Defining a metagenomic threshold for detecting low abundances of Providencia alcalifaciens in canine faecal samples.

Introduction: Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. Providencia alcalifaciens is suspected to play a role in the disease as it was commonly found in dogs suffering from AHDS during a Norwegian outbreak in 2019. The role of this bacterium as a constituent of the canine gut microbiota is unknown, hence this study set out to investigate its occurrence in healthy dogs using metagenomics. Materials and methods: To decrease the likelihood of false detection, we established a metagenomic threshold for P. alcalifaciens by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for P. alcalifaciens was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for P. alcalifaciens. Finally, the metagenomic threshold was used to determine the occurrence of P. alcalifaciens in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project. Results: The metagenomic assay and qPCR had a detection limit of 1.1x103 CFU P. alcalifaciens per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique k-mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, P. alcalifaciens was found in only 1.1% (95% CI [0.0, 6.8]) of the samples, and then in low relative abundances (median: 0.04%; range: 0.00 to 0.81%). The sensitivity of the qPCR and shotgun metagenomics assay was low, as only 40% of culture-positive samples were also positive by qPCR and metagenomics. Discussion: Using our detection limit, the occurrence of P. alcalifaciens in faecal samples from healthy dogs was low. Given the low sensitivity of the metagenomic assay, these results do not rule out a significantly higher occurrence of this bacterium at a lower abundance.

An Official Outbreak Investigation of Acute Haemorrhagic Diarrhoea in Dogs in Norway Points to Providencia alcalifaciens as a Likely Cause.

An outbreak investigation was initiated in September 2019, following a notification to the Norwegian Food Safety Authority (NFSA) of an unusually high number of dogs with acute haemorrhagic diarrhoea (AHD) in Oslo. Diagnostic testing by reporting veterinarians had not detected a cause. The official investigation sought to identify a possible common cause, the extent of the outbreak and prevent spread. Epidemiological data were collected through a survey to veterinarians and interviews with dog owners. Diagnostic investigations included necropsies and microbiological, parasitological and toxicological analysis of faecal samples and food. In total, 511 dogs with acute haemorrhagic diarrhoea were registered between 1 August and 1 October. Results indicated a common point source for affected dogs, but were inconclusive with regard to common exposures. A notable finding was that 134 of 325 faecal samples (41%) cultured positive for Providencia alcalifaciens. Whole genome sequencing (WGS) of 75 P. alcalifaciens isolates from 73 dogs revealed that strains from 51 dogs belonged to the same WGS clone. Findings point to P. alcalifaciens as implicated in the outbreak, but investigations are needed to reveal the pathogenic potential of P. alcalifaciens in dogs and its epidemiology.

Trueperella pecoris sp. nov. isolated from bovine and porcine specimens.

A novel Gram-stain-positive bacterium was isolated from a purulent bovine milk sample, the bovine placenta from an abortion, the udder secretion of a heifer and the lung of a pig that had succumbed from suppurative bronchopneumonia in Switzerland from 2015 to 2019. The strains grew best under aerobic conditions with 5 % CO2 and colonies were non-haemolytic and greyish-white. They were non-motile and negative for catalase and oxidase. The genomes of the four strains 19M2397T, 15A0121, 15IMD0307 and 19OD0592 were obtained by sequencing. The results of phylogenetic analyses based on the 16S rRNA gene grouped them within the genus Trueperella in the family Arcanobacteriaceae. The genomes had DNA G+C contents of 61.2-62.2 mol% and showed digital DNA-DNA hybridization (dDDH) values of 21.4-22.8 % and average nucleotide identity (ANI) values of approximately 77 % to their closest relatives Trueperella pyogenes and Trueperella bernardiae. With respect to the presence in different livestock species we propose the name Trueperella pecoris sp. nov. The type strain is 19M2397T (=CCOS 1952T=DSM 111392T), isolated from the udder secretion of a heifer diagnosed with summer mastitis in 2019.

Atopic dermatitis in West Highland white terriers - Part III: early life peripheral blood regulatory T cells are reduced in atopic dermatitis.

