Impact of the early-life skin microbiota on the development of canine atopic dermatitis in a high-risk breed birth cohort.

Canine atopic dermatitis (CAD) is a prevalent inflammatory skin disease of dogs worldwide. Certain breeds such as the West Highland White Terriers (WHWT) are predisposed to suffer from CAD. Microbial dysbiosis is known to play a significant role in the pathogenesis of the disease, which is similar to its human counterpart, atopic dermatitis (AD). To date, no large cohort-study has been conducted in a predisposed dog breed to study the impact of the early-life microbiota on the development of CAD, as well as the possible implication of factors such as hygiene and access to the outdoors. In this study skin samples of 143 WHWT, including 109 puppies up to three weeks old and 34 parent dogs, from 17 breeders, were subjected to 16S rRNA gene and ITS2 amplicon sequencing to disclose the bacterial and fungal oral and skin microbiota, respectively. The oral samples served as a control group to confirm differences between haired and mucosal surfaces. The cutaneous microbiota differed between sample sites and age of the dogs. The season of sampling, geographical origin as well as hygiene status of the household and the access to the outdoors shaped the skin microbiota of the puppies significantly. However, we found that the individual early-life microbiota did not predispose for the later development of CAD.

Detection of specific Treponema species and Dichelobacter nodosus from digital dermatitis (Mortellaro's disease) lesions in Swiss cattle.

INTRODUCTION: The aim of this study was to determine the prevalence of the three Treponema species as well as D. nodosus in Digital dermatitis (DD) and slurry of Swiss cattle using PCR. A total of 86 specimens from 24 farms were enrolled in the study. Slurry samples from 21 DD-affected and one unaffected farm were collected to assess the potential of environmental transmission. Nested and real-time PCR were performed from the specimens to detect Treponema species and D. nodosus, respectively. The DD-stages were positive for at least one or more of the DD-associated Treponema species in 50 of 61 cases (82.0%) and in 9 of 25 cases (36.0%) in unaffected animals. Infected animals with small focal active lesions showed a significantly lower prevalence (14.8%) compared to the other DD stages (67.2%; P=0.011). Most prevalent was T. phagedenis (65.1%). D. nodosus was detected in 51.8% of clinical DD lesions and 24.1% in unaffected cases, but its presence was not significantly associated with the various DD-stages. All samples positive for D. nodosus contained the acid protease gene aprB2 but were negative for aprV2, the latter associated with virulence in sheep foot rot. Control farms were negative for all DD-associated Treponema species while positive for aprB2 and negative for aprV2. The presence of aprB2 suggests it is ubiquitous in the animal environment. With respect to the slurry samples, three out of 21 specimens (14.3%) were positive for one or more of the DD-associated Treponema species and eleven out of 21 specimens (52.4%) were positive for aprB2 and negative for aprV2 of D. nodosus. In conclusion, an association was found between the presence of clinical DD and specific Treponema species, while for D. nodosus no such link with DD lesions could be observed.

