Management and housing factors associated with paratuberculosis-positive herds in small structured alpine cattle husbandry.

Paratuberculosis (Johne´s disease) is a world-wide cattle disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), associated with substantial economic losses. Purchase of subclinically infected animals or contact with animals and equipment of infected farms are known risk factors for disease transmission among herds. The aim of the present study was to identify specific management factors in Austria that triggered a MAP-positive herd status and to evaluate known risk factors for the transmission in cattle in small structured alpine agricultural systems. The agriculture in the Austrian province of Tyrol is characterized by smallholder structures, including shared alpine pastures and traditional barn management techniques. The data from an extensive survey with 50 questions in 2013/2014 and the development of the MAP herd status of 5592 cattle farms by taking feces and blood samples were examined and statistically evaluated. MAP herd status was determined by combining the results of boot swab samples, manure samples, pooled and individual feces samples as well as serological antibody testing by ELISA. The statistical analysis (odds ratio; OR) showed that the use of milk replacers for calf feeding (p = 0.047, OR=0.472) and the use of straw as bedding material for cows (p = 0.032, OR=0.625) were associated with a decreased chance of being a MAP-positive herd. Further, housing cows in deep litter systems (p = 0.028, OR=2.232), the presence of slurry channels (p = 0.028, OR=1.411) and the use of solid manure in young cattle (p = 0.041, OR=1.744) were associated with an increased OR for being MAP-positive. Surprisingly, sharing of lowland pastures (p = 0.564, OR=1.080), alpine pastures (p = 0.419, OR=1.143) or farm equipment (p = 0.733, OR=0.963) and farm size (p = 0.425) had no significant influence on the MAP herd status. The identified differences compared with previously published results in respect of MAP spread in cattle might be attributed to the traditional agricultural structures, including small family-based farms and common pasture during summer in alpine regions. Results of this study contribute to the understanding of the spread of MAP in cattle farming in alpine regions.

Dogs as carriers of virulent and resistant genotypes of Clostridioides difficile.

While previous research on zoonotic transmission of community-acquired Clostridioides difficile infection (CA-CDI) focused on food-producing animals, the present study aimed to investigate whether dogs are carriers of resistant and/or virulent C. difficile strains. Rectal swabs were collected from 323 dogs and 38 C. difficile isolates (11.8%) were obtained. Isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing (WGS) and a DNA hybridization assay. Multilocus sequence typing (MLST), core genome MLST (cgMLST) and screening for virulence and antimicrobial resistance genes were performed based on WGS. Minimum inhibitory concentrations for erythromycin, clindamycin, tetracycline, vancomycin and metronidazole were determined by E-test. Out of 38 C. difficile isolates, 28 (73.7%) carried genes for toxins. The majority of isolates belonged to MLST sequence types (STs) of clade I and one to clade V. Several isolates belonged to STs previously associated with human CA-CDI. However, cgMLST showed low genetic relatedness between the isolates of this study and C. difficile strains isolated from humans in Austria for which genome sequences were publicly available. Four isolates (10.5%) displayed resistance to three of the tested antimicrobial agents. Isolates exhibited resistance to erythromycin, clindamycin, tetracycline and metronidazole. These phenotypic resistances were supported by the presence of the resistance genes erm(B), cfr(C) and tet(M). All isolates were susceptible to vancomycin. Our results indicate that dogs may carry virulent and antimicrobial-resistant C. difficile strains.

Sonographic and histological udder parenchyma changes after intramammary infection of sheep with Mycoplasma agalactiae.

INTRODUCTION: The sonographic findings of the udder parenchyma and udder lymph nodes in 30 lactating sheep after experimental infection with Mycoplasma agalactiae are described. The objective of the study was to describe infection related changes in the udder parenchyma and udder lymph nodes using physical, sonographic, and histological examination and to detect associations between sonographic and histological changes of the tissues. Animals were intramammarily infected with different mutant cocktails and the wild type PG2. One group served as a negative control. A 15 MHz linear transducer (Esaote MyLab 30 CV, Esaote, Florence, Italy) was used for sonographic examinations. Compared with the uninfected control group with homogeneously granular parenchyma, the udder lymph nodes were larger and the udder parenchyma was more inhomogeneous and partially hyperechoic. The corresponding histological findings in infected mammary glands comprised proliferation of interstitial connective tissue, non-purulent interstitial mastitis, and purulent galactophoritis. The infected udder lymph nodes showed reactive hyperplasia. The findings obtained in this study may improve the diagnosis of Mycoplasma mastitis in sheep.

