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.

Laboratory diagnosis of ruminant abortion in Europe.

Abortion in ruminants is a major cause of economic loss worldwide, and the management and control of outbreaks is important in limiting their spread, and in preventing zoonotic infections. Given that rapid and accurate laboratory diagnosis is central to controlling abortion outbreaks, the submission of tissue samples to laboratories offering the most appropriate tests is essential. Direct antigen and/or DNA detection methods are the currently preferred methods of reaching an aetiological diagnosis, and ideally these results are confirmed by the demonstration of corresponding macroscopic and/or histopathological lesions in the fetus and/or the placenta. However, the costs of laboratory examinations may be considerable and, even under optimal conditions, the percentage of aetiological diagnoses reached can be relatively low. This review focuses on the most commonly occurring and important abortifacient pathogens of ruminant species in Europe highlighting their epizootic and zoonotic potential. The performance characteristics of the various diagnostic methods used, including their specific advantages and limitations, are discussed.

Staphylococcus aureus host range and human-bovine host shift.

Staphylococcus aureus is a major agent of bovine mastitis. The concomitant emergence of pig-associated methicillin-resistant S. aureus (MRSA) in human carriage and infection requires a reexamination of the host range and specificity of human- and cow-associated S. aureus strains, something which has not been systematically studied previously. The genetic relatedness of 500 S. aureus isolates from bovine mastitis cases, 57 isolates from nasal carriage of farmers, and 133 isolates from nonfarmers was determined by amplified fragment length polymorphism (AFLP) analysis and spa typing. Multilocus sequence typing (MLST) was conducted on a subset of isolates to match AFLP clusters with MLST clonal complexes (CCs). This data set allowed us to study host range and host specificity and to estimate the extent of bovine-to-human transmission. The genotype compositions of S. aureus isolates from farmers and nonfarmers were very similar, while the mastitis isolates were quite distinct. Overall, transmission was low, but specific genotypes did show increased cow-to-human transmission. Unexpectedly, more than one-third of mastitis isolates belonged to CC8, a lineage which has not been considered to be bovine mastitis associated, but it is well known from human carriage and infection (i.e., USA300). Despite the fact that we did detect some transmission of other genotypes from cows to farmers, no transmission of CC8 isolates to farmers was detected, except for one tentative case. This was despite the close genetic relatedness of mastitis CC8 strains to nonfarmer carriage strains. These results suggest that the emergence of the new bovine-adapted genotype was due to a recent host shift from humans to cows concurrent with a loss of the ability to colonize humans. More broadly, our results indicate that host specificity is a lineage-specific trait that can rapidly evolve.

Link between genotype and antimicrobial resistance in bovine mastitis-related Staphylococcus aureus strains, determined by comparing Swiss and French isolates from the Rhône Valley.

Staphylococcus aureus is a major bovine mastitis pathogen. Although the reported antimicrobial resistance was generally low, the emergence of new genetic clusters in bovine mastitis requires examination of the link between antimicrobial resistance and genotypes. Here, amplified fragment length polymorphism (AFLP) profiles and standard antimicrobial resistance profiles were determined in order to characterize a total of 343 S. aureus cow mastitis isolates from two geographically close regions of Switzerland and France. AFLP profiles revealed similar population compositions in the two regions, with 4 major clusters (C8, C20, C97, and C151), but the proportions of isolates in each cluster significantly diverged between the two countries (P = 9.2 × 10⁻9). Antimicrobial resistance was overall low (< 5% resistance to all therapeutically relevant molecules), with the exception of penicillin resistance, which was detected in 26% of the isolates. Penicillin resistance proportions differed between clusters, with only 1 to 2% of resistance associated with C20 and C151 and up to 70% associated with bovine C97. The prevalence of C20 and C8 was unexpectedly high and requires further investigation into the mechanism of adaptation to the bovine host. The strong association of penicillin resistance with few clusters highlights the fact that the knowledge of local epidemiology is essential for rational choices of antimicrobial treatment in the absence of susceptibility testing. Taken together, these observations argue in favor of more routine scrutiny of antimicrobial resistance and antibiotic-resistant clones in cattle and the farm environment.

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.

Detection of Dichelobacter nodosus in wild ungulates (Capra ibex ibex and Ovis aries musimon) and domestic sheep suffering from foot rot using a two-step polymerase chain reaction.