INTRODUCTION: La Dermatite digitée (DD) chez les bovins est une maladie infectieuse podale multifactorielle, qui est devenue un problème émergent pour le bien-être animal et pour l'économie au niveau mondial. Trois espèces de Treponema, T. pedis, T. medium et T. phagedenis, sont associées avec la DD. Cependant, leur prévalence est inconnue en Suisse. Il a également été rapporté que Dichelobacter nodosus pouvait contribuer au développement de la DD. Le but de cette étude a été de déterminer la prévalence des trois espèces de Treponema ainsi que de D. nodosus dans des lésions de DD et du lisier de vaches suisses, en utilisant des techniques basées sur la PCR. Vingt-deux exploitations avec de la DD clinique et deux exploitations sans signes cliniques de DD ont été inclues dans l'étude. Un total de 86 échantillons de cas de DD ont été prélevés (M1, n=15; M2, n=19; M3, n=9; M4, n=2, M4.1, n=16 and M5, n=25) en utilisant des coton-tiges secs et stériles. De plus, afin d'évaluer le potentiel de transmission par l'environnement, des échantillons de lisier ont été prélevés sur des exploitations atteintes de DD (n=21) et sur une exploitation à stabulation libre exempte de DD. La PCR nichée et la PCR en temps réel ont ensuite été utilisées sur l'ADN extrait des échantillons afin de détecter les espèces de Treponema et D. nodosus, respectivement. Les associations entre la présence d'espèces de Treponema et de D. nodosus avec le statut DD des animaux ont été évaluées avec le test Pearson's Chi-Square. Les stades de DD (M1 à M4.1) et M5 (peau saine) étaient positifs pour au moins une ou plusieurs des espèces de Treponema associées à la DD dans 50 de 61 cas (82.0%) et 9 de 25 cas (36.0%), respectivement. Les lésions M1 ont montré une prévalence nettement inférieure (14.8%) comparé aux autres stades de DD (M2, M3, M4 et M4.1; 67.2%; P=0.011). T. phagedenis était prédominant (65.1%). D. nodosus a été détecté dans 51.8% des lésions cliniques de DD (M1 à M4) et 24.1% des échantillons M5, mais sa présence n'était pas associée significativement avec les divers stades de DD. Tous les échantillons positifs pour D. nodosus contenaient le gène de la protéase acide aprB2, mais étaient négatifs pour aprV2, un gène associé à la virulence dans le piétin des moutons. Les exploitations de contrôle étaient négatives pour toutes les espèces de Treponema associées à la DD, mais positives pour aprB2 et négatives pour aprV2. La présence du gène aprB2 suggère qu'il est ubiquitaire dans l'environnement des animaux et n'est pas une association en soi avec le piétin des moutons. En ce qui concerne les échantillons de lisier, trois des 21 échantillons (14.3%) étaient positifs pour au moins une ou plusieurs espèces de Treponema associées à la DD et onze des 21 échantillons (52.4%) étaient positifs pour aprB2 et négatifs pour aprV2 de D. nodosus. En conclusion, une association a été trouvée entre la présence de DD clinique et des espèces de Treponema spécifiques, alors que pour D. nodosus aucun lien avec des lésions de DD n'a pu être observé. Cette étude démontre la présence des trois espèces de Tréponèmes chez les bovins suisses et facilite la compréhension de l'implication de Treponema spécifiques dans les lésions de DD.

Bacterial, fungal, parasitological and pathological analyses of abortions in small ruminants from 2012-2016.

INTRODUCTION: Abortion in small ruminants presents a clinical and economic problem with legal implications regarding animal health and zoonotic risk by some of the abortive pathogens. Several bacteria, fungi and parasites can cause abortion, but cost-orientated routine diagnostics only cover the most relevant epizootic agents. To cover a broad-range of common as well as underdiagnosed abortifacients, we studied 41 ovine and 36 caprine abortions by Stamp's modification of the Ziehl-Neelsen stain, culture for classical and opportunistic abortive agents, real-time PCR for C. burnetii, C. abortus, pathogenic Leptospira spp., Toxoplasma gondii and Neospora caninum. When the dam's serum was available detection of antibodies against B. melitensis, C. burnetii, C. abortus and Leptospira spp. was performed. In 37 cases sufficient placental tissue was available for pathological and histopathological examination. From the 77 cases 11 (14.3%) were positive by staining whereas real-time PCR detected C. burnetii and C. abortus in 49.3% and 32.5% of the cases. Antibodies against C. abortus and Leptospira spp. (33.3 and 26.7%) were detected. In 23.4% a bacterial culturable pathogen was isolated. Fungal abortion was confirmed in 1.3% of cases. A single abortive agent was identified in 44.2% of the cases and in 31.2% multiple possible abortifacients were present. Our study shows that the highest clarification rate can only be achieved by a combination of methods and evidences the role that multi-infections play as cause of abortion.