INTRODUCTION: Les constatations échographiques sur le parenchyme de la mamelle et des ganglions lymphatiques de la mamelle chez 30 brebis en lactation après une infection expérimentale avec Mycoplasma agalactiae sont décrits. L'objectif de l'étude était de décrire les modifications liées à l'infection dans le parenchyme mammaire et les ganglions lymphatiques de la mamelle à l'aide d'un examen physique, échographique et histologique et de détecter les associations entre les altérations échographiques et histologiques des tissus. Les animaux ont été infectés par voie intramammaire avec différents cocktails de mutants et le type sauvage PG2. Un groupe a servi de contrôle négatif. Une sonde linéaire de 15 MHz (Esaote MyLab 30 CV, Esaote, Florence, Italie) a été utilisé pour les examens échographiques. Comparativement au groupe témoin non infecté avec un parenchyme granulaire homogène, les ganglions lymphatiques de la mamelle étaient plus gros et le parenchyme de la mamelle était plus inhomogène et partiellement hyperéchogène. Les résultats histologiques correspondants dans les glandes mammaires infectées comprenaient une prolifération de tissu conjonctif interstitiel, une mammite interstitielle non purulente et une galactophorite purulente. Les ganglions lymphatiques de la mamelle infectée présentaient une hyperplasie réactive. Les résultats obtenus dans cette étude peuvent améliorer le diagnostic de la mammite à Mycoplasma chez le mouton.

A novel video-endoscope-guided myringotomy technique in dogs: Investigation in the value of vertical access to the tympanic membrane from beneath the patient - a pilot study.

Myringotomy is a well-accepted method for diagnosing and treating otitis media in dogs having an intact tympanic membrane. In a recent study, the contamination rate of middle ear aspirates from the external ear canal via myringotomy was 67%. To evaluate the iatrogenic contamination rate of the middle ear aspirates by material from the ear canal, using a novel technique: Vertical access to the tympanic membrane from beneath the patient. Thirty-six ears from 20 canine Fresh cadavers with a CT scan negative for otitis externa and otitis media were video-otoscopically flushed with the ear upside. The instillation of the fluorescent dye into the ear canal and immediate retrieval were performed. With the patient positioned on a custom-made fenestrated table plate, a modified video-otoscopically guided myringotomy approaching the tympanum vertically from underneath, was performed. Contamination rates were assessed by the visual detection of fluorescent dye within the aspirated fluid, either by yellow staining solely, or a positive fluorescence test. Cytology and microbial cultivation were accomplished. Middle ear sample contamination by the material from the ear canal was identified in 2 of 36 (5.55%) ears. Neither a change in colour nor fluorescence was detected in 34 of 36 (94.44%) middle ear samples. Sixteen of 36 (44.44%) external ear canal samples and 4 of 36 (11.11%) middle ear aspirates had positive bacterial culture. This novel technique is a promising method for middle ear material collection in patients with concurrent otitis externa.

Diversity of methicillin-resistant coagulase-negative Staphylococcus spp. and methicillin-resistant Mammaliicoccus spp. isolated from ruminants and New World camelids.

