Epidemiological study of Coxiella burnetii in dairy cattle and small ruminants in Québec, Canada.

The bacterium Coxiella burnetii (C. burnetii) can infect a wide range of animals, most notably ruminants where it causes mainly asymptomatic infections and, when clinical, it is associated with reproductive disorders such as abortion. It is also the etiological agent of Q fever in humans, a zoonosis of increasingly important public health concern. A cross-sectional study was performed to estimate the apparent prevalence and spatial distribution of C. burnetii positivity in dairy cattle and small ruminant herds of two regions of Québec, Canada, and identify potential risk factors associated with positivity at animal and herd levels. In dairy cattle herds, individual fecal samples and repeated bulk tank milk samples (BTM) were collected. In small ruminant herds, serum and feces were sampled in individual animals. ELISA analyses were performed on serum and BTM samples. Real-time quantitative PCR (qPCR) was done on fecal and BTM samples. An animal was considered C. burnetii-positive when at least one sample was revealed positive by ELISA and/or qPCR, while a herd was considered C. burnetii-positive when at least one animal inside that herd was revealed positive. None of the 155 cows had a qPCR-positive fecal sample, whereas 37.2 % (95 % CI = 25.3-49.1) of the 341 sheep and 49.2 % (95 % CI = 25.6-72.7) of the 75 goats were C. burnetii-positive. The apparent prevalence of C. burnetii-positive herds was 47.3 % (95 % CI = 35.6-59.3) in dairy cattle herds (n = 74), 69.6 % (95 % CI = 47.1-86.8) in sheep flocks (n = 23) and 66.7 % (95 % CI = 22.3-95.7) in goat herds (n = 6). No spatial cluster of positive herds was detected. At the individual level, the only significant association with positivity in multivariable regressions was higher parity number in small ruminants. At the herd level, the use of calving group pen, the distance to the closest positive bovine herd, and small ruminant herd density in a 5 km radius were associated with dairy cattle herd positivity, whereas small ruminant herds with more than 100 animals and with a dog on the farm had greater odds of C. burnetii positivity. Our study shows that the infection is frequent on dairy cattle and small ruminant herds from the two studied regions and that some farm and animal characteristics might influence the transmission dynamics of the C. burnetii infection.

Prevalence of shedding and antibody to Coxiella burnetii in post-partum dairy cows and its association with reproductive tract diseases and performance: A pilot study.

The bacterium Coxiella burnetii has been associated with reproduction disorders in dairy cattle. A cross-sectional study was conducted in Québec, Canada, to estimate the prevalence of C. burnetii in dairy cows from C. burnetii RT-PCR-positive and/or ELISA-positive herds. As a secondary objective, the associations between C. burnetii-positivity and three reproductive outcomes (purulent vaginal discharge, cytological endometritis, and success at first service) were assessed. A total of 202 post-parturient dairy cows from nine herds were sampled at 35 ± 7 days in milk. Vaginal mucus and composite milk were collected from each cow and screened for the presence of C. burnetii by real-time PCR (RT-PCR) and ELISA, respectively. Purulent vaginal discharge and cytological endometritis were evaluated using a Metricheck device and a modified cytobrush, respectively. The first insemination postpartum was done following an ovulation synchronization protocol around 70 days in milk, and success at first service was recorded. Multilevel logistic regressions adjusted for parity were used to model purulent vaginal discharge, cytological endometritis and success at first service according to C. burnetii cow status. All 202 RT-PCR-assayed vaginal samples were C. burnetii-negative. A positive result for anti-C. burnetii antibodies detection in composite milk was obtained in 25/202 samples and a doubtful result in 4/202 samples. After adjustment for sampling weights, the 202 ELISA-assayed composite milk samples gave an estimated overall prevalence of C. burnetii positive cows of 12.9 % (CI = 6.1-19.6 %) and of doubtful cows of 1.4 % (CI = 0.0-3.3 %). The proportion of ELISA-positive cows was lower in first parity (0%) compared to second (17.1 %) or third parity cows (20.0 %). The associations between ELISA positivity and reproductive outcomes were not statistically significant, perhaps due to the limited sample size, but could be used as pilot estimate for large-scale studies investigating the impact of C. burnetii infection on reproduction disorders in dairy cattle.

Prevalence and risk factors for Campylobacter spp., Salmonella spp., Coxiella burnetii, and Newcastle disease virus in feral pigeons (Columba livia) in public areas of Montreal, Canada.

