The relationship between socioeconomic indices and potentially zoonotic pathogens carried by wild Norway rats: a survey in Rhône, France (2010-2012).

Leptospira interrogans, hantaviruses (particularly Seoul virus), hepatitis E virus (HEV), and Toxoplasma gondii are rat-associated zoonoses that are responsible for human morbidity and mortality worldwide. This study aimed to describe the infection patterns of these four pathogens in wild rats (Rattus norvegicus) across socioeconomic levels in neighbourhoods in Lyon, France. The infection or exposure status was determined using polymerase chain reaction or serology for 178 wild rats captured in 23 locations; additionally, confirmatory culture or mouse inoculation was performed. Multivariate logistic regression analyses were used to investigate whether morphological and socioeconomic data could predict the infection status of the rats. This study revealed that the rat colony's age structure may influence the prevalence of L. interrogans, hantavirus, and HEV. In addition, areas with high human population densities and low incomes may be associated with a greater number of infected rats and an increased risk of disease transmission.

Comparison of two real-time RT-PCR assays for differentiation of C-strain vaccinated from classical swine fever infected pigs and wild boars.

Classical swine fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance.

Development of improved analytical methods for use in animal health and in foodborne disease surveillance for source attribution.

Considering the 'One Health' principles, the links between animal and human health are very strong. Both domestic and wild animals are sources of infectious agents that cause diseases in humans. Poor animal health may also indirectly affect human health, through reduced access to food. A large number of infectious diseases of animals, the transboundary animal diseases, spread rapidly across borders. Robust and accurate diagnostic assays are needed to detect the infectious agents rapidly and to limit their spread. A large arsenal of novel assays has been developed during the last three decades, with a tremendous impact on the detection of infectious agents. The new diagnostic methods are mostly laboratory-based and expensive, requiring sophisticated equipment and special skills. However, rapid and cheap field-based assays have also been developed. Herein, the authors give several examples of the development of novel assays, with special focus on the 'One Health' principles.

Detection of herpesvirus DNA in Arctic foxes (Vulpes lagopus; syn. Alopex lagopus) with fatal encephalitis.

A captive breeding programme for the Fennoscandian Arctic fox (Vulpes lagopus; syn. Alopex lagopus) failed due to fatal encephalitis. The aim of this study was to identify the causative agent. Viral nucleic acid was detected by PCR and in situ hybridization in the brain of affected foxes. The results suggest that a herpesvirus might be the causative agent. Whether this infection also occurs in free-living Arctic foxes is unknown.

Molecular epidemiology of hepatitis E virus in humans, pigs and wild boars in Sweden.

Hepatitis E infections in humans are usually acquired in endemic countries in Asia or Africa. In Sweden 17 cases infected in Europe, between 1993 and 2009, were identified. All had clinical hepatitis E with unknown source of infection. Hepatitis E virus (HEV) was identified in faecal samples from 63 piglets in 12 pig farms in Sweden. HEV was also identified in blood from 13 out of 159 investigated Swedish wild boars from nine counties. Partial HEV genomes from humans, pigs and wild boars were sequenced and compared by phylogeny. The results showed close relatedness between HEV strains from piglets from the same farm and from wild boars from the same county. HEV strains from humans showed relatedness with strains from pigs and wild boars from the same county. This study showed that HEV strains form geographical clusters in the phylogenetic tree. The methods used in this study may thus be used for tracing the origin of an infecting strain. Furthermore, this study indicated that there are endemic sources of human HEV infections in Sweden.

Endemic hepatitis E in two Nordic countries.

Antibodies against hepatitis E virus (anti-HEV) were found in 248 Swedish and Danish patients between 1993 and 2007. Most patients were symptomatic and tested for anti-HEV due to travel abroad. Among patients with known country of infection, most were infected in Asia, mainly on the Indian subcontinent. However, 29 patients were infected in Europe, nine of these had HEV IgM and/or HEV RNA in serum. In sera from 65 of 141 tested patients HEV RNA could be detected, and 63 strains could be typed by limited sequencing within ORF2. HEV RNA was found in sera from 71% of the patients with HEV IgM and IgG and in 18% of the patients with only detectable HEV IgG. It was also found up to three weeks after the onset of disease in 67% of the patients with known date of onset. Patients infected in Europe were infected by genotype 3, and were older than those infected by genotype 1 (mean age 55.3 vs 30 years, p<0.001). Since it is known that genotype 3 can infect domestic pigs, HEV strains from 18 piglets in 17 herds in Sweden and Denmark were sequenced. Phylogenetic analyses of the genotype 3 strains showed geographical clades and high similarity between strains from patients and pigs from the same area. There are thus autochthonous hepatitis E cases in Scandinavia, and there are probably many undiagnosed ones. Patients with hepatitis of unknown etiology should therefore be investigated for anti-HEV even if they have not been outside Europe, since infections acquired from pigs or other animals should be taken into consideration.

Detection and genetic characterisation of Hepatitis E virus in Czech pig production herds.