Information about livestock carrying methicillin-resistant coagulase-negative staphylococci and mammaliicocci (MRCoNS/MRM) is scarce. The study was designed to gain knowledge of the prevalence, the phenotypic and genotypic antimicrobial resistance and the genetic diversity of MRCoNS/MRM originating from ruminants and New World camelids. In addition, a multi-locus sequence typing scheme for the characterization of Mammaliicoccus (formerly Staphylococcus) sciuri was developed. The study was conducted from April 2014 to January 2017 at the University Clinic for Ruminants and the Institute of Microbiology at the University of Veterinary Medicine Vienna. Seven hundred twenty-three nasal swabs originating from ruminants and New World camelids with and without clinical signs were examined. After isolation, MRCoNS/MRM were identified by MALDI-TOF, rpoB sequencing and typed by DNA microarray-based analysis and PCR. Antimicrobial susceptibility testing was conducted by agar disk diffusion. From all 723 nasal swabs, 189 MRCoNS/MRM were obtained. Members of the Mammaliicoccus (M.) sciuri group were predominant (M. sciuri (n = 130), followed by M. lentus (n = 43), M. fleurettii (n = 11)). In total, 158 out of 189 isolates showed phenotypically a multi-resistance profile. A seven-loci multi-locus sequence typing scheme for M. sciuri was developed. The scheme includes the analysis of internal segments of the house-keeping genes ack, aroE, ftsZ, glpK, gmk, pta1 and tpiA. In total, 28 different sequence types (STs) were identified among 92 selected M. sciuri isolates. ST1 was the most prevalent ST (n = 35), followed by ST 2 (n = 15), ST3 and ST5 (each n = 5), ST4 (n = 3), ST6, ST7, ST8, ST9, ST10 and ST11 (each n = 2).

Polyarthritis caused by Erysipelothrix rhusiopathiae in three Austrian sheep flocks - diagnosis, treatment and management measures.

INTRODUCTION: Polyarthritis caused by Erysipelothrix rhusiopathiae is a well-known disease in pigs, and ovine erysipelas infection also commonly affects two-to-six month-old lambs. This report describes case histories of three sheep flocks where lambs exhibited swollen joints and lameness. Special emphasis was given to clinical and diagnostic imaging findings, synovia sampling and the treatment regime. Lambs with only mild lameness, liquid serofibrinous joint effusion and lambs showing no bone involvement, as revealed by ultrasonography or radiography, were treated with systemically administered antibiotics selected from results of antimicrobial susceptibility testing of E. rhusiopathiae isolated from synovial samples, and non-steroidal anti-inflammatory drugs. Lambs with severe lameness and severely swollen joints were euthanized, and routine necropsy was undertaken with a focus on the joints. Further, a herd-specific autogenous vaccine was produced by a specialized laboratory. In conclusion, E. rhusiopathiae infection should be considered as a differential diagnosis in herds associated with lameness and polyarthritis in lambs aged between two up to 17 months.

INTRODUCTION: La polyarthrite causée par Erysipelothrix rhusiopathiae est une maladie bien connue chez le porc. Chez les ovins, l'infection touche le plus souvent les agneaux âgés de deux à six mois. Ce rapport de cas décrit trois troupeaux de moutons où des agneaux présentaient des articulations enflées et une boiterie. Un accent particulier a été mis sur la clinique, les résultats de l'imagerie diagnostique, les prélèvements de synovie et le mode de traitement. Les agneaux présentant uniquement une légère boiterie, des épanchements articulaires séro-fibrineux et ceux ne présentant pas d'atteinte osseuse, révélée par échographie ou radiographie, ont été traités avec des antibiotiques administrés par voie systémique, sélectionnés à partir des résultats de la sensibilité d'E. Rhusiopathiae isolé sur les échantillons synoviaux, ainsi qu'avec des anti-inflammatoires non stéroïdiens. Les agneaux présentant une boiterie sévère et des articulations gravement enflées ont été euthanasiés et une autopsie de routine a été réalisée avec un accent particulier mis sur les articulations. De plus, un vaccin autogène spécifique au troupeau a été produit par un laboratoire. En conclusion, l'infection à E. rhusiopathiae doit être considérée comme un diagnostic différentiel dans les troupeaux où l'on constate des boiteries et des polyarthrites chez les agneaux âgés de 2 à 17 mois.

Myringotomy in dogs: Contamination rate from the external ear canal - a pilot study.