BACKGROUND: Regulatory T (Treg) cells are involved in homeostasis of immune regulation and suppression of inflammation and T-cell polarisation. Current knowledge regarding the role of Treg cells in the initiation of allergic disease is limited for both people and dogs. OBJECTIVES: To explore the role of circulating Treg cells and their possible influencing factors, on the development of atopic dermatitis (AD). METHODS AND MATERIALS: This study followed part of a birth cohort of West Highland white terrier dogs and classified them according to eventual clinical signs of AD (i.e. allergic versus healthy). The Treg phenotypes were assessed longitudinally by flow cytometry at 3, 3-12 and 12-36 months of age, and associated with development of AD. Different early life antigenic factors [endotoxins and allergens in house dust, Toxocara canis-specific immunoglobulin (Ig)E/IgG, allergen-specific and total IgE, skin microbiota] were measured at three months of age, and a possible association with Treg cell levels was assessed. RESULTS: The percentages of CD4+ CD25+ Foxp3+ Treg cells in healthy dogs were significantly higher at in 3-month-old (mean 4.5% healthy versus 3.3% allergic; P = 0.021) and <1-year-old (4.0% healthy versus 2.9% allergic; P = 0.028) dogs when compared to percentages of Treg cells in dogs that developed AD. There was a significantly positive correlation between the relative abundance of Lachnospiraceae on the skin and CD4+ CD25+ Foxp3+ Treg cells in puppies that became allergic (r = 0.568, P = 0.017). CONCLUSION AND CLINICAL IMPORTANCE: Further large-scale studies are needed to identify the practical value of these findings in AD diagnosis, treatment and prevention.

Evaluation of an investigative model in dairy herds with high calf perinatal mortality rates in Switzerland.

The objective of this study was to evaluate an investigative model which encompassed the risk factors, incidence, timing and causes of perinatal mortality (PM) (0-48 h) on high risk dairy farms (PM of >5% in the previous year) in Switzerland. This pilot-study was carried out on 47 predominantly Holstein PM calves from 21 dairy farms, between September 2016 and January 2018. Gross pathological examinations of calves and placentae as well as histopathological examinations of internal organs and placental tissue were performed. Further investigations included microbiological examinations: broad-spectrum bacterial and fungal culture, detection of Chlamydia abortus, Coxiella burnetii, pathogenic Leptospira spp. and Neospora caninum by real-time PCR (qPCR) and of bovine viral diarrhoea virus (BVDV) by Ag-ELISA. Maternal blood samples were used for serology of bovine herpesvirus 1 (BHV-1), Brucella abortus, Chlamydia abortus, Coxiella burnetii and nine pathogenic leptospiral serovars and the evaluation of trace element status. A questionnaire was completed with the farmer, which included general farm characteristics and case-related data. Inbreeding coefficients (IC) were calculated for pure-bred matings. At the farm-level, the PM rate was 10.0% (5.3-28.2%) and at the cow-level, 11.5%. These values, from high-risk farms, were approximately five-times higher than the contemporary national bovine PM rate (2.3%) in Switzerland. The risk factors associated with these high PM rates were the self-selection of high risk herds, the high proportion of primiparae in these herds (45%) and the evidence of widespread pathogenic infections on these farms (exposure: 67% of herds, 53% of dams; infection: 57% of herds, 45% of calves). The majority (68.1%) of calves died intrapartum. The most commonly diagnosed initiating/ultimate cause of death (UCOD) was infection (34%) of which Coxiella burnetii was the most frequently detected pathogen, by antigen. The most frequently diagnosed proximate cause of death (PCOD) was asphyxia (44.7%), though multiple PCOD was also common (21.3%). This study was the first detailed investigation of bovine PM in Switzerland. Infectious causes were diagnosed more frequently than expected. While the findings from these high PM Swiss herds may have limited external validity, the investigative model adopted and the detailed research methodologies employed can be replicated and re-evaluated, respectively, in future studies on PM internationally.

Bovine leptospirosis in abattoirs in Uganda: Molecular detection and risk of exposure among workers.