Feral pigeons (Columbia livia) can harbor a range of zoonotic pathogens. A transversal study was undertaken to estimate the prevalence of feral pigeons infected by various pathogens in public areas in Montreal, Quebec. Cloacal swabs from captured birds were cultured for Salmonella spp. and Campylobacter spp. and tested by real-time polymerase chain reaction (RT-PCR) for the detection of Coxiella burnetii. An oropharyngeal swab was also submitted to real-time reverse-transcription polymerase chain reaction (RRT-PCR) for the detection of Newcastle disease virus. Among the 187 pigeons tested from 10 public areas, 9.1% (95% CI: 3.0 to 15.2) were positive for Campylobacter spp. with all strains identified as Campylobacter jejuni. The Campylobacter status of birds was not associated with individual characteristics of birds, with the exception of body score. None of the pigeons tested positive for the other pathogens. Direct or indirect contacts with feral pigeons may constitute a potential risk for Campylobacter infection in humans.

Les pigeons sauvages (Columbia livia) peuvent être porteurs d'une variété d'agents pathogènes zoonotiques. Une étude transversale a été réalisée dans le but d'estimer la prévalence de pigeons sauvages infectés par différents agents pathogènes dans des aires publiques de la ville de Montréal, Québec (Canada). Des écouvillons cloacaux d'oiseaux capturés ont été cultivés pour Salmonella spp. et Campylobacter spp. et testés par une réaction en chaîne par polymérase en temps réel (RT-PCR) pour la détection de Coxiella burnetii. Des écouvillons oropharyngés ont également été testés par une réaction en chaîne par polymérase en temps réel après transcription inverse (RRT-PCR) pour la détection du virus de la maladie de Newcastle. Parmi les 187 pigeons testés provenant de 10 aires publiques, 9,1 % (IC 95 % : 3,0­15,2) étaient positifs à Campylobacter spp.; toutes les souches ont été identifiées en tant que Campylobacter jejuni. L'infection par Campylobacter n'était pas associée aux caractéristiques individuelles des oiseaux, à l'exception de l'état de chair. Aucun pigeon n'était positif aux autres agents pathogènes. Le contact direct ou indirect avec des pigeons sauvages peut représenter un risque potentiel pour les infections à Campylobacter jejuni chez l'humain.(Traduit par les auteurs).

Quantification of E. coli O157 and STEC in feces of farm animals using direct multiplex real time PCR (qPCR) and a modified most probable number assay comprised of immunomagnetic bead separation and qPCR detection.

To better understand Escherichia coli O157:H7 on-farm transmission dynamics requires sensitive methods for quantification of a broad range of concentrations of target organisms. For this purpose, a multiplex real time PCR (qPCR) assay was developed for quantification of O157 E. coli from 1g fecal samples of cattle and other animal species, targeting the Shiga toxin genes (stx1 and stx2) and the O157 somatic antigen gene, per. The multiplex qPCR assay provided specific detection across a broad range of bacterial concentrations with a lower limit of detection (LOD) of 10(1) genome copies which is equivalent to 10(1) bacteria. However, the LOD, when direct qPCR was applied to quantification of the targets in the feces of dairy cattle, was 10(3) genome copies per gram of feces. Enumeration below the threshold for direct qPCR was performed using a modified most probable number (mMPN) method whereby E. coli O157 in enriched samples was isolated using immunomagnetic bead separation (IMS) and detected using qPCR, thus reducing the time and logistic constraints of biochemical/serological/gel analysis. Application of the mMPN (IMS/qPCR) assay to samples that were negative when tested using direct qPCR alone permitted quantification of low levels of E. coli O157 below levels detectable with direct qPCR. The direct qPCR and mMPN (IMS/qPCR) assays were applied to fecal samples from dairy, beef, swine and poultry feces. This approach can be employed to gain a better understanding of the patterns of infection in animals for analysis of on-farm transmission dynamics, for evaluating the effects of on-farm control strategies and for risk assessment in public health.

Emergence of a new swine H3N2 and pandemic (H1N1) 2009 influenza A virus reassortant in two Canadian animal populations, mink and swine.

A swine H3N2 (swH3N2) and pandemic (H1N1) 2009 (pH1N1) influenza A virus reassortant (swH3N2/pH1N1) was detected in Canadian swine at the end of 2010. Simultaneously, a similar virus was also detected in Canadian mink based on partial viral genome sequencing. The origin of the new swH3N2/pH1N1 viral genes was related to the North American swH3N2 triple-reassortant cluster IV (for hemagglutinin [HA] and neuraminidase [NA] genes) and to pH1N1 for all the other genes (M, NP, NS, PB1, PB2, and PA). Data indicate that the swH3N2/pH1N1 virus can be found in several pigs that are housed at different locations.

Identification of a novel herpesvirus associated with cutaneous ulcers in a fisher (Martes pennanti).