INTRODUCTION: Les avortements représentent un problème à la fois clinique et économique avec des conséquences en matière d'épizooties et un risque de zoonose pour certains agents. Diverses bactéries, champignons et parasites peuvent causer des avortements mais le diagnostic de routine, orienté sur les coûts, se concentre sur les principaux agents épizootiques. Afin d'avoir une vision large sur les agents d'avortements les plus fréquents et sur ceux qui sont sous-diagnostiqués, nous avons examinés 41 avortements de moutons et 36 de chèvres au moyen d'une coloration de Ziehl-Neelsen modifiée selon Stamp, de cultures ciblant les agents d'avortements classiques et opportunistes, d'une PCR en temps réel ciblant C. burnetii, C. abortus, les leptospires pathogènes, Toxoplasma gondii et Neospora caninum. Lorsque du sérum de la mère était disponible, nous avons procédé à une recherche d'anticorps contre B. melitensis, C. burnetii, C. abortus et Leptospira spp. Dans 37 cas, on disposait d'assez de tissu placentaire pour des examens pathologiques. Sur les 77 cas, 11 (14.3%) étaient positifs à la coloration alors que la PCR en temps réel démontrait la présence de C. burnetii et de C. abortus dans 49.3% respectivement 32.5% des cas. On a trouvé des anticorps contre C. abortus und Leptospira spp. dans 33.3% respectivement 26.7% des cas. Dans 23.4% des cas, on a pu mettre en évidence des pathogènes bactériens cultivables. Un avortement mycotique a été confirmé dans 1.3% des cas. Dans 44.2% des cas, un seul agent abortif était présent et dans 31.2% des cas, on trouvait plusieurs agents potentiels. Notre étude indique que le plus haut taux de diagnostic ne peut être atteint qu'en combinant diverses méthodes et montre le rôle possible de multi infections dans l'origine des avortements.

Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates.

Yersinia enterocolitica 4/O:3 is the most important human pathogenic bioserotype in Europe and the predominant pathogenic bioserotype in slaughter pigs. Although many studies on the virulence of Y. enterocolitica strains have showed a broad spectrum of detectable factors in pigs and humans, an analysis based on a strict comparative approach and serving to verify the virulence capability of porcine Y. enterocolitica as a source for human yersiniosis is lacking. Therefore, in the present study, strains of biotype (BT) 4 isolated from Swiss slaughter pig tonsils and feces and isolates from human clinical cases were compared in terms of their spectrum of virulence-associated genes (yadA, virF, ail, inv, rovA, ymoA, ystA, ystB and myfA). An analysis of the associated antimicrobial susceptibility pattern completed the characterization. All analyzed BT 4 strains showed a nearly similar pattern, comprising the known fundamental virulence-associated genes yadA, virF, ail, inv, rovA, ymoA, ystA and myfA. Only ystB was not detectable among all analyzed isolates. Importantly, neither the source of the isolates (porcine tonsils and feces, humans) nor the serotype (ST) had any influence on the gene pattern. From these findings, it can be concluded that the presence of the full complement of virulence genes necessary for human infection is common among porcine BT 4 strains. Swiss porcine BT 4 strains not only showed antimicrobial susceptibility to chloramphenicol, cefotaxime, ceftazidime, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, tetracycline and trimethoprim but also showed 100% antibiotic resistance to ampicillin. The human BT 4 strains revealed comparable results. However, in addition to 100% antibiotic resistance to ampicillin, 2 strains were resistant to chloramphenicol and nalidixic acid. Additionally, 1 of these strains was resistant to sulfamethoxazole. The results demonstrated that Y. enterocolitica BT 4 isolates from porcine tonsils, as well as from feces, show the same virulence-associated gene pattern and antibiotic resistance properties as human isolates from clinical cases, consistent with the etiological role of porcine BT 4 in human yersiniosis. Thus, cross-contamination of carcasses and organs at slaughter with porcine Y. enterocolitica BT 4 strains, either from tonsils or feces, must be prevented to reduce human yersiniosis.

Nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) among Swiss veterinary health care providers: detection of livestock- and healthcare-associated clones.

