Overview of spatio-temporal distribution inferred by multi-locus sequence typing of Taylorella equigenitalis isolated worldwide from 1977 to 2018 in equidae.

The accurate identification of Taylorella equigenitalis strains is essential to improve worldwide prevention and control strategies for contagious equine metritis (CEM). This study compared 367 worldwide equine strains using multilocus sequence typing according to the geographical origin, isolation year and equine breed. The strains were divided into 49 sequence types (STs), including 10 described for the first time. Three major and three minor clonal complexes (CCs), and 11 singletons, were identified. The genetic heterogeneity was low (0.13 STs/strain) despite the wide diversity of geographical origins (n = 16), isolation years (1977-2018) and equine breeds (n = 18). It was highest outside Europe and in the 1977-1997 period; current major STs and CCs already existed before 1998. Previous data associated the major CC1 with the first CEM outbreaks in 1977-1978 in the United Kingdom, Australia and the United States, and revealed its circulation in France. Our study confirms its circulation in France over a longer period of time (1992-2018) and its distribution in Spain and Germany but not throughout Europe. In addition to CC1, relationships between non-European and European countries were observed only through ST4, ST17 and ST30. Within Europe, several STs emerged with cross-border circulation, in particular ST16 and ST46 from the major complexes CC2 and CC8. These results constitute a baseline for monitoring the spread of CEM outbreaks. A retrospective analysis of a higher number of strains isolated worldwide between 1977 and the early 2000s would be helpful to obtain an exhaustive picture of the original CEM situation.

An ADAMTS3 missense variant is associated with Norwich Terrier upper airway syndrome.

In flat-faced dog breeds, air resistance caused by skull conformation is believed to be a major determinant of Brachycephalic Obstructive Airway Syndrome (BOAS). The clinical presentation of BOAS is heterogeneous, suggesting determinants independent of skull conformation contribute to airway disease. Norwich Terriers, a mesocephalic breed, are predisposed to Upper Airway Syndrome (UAS), a disease whose pathological features overlap with BOAS. Our health screening clinic examined and scored the airways of 401 Norwich terriers by laryngoscopy. Genome-wide association analyses of UAS-related pathologies revealed a genetic association on canine chromosome 13 (rs9043975, p = 7.79x10-16). Whole genome resequencing was used to identify causal variant(s) within a 414 kb critical interval. This approach highlighted an error in the CanFam3.1 dog assembly, which when resolved, led to the discovery of a c.2786G>A missense variant in exon 20 of the positional candidate gene, ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). In addition to segregating with UAS amongst Norwich Terriers, the ADAMTS3 c.2786G>A risk allele frequency was enriched among the BOAS-susceptible French and (English) Bulldogs. Previous studies indicate that ADAMTS3 loss of function results in lymphoedema. Our results suggest a new paradigm in the understanding of canine upper airway disease aetiology: airway oedema caused by disruption of ADAMTS3 predisposes dogs to respiratory obstruction. These findings will enhance breeding practices and could refine the prognostics of surgical interventions that are often used to treat airway obstruction.

Seroprevalence survey for Salmonella Abortusovis infection in Swiss sheep flocks.

Between 1976 and 2003, no infections with Salmonella Abortusovis had been officially recorded in Switzerland. Since then, however, several sheep flocks were infected and suffered massive fetal losses suggesting a re-emergence of the disease. Therefore, the aim of this study was to assess the epidemiological situation of S. Abortusovis infection in sheep in this country. A representative serum sample collected in 2007 in the context of certifying Brucella freedom included sera from 578 flocks with a total of 8426 sheep from all regions in Switzerland and the Principality of Liechtenstein. Sera were tested by ELISA for the presence of antibodies specific for S. Abortusovis. The cantonal seroprevalence was estimated at the sheep as well as the flock-level by taking into account (a) all flocks with one or more seropositive sheep (Flock 1+) and (b) only the flocks with two or more seropositive sheep (Flock 2+). Flocks with seropositive sheep were found throughout the country with an overall sheep-level prevalence of 1.7%. At the flock-level, overall prevalences of 16.3% and 5.0% were found for Flock 1+ and Flock 2+ definitions, respectively. Significant sheep-level clusters were located in the cantons of Bern, the Valais and Graubünden, while significant flock-level clusters (Flock 1+ and Flock 2+) were located in the canton of Graubünden only. Our results indicate that exposure of Swiss sheep flocks to S. Abortusovis is wide-spread.

Use of an indirect enzyme-linked immunosorbent assay for detection of antibodies in sheep naturally infected with Salmonella Abortusovis.