On December 8, 2008, a male fisher (Martes pennanti) housed in a quarantine enclosure at the St-Félicien Zoo was found dead with multiple skin ulcers on the muzzle and plantar pads. At necropsy, no major findings were found, and a specific cause of death was not determined microscopically. However, at the borders of ulcerated sites, there were increased numbers of koilocytes, with perinuclear vacuolation and nuclear enlargement. A pan-herpesvirus nested polymerase chain reaction (PCR) assay was conducted, and an expected PCR product of 230 nucleotides was obtained within tissues collected from around the skin ulcers. Other tissues, including intestines and pool of lung, liver, and kidney, tested negative. The obtained PCR amplicon was sequenced and was highly related to the partial viral DNA polymerase (DPOL) gene of Mustelid herpesvirus 1. Virus isolation was negative, and no virion was detected by electron microscopy. The pathogenic potential of this novel herpesvirus and its role in the death of the fisher are unknown.

Emergence of a new type of porcine circovirus in swine (PCV): a type 1 and type 2 PCV recombinant.

In late September 2008, tissue samples from piglets experiencing an acute outbreak of porcine reproductive and respiratory syndrome (PRRS) were submitted to the Veterinary diagnostic service of the University of Montreal. Several diagnostic assays were performed including a multiplex real-time quantitative PCR assay (mrtqPCR) for the detection and differentiation of porcine circovirus (PCV) type 2a and 2b genotypes in the lung and lymph nodes. The pig samples were found to be positive for PCV2a using the mrtqPCR but odd results were obtained. The Ct values obtained with mrtqPCR probes targeting the ORF1 and ORF2 of PCV2 were not as expected which suggested the presence of genomic variations in the PCV2 viral genome. Ultimately, a total of three diagnostic cases with mrtqPCR unusual results were investigated. After virus isolation and sequence analyses, a new type of PCV was identified in those three cases. Based on sequence analyses, this new PCV genome contains the ORF1 of PCV1 and the ORF2 of PCV2a and its entire viral genome nucleotide identity compared to PCV1, PCV2a and 2b are 86.4%, 88.7% and 86.5%, respectively. It is proposed to name this new PCV by taking into account the nomenclature of Segales et al. (2008) and by indicating the origin of the ORF1 at first and the origin of the ORF2 in second. Consequently, the name proposed for this new PCV is PCV1/2a. The prevalence of PCV1/2a seems to be very low in Quebec, Canada (2.5% of PCV positive cases), and its origin is now in debate.

Characterization of a Canadian mink H3N2 influenza A virus isolate genetically related to triple reassortant swine influenza virus.

In 2007, an H3N2 influenza A virus was isolated from Canadian mink. This virus was found to be phylogenetically related to a triple reassortant influenza virus which emerged in Canadian swine in 2005, but it is antigenically distinct. The transmission of the virus from swine to mink seems to have occurred following the feeding of animals with a ration composed of uncooked meat by-products of swine obtained from slaughterhouse facilities. Serological analyses suggest that the mink influenza virus does not circulate in the swine population. Presently, the prevalence of influenza virus in Canadian farmed and wild mink populations is unknown. The natural occurrence of influenza virus infection in mink with the presence of clinical signs is a rare event that deserves to be reported.

Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey.

By the end of 2004, the Canadian swine population had experienced a severe increase in the incidence of Porcine circovirus-associated disease (PCVAD), a problem that was associated with the emergence of a new Porcine circovirus-2 genotype (PCV-2b), previously unrecovered in North America. Thus, it became important to develop a diagnostic tool that could differentiate between the old and new circulating genotypes (PCV-2a and PCV-2b, respectively). Consequently, a multiplex real-time quantitative polymerase chain reaction (mrtqPCR) assay that could sensitively and specifically identify and differentiate PCV-2 genotypes was developed. A retrospective epidemiologic survey that used the mrtqPCR assay was performed to determine if cofactors could affect the risk of PCVAD. From 121 PCV-2-positive cases gathered for this study, 4.13%, 92.56%, and 3.31% were positive for PCV-2a, PCV-2b, and both genotypes, respectively. In a data analysis using univariate logistic regressions, the PCVAD-compatible (PCVAD/c) score was significantly associated with the presence of Porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV viral load, PCV-2 viral load, and PCV-2 immunohistochemistry (IHC) results. Polytomous logistic regression analysis revealed that PCVAD/c score was affected by PCV-2 viral load (P = 0.0161) and IHC (P = 0.0128), but not by the PRRSV variables (P > 0.9), which suggests that mrtqPCR in tissue is a reliable alternative to IHC. Logistic regression analyses revealed that PCV-2 increased the odds ratio of isolating 2 major swine pathogens of the respiratory tract, Actinobacillus pleuropneumoniae and Streptococcus suis serotypes 1/2, 1, 2, 3, 4, and 7, which are serotypes commonly associated with clinical diseases.