We screened a total of 340 veterinarians (including general practitioners, small animal practitioners, large animal practitioners, veterinarians working in different veterinary services or industry), and 29 veterinary assistants for nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) at the 2012 Swiss veterinary annual meeting. MRSA isolates (n = 14) were detected in 3.8 % (95 % CI 2.1 - 6.3 %) of the participants whereas MRSP was not detected. Large animal practitioners were carriers of livestock-associated MRSA (LA-MRSA) ST398-t011-V (n = 2), ST398-t011-IV (n = 4), and ST398-t034-V (n = 1). On the other hand, participants working with small animals harbored human healthcare-associated MRSA (HCA-MRSA) which belonged to epidemic lineages ST225-t003-II (n = 2), ST225-t014-II (n = 1), ST5-t002-II (n = 2), ST5-t283-IV (n = 1), and ST88-t186-IV (n = 1). HCA-MRSA harbored virulence factors such as enterotoxins, ß-hemolysin converting phage and leukocidins. None of the MRSA isolates carried Panton-Valentine leukocidin (PVL). In addition to the methicillin resistance gene mecA, LA-MRSA ST398 isolates generally contained additional antibiotic resistance genes conferring resistance to tetracycline [tet(M) and tet(K)], trimethoprim [dfrK, dfrG], and the aminoglycosides gentamicin and kanamycin [aac(6')-Ie - aph(2')-Ia]. On the other hand, HCA-MRSA ST5 and ST225 mainly contained genes conferring resistance to the macrolide, lincosamide and streptogramin B antibiotics [erm(A)], to spectinomycin [ant(9)-Ia], amikacin and tobramycin [ant(4')-Ia], and to fluoroquinolones [amino acid substitutions in GrlA (S84L) and GyrA (S80F and S81P)]. MRSA carriage may represent an occupational risk and veterinarians should be aware of possible MRSA colonization and potential for developing infection or for transmitting these strains. Professional exposure to animals should be reported upon hospitalization and before medical intervention to allow for preventive measures. Infection prevention measures are also indicated in veterinary medicine to avoid MRSA transmission between humans and animals, and to limit the spread of MRSA both in the community, and to animal and human hospitals.

[Occurrence of Clostridium perfringens type A and type C in piglets of the Swiss swine population]. / Vorkommen von Clostridium perfringens Typ A und Typ C bei Saugferkeln in der schweizerischen Schweinepopulation.

Necrotizing enteritis (NE) of newborn piglets still represents an economical problem in Swiss pig breeding and production. The aim of our study was to identify risk factors for NE and evaluate the prevalence of C. perfringens with the toxingenes cpb and cpb2 in Swiss pig breeding farms. The prevalence of theses C. perfringens was investigated using fecal swabs followed by bacteriological culturing and genotyping. Close proximity to other breeding farms and large herd sizes were shown to predispose to NE. C. perfringens type C, carrying the genes cpa, cpb and cpb2 were frequently identified in herds with acute outbreaks of NE. Farms not affected by NE or those using prophylactic vaccination against NE were predominantly positive for C. perfringens type A strains with cpb2 and showed much lower prevalence of C. perfringens type C, compared to acutely affected herds. Our results demonstrate that C. perfringens type A strains with cpb2 are not associated with NE. Besides typical necropsy finding, only the identification of cpb can be used for the diagnosis of NE in affected herds.

Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems.

We describe the development of a novel PCR assay for the rapid detection of members of the Brucella genus, and the differentiation between six recognized Brucella species. The assay has proven to be highly specific with the additional advantage of being suitable for use with both conventional and real-time PCR.

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates.

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.

Discovery of insertion element ISCfe1: a new tool for Campylobacter fetus subspecies differentiation.

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis (CFV) and C. fetus subsp. fetus (CFF). CFV is the causative agent of bovine genital campylobacteriosis, a highly contagious venereal disease that may lead to serious reproductive problems, including sterility and abortion. In contrast, CFF can be isolated from the gastrointestinal tract of a wide range of host species, is associated with abortion in sheep and cattle, and can also be isolated from local and systemic infections in humans. Despite differences in host and niche preferences, microbiological differentiation of the two subspecies of C. fetus is extremely difficult. This study describes the identification of a new insertion element, ISCfe1, which is present exclusively in CFV strains, with highly conserved specific ISCfe1 insertion sites. The results are useful for identification and differentiation of the two C. fetus subspecies and will help in understanding the evolution and pathogenesis of C. fetus.

Sequential development of resistance to fluoroquinolones and erythromycin in an isolate of Campylobacter jejuni.

A severe episode of Campylobacter jejuni gastroenteritis in a patient with HIV infection was treated with ciprofloxacin and, because of therapeutic failure, subsequently with roxithromycin. After treatment, C. jejuni was again isolated from feces and shown to be resistant to both drugs. We present molecular evidence of the sequential development of both types of resistance in the patient isolate. To our knowledge, this is the first case with documented evidence showing sequential emergence of resistance to fluoroquinolones and erythromycin in a strain of C. jejuni during treatment.