Leptospirosis is a zoonotic bacterial disease reported worldwide. In Uganda, seropositivity has been reported in both humans and domesticated animals, including cattle. However, it remains unknown whether cattle are shedding leptospires and thus acting as potential source for human leptospirosis. We conducted this cross-sectional study in two cattle abattoirs in Kampala, Uganda between June and July 2017. Kidney and urine samples from 500 cattle sourced from across the country were analysed by real-time PCR to establish the prevalence of Leptospira-positive cattle and risk of exposure to abattoir workers. The species of infecting Leptospira was determined by amplification of secY gene and compared to reference sequences published in GenBank. Of 500 cattle tested, 36 (7.2%) had Leptospira DNA in their kidneys (carriers), 29 (5.8%) in their urine (shedders); with an overall prevalence (kidney and/or urine) of 8.8%. Leptospira borgpetersenii was confirmed as the infecting species in three cattle and Leptospira kirschneri in one animal. Male versus female cattle (OR = 3, p-value 0.003), exotic versus local breeds (OR = 21.3, p-value 0.002) or cattle from Western Uganda (OR = 4.4, p-value 0.001) and from regions across the border (OR = 3.3, p-value 0.032) versus from the central region were more likely to be Leptospira-positive. The daily risk of exposure of abattoir workers to ≥1 (kidney and/or urine) positive carcass ranged from 27% (95% credibility interval 18.6-52.3) to 100% (95% CI 91.0-100.0), with halal butchers and pluck inspectors being at highest risk. In conclusion, cattle slaughtered at abattoirs in Uganda carry and shed pathogenic Leptospira species; and this may pose occupation-related risk of exposure among workers in these abattoirs, with workers who handle larger numbers of animals being at higher risk.

Serological detection of anti-leptospiral antibodies in outdoor cats in Switzerland.

Leptospirosis is endemic in Switzerland affecting a broad range of hosts. The aim of this study was to estimate the exposure of cats to Leptospira in Switzerland. Plasma samples from 107 outdoor cats with an array of clinical problems were tested via microscopic agglutination test for the presence of anti-leptospiral antibodies against 12 serovars of 9 serogroups. Using a reciprocal cut-off titre of 1:100, an overall seroprevalence of 10.3% (95%CI 5.2-17.7) was observed. Seroreactivity against serovars Bratislava (n = 6), Australis (2), Pomona (3) and Copenhageni (1) was detected with reciprocal titres ranging from 1:100 to 1:800. The serologic status of the cats was independent of the disease group (p = 0.62). These results show that cats in Switzerland are commonly exposed to Leptospira and confirm the importance of serogroup Australis in this region.

Limited added value of fungal ITS amplicon sequencing in the study of bovine abortion.

Bovine mycotic abortion is sporadic and caused by different ubiquitous and opportunistic fungi. Recently, a broad spectrum of bacterial opportunists involved in bovine abortion was revealed by 16S rRNA gene amplicon sequencing. We hypothesized that fungal organisms potentially involved in bovine abortion also might remain undetected by conventional culture. In this retrospective study, we therefore applied fungal internal transcribed spacer 2 (ITS2) region amplicon sequencing to 74 cases of bovine abortion submitted to our diagnostic service. The investigation was complemented by fungal culture and, retrospectively, by data from bacteriological, virological and parasitological analyses and histopathological examination of placentas. Fungal DNA was found in both the placentas and abomasal contents, with 92 fungal genera identified. In 18 cases, >75% of the reads belonged to one specific fungal genus: Candida (n = 7), Malassezia (n = 4), Cryptococcus (n = 3), unidentified Capnodiales (n = 3), Actinomucor (n = 1), Cystofilobasidium (n = 1), Penicillium (n = 1), Verticillum (n = 1) and Zymoseptoria (n = 1) with one case harboring two different genera. By culture, in contrast, fungal agents were detected in only 6 cases. Inflammatory and/or necrotizing lesions were found in 27/40 histologically assessed placentas. However, no lesion-associated fungal structures were detected in HE- and PAS-stained specimens. Complementary data revealed the presence of one or more non-fungal possible abortifacient: Chlamydiales, Coxiella burnetii, Leptospira spp., Campylobacter fetus subsp. fetus, Streptococcus uberis, Escherichia coli, Streptococcus pluranimalium, Bacillus licheniformis, Campylobacter fetus subsp. fetus, Serratia marcescens, Trueperella pyogenes, Schmallenbergvirus, Neospora caninum. The mycobiota revealed by sequencing did not differ between cases with or without a possible infectious etiology. Our study suggests that amplicon sequencing of the ITS2 region from DNA isolated from bovine abortion does not provide additional information or new insight into mycotic abortion and without complementary analyses may easily lead to a false interpretation of the role of fungal organisms in bovine abortion.

Neglected zoonotic agents in cattle abortion: tackling the difficult to grow bacteria.

