Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both.

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed "B. pulli" also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.

Detection of economically important viruses in boar semen by quantitative RealTime PCR technology.

The objective of this study was to develop quantitative real-time polymerase chain reaction (ReTi-PCR) tests for the detection of five economically important viruses in swine semen namely, pseudorabies virus (PRV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each ReTi-PCR test was validated for specificity, analytical sensitivity (detection limits), and experimental infection studies were performed to compare the conventional virus isolation methods with the newly developed ReTi-PCR tests. All five developed ReTi-PCR tests are very rapid compared to virus isolation, highly specific, and even more sensitive (lower detection limits) than conventional virus isolation methods for the detection of mentioned viruses in semen. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by ReTi-PCR tests than by virus isolations. The high throughput of these rapid ReTi-PCR tests makes it possible to screen large number of semen samples for the presence of viruses prior to insemination. This is a substantial advantage, in particular for boar semen the quality of which deteriorates quickly after storage. In general, the newly developed ReTi-PCR tests are valuable tools for the early, reliable and rapid detection of five economically important viruses, namely PRV, CSFV, FMDV, SVDV, and PRRSV in boar semen. These ReTi-PCR tests will improve the control of viral diseases transmitted via semen.

Evaluation of tests for antibodies against bovine herpesvirus 1 performed in national reference laboratories in Europe.

Sets of serum and milk samples were collected from various countries and prepared, lyophilised and distributed by 1 laboratory to 12 reference laboratories in Europe. The serum sets contained the three European bovine herpesvirus 1 (BHV1) reference serum samples (EU1, EU2 and EU3), serum samples from naturally and experimentally BHV1-infected cattle, from vaccinated, and vaccinated-challenged cattle, from uninfected cattle, and a series of serum dilutions. In addition, sets of milk samples were distributed. The samples were tested for antibodies against BHV1 in virus neutralisation tests, in gB-specific ELISAs, in indirect ELISAs and in gE-specific ELISAs. It was found that the virus neutralisation test and the gB-specific ELISAs were most sensitive for the detection of antibodies in serum, whereas for assaying milk samples the indirect ELISAs were the tests of choice. The results show that the quality of most laboratories appeared to be adequate, but that one laboratory performed considerably below an acceptable level of quality. Four samples from the panel have been proposed that might be selected as reference sera in addition to the three European reference samples.

Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR.

BACKGROUND: Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose. METHODS: Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene. RESULTS: The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log105 to log107 Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 104-107 mycobacterial genomes per gram of lymph nodes were detected. CONCLUSIONS: The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.

Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker.

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.

Genetic diversity of porcine circovirus type 2 (PCV2) in the Romanian wild boar population.

In this study, we have analyzed 23 PCV2 ORF2 sequences recovered from wild boar population in Romania. The PCV2 sequences were originated from different geographical regions in Romania, and collected between 2008 and 2009 during the classical swine fever virus (CSFV) surveillance campaign. Complete open reading frame 2 (ORF2) nucleotide sequences were obtained and compared with sequences mainly from European and Asian isolates. The Romanian sequences were identified as belonging to previously described clusters 2a and 2b, with high degree of heterogeneity (PCV2 ORF2 nucleotide homology ranged between 90.1% and 100%). Interestingly, for cluster 2a, the majority of the sequences (8 from a total number of 9) clustered mainly with the Asian isolates (especially China, but also India and South Korea), with three exceptions from Europe previously reported in Germany, Belgium and The Netherlands.

Estimation of hepatitis E virus transmission among pigs due to contact-exposure.

Locally acquired hepatitis E in humans from industrialized countries has been repeatedly suggested to originate from pigs. Pigs may serve as a reservoir of hepatitis E virus (HEV) for humans when a typical infected pig causes on average more than one newly infected pig, a property that is expressed by the basic reproduction ratio R(0). In this study, R(0) for HEV transmission among pigs was estimated from chains of one-to-one transmission experiments in two blocks of five chains each. Per chain, susceptible first-generation contact pigs were contact-exposed to intravenously inoculated pigs, subsequently susceptible second-generation contact pigs were contact-exposed to infected first-generation contact pigs, and lastly, susceptible third-generation contact pigs were contact-exposed to infected second-generation contact pigs. Thus, in the second and third link of the chain, HEV-transmission due to contact with a contact-infected pig was observed. Transmission of HEV was monitored by reverse transcriptase polymerase chain reaction (RT-PCR) on individual faecal samples taken every two/three days. For susceptible pigs, the average period between exposure to an infectious pig and HEV excretion was six days (standard deviation: 4). The length of HEV-excretion (i.e. infectious period) was estimated at 49 days (95% confidence interval (CI): 17-141) for block 1 and 13 days (95% CI: 11-17) for block 2. The R0 for contact-exposure was estimated to be 8.8 (95% CI: 4-19), showing the potential of HEV to cause epidemics in populations of pigs.