Myringotomy for sample collection from the middle ear cavity for cytology and bacterial culture is considered a routine method to diagnose otitis media in dogs. The objective of this study was to determine the rate of contamination of middle ear aspirates with material from the external ear canal obtained by video-otoscopic guided myringotomy. In canine cadavers (n = 17) free from otitis externa the external ear canals were flushed under video-otoscopic control and a fluorescent dye was instilled. After removal of residual fluid a myringotomy was performed. If air was aspirated, 1 mL of saline was instilled through the same myringotomy needle into the middle ear cavity and re-aspirated. Contamination from the external ear canal was demonstrated by positive fluorescence of the aspirate. Bacterial cultures and cytological examinations of the external ear canals and middle ear cavities were performed. Data from 28 ears under investigation were included. In 19 of 28 middle ear aspirates (67.9%), clear yellow fluorescent fluid was obtained, indicating a contamination from the external ear canal. Microorganisms were detected in 4 of 26 middle ear samples (15.4%) and in 15 of 26 external ear canals (57.7%). Sample collection by myringotomy in this study was associated with a high contamination rate, implying that the suitability of this method for detection of otitis media in patients with concurrent otitis externa is questionable. Furthermore, the potential for iatrogenic spread of pathogenic microorganisms into the middle ear cavity needs to be considered.

[Diagnostic procedure after abortions in sows after simultaneous infection with leptospira and chlamydia]. / Diagnostische Abklärung von Aborten bei Zuchtsauen nach Leptospiren- und Chlamydieninfektion.

INTRODUCTION: In a farrowing farm 2 first parity sows aborted on day 95 and day 110 of gestation due to an infection with leptospira and chlamydia. The double infection was diagnosed by PCR examination of abortion material. Serum samples of both sows and additional 8 sows taken three weeks after abortions were sent to two different labs for serological examination for antibodies against leptospira and chlamydia using a microagglutination test and a complement fixation test, respectively. In both labs the tests for antibodies against chlamydia were negative. Titers against diverse leptospira serovars varied between both labs and were low, so that they were not indicative for the involvement of the two pathogens regarding abortion. This case report indicates the diagnostic difficulties of direct and indirect detection methods for leptospira and chlamydia to assess the impact of these pathogens on observed reproductive failure.

Diversity of methicillin-resistant Staphylococcus aureus (MRSA) isolated from Austrian ruminants and New World camelids.

The aim of this study was to determine the prevalence, the antimicrobial resistance patterns and the genetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) from Austrian ruminants and New World camelids that were treated at the University of Veterinary Medicine, Vienna. Between April 2014 and January 2017, 723 nasal swabs originating from ruminants and New World camelids were examined. MRSA isolates were characterized by mecA/mecA1/mecC PCRs and by DNA microarray analysis. They were genotyped by spa typing, dru typing, MLST and MLVA. Glycopolymer fingerprinting by FTIR spectroscopy was also performed. Antimicrobial susceptibility testing was conducted by agar disk diffusion. Twelve MRSA isolates were mecA-positive, whereas three were mecC-positive. The MRSA isolates carried five different SCCmec elements, and belonged to three sequence types (ST45, ST130, ST398). The MRSA isolates displayed seven different resistance phenotypes. The present study describes for the first time mecC-carrying MRSA isolates originating from domesticated animals in Austria. More systematic studies are needed to unravel the role of ruminants and New World camelids as reservoirs for MRSA as a potential risk for zooanthropogenic transmission.

Pyogranulomatous panniculitis in ferrets (Mustela putorius furo) with intralesional demonstration of Pseudomonas luteola.

One ferret (Mustela putorius furo) from Finland and two ferrets from Austria, aged 1-4.5 years and of both genders, were presented with pyogranulomatous subcutaneous inflammation affecting the inguinal, preputial and femoral regions, respectively. Histologically, microorganisms were detected within the lesions. The organisms had a capsule that stained positively by the periodic acid-Schiff reaction. Pseudomonas spp. were cultured from the lesions in two cases. In the third case, electron microscopy revealed a prokaryotic organism surrounded by an electron lucent matrix. 16S rRNA gene sequencing showed highest sequence homology to Pseudomonas luteola in all three cases. In combination with recent reports of pleuropneumonia in ferrets due to P. luteola infection, these cases might indicate a predisposition of ferrets for infection by these bacteria.

Multiple strain infections and high genotypic diversity among Mycobacterium avium subsp. paratuberculosis field isolates from diseased wild and domestic ruminant species in the eastern Alpine region of Austria.

