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.

[Abortions and stillbirths caused by Coxiella burnetii in goats]. / Aborte und Totgeburten bei Ziegen unter besonderer Berücksichtigung von Coxiella burnetii.

INTRODUCTION: Coxiellosis, caused by the bacterium Coxiella burnetii, is a reportable disease in animals and humans in Switzerland. The number of cases in farm animals and humans has risen continuously in recent years. The aim of this work was to investigate abortions and stillbirths in goats with a focus on C. burnetii, to identify excretory routes which pose a zoonotic risk and the excretion time after an acute infection. Besides the submitted fetuses, does were screened with a serological antibody test. In addition, excretion via milk, faeces and vaginal mucus were investigated in dams with fetuses tested positive for C. burnetii at 14-day intervals.C. burnetii were isolated in 8 cases (3× in the placenta, 2× in the abomasum, 3× in the placenta and abomasum) of 13 examined stillbirths/abortions. Ten abomasums of goat kids and 8 placentas were examined using modified Ziehl-Neelsen staining (ZN) according to Stamp simultaneously with a real-time PCR. Four of 18 samples were false negative using modified ZN staining according to Stamp in contrast to real-time PCR. Seven does had serum antibodies against Coxiella. The excretion of C. burnetii persisted for 63 days in the milk, for 96 days in the vaginal mucus and for 96 respectively 114 days in two does monitored extensively. Intermittent excretion could also be observed in the milk during these 63 days. The present study showed that confirmation of disease, respectively transmission cannot be based on a single test. Only combined serological antibody test and real-time PCR examinations of birth material, milk, feces and vaginal mucus can result in a conclusive diagnosis. In addition, the examination using modified ZN staining according to Stamp is less sensitive and specific than the real-time PCR examination.

INTRODUCTION: La coxiellose, causée par la bactérie Coxiella burnetii, est une maladie à déclaration obligatoire en Suisse qui touche les animaux et les humains. Le nombre de cas chez les animaux de rente et les humains n'a cessé d'augmenter ces dernières années. Le but de ce travail était d'étudier les avortements et la mortalité périnatale chez les chèvres avec un focus sur C. burnetii, d'en identifier les voies d'excrétion qui présentent un risque zoonotique et de déterminer le temps d'excrétion après une infection aiguë. Pour ce faire, des examens sérologiques d'anticorps ont été effectués sur les mères en parallèle des examens sur les fœtus envoyés. L'excrétion par le lait, les selles et les sécrétions vaginales ont été examinées à intervalles de 14 jours sur les mères dont les fœtus ont été testés positifs à C. burnetii. Sur les 13 mort-nés et avortements examinés, C. burnetii a été isolés dans 8 échantillons (3× dans le placenta, 2× dans la caillette, 3× dans le placenta et la caillette). Dix caillettes de chevr­eaux et 8 placentas ont été simultanément examinés en utilisant une coloration Ziehl-Neelsen (ZN), modifiée selon Stamp, et un real-time PCR. Sur les 18 échantillons examinés, 4 échantillons ont donné des faux négatifs en utilisant la coloration Ziehl-Neelsen modifiée par rapport à la real-time PCR. La sérologie a dévoilé que 7 femelles présentaient des anticorps contre Coxiella. Pour 2 femelles, suivies durant une période plus longue, l'excrétion de C. burnetii dans le lait a persisté durant 63 jours, dans les sécrétions vaginales durant 96 jours pour les 2 femelles et dans les selles durant 96 et 114 jours respectivement. Une excrétion intermittente par le lait a également pu être observée durant les 63 jours. Cette étude a démontré que la mise en évidence de la maladie respectivement de l'excrétion ne peut pas être assurée sur la base d'un seul test. Seul la combinaison de la sérologie et des examens au moyen de la real-time PCR sur les arrière-faix, le lait, les selles et les sécrétions vaginales peuvent aboutir à un diagnostic concluant. De plus, l'examen au moyen de la coloration ZN modifiée selon Stamp est moins sensible et moins spécifique que la real-time PCR.

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.

Diagnostic value of two commercial chromatographic "patient-side" tests in the diagnosis of acute canine leptospirosis.

OBJECTIVES: To determine the diagnostic performance of two patient-side tests (RDT-1: Test-it™ and RDT-2 Witness®Lepto) in the early diagnosis of canine leptospirosis. METHODS: Retrospective study of 108 dogs with leptospirosis and 53 controls. Leptospirosis was diagnosed based on compatible clinical and clinicopathologic signs and either a single microscopic agglutination test titre_ >800 (n=49), seroconversion (n=53), positive urine real time PCR (RT-PCR) (n=1), evidence of spirochaetes in silver-stained tissues (n=1) or a combination of these (n=4). Leptospirosis was excluded in dogs with a convincing alternative diagnosis and single microscopic agglutination testing titres _<200 (n=46) or lack of seroconversion (n=7). Indices of diagnostic accuracy of the rapid diagnostic tests were calculated by comparing admission rapid diagnostic test results to the final disease status. RESULTS: Rapid diagnostic test-1 was performed in 118 dogs, rapid diagnostic test-2 in 69 dogs and both tests in 26 dogs. Weak positive results occurred frequently representing 22·6% (rapid diagnostic test-1) and 32·3% (rapid diagnostic test-2) of all positive tests in dogs with leptospirosis. If weak positive rapid diagnostic tests were considered positive, rapid diagnostic test-1 and rapid diagnostic test-2 had sensitivities of 82 and 76%, specificities of 91 and 100%, positive predictive values of 94% and 100% and negative predictive values of 73% and 74%, respectively. There were some technical problems with rapid diagnostic test-1. CLINICAL SIGNIFICANCE: The diagnostic performance of the rapid diagnostic tests is similar to that reported for the microscopic agglutination test. Both can support a diagnosis of leptospirosis with high specificity but leptospirosis cannot be excluded based on a negative admission test result. Both RDTs are useful in conjunction with other confirmatory tests.

Bacteriological diagnosis and molecular strain typing of Mycobacterium bovis and Mycobacterium caprae.

The primary isolation of a Mycobacterium sp. of the Mycobacterium tuberculosis complex from an infected animal provides a definitive diagnosis of tuberculosis. However, as Mycobacterium bovis and Mycobacterium caprae are difficult to isolate, particularly for animals in the early stages of disease, success is dependent on the optimal performance of all aspects of the bacteriological process, from the initial choice of tissue samples at post-mortem examination or clinical samples, to the type of media and conditions used to cultivate the microorganism. Each step has its own performance characteristics, which can contribute to sensitivity and specificity of the procedure, and may need to be optimized in order to achieve the gold standard diagnosis. Having isolated the slow-growing mycobacteria, species identification and fine resolution strain typing are keys to understanding the epidemiology of the disease and to devise strategies to limit transmission of infection. New technologies have emerged that can now even discriminate different isolates from the same animal. In this review we highlight the key factors that contribute to the accuracy of bacteriological diagnosis of M. bovis and M. caprae, and describe the development of advanced genotyping techniques that are increasingly used in diagnostic laboratories for the purpose of supporting detailed epidemiological investigations.

Evaluation of two cocktails containing ESAT-6, CFP-10 and Rv-3615c in the intradermal test and the interferon-γ assay for diagnosis of bovine tuberculosis.

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n=23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p>0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p>0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis.