Stability of canine urine samples under different storage conditions.

The stability of canine urine samples is essential when the samples cannot be analyzed immediately. The objective of this study was to investigate the stability of canine urine samples at room temperature and under refrigerated conditions. Samples from 20 dogs were collected, divided, and stored at 4°C and 20°C. The samples were examined up to 48 h after collection for specific gravity, pH, protein, bilirubin, glucose, ketones, and sediment and at 4 h and 24 h for bacterial growth. Specific gravity and all chemistry parameters were stable for a minimum of 48 h in 90% of samples. The sediment was stable, apart from crystals. The bacterial growth of 3 bacterial species tested in vitro, as well as the clinical samples, was mostly constant over 24 h at the refrigerated temperature. In urine samples stored at room temperature, the total number of aerobic growing bacteria was increasing. The results of our study showed that routinely measured parameters were stable in unpreserved urine for a minimum of 4 h and up to 48 h in most cases. If it is not possible to culture urine immediately, it is recommended that urine samples be stored at 4°C for a period of up to 24 h.

La stabilité des échantillons d'urine canins est essentielle lorsque les échantillons ne peuvent être analysés immédiatement. L'objectif de la présente étude était d'examiner la stabilité d'échantillons d'urine canins à température ambiante et réfrigérés. Des échantillons provenant de 20 chiens furent prélevés, divisés et entreposés à 4 °C et 20 °C. Les échantillons furent examinés jusqu'à 48 h après le prélèvement pour la gravité spécifique, le pH, les protéines, la bilirubine, le glucose, les cétones et le sédiment, et à 4 h et 24 h pour la croissance bactérienne. La gravité spécifique et tous les paramètres chimiques étaient stables pour un minimum de 48 h dans 90 % des échantillons. Le sédiment était stable, sauf pour les cristaux. La croissance bactérienne de trois espèces bactériennes testée in vitro, ainsi que dans les échantillons cliniques, était généralement constante sur une période de 24 h à la température de réfrigération. Dans les échantillons d'urine entreposés à la température ambiante, le nombre total de bactérie aérobie augmentait. Les résultats de notre étude démontrent que les paramètres mesurés de routine sont stables dans de l'urine sans agent de préservation pour un minimum de 4 h et jusqu'à 48 h dans la majorité des cas. S'il n'est pas possible de mettre l'urine en culture immédiatement, il est recommandé que les échantillons d'urine soient entreposés à 4 °C pour une période allant jusqu'à 24 h.(Traduit par Docteur Serge Messier).

Horses as a Crucial Part of One Health.

One Health (OH) is a crucial concept, where the interference between humans, animals and the environment matters. This review article focusses on the role of horses in maintaining the health of humans and the environment. Horses' impact on environmental health includes their influence on soil and the biodiversity of animal and plant species. Nevertheless, the effect of horses is not usually linear and several factors like plant-animal coevolutionary history, climate and animal density play significant roles. The long history of the relationship between horses and humans is shaped by the service of horses in wars or even in mines. Moreover, horses were essential in developing the first antidote to cure diphtheria. Nowadays, horses do have an influential role in animal assisted therapy, in supporting livelihoods in low income countries and as a leisure partner. Horses are of relevance in the spillover of zoonotic and emerging diseases from wildlife to human (e.g., Hendra Virus), and in non-communicable diseases (e.g., post-traumatic osteoarthritis in horses and back pain in horse riders). Furthermore, many risk factors-such as climate change and antimicrobial resistance-threaten the health of both horses and humans. Finally, the horse is a valuable factor in sustaining the health of humans and the environment, and must be incorporated in any roadmap to achieve OH.

Efficacy of dairy on-farm high-temperature, short-time pasteurization of milk on the viability of Mycobacterium avium ssp. paratuberculosis.

Feeding pasteurized milk to suckling calves is a popular practice used increasingly on dairy farms. Waste milk is frequently fed to calves because of its high nutritional value and economic benefits compared to milk replacement products. However, one of the disadvantages of feeding waste milk is the potential for exposure to a high number of bacterial contaminants, which may lead to serious illnesses or infections in calves. One of these contaminants is Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of Johne's disease (paratuberculosis). The transmission and distribution of paratuberculosis in dairy herds occurs mostly through the feeding newborn calves with contaminated colostrum or milk, because this age group is believed to be most susceptible to infection. To reduce the risk of transmission of pathogens, on-farm pasteurization of milk has become increasingly popular. In this study, we analyzed the efficacy of a new commercial high-temperature, short-time pasteurizer (73.5°C for 20 to 25 s) in terms of MAP inactivation under experimental on-farm conditions. The pasteurizer uses a newly developed steam-heating technique, allowing for the pasteurization of the transition milk without clumping. In 3 independent trials, we spiked fresh raw milk samples to a level of 107 or 104 viable MAP cells/mL before pasteurization. We examined the thermal inactivation and viability of MAP using culture and a D29 bacteriophage-based assay. To verify the identity and number of MAP cells, we also performed PCR assays. Pasteurization of the inoculated milk (107 and 104 MAP cells/mL) resulted in a remarkable reduction in viable MAP cells. The mean inactivation rate of MAP ranged from 0.82 to 2.65 log10 plaque-forming units/mL, depending on the initial MAP amount inoculated and the addition of conservative agents to the pasteurized milk. Nevertheless, approximately 103 MAP cells/mL remained viable and could be transferred to calves after high-temperature, short-time pasteurization of milk.

Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples.

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from "sample in" to "result out" was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases.

Detection of Mycobacterium avium subsp. paratuberculosis in non-human primates.

BACKGROUND: Due to a sporadic occurrence of Mycobacterium avium subsp. paratuberculosis (MAP) in non-human primates (NHP), the susceptibility of different NHP to MAP should be investigated. METHODS: Fecal and tissue samples (ileum, ileocecal lymph node, bone marrow) of 20 animals (seven species) were analyzed by IS900-based PCRs and sequenced. Samples of MAP PCR positive NHP were further cultivated. RESULTS: MAP DNA was detectable in two animals; the ileum of a cottontop tamarin and the bone marrow of a common marmoset. Cultivation of MAP failed. Sequence analysis revealed 100% homology to the MAP-K10 sequence. Pathohistological examinations offered no direct correlation to a MAP infection. CONCLUSIONS: MAP was detected for the first time in a common marmoset. But as both NHP suffered from other diseases, an asymptomatic infection with MAP was assumed. The detection of MAP in the bone marrow might play a role in establishing latent paratuberculosis, as known from tuberculosis.

DETECTION OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN ROCK HYRAXES ( PROCAVIA CAPENSIS) IMPORTED FROM SOUTH AFRICA.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic, progressive, and consecutively fatal enteritis, especially in ruminants. MAP distribution among wildlife is not yet clear. In this study, three wild-born rock hyraxes ( Procavia capensis) had been imported from South Africa to a German zoological garden. During the quarantine period, four young animals were born. The wild-born animals showed symptoms of mild diarrhea shortly after their arrival in the zoological garden, but all routine parasitological and bacteriologic tests performed were negative. Therefore, the animals were additionally tested for MAP infection. MAP DNA was detected by seminested PCR (snPCR) in a pooled fecal sample of the seven animals. Subsequent PCR analysis of the individual feces samples confirmed the excretion of MAP in two rock hyraxes (one wild-born and one born in captivity). Sequence analysis of the corresponding 278-bp amplicons revealed 100% homology to the reference MAP-K10 IS900 sequence. No antibody response against MAP was detected in the individual serum samples. MAP-specific postmortem lesions were not observed by gross pathology and histology, neither after death nor after euthanization of the animals. Nevertheless, MAP was detected by snPCR and culture in the gastrointestinal tract, urogenital tract, cardiovascular system, and/or respiratory system of three other animals of the group (one wild-born and two born in captivity). This study is the first report confirming MAP occurrence in rock hyraxes. Therefore, it is recommended that veterinarians and zoo employees consider rock hyraxes as a possible source of MAP infection for domestic livestock in South Africa and the valuable animal stock of zoological facilities.

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: The detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%. CONCLUSIONS/SIGNIFICANCE: The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.

Distribution of Mycobacterium avium ssp. paratuberculosis in a German zoological garden determined by IS900 semi-nested and quantitative real-time PCR.

Little data concerning the distribution of Mycobacterium avium ssp. paratuberculosis (MAP) in zoological gardens is available. The presence of MAP in captured wildlife might provide further information on non-ruminant hosts and expand the list of animals susceptible to MAP being potential sources of MAP transmission. Therefore, a German zoological garden with recent history of clinical paratuberculosis in Barbary sheep (Ammotragus lervia) and an alpaca (Lama pacos) was selected to estimate the distribution of MAP infections in 21 mammalian and avian species. Pooled faecal samples from individual animals of each species were tested for the presence of MAP. A previously developed IS900 semi-nested PCR (snPCR) assay, amplifying a 587 bp and a 278 bp fragment, was used for the detection of MAP-DNA. Based on this snPCR, in 14 out of the 21 pooled faecal samples MAP-DNA was detected. MAP positive snPCR results were observed in ruminants and camelids as well as in non-ruminants such as equines, primates, rodents, and birds. Moreover, a quantitative real-time PCR demonstrated that the concentration of MAP-DNA was within the range of 2.2 × 10(3)-9.6 × 10(6) MAP-DNA equivalents per gram faeces. The highest amount was shed by primates such as Black-and-white ruffed lemurs (Varecia variegata) and Cottontop tamarins (Saguinus oedipus). This is the first survey investigating the presence of MAP in a German zoo, which includes non-ruminants. The results of the present study confirm the wide host range of MAP and demonstrate that MAP occurs more frequently in zoo animals than expected. In order to restrict further spread of MAP in European zoos, additional investigations regarding the existing transmission pathways of MAP in zoos are recommended.