Prevalence of pathogenic Yersinia enterocolitica in slaughter-aged pigs during a one-year survey, 2010-2011, France.

The prevalence of pathogenic Yersinia enterocolitica in French slaughter-aged pigs was estimated by sampling 3120 pigs from 96 batches in 16 slaughterhouses from January 2010 to February 2011. Respectively, 36 batches (20 pigs/batch) and 60 batches (40 pigs/batch) were considered during the cold period and the warm period. Tonsils were swabbed before the chilling step. Pathogenic Y. enterocolitica was detected after enrichment in ITC and streaking on CIN and YeCM media. Typical isolates were confirmed as Y. enterocolitica and biotyped by biochemical tests as described in the ISO 10273:2003 method. Of the tested pigs, 13.7% (CI95% [10.1-17.3]) were found positive for pathogenic Y. enterocolitica and 74.3% (CI95% [64.8-83.8]) of the pig batches contained at least one positive pig. The percentage of positive pigs per batch was generally low; 60.3% of positive batches contained fewer than 5 positive pigs. The prevalence of the pathogen at the batch level remained unchanged throughout this one-year study, but the prevalence in pigs was significantly higher during the warm period than during the cold period. Biotype 4 was the most prevalent biotype among the 827 isolated strains (91.9% of the isolates), followed by biotype 3 (7.25% of the isolates). Six isolates were of biotype 5 and one of biotype 2. Biotype 4 was found in all the 16 participating slaughterhouses, biotype 3 in ten slaughterhouses and biotype 5 in four. This study provides valuable recent figures for the prevalence of pathogenic Y. enterocolitica in French pigs. It also highlights the seasonal aspect of the carriage of this pathogen by pigs, a pattern which differs from those in other countries.

Length variations in the NA stalk of an H7N1 influenza virus have opposite effects on viral excretion in chickens and ducks.

A deletion of ∼20 amino acids in the stalk of neuraminidase is frequently observed upon transmission of influenza A viruses from waterfowl to domestic poultry. A pair of recombinant H7N1 viruses bearing either a short- or long-stalk neuraminidase was genetically engineered. Inoculation of the long-stalk-neuraminidase virus resulted in a higher cloacal excretion in ducks and led conversely to lower-level oropharyngeal excretion in chickens, associated with a higher-level local immune response and better survival. Therefore, a short-stalk neuraminidase is a determinant of viral adaptation and virulence in chickens but is detrimental to virus replication and shedding in ducks.

Sequential autoprocessing of Marek's disease herpesvirus protease differs from that of other herpesviruses.

Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I)(P3)-X(P2)-A(P1)/S(P1') (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: M(P3)-N(P2)-A(P1)/S(P1'). Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.

Single-nucleotide polymorphisms in two Marek's disease virus genes (Meq and gD): application to a retrospective molecular epidemiology study (1982-1999) in France.

Marek's disease virus (MDV) is a herpesvirus that causes a lymphoproliferative disease in chickens. Vaccines against MDV are available, but the virus is gradually becoming more virulent. A molecular epidemiology study of MDV was carried out by assessing nucleotide variation in two different genes, Meq and gD, in 68 French field isolates circulating from 1982 to 1999, compared with reference strains. Viral DNA was amplified by nested PCR and sequenced directly. Comparison of the nucleotide sequences revealed a high nucleotide sequence identity (98 %). Single-nucleotide polymorphisms were identified, leading to the identification of three gene alleles for gD and six for Meq. Nine combinations of alleles were identified. A majority of French isolates (60.5 %) clustered in the C1 type, which has been present for over 17 years. Waves of non-C1-type isolates appeared when vaccine efficacy decreased. Furthermore, specific discriminating sequences were obtained for the CVI-988 vaccine strain.

Detection of avian oncogenic Marek's disease herpesvirus DNA in human sera.

