was observed. Although short and long proteins were indicated as factors influencing virus pathogenicity, the role of identified sequence differences in infectivity determination remain unclear.