An indirect enzyme-linked immunosorbent assay (ELISA) was modified and validated to detect antibodies against Salmonella Abortusovis in naturally infected sheep. The ELISA was validated with 44 positive and 45 negative control serum samples. Compared with the immunoblot, the sensitivity and specificity of the assay were 98% and 100%, respectively. To follow antibody levels over time, samples from 12 infected ewes were collected at 1, 3, and 10 months after abortion. All animals showed antibody levels above the cutoff value throughout the observation period. One and 3 months after abortion, high antibody levels could be detected in all but one animal, whereas after 10 months, 9 animals had markedly lower but still positive antibody levels. The test characteristics and evidence for the persistence of detectable antibody levels in all infected animals for up to 10 months indicates that the ELISA can be used for herd surveillance testing.

Diagnosis by culture and PCR of Salmonella abortusovis infection under clinical conditions in aborting sheep in Switzerland.

Since 2003 eleven Swiss sheep flocks were affected by abortion storms due to Salmonella abortusovis, an infection which had not been reported in this country for decades although cases of salmonellosis are notifiable in Switzerland. This raised doubts about the adequacy of the currently used diagnostic tools and the origin of this infection. Therefore, PCR was tested for its potential as a more rapid and more reliable method for diagnosing S. abortusovis infections under field conditions. Fecal and vaginal samples were collected at different times after abortion and PCR was used to detect bacterial DNA. Bacteria were isolated by conventional culture techniques. For determining their origin they were analyzed by pulsed field gel electrophoresis (PFGE) and compared to isolates from Germany and France. Sequencing of randomly selected amplicons allowed confirming the specificity of the result. PCR was more sensitive because it allowed detecting S. abortusovis DNA up to three months after infection even in samples that were negative by culture. Escherichia coli from the digestive tract of sheep could inhibit the growth of S. abortusovis in vitro suggesting that the lower sensitivity of diagnosis by bacterial culture may in part be due to growth inhibition of S. abortusovis by resident bacteria. Results of PFGE indicated that the Swiss strains were closely related among themselves but distinct from German and French strains suggesting the presence of an autochthonous infection.

Is interleukin-4delta3 splice variant expression in bovine tuberculosis a marker of protective immunity?

Splice variants of the interleukin-4 (IL-4) cytokine gene have been described for humans, mice, and cattle. IL-4 splice variants have been shown to inhibit IL-4-mediated cellular responses and thus act as IL-4 antagonists. Recent work has highlighted the possibility of a correlation between IL-4 splice variants and protection against clinical tuberculosis. In this study we investigated the potential role of IL-4 splice variants IL-4delta2 and IL-4delta3 in cattle with bovine tuberculosis, using quantitative real-time reverse transcription-PCR. For this analysis we used naturally exposed tuberculin skin test-positive field reactor cattle, uninfected control cattle, and cattle from two experimental models of protective immunity against Mycobacterium bovis: (i) vaccination with M. bovis BCG and challenge with virulent M. bovis and (ii) infection with M. bovis and treatment with isoniazid (INH) prior to rechallenge. The cytokine levels of field reactor cattle were compared to the levels of uninfected controls, while in kinetic studies of BCG vaccination and INH treatment we compared pre-experimental values with sequential samples for each individual animal. The data revealed a significant increase in IL-4delta3 mRNA expression in field reactor cattle, which had no visible pathology compared to cattle with gross pathology typical of bovine tuberculosis. Increased IL-4delta3 expression in both cattle models of protective immunity (BCG vaccination and INH treatment) was transient over time, reaching significance in the INH treatment model. Our results support the hypothesis that IL-4delta3 is involved in protective immunity against M. bovis infection in cattle and are in accordance with clinical studies that have suggested a role for IL-4 splice variants in protective immunity in tuberculosis.

Regulation of bovine IL-12R beta 2 subunit mRNA expression in bovine lymph node cells.

The beta 2 subunit of the interleukin (IL)-12 receptor (IL-12R beta 2) has been shown to play an essential role in differentiation of T helper 1 (Th1) cells in the murine and human system, and antibodies raised against IL-12R beta 2 recognized this molecule on human Th1 but not Th2 cells. However, while the cytokines secreted by clones of murine cells allowed the definition of distinct T helper cell subsets, bovine clones with polarized Th1 and Th2 cytokine profiles were rarely found. This raised important questions about the regulation of immune responses in cattle. We therefore cloned bovine IL-12R beta2 (boIL-12R beta 2) DNA complementary to RNA (cDNA) from the start codon to the 3' end of the mRNA. Comparison of boIL-12R beta 2 cDNA with human and murine IL-12R beta 2 cDNA sequences revealed homologies of 85 and 78%, respectively. The deduced protein sequence showed the hallmark motifs of the cytokine receptor superfamily including the four conserved cysteine residues, the WSXWS motif and fibronectin domains in the extracellular part as well as a STAT4 binding site in the intracellular part of the molecule. Using real-time reverse transcription-polymerase chain reaction, upregulation of mRNA expression of this molecule could be demonstrated in cultured bovine lymph node cells stimulated with phytohemagglutinin. Furthermore, cells with upregulated boIL-12R beta 2 mRNA responded with enhanced expression of interferon gamma to treatment with interleukin 12.