Johne's disease, or paratuberculosis, is a chronic fatal ruminant gastroenteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP) whose foodborne zoonotic potential and association with Crohn's disease are still under debate. The disease is widespread but its epidemiology and epizootiology remains elusive. Wildlife is suspected to play a major role. After a surge in MAP seroprevalence in Austrian cattle, paratuberculosis was declared a notifiable disease in Austria in 2006. At the same time a rise in MAP cases in wild ruminant populations in the Austrian province of Styria was reported. All five autochthonous ruminants were affected. Genetic analysis of isolates, yielded numerous genotypes (>15) and several multiple strain infections (15%) across host species. Identical MIRU-VNTR profiles were identified in different species and sampling locations. On the other hand varying MIRU-VNTR profiles were revealed at the same location and in conspecifics. Our data, taken together with earlier epidemiological studies on MAP and other mycobacteria, raised concerns about the organisms' ecology. Constraints regarding in vitro culture of this highly fastidious organism potentially bias our current understanding of its epidemiology. We suggest that MAP infections could be polyclonal and question the informative value of genotyping a single MAP colony derived from a single specimen for epidemiological analysis of MAP.

Herd-level risk factors for the seropositivity to Mycoplasma hyopneumoniae and the occurrence of enzootic pneumonia among fattening pigs in areas of endemic infection and high pig density.

The aim of the present study was to identify potential risk factors for the occurrence of enzootic pneumonia (EP) in herds situated in a region of high pig density, where a majority of herds is endemically infected with Mycoplasma hyopneumoniae. Between 2006 and 2010, overall 100 herds were enrolled in a case-control study. Data were collected through personal interview with the farmers, clinical examination of pigs and their environments, and serological testing for M. hyopneumoniae, swine influenza virus and porcine reproductive and respiratory syndrome virus. There were 40 case herds (coughing index high, seroprevalence high) with a mean coughing index of 4.3 and a seroprevalence of 86.6%. There were two control groups. Control group I consisted of 25 herds (coughing index low, seroprevalence low) with mean values of 0.7 and 11.2%, and 35 herds were allocated to control group II (coughing index low, seroprevalence high) where the mean coughing index was 0.9 and seroprevalence 86.3%. Case herds and control II herds had an increased age of piglets at weaning compared to control I herds. Any contact between fattening pigs of different age during restocking of compartments increased the risk for the occurrence of EP in a herd. Finally, farms that use living animals for the exposure to gilts during the acclimatization and farms that had increased number of weaned piglets per sow and year were less likely to test positive for M. hyopneumoniae and less likely to develop clinical symptoms of EP in fattening pigs.

[Evaluation of the efficacy of a combination therapy of an antibiotic and a NSAID following an experimental Haemophilus parasuis infection in nursery piglets]. / Wirksamkeitsbeurteilung einer Kombinationstherapie aus Antibiose und NSAID nach experimenteller Haemophilus-parasuis-Infektion bei Absetzferkeln.

OBJECTIVE: The aim of the present study was to evaluate the combination therapy of an antibiotic (enrofloxacine-arginine, Baytril® RSi) and a non-steroidal anti-inflammatory drug (NSAID, ketoprofen, Dinalgen® 60 mg/ml) against a Haemophilus parasuis (HPS) infection in nursery piglets. MATERIAL AND METHODS: Forty-eight 3-week-old pigs were divided into four groups (group 1: non-infected controls; group 2: HPS infection; group 3: HPS infection/Baytril® RSi; group 4: HPS infection/Baytril® RSi/Dinalgen®) and housed within the isolation facility. After an acclimatization period of 10 days, the piglets in groups 2-4 were intratracheally infected with 1 x 107 colony forming units (CFU) HPS serovar 5, whereas animals of group 1 received physiological saline. Total clinical scores and joint scores were calculated daily after clinical examination. Seven days after the infection, piglets were humanely euthanized. At necropsy, pathological findings on serosal surfaces were scored according to severity and extension. RESULTS: Group 1 had the lowest clinical and pathological scores, followed by groups 4, 3 and 2. Piglets treated with the combination of an antibiotic and an NSAID showed the lowest body temperatures (significant). The average daily weight gain (ADWG) was not significantly different between the groups, but piglets of group 4 tended to reach a higher mean ADWG (340.5 g/d) than animals of the non-infected group 1 (323.8 g/d), the Baytril® RSi-treated group 3 (278.0 g/d) and the positive control group 2 (247.0 g/d). Piglets of the positive control group (group 2) achieved the highest values in the clinical, joint and serositis scores. CONCLUSION: The study demonstrated that a simultaneous treatment with enrofloxacine-arginine (Baytril® RSi) and ketoprofen had a superior therapeutic effect compared to a single antibiotic treatment with Baytril® RSi in nursery piglets experimentally infected with HPS.