Severe keratinous hoof afflictions have been recorded in ibex (Capra ibex ibex) since 1995 and more recently in mouflon (Ovis aries musimon) in Switzerland. Based on clinical observations and comparison with diseases known to affect domestic ungulates, it was hypothesized these wild ungulates were affected by foot rot associated with infection with Dichelobacter nodosus. Dichelobacter nodosus has been shown to be the essential pathogen for initiation and establishment of foot rot, a highly contagious foot disease of sheep and goats. Because these bacteria could not be cultivated from affected ibex, we developed a nested polymerase chain reaction that allowed detection of D. nodosus without culture. Using this assay, we were able to diagnose D. nodosus infections of ibex, mouflon, and domestic sheep in natural outbreaks. From these results we conclude that D. nodosus plays an etiological role in foot rot not only in domestic but also in wild Caprinae.

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.

Comparison of itraconazole and fluconazole treatments in a murine model of coccidioidal meningitis.

Coccidioidal meningitis (CM) is a devastating disease that requires long-term therapy and for which there is little hope of a cure. A model was used to compare the efficacies of itraconazole and fluconazole. CD-1 mice were infected intrathecally with 30 to 36 viable arthroconidia of Coccidioides. Oral therapy with cyclodextrin (control) or itraconazole or fluconazole at 10, 25, or 50 mg/kg of body weight twice daily (BID) was given for 12 days, from day 3 of infection. Treatment with both antifungals at all doses prolonged survival compared with that of the control treatment (P < 0.01 to 0.0001). At 50 mg/kg, itraconazole and fluconazole were equivalent, whereas itraconazole at 10 or 25 mg/kg prolonged survival compared to that achieved with fluconazole at these dosages (P < 0.05 and 0.01, respectively). Early histologic analysis (10 days of treatment) with 50 mg/kg BID itraconazole or fluconazole showed suppression of CM in all five animals per group; in quantitative cultures, three of three animals from each group had no detectable infection in the brain, spinal cord, or a site of secondary infection, the lungs. In contrast, four of seven controls showed mild to severe meningitis, with arteritis detected in three animals. In a short-term organ clearance study, 5 days of treatment with 10 or 50 mg/kg BID itraconazole or fluconazole reduced the tissue burdens in the brain and spinal cord compared to the tissue burdens in the controls (P < 0.02 to 0.0003). Fluconazole at 10 mg/kg did not reduce the fungal burden in secondary sites, the lungs and kidneys, whereas this itraconazole dose was more effective in clearing the fungi from both organs (P < 0.05 and P < 0.001, respectively). At 50 mg/kg, itraconazole and fluconazole were equivalent in clearing the fungi from the brain and kidney, but itraconazole was superior to fluconazole in clearing the fungi from the spinal cord and lungs (P < 0.05). Thus, both itraconazole and fluconazole were effective at controlling CM, but neither eliminated Coccidioides from tissues. Overall, itraconazole was more efficacious on an mg/kg basis; at high doses they were similarly effective.

Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.

The role of B- and T-cell immunity in toltrazuril-treated C57BL/6 WT, microMT and nude mice experimentally infected with Neospora caninum.

Neospora caninum is a protozoan parasite predominantly known for causing abortion in cattle and neuromuscular disease in dogs. So far, no efficient metaphylactic chemotherapy has been developed. In preliminary studies, toltrazuril had been successfully used against experimental neosporosis in mice and calves. In the present study, we used immunocompetent and immunodeficient mouse strains to address the role of immunity in supporting the chemotherapy of experimental N. caninum infection. WT, microMT and athymic nude mice were intraperitoneally inoculated with 1x10(6) Nc-1 tachyzoites. The drug was administered in the drinking water for 6 consecutive days so as to obtain a daily dose of approximately 20 mg toltrazuril/kg body weight. The course of infection was monitored by clinical, histological and immunohistochemical means, as well as by the search for parasite DNA using PCR-analyses of various organs. In immunocompetent WT mice, treatment proved to be of high efficacy by abrogation of any lesion formation or PCR-positivity in medicated C57BL/6 mice and a significant reduction of lesion formation or PCR-positivity in BALB/c animals. Similarly, treated microMT mice exhibited a significant reduction in cerebral lesion formation as well as in parasite DNA detectability by PCR when compared to untreated animals. Athymic nude mice, however, did not respond to treatment in that only a delay of the parasite dissemination was achieved, and nude mice still showed the neosporosis disease symptoms, although later than untreated animals. We conclude that treatment with toltrazuril appears to act parasitostatically rather than parasitocidically. This is supported by the fact that: (1) although the lack of B-cells did not impair the effect of toltrazuril, (2) the lack of T-cells did not allow for a full efficacy of treatment. Therefore, chemotherapy with toltrazuril against experimental infections with N. caninum requires the support of T-cell immunity in order to be successful.