The emergence of a new strain of porcine circovirus-2 in Ontario and Quebec swine and its association with severe porcine circovirus associated disease--2004-2006.

In the late fall of 2004 more severe lesions of porcine circovirus-2 associated disease (PCVAD) than usual occurred during an outbreak of porcine circovirus-2 (PCV-2) infection in Ontario nursery and grower/finisher pigs. The lesions were of unprecedented severity and included diffuse bronchointerstitial pneumonia, granulomatous enteritis, vasculitis, interstitial nephritis, and new lesions of splenic infarction. Some affected herds had up to 50% mortality. The outbreak correlated with the sudden emergence of a variant PCV-2, with PCR restriction fragment length polymorphism (RFLP) type 321. Phylogenetic comparison of ORF2 sequences and full genome sequences showed the new variant to be different from the previously dominant RFLP type 422 viruses, and similar to viruses that had occurred in France and other European and Asian countries. A subsequent retrospective study showed a statistically significant increase in the frequency of histological lesions in lymph node, spleen, lung, small intestine, colon and kidney, for pigs spontaneously infected with RFLP type 321, compared with the older RFLP type 422 strain. Viral burden, based on IHC staining in lymph node, also showed a statistically significant increase in pigs infected with the newer variant RFLP type 321, compared with the older RFLP type 422 strain. This enhanced virulence in pigs infected with PCV-2 RFLP type 321 strain may be related to the genetic differences in this new strain of PCV-2. This virus is now the dominant strain of PCV-2 virus found in Ontario and Quebec swine.

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada.

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed.

Molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) pathogenicity islands in F165-positive E. coli strain from a diseased animal.

Septicemic Escherichia coli 4787 (O115: K-: H51: F165) of porcine origin possess gene clusters related to extraintestinal E. coli fimbrial adhesins. This strain produces two fimbriae: F165(1) and F165(2). F165(1) (Prs-like) belongs to the P fimbrial family, encoded by foo operon and F165(2) is a F1C-like encoded by fot operon. Data from this study suggest that these two operons are part of two PAIs. PAI I(4787) includes a region of 20 kb, which not only harbors the foo operon but also contains a potential P4 integrase gene and is located within the pheU tRNA gene, at 94 min of the E. coli chromosome. PAI II(4787) includes a region of over 35 kb, which harbors the fot operon, iroBCDEN gene clusters, as well as part of microcin M genes and nonfunctional mobility genes. PAI II(4787) is found between the proA and yagU at 6 min of the E. coli chromosome.

Natural West Nile virus infection in a captive juvenile Arctic wolf (Canis lupus).

A case of West Nile virus (WNV) infection in a captive 4-month-old Arctic wolf (Canis lupus) is described. The animal had vomiting, anorexia, and ataxia before death. Histopathology revealed multifocal severe renal lymphoplasmacytic vasculitis, mostly affecting small arterioles, with fibrinoid degeneration of some vessel walls. Many small foci of gliosis were detected in the cerebral cortex. West Nile virus was demonstrated in the kidneys and cerebrum by immunohistochemistry and polymerase chain reaction. The described renal changes represent a novel pathological finding of WNV infection.

High-level heterologous expression and secretion in Streptomyces lividans of two major antigenic proteins from Mycobacterium tuberculosis.

Two major antigens from Mycobacterium tuberculosis were produced by Streptomyces lividans as secreted extracellular proteins. An expression-secretion vector had been constructed that contained the promoter of xylanase A and the signal sequence of cellulase A. The latter contained two initiation codons preceded by a Shine-Dalgarno sequence plus eight nucleotides complementary to the 16S rRNA. The genes encoding the 38-kDa (Rv0934) and 19-kDa (Rv3763) proteins, respectively, were amplified by polymerase chain reaction and cloned into that vector. The recombinant proteins were then purified from the culture supernatants of the clones. The yields after purification were 80 mg/L for the 38-kDa protein and 200 mg/L for the 19-kDa protein. Sequence analysis of the N-terminal sequences showed a deletion of seven or eight amino acids for the 38-kDa protein, while in the 19-kDa protein 22 or 23 amino acids were lost, as compared with the respective wild-type proteins. However, the 19 kDa recombinant protein had the same N-terminal sequence as the one recovered from the M. tuberculosis culture supernatant. The high yields obtained for these two proteins demonstrated the potential of S. lividans as an alternative host for the production of recombinant proteins from M. tuberculosis. The culture conditions have yet to be worked out to minimize proteolytic degradation and to recover intact products.