Cooled storage of canine semen: in vitro effects of different concentrations of an antibiotic combination on growth of mollicutes.

The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n = 20). Semen aliquots were diluted with Tris-citric acid-fructose-egg yolk extender containing either 1.0 g/l streptomycin and 0.6 g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS-1: 0.25, 0.05, 0.15 and 0.3; GTLS-2: 0.5, 0.1, 0.3 and 0.6; GTLS-3: 1.0, 0.2, 0.6 and 1.2 g/l). Samples were assessed for motility and membrane integrity by computer-assisted sperm analysis immediately after dilution and at 24, 48 and 72 h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72 h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS-3 (control vs GTLS-1 and GTLS-2 p < 0.05, control vs GTLS-3 p < 0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1-3 and percentage of membrane-intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p < 0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled-stored dog semen.

Detection of pathogens in Boidae and Pythonidae with and without respiratory disease.

Respiratory diseases in boid snakes are common in captivity, but little information is available on their aetiology. This study was carried out to determine the occurrence of lung associated pathogens in boid snakes with and without respiratory signs and/or pneumonia. In total, 80 boid snakes of the families Boidae (n = 30) and Pythonidae (n = 50) from 48 private and zoo collections were included in this survey. Husbandry conditions were evaluated using a detailed questionnaire. All snakes were examined clinically and grouped into snakes with or without respiratory signs. Tracheal wash samples from all snakes were examined bacteriologically as well as virologically. All snakes were euthanased, and a complete pathological examination was performed. Respiratory signs and pneumonia were detected more often in pythons than in boas. An acute catarrhal pneumonia was diagnosed more often in snakes without respiratory signs than in snakes with respiratory signs, which revealed fibrinous and fibrous pneumonia. Poor husbandry conditions are an important trigger for the development of respiratory signs and pneumonia. Different bacterial pathogens were isolated in almost all snakes with pneumonia, with Salmonella species being the most common. Ferlavirus (formerly known as ophidian paramyxovirus)-RNA was detected only in pythons. Inclusion body disease was rarely seen in pythons but often in boas. Adenovirus and Mycoplasma were other pathogens that were diagnosed in single snakes with pneumonia. In living boid snakes with respiratory signs, tracheal wash samples were found to be a useful diagnostic tool for the detection of viral and bacterial pathogens.

Discrepancy between minimal inhibitory concentration to enrofloxacin and mutations present in the quinolone-resistance determining regions of Mycoplasma gallisepticum field strains.

Molecular characterization of the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV in 93 Mycoplasma gallisepticum field strains isolated in different geographic regions revealed discrepancies between minimal inhibitory concentration values and presence of amino acid substitutions within the QRDRs of GyrA and ParC in 9/93 (10%) strains. This may delimitate applicability of a gene-based assay to detect fluoroquinolone resistance in this avian pathogen.

Multilocus sequence typing of Mycoplasma agalactiae.

Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. We have developed a multilocus sequence typing (MLST) scheme using the sequenced genomes of the M. agalactiae strains PG2 and 5632. An MLST scheme based on the genes gltX, metS, gyrB, tufA and dnaA was designed and in total 3468 bp of sequence were analysed for each strain. MLST offers a highly discriminatory typing method for M. agalactiae and was capable of subdividing 53 strains into 17 distinct sequence types, largely according to geographical origin. MLST detected unexpected diversity in recent isolates from Spain, identifying two novel outliers, and enabled typing of novel Mongolian isolates for the first time. Genetic diversity in the sequenced regions was largely due to mutation, with recombination playing a much smaller role. A web-accessible database has been set up for this MLST scheme for M. agalactiae: http://pubmlst.org/magalactiae/. MLST offers a robust, objective molecular epidemiological tool for M. agalactiae that that enables interlaboratory comparison of data.