Echinococcus multilocularis proliferation in mice and respective parasite 14-3-3 gene expression is mainly controlled by an alphabeta CD4 T-cell-mediated immune response.

The role of specific B lymphocytes and T-cell populations in the control of experimental Echinococus multilocularis infection was studied in micro MT, nude, T-cell receptor (TCR)-beta(-/-), major histocompatibility complex (MHC)-I(-/-) and MHC-II(-/-) mice. At 2 months postinfection, the parasite mass was more than 10 times higher in nude, TCR-beta(-/-) and MHC-II(-/-) mice than in infected C57BL/6 wild-type (WT) mice, and these T-cell-deficient mice started to die of the high parasite load at this time-point. In contrast, MHC-I(-/-) and micro MT mice exhibited parasite growth rates similar to those found in WT controls. These findings clearly point to the major role that CD4(+) alphabeta(+) T cells play in limiting the E. multilocularis proliferation, while CD8(+) T and B cells appeared to play a minor role in the control of parasite growth. In the absence of T cells, especially CD4(+) or alphabeta(+) T cells, the cellular immune response to infection was impaired, as documented by the lack of hepatic granuloma formation around the parasite and by a decreased splenocyte responsiveness to concanavalin A (Con A) and parasite antigen stimulation. Surprisingly, in T-cell-deficient mice, the ex vivo expression of interferon-gamma (IFN-gamma) and other inflammatory cytokines (except for interleukin-6) were increased in association with a high parasite load. Thus, the relative protection mediated by CD4(+) alphabeta(+) T cells against E. multilocularis infection seemed not be IFN-gamma dependent, but rather to rely on the effector's function of CD4(+) alphabeta(+) T cells. The local restriction of parasite germinal cell proliferation was reflected by a regulatory effect on the expression of 14-3-3 protein within the parasite tissue in T-cell-deficient mice. These results provide a strong indication that the CD4(+) alphabeta(+) T-cell-mediated immune response contributes to the control of the parasite growth and to the regulation of production of the parasite 14-3-3 protein in metacestode tissues.

Inducible nitric oxide synthase deficiency in mice increases resistance to chronic infection with Echinococcus multilocularis.

The production of nitric oxide (NO) by intraperitoneal macrophages of mice during secondary infection with Echinococcus multilocularis mediates immunosuppression at early and late stages of infection. We addressed the role of NO in host resistance against this extracellular metazoan parasite by infecting inducible nitric oxide synthase knockout ((iNOS-KO) mice (of the C57BL/6 background) with 100 metacestode vesicles. The parasite weight was significantly lower in iNOS-KO mice when compared with wild-type (WT) mice at 4 months postinfection (late stage), thus demonstrating that iNOS deficiency confers a certain degree of resistance against persistent chronic infection. However, histological analysis of periparasitic tissue showed no differences between WT and iNOS-KO mice, as both exhibited granuloma formation and the presence of giant cells. Together with histology, the production of a high level of interferon-gamma (IFN-gamma) in infected iNOS-KO mice upon stimulation with concanavalin A (Con A) and VF-antigen indicated normal T-cell signalling in these animals. As expected, peritoneal exudate cells (PEC) from infected iNOS-KO mice produced no detectable NO, while the PEC from infected WT mice produced high levels of NO after stimulation with lipopolysaccharide (LPS) and parasite protein or carbohydrate antigen, or even without in vitro stimulation. Consequently, the high level of NO production observed during chronic infection in WT mice appears to contribute more to immunosuppression than to limitation of parasite growth. This is also reflected by the fact that splenocyte proliferation was significantly higher and parasite masses lower in iNOS-KO mice (at 1 and 4 months postinfection) than in WT mice.

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.