BACKGROUND: Coxiella burnetii, Chlamydia abortus and Leptospira spp. are difficult to grow bacteria that play a role in bovine abortion, but their diagnosis is hampered by their obligate intracellular lifestyle (C. burnetii, C. abortus) or their lability (Leptospira spp.). Their importance is based on the contagious spread in food-producing animals, but also as zoonotic agents. In Switzerland, first-line routine bacteriological diagnostics in cattle abortions is regulated by national law and includes only basic screening by staining for C. burnetii due to the high costs associated with extended spectrum analysis. The aim of this study was to assess the true occurrence of these zoonotic pathogens in 249 cases of bovine abortion in Switzerland by serology (ELISA for anti-C. burnetii and C. abortus antibodies and microscopic agglutination test for anti-Leptospira spp. antibodies), molecular methods (real-time PCR and sequencing of PCR products of Chlamydiales-positive cases), Stamp's modification of the Ziehl-Neelsen (mod-ZN) stain and, upon availability of material, by histology and immunohistochemistry (IHC). RESULTS: After seroanalysis the prevalence was 15.9% for C. burnetii, 38.5% for C. abortus and 21.4% for Leptospira spp. By real-time PCR 12.1% and 16.9% of the cases were positive for C. burnetii and Chlamydiales, respectively, but only 2.4% were positive for C. burnetii or Chlamydiales by mod-ZN stain. Sequencing of PCR products of Chlamydiales-positive cases revealed C. abortus in 10% of cases and the presence of a mix of Chlamydiales-related bacteria in 5.2% of cases. Pathogenic Leptospira spp. were detected in 5.6% of cases. Inflammatory lesions were present histologically in all available samples which were real-time PCR-positive for Chlamydiales and Leptospira spp. One of 12 real-time PCR-positive cases for C. burnetii was devoid of histological lesions. None of the pathogens could be detected by IHC. CONCLUSION: Molecular detection by real-time PCR complemented by histopathological analysis is recommended to improve definitive diagnosis of bovine abortion cases and determine a more accurate prevalence of these zoonotic pathogens.

Cross-Sectional Serological Survey for Leptospira spp. in Beef and Dairy Cattle in Two Districts in Uganda.

Seroprevalence of Leptospira spp. in cattle is unknown in Uganda. The aim of this study was to estimate the seroprevalence of L. interrogans Icterohaemorrhagiae, Pomona, L. kirschneri Butembo, Grippotyphosa, L. borgpetersenii Nigeria, Hardjo, Wolfii, and Kenya and an overall seroprevalence in cattle from Kole and Mbale districts. Two hundred-seventy five bovine sera from 130 small holder farms from Kole (n = 159) and Mbale (n = 116), collected between January and July 2015, were tested for antibodies against eight Leptospira strains by Microscopic Agglutination Test. A titer of ≥100 was considered seropositive, indicating past exposure. Overall, the seroprevalence was 19.27% (95% CI 14.9-24.5%). Pomona seroprevalence was highest with 9.45% (6.4-13.7%), followed by Kenya 5.09% (2.9-8.6%), Nigeria 4.00% (2.1-7.2%), Wolfii 3.27% (1.6-6.3%), Butembo 1.86% (0.7-4.4%), Hardjo 1.45% (0.5-3.9%), and Icterohaemorragiae and Grippotyphosa with less than 1% positive. Seroprevalence did not differ between districts and gender (p ≥ 0.05). Seven animals had titers ≥400. Cross-reactions or exposure to ≥1 serovar was measured in 43% of serum samples. Seroprevalence of 19% implies exposure of cattle to leptospires.

Amplicon sequencing of bacterial microbiota in abortion material from cattle.

Abortions in cattle have a significant economic impact on animal husbandry and require prompt diagnosis for surveillance of epizootic infectious agents. Since most abortions are not epizootic but sporadic with often undetected etiologies, this study examined the bacterial community present in the placenta (PL, n = 32) and fetal abomasal content (AC, n = 49) in 64 cases of bovine abortion by next generation sequencing (NGS) of the 16S rRNA gene. The PL and AC from three fetuses of dams that died from non-infectious reasons were included as controls. All samples were analyzed by bacterial culture, and 17 were examined by histopathology. We observed 922 OTUs overall and 267 taxa at the genus level. No detectable bacterial DNA was present in the control samples. The microbial profiles of the PL and AC differed significantly, both in their composition (PERMANOVA), species richness and Chao-1 (Mann-Whitney test). In both organs, Pseudomonas was the most abundant genus. The combination of NGS and culture identified opportunistic pathogens of interest in placentas with lesions, such as Vibrio metschnikovii, Streptococcus uberis, Lactococcus lactis and Escherichia coli. In placentas with lesions where culturing was unsuccessful, Pseudomonas and unidentified Aeromonadaceae were identified by NGS displaying high number of reads. Three cases with multiple possible etiologies and placentas presenting lesions were detected by NGS. Amplicon sequencing has the potential to uncover unknown etiological agents. These new insights on cattle abortion extend our focus to previously understudied opportunistic abortive bacteria.