Detection of Mycoplasma bovis by in-situ hybridization and expression of inducible nitric oxide synthase, nitrotyrosine and manganese superoxide dismutase in the lungs of experimentally-infected calves.

Pneumonic lesions occurring in calves after respiratory infection with Mycoplasma bovis are characterized by subacute or chronic suppurative bronchopneumonia with multiple foci of necrosis and by persistence of M. bovis antigen, which is frequently associated with phagocytes at the periphery of the necrotic foci. The aims of this study were: (1) to investigate the expression of inducible nitric oxide synthase (iNOS), nitrotyrosine (NT) and manganese superoxide dismutase (Mn-SOD) in the lung lesions of calves infected experimentally with M. bovis, and (2) to analyse the distribution and localization of M. bovis DNA by in-situ hybridization and correlate these findings with the immunohistochemical detection of M. bovis antigen. Phagocytic cells infiltrating the lung tissue were characterized using the markers CD68, S100A8 and S100A9. Lung tissue from 18 infected calves and three non-infected controls were examined. All infected calves had an increased number of cells expressing iNOS, NT and Mn-SOD in the inflamed lung tissue. These molecules were most strongly expressed by macrophages demarcating necrotic areas, by altered bronchiolar epithelial cells and by macrophages within obliterated bronchioles. Co-localization of M. bovis DNA, M. bovis antigen and macrophages expressing iNOS, NT and Mn-SOD was observed. These findings suggest that the generation of reactive oxygen and nitrogen species is involved in the development of severe chronic lung damage in M. bovis infection.

Prevalence of potentially pathogenic bacteria as genital pathogens in dairy cattle.

Bacteria on the genital mucosa have been studied less in healthy, non-puerperal cows than in cows with puerperal endometritis. We have thus analysed bacteria in swabs from the vagina and cervix of post-puerperal cattle (n = 644). Out of the animals, 6.8% had aborted within the last 12 months, 2.6% and 11.6% showed signs of vaginitis and endometritis, respectively. In 17.2% of cervical swabs pathogenic gram-positive and in 11.5% pathogenic gram-negative bacteria were found. Arcanobacterium pyogenes was isolated from 41.3% of cows with endometritis and from 3.5% without endometritis (p < 0.05). From 12.5% of cows with abortion but from no cow without abortion, Staphylococcus aureus was recovered (p < 0.05). Out of 383 vaginal swabs, 88.3% were positive. In 3.4% of swabs pathogenic gram-positive and in 16.7% pathogenic gram-negative microorganisms were found. The percentage of positive vaginal swabs did not differ between pregnant and non-pregnant animals. In the genital tract, the percentage of swabs positive for normal mucosal bacteria decreased from caudally to cranially (p < 0.05). Pathogenic bacteria were found more often in cervical than in vaginal swabs (p < 0.05). In conclusion, bacteria on the vaginal and cervical mucosa in cattle involve a wide range of species. In animals without endometritis or vaginitis, colonization of the mucosa rather than infection has to be assumed.

Infections with weakly haemolytic Brachyspira species in pigs with miscellaneous chronic diseases.

The prevalence of infections with different Brachyspira species was assessed in 202 pigs with various chronic herd problems using different methods. Twenty-seven pigs (13.4%) were positive for Brachyspira spp. with at least one of the methods used. The highest number of positives was identified with mucosal scraping-PCR (23), followed by PET-PCR (22) and bacteriological-biochemical analysis (15). With the exception of three cases of B. pilosicoli infections, only weakly pathogenic Brachyspira species were identified. The majority was B. murdochii, followed by B. innocens and B. intermedia. Concurrent infections with two or more Brachyspira species were common and accounted for 37.1% of the total. Presence of weakly haemolytic Brachyspira was associated with wasting and diarrhoea in a number of cases. This investigation shows that infections with weakly haemolytic Brachyspira spp. may contribute to colonic pathology in pigs with chronic herd problems and that mixed infections seem to occur more frequently than previously noticed.