The objective of the present study was to carry out a small surveillance programme in Czech pig production herds using the nested reverse transcriptase-polymerase chain reaction (nRT-PCR) technique to trace Hepatitis E virus (HEV) in different biological samples and to characterise the detected swine HEV isolates by phylogenetic analysis. A total of 32 piglets from 11 herds clinically suspected of postweaning multisystemic wasting syndrome (PMWS) were examined. Bile, liver tissue and serum samples were collected from each animal. Due to the high genetic variability of HEV, three sets of primers targeting each of the open reading frames (ORFs) of its genome were used. HEV RNA was most frequently detected in the bile samples (40.0%), followed by liver tissue (16.1%) and serum (3.2%). Seven (63.6%) of the 11 monitored farms were found to have at least one HEV RNA positive piglet. Specific 242 bp sequences within the ORF1 coding non-structural proteins were sequenced and phylogenetically analysed. Phylogenetic analysis using the neighbor-joining and maximum parsimony method confirmed that all detected Czech swine HEV isolates belonged to genotype III. Comparison of the Czech swine HEV isolates with corresponding sequences of swHEV available in GenBank failed to find any 100% homologous HEV isolate.

Evidence for the presence of hepatitis E virus in pigs in the United Kingdom.

Samples of serum, tissue and faeces from two pig herds in England were examined for hepatitis E virus by reverse-transcriptase PCR (RT-PCR), and a virus strain from each herd was partially sequenced. Eleven of 42 faecal samples and 16 of 21 tissue samples from two pigs were positive for the virus by RT-PCR. Analysis of two unique but closely related nucleotide sequences obtained from the two herds showed that the viruses clustered in genotype III (6) with a human strain of the virus from an autochthonously acquired case of acute hepatitis in the UK. An ELISA based on recombinant open reading frame 2 (ORF-2) was used to detect antibodies to hepatitis E virus in 256 pig sera from the UK; 85.5 per cent of the samples were positive, compared with 58 per cent of similar samples from Swedish pigs and 23.5 per cent of samples from Dutch pigs.

Identification and sequence analysis of the glycoprotein B gene of porcine cytomegalovirus.

Porcine cytomegalovirus (PCMV) is one of the pathogens that should be eliminated from pigs intended for use as organ donors in xenotransplantation. For this purpose, reliable diagnostic test systems are needed. To provide a basis for this goal and to analyse the evolutionary relationships of PCMV within the herpesvirus family, the putative glycoprotein B (gB) gene of PCMV was identified by assuming gene colinearity and a relative conservation of nucleotide sequences in comparison with closely related herpesviruses. Using this approach the complete nucleotide sequence of the PCMV gB gene was determined. A protein of 860 amino acids was deduced and a putative cleavage site, conserved cysteine residues, as well as potential N-terminal glycosylation motifs were identified. In a comparison of PCMV gB with the corresponding region of other herpesviruses, the highest identities were found with human herpesviruses 6 and 7 (HHV-6 and 7; 43.4% and 42.6%, respectively). Also in phylogenetic analysis, the PCMV gB clustered with HHV-6 and HHV-7. Between the complete gB sequences of five different PCMV strains and isolates from the United Kingdom, Germany, Spain, Japan and Sweden, differences of 3.4% were found, indicating a considerable intra-species variation. The characterisation of the protein deduced from the identified gene provides further evidence that this is indeed the gB gene of PCMV and provides important taxonomical information regarding PCMV. The identification of the gB gene should facilitate the development of sensitive and robust diagnostic methods for the PCMV screening of pigs.

Characterization of the DNA polymerase loci of porcine cytomegaloviruses from diverse geographic origins.

Porcine cytomegalovirus (PCMV) is an undesired pathogen in pigs intended for use as organ donors in xenotransplantation. In the present work, we characterized the first set of genes of PCMV. From a German isolate, the DNA polymerase (DPOL) locus was amplified and two complete open reading frames (ORF) as well as two partial ORFs including the complete DPOL gene and the 3'-end of the glycoprotein gB gene were sequenced. The deduced amino acid sequences showed the highest identities with the respective proteins of the betaherpesviruses, in particular those (ORFs 36-39) of the human herpesviruses 6 and 7 (HHV-6 and -7). In phylogenetic analysis, PCMV clustered also with HHV-6 and HHV-7. On this basis, PCMV could be firmly classified to the Betaherpesvirinae and tentatively assigned to the genus Roseolovirus. In addition to the German isolate, the DPOL gene was analysed from a British and a Japanese strain as well as a Spanish isolate. Differences of 0.4 to 1% were found on the nucleotide and the amino acid level. On the basis of the conserved regions, primer pairs were selected for PCR which detected PCMV in blood and tissue samples from four European countries. Therefore, these are the first nucleic acid-based test systems which were shown to universally detect PCMV. The application of these assays to organs of domestic pigs from Germany revealed a PCMV prevalence of > 50%.

Detection of challenge virus in fetal tissues by nested PCR as a test of the potency of a porcine parvovirus vaccine.

To estimate the potency of a porcine parvovirus (PPV) vaccine, three vaccinated and three non-vaccinated pregnant gilts were infected with PPV and the distribution of the virus was studied in the tissues of their 51 fetuses. Virus detection was attempted using haemagglutination (HA) and immunofluorescence (IF) assays, as well as by standard (single) and nested polymerase chain reactions (PCR). None of the detection methods yielded positive results when used to test for the presence of virus in suspensions of organs from the fetuses from the vaccinated gilts. However, the virus was detected in the fetuses from non-vaccinated gilts as follows: HA was positive in 14 cases out of 23 (60.8%), IF in 16/23 (69.5%), standard PCR in 12/20 (60%), and the nested PCR in 19/23 (82.6%). Although the correlation among the results of various methods of virus detection was rather close (r < 0.83), the sensitivity of the nested PCR was the highest, both when testing dilutions of PPV and when analysing the fetal organs. The nested PCR therefore provides a reliable approach for studies of virus distribution in fetal organs, with special reference to potency tests on vaccines.