Macrococcus canis and M. caseolyticus in dogs: occurrence, genetic diversity and antibiotic resistance.

BACKGROUND: The discovery of a new Macrococcus canis species isolated from skin and infection sites of dogs led us to question if Macrococcus spp. are common in dogs and are resistant to antibiotics. HYPOTHESIS/OBJECTIVES: To evaluate the occurrence of Macrococcus spp. in dogs, determine antibiotic resistance profiles and genetic relationships. ANIMALS: One hundred and sixty two dogs (mainly West Highland white terriers and Newfoundland dogs) were screened for the presence of Macrococcus, including six dogs with Macrococcus infections. METHODS: Samples were taken from skin, ear canal and oral mucosa using swabs. Macrococci were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, 16S rRNA sequencing and nuc-PCR. Minimal inhibitory concentrations of 19 antibiotics were determined using broth microdilution. Resistance mechanisms were identified by microarray and sequencing of the fluoroquinolone-determining region of gyrA and grlA. Sequence type (ST) was determined by multilocus sequence typing. RESULTS: Out of the 162 dogs, six harboured M. caseolyticus (n = 6) and 13 harboured M. canis (n = 16). Six isolates of M. canis and one of M. caseolyticus were obtained from infection sites. The 22 M. canis strains belonged to 20 different STs and the seven M. caseolyticus strains to three STs. Resistance to antibiotics was mostly associated with the detection of known genes, with mecB-mediated meticillin resistance being the most frequent. CONCLUSION AND CLINICAL IMPORTANCE: This study gives some insights into the occurrence and genetic characteristics of antibiotic-resistant Macrococcus from dogs. Presence of M. canis in infection sites and resistance to antibiotics emphasized that more attention should be paid to this novel bacteria species.

Clinical, serological and echocardiographic examination of healthy field dogs before and after vaccination with a commercial tetravalent leptospirosis vaccine.

BACKGROUND: Leptospirosis is a re-emerging bacterial zoonosis caused by spirochetes of the genus Leptospira. Severe disease has been reported in dogs in Europe despite vaccination with bivalent Leptospira vaccines. Recently, a tetravalent canine Leptospira vaccine (Nobivac® L4) was licenced in Europe. The goal of this study was to investigate clinical signs, microscopic agglutination test (MAT) titres, haematology, blood biochemistry, cardiac (c) Troponin I levels and echocardiography before and after vaccination with this tetravalent vaccine. Forty-eight healthy dogs were prospectively enrolled and vaccinated twice, 3-4 weeks apart (T0 and T1). Before vaccination (T0) and 16-31 days after the second vaccination (T2), MAT (n = 48), haematology (n = 48), blood biochemistry (n = 36) and cTroponin I measurements (n = 29) were performed, and MAT was repeated 347-413 days after the second vaccination (T3, n = 44). Echocardiography was performed before the first and second vaccination (T0 and T1, n = 24). RESULTS: Mild and transient clinical signs within 5 days following the first and second vaccination occurred in 23% and 10% of the dogs, respectively. Before the first vaccination (T0), all dogs showed negative MAT titres for the tested serovars except for Canicola (50% with titres 100-400). At T2, positive MAT titres to the serovars Canicola (100%), Australis (89%), Grippotyphosa (86%), Bratislava (60%), Autumnalis (58%), Copenhageni (42%), Pomona (12%), Pyrogenes (8%) and Icterohaemorrhagiae (2%) were found. Median to high titres (≥ 400) were most common to the serovar Canicola (92%) and less common to the serovars Australis (41%), Grippotyphosa (21%), Bratislava (12%), Autumnalis (4%), Pyrogenes (4%) and Pomona (2%). At T3, positive MAT titres (titre range: 100-400) were found in 2-18% of the dogs to serovars of the vaccine serogroups and in 2-18% of the dogs to the non-vaccine serovars Pomona, Autumnalis, Pyrogenes and Ballum. Haematology, blood biochemistry, cTroponin I levels and echocardiography results did not change significantly following vaccination. CONCLUSIONS: Clinical signs following vaccination with Nobivac® L4 were transient and mild in all cases. Seroconversion differed considerably among individual dogs and among the vaccine serogroups.

Canine leptospirosis in Switzerland-A prospective cross-sectional study examining seroprevalence, risk factors and urinary shedding of pathogenic leptospires.

Leptospirosis is an important worldwide zoonosis. While human leptospirosis remains rare in Switzerland, the incidence of canine leptospirosis is unusually high compared to other European countries. The aims of this cross-sectional study were to determine the exposure of asymtomatic dogs to pathogenic Leptospira in Switzerland, to characterise risk factors associated with seropositivity and to determine the prevalence of urinary shedding. Sampling was stratified to cover the whole of Switzerland. Sera were tested by microscopic agglutination test for antibodies against a panel of 12 serovars. Urine was tested for pathogenic Leptospira using a LipL32 real-time PCR. Of 377 sera, 55.7% (95%CI 51.2-60.7) showed a reciprocal MAT titre of ≥1:40 and 24.9% (95%CI 20.7-29.4) of ≥1:100 to at least one serovar. Seropositivity (MAT ≥1:100) was most common to serovars Australis (14.9%; 95% CI 11.4-18.6) and Bratislava (8.8%; 95%CI 6.1-11.7), followed by Copenhageni (6.1%; 95%CI 3.7-8.5), Canicola (5%; 95%CI 2.9-7.4), Grippotyphosa (4.5%; 95%CI 2.7-6.9), Pomona (4%; 95%CI 2.1-6.1), Autumnalis (2.7%; 95%CI 1.3-4.2) and Icterohaemorrhagiae (1.6%; 95%CI 0.5-2.9). In unvaccinated dogs (n=84) the prevalence of a MAT titre ≥100 was 17.9% (95%CI 10.7-26.2), with a similar distribution of reactive serovars. Variables associated with seropositivity (≥1:40) to any serovar included age (OR 1.29/year; 95%CI: 1.1-1.5) and bioregion with higher risks in the regions Northern Alps (OR 14.5; 95%CI 2.2-292.7), Central Plateau (OR 12.3; 95%CI 2.0-244.1) and Jura (OR 11.2; 95%CI 1.7-226.7) compared to Southern Central Alps. Dogs living with horses were significantly more likely to have antibodies to serovar Bratislava (OR 4.68;95%CI 1.2-17.2). Hunting was a significant risk factor for seropositivtiy to serovar Grippotyphosa (OR 8.03; 95%CI 1.6-30.8). Urine qPCR positivity was uncommon (1/408 dogs; 0.2%; 95% CI0-0.7). These results demonstrate that dogs in Switzerland are commonly exposed to pathogenic Leptospira; however, the risk of dogs contributing to the spread of Leptospira in the environment appears low.

Macrococcus canis sp. nov., a skin bacterium associated with infections in dogs.

Gram-stain-positive cocci were isolated from miscellaneous sites of the skin of healthy dogs as well as from infection sites in dogs. The closest relative by sequencing of the 16S rRNA gene was Macrococcus caseolyticus with 99.7 % sequence identity, but compared with M. caseolyticus, the novel strains shared only 90.8 to 93.5 % DNA sequence identity with cpn60, dnaJ, rpoB and sodA partial genes, respectively. The novel strains also exhibited differential phenotypic characteristics from M. caseolyticus, and the majority displayed a visible haemolysis on sheep blood agar, while M. caseolyticus did not have any haemolytic activity. They generated different matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS spectral profiles compared with the other species of the genus Macrococcus. Furthermore, strain KM 45013T shared only 53.7 % DNA-DNA relatedness with the type strain of M. caseolyticus, confirming that they do not belong to the same species. The DNA G+C content of strain KM 45013T was 36.9 mol%. The most abundant fatty acids were C14 : 0, C18 : 3ω6c (6, 9, 12) and C16 : 0 n alcohol. MK-6 was the menaquinone type of KM 45013T. Cell-wall structure analysis revealed that the peptidoglycan type was A3α l-Lys-Gly2-l-Ser. Based on genotypic and chemotaxonomic characteristics, we propose to classify these strains within a novel species of the genus Macrococcus for which the name Macrococcus canis sp. nov. is proposed. The type strain is KM 45013T (=DSM 101690T=CCOS 969T=CCUG 68920T).

Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing.

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.

Overview and phylogeny of Mycobacterium tuberculosis complex organisms: implications for diagnostics and legislation of bovine tuberculosis.

Members of the Mycobacterium tuberculosis complex (MTBC) cause a serious disease with similar pathology, tuberculosis; in this review, bovine tuberculosis will be considered as disease caused by any member of the MTBC in bovids. Bovine tuberculosis is responsible for significant economic loss due to costly eradication programs and trade limitations and poses a threat to both endangered and protected species as well as to public health. We here give an overview on all members of the MTBC, focusing on their isolation from different animal hosts. We also review the recent advances made in elucidating the evolutionary and phylogenetic relationships of members of the MTBC. Because the nomenclature of the MTBC is controversial, its members have been considered species, subspecies or ecotypes, this review discusses the possible implications for diagnostics and the legal consequences of naming of new species.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group.

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

A Bayesian approach to study the risk variables for tuberculosis occurrence in domestic and wild ungulates in South Central Spain.

BACKGROUND: Bovine tuberculosis (bTB) is a chronic infectious disease mainly caused by Mycobacterium bovis. Although eradication is a priority for the European authorities, bTB remains active or even increasing in many countries, causing significant economic losses. The integral consideration of epidemiological factors is crucial to more cost-effectively allocate control measures. The aim of this study was to identify the nature and extent of the association between TB distribution and a list of potential risk factors regarding cattle, wild ungulates and environmental aspects in Ciudad Real, a Spanish province with one of the highest TB herd prevalences. RESULTS: We used a Bayesian mixed effects multivariable logistic regression model to predict TB occurrence in either domestic or wild mammals per municipality in 2007 by using information from the previous year. The municipal TB distribution and endemicity was clustered in the western part of the region and clearly overlapped with the explanatory variables identified in the final model: (1) incident cattle farms, (2) number of years of veterinary inspection of big game hunting events, (3) prevalence in wild boar, (4) number of sampled cattle, (5) persistent bTB-infected cattle farms, (6) prevalence in red deer, (7) proportion of beef farms, and (8) farms devoted to bullfighting cattle. CONCLUSIONS: The combination of these eight variables in the final model highlights the importance of the persistence of the infection in the hosts, surveillance efforts and some cattle management choices in the circulation of M. bovis in the region. The spatial distribution of these variables, together with particular Mediterranean features that favour the wildlife-livestock interface may explain the M. bovis persistence in this region. Sanitary authorities should allocate efforts towards specific areas and epidemiological situations where the wildlife-livestock interface seems to critically hamper the definitive bTB eradication success.

Eradication of bovine tuberculosis at a herd-level in Madrid, Spain: study of within-herd transmission dynamics over a 12 year period.

BACKGROUND: Eradication of bovine tuberculosis (bTB) through the application of test-and-cull programs is a declared goal of developed countries in which the disease is still endemic. Here, longitudinal data from more than 1,700 cattle herds tested during a 12 year-period in the eradication program in the region of Madrid, Spain, were analyzed to quantify the within-herd transmission coefficient (ß) depending on the herd-type (beef/dairy/bullfighting). In addition, the probability to recover the officially bTB free (OTF) status in infected herds depending on the type of herd and the diagnostic strategy implemented was assessed using Cox proportional hazard models. RESULTS: Overall, dairy herds showed higher ß (median 4.7) than beef or bullfighting herds (2.3 and 2.2 respectively). Introduction of interferon-gamma (IFN-γ) as an ancillary test produced an apparent increase in the ß coefficient regardless of production type, likely due to an increase in diagnostic sensitivity. Time to recover OTF status was also significantly lower in dairy herds, and length of bTB episodes was significantly reduced when the IFN-γ was implemented to manage the outbreak. CONCLUSIONS: Our results suggest that bTB spreads more rapidly in dairy herds compared to other herd types, a likely cause being management and demographic-related factors. However, outbreaks in dairy herds can be controlled more rapidly than in typically extensive herd types. Finally, IFN-γ proved its usefulness to rapidly eradicate bTB at a herd-level.