The avian herpesvirus Marek's disease virus (MDV) has a worldwide distribution and is responsible for T-lymphoma in chickens. The question as to whether MDV poses a public health hazard to humans was first raised when the virus was isolated in 1967. However, no irrefutable results have been obtained in immunological and virological studies. We used a nested-PCR to detect MDV DNA in human serum samples. A total of 202 serum samples from individuals exposed and not exposed to poultry was tested by nested-PCR for a target sequence located in the MDV gD gene. The assay system was specific and sensitive, making it possible to detect a single copy of the target sequence. Forty-one (20%) of the 202 serum samples tested positive for MDV DNA. The prevalence of MDV DNA was not significantly different in the group exposed to poultry and the group not exposed to poultry. There was also no difference due to age or sex. Alignment of the 41 gD sequences amplified from human sera with eight gD sequences amplified from MDV-infected chicken sera showed a maximum nucleotide divergence of 1.65%. However, four 'hot-spot' mutation sites were identified, defining four groups. Interestingly, two groups contained only human MDV-gD sequences. The status of the MDV genome detected in human blood is discussed.

Serological conservation and location of the adhesin of avian Escherichia coli type 1 fimbriae.

By inoculation of mice with purified type 1-like fimbriae isolated from an avian Escherichia coli strain, a monoclonal antibody (mAb G5) was obtained. mAb G5 reacted in an enzyme-linked immunosorbent assay (ELISA) with type 1-like and type 1A fimbriae differing in the molecular masses of their major fimbrial subunit and isolated from several avian E. coli strains. The specificity of mAb G5 for type 1 fimbriae was assessed in a whole bacteria ELISA with 16 reference E. coli strains expressing different types of fimbriae. Immunoblotting experiments showed that mAb G5 recognized the 29 kDa minor component of reference type 1A fimbriae which has been identified as the adhesin. mAb G5 also recognized the 29 kDa component of type 1-like and type 1A fimbriae expressed by avian E. coli strains, suggesting that the adhesin is antigenically conserved among these fimbriae. Immunoelectron microscopic studies gave evidence that the adhesin could be located mainly at the tip or both at the tip and along the fimbriae, depending on the strain.

Transformed and tumorigenic phenotypes induced by avian retroviruses containing the v-mil oncogene.

Avian retrovirus MH2 contains two oncogenes, v-mil and v-myc. We have previously shown that a spontaneous mutant of MH2 (PA200-MH2), expressing only the v-mil oncogene, is able to induce proliferation of quiescent neuroretina cells. In this study, we investigated the transforming and tumorigenic properties of v-mil. PA200 induced fibrosarcomas in about 60% of the injected chickens, whereas inoculation of MH2 resulted mainly in the appearance of kidney carcinomas. Analysis of several parameters of transformation showed that PA200, in contrast to MH2, induced only limited in vitro transformation of fibroblasts and neuroretina cells. These results suggest that v-myc is the major transforming and tumorigenic gene in MH2-infected cells. This low in vitro transforming capacity differentiates v-mil not only from other avian oncogenes, but also from the homologous murine v-raf gene.

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus.

Expression of the src gene of Rous sarcoma virus in chicken embryo neuroretinal cells results in morphological transformation and sustained proliferation of this normally resting cell population. PA101 and PA104 are two mutants of Rous sarcoma virus which induce neuroretinal cell proliferation in the absence of morphological transformation. Their mitogenic property is temperature sensitive, and they both encode p60src proteins with low kinase activity. To study the role of the mitogenic function and protein kinase activity of p60src in tumorigenesis, we investigated the oncogenicity of PA101 and PA104. Both mutants were less tumorigenic than wild-type virus when injected into chicks. Tumorigenicity was further assayed by inoculating infected chicken embryo fibroblasts and neuroretinal cells onto the chorioallantoid membrane of embryonated duck eggs. This system provides a nonpermissive and immunodeficient environment for xenogenic cell grafting and allows the study of cell tumorigenicity within a temperature range of 37 to 39.5 degrees C. Chicken embryo fibroblasts and neuroretinal cells infected with PA101 were as tumorigenic as wild type-infected cells at 37 degrees C, but tumor development was significantly reduced at 39.5 degrees C. In contrast, both cell types infected with PA104 displayed sharply reduced tumorigenicity. Cell cultures derived from PA101 tumors induced on the chorioallantoid membrane were similar to the corresponding cells maintained in vitro in terms of morphology, production of plasminogen activator, relative amounts of phosphotyrosine in total cellular proteins, and phosphorylation of 34,000-molecular-weight protein. These results indicate that the expression of the mitogenic function of src does not account per se for cell tumorigenicity and that tumor formation is compatible with low levels of p60src protein kinase activity.