Epidemiological tracing of bovine tuberculosis in Switzerland, multilocus variable number of tandem repeat analysis of Mycobacterium bovis and Mycobacterium caprae.

BACKGROUND: After 15 years of absence, in 2013 bovine tuberculosis (bTB), caused by Mycobacterium (M.) bovis and M. caprae, reemerged in the Swiss dairy cattle population. In order to identify the sources of infection as well as the spread of the agents, molecular-epidemiologic tracing by MIRU-VNTR analysis in combination with spoligotyping was performed. A total of 17 M. bovis and 7 M. caprae isolates were cultured from tuberculous bovine lymph nodes and analyzed with a set of 49 genetic markers by using automated capillary electrophoresis. RESULTS: The outbreak in the western part of Switzerland was caused by M. bovis spoligotype SB0120. With the exception of four single-locus variations observed in MIRU 20, the MIRU-VNTR profiles of the 17 M. bovis isolates were identical, indicating a single source of infection. M. bovis detected in one archival bovine specimen from the outbreak region showed an identical MIRU-VNTR profile, suggesting persistence of the agent in a dairy herd for nearly fifteen years. The outbreak in the eastern part of Switzerland was caused by M. caprae spoligotype SB0418. All Swiss M. caprae isolates showed the Lechtal-type MIRU-VNTR profile, described as endemic in wild ruminants and in dairy cattle in Austrian bordering regions. This suggests the agent was most likely introduced by Swiss dairy cattle summering on Austrian pastures. CONCLUSIONS: The present study is the first MIRU-VNTR analysis of Swiss bTB mycobacterial isolates. The genotyping assay was found to be highly discriminating and suitable for the epidemiological tracing of further outbreaks. These findings will contribute to the development of an international MIRU-VNTR database aiming to improve bTB surveillance.

A novel multiplex qPCR targeting 23S rDNA for diagnosis of swine dysentery and porcine intestinal spirochaetosis.

BACKGROUND: A multiplex qPCR targeting a 128 bp region on the 23S rDNA gene was developed for detection of Brachyspira (B.) hyodysenteriae and B. pilosicoli, the agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), together with a triplet of apathogenic Brachyspira spp. (B. innocens, B. intermedia, B. murdochii) in porcine feces. The multiplex qPCR was evaluated against a duplex PCR (La et al., J Clin Microbiol 41:3372-5, 2003). RESULTS: Using DNA extracted from fecal culture, the multiplex qPCR showed excellent agreement with the duplex PCR (κ = 0.943 and 0.933). In addition, thanks to the three probes whereof one detecting the apathogenic Brachyspria spp., a more diversified overview of the brachyspiral flora in porcine fecal samples can be delivered as a part of the routine diagnostic. The multiplex qPCR with a limit of detection of 5-10 genomic equivalents (GE) per reaction (6 × 102 GE per gram) allows reliable detection of Brachyspira species directly from fecal swab DNA. In line with this, analysis of 202 fecal swabs in comparison with culture-based qPCR showed a high agreement for the causative agents of SD (B.hyodysenteriae: κ = 0.853, sensitivity 87% specificity 98%). CONCLUSION: The novel multiplex qPCR is robust and has a high analytical sensitivity and is therefore suitable for high-throughput screening of porcine fecal swabs for the causative agents of SD. This assay can therefore be used for the direct proof of the pathogenic B. spp. in fecal swabs within the scope of a monitoring program.

First screening for Brachyspira hampsonii in Swiss pigs applying a new high resolution melting assay.

A new High Resolution Melting (HRM) assay was developed for the rapid detection of Brachyspira (B.) hampsonii. B. hampsonii occurs in different European countries, however, until today it has not been encountered in Switzerland. Four B. hampsonii reference strains were used to develop the HRM assay: B. hampsonii clade I ATCC BAA2463 and clade II ATCC BAA2464 strain, as well as two isolated strains P280/1 from the UK and the German isolate 5369-1x/12. A conserved region of the nox gene was used to design B. hampsonii-specific primers. The HRM melting curves for the four reference strains showed reproducible difference graphs with distinct differences between the four strains based on a slight variation between the four amplicon sequences. In addition, DNA from 22 B. hampsonii strains representing four genetic B. hampsonii groups was used to validate the method. Melting temperatures in the interval between 73.1 and 74°C were obtained for all B. hampsonii strains and allow differentiating B. hampsonii from other Brachyspira species. In total 897 Swiss porcine fecal Brachyspira isolates, cultured between 2009 and 2015, were analysed by the HRM protocol. B. hampsonii was not detected among these Swiss Brachyspira isolates. In conclusion, the rapid and low-cost HRM approach allows a sensitive and specific identification of B. hampsonii.

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis.

BACKGROUND: Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP. RESULTS: By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%). CONCLUSIONS: There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.

Risk factors for invasive reptile-associated salmonellosis in children.

Reptile-associated salmonellosis (RAS) in children has been reported primarily due to direct contact with turtles, but recently also due to indirect contact with more exotic reptiles, causing disease in infants. To evaluate risk factors for RAS, we reviewed the RAS cases published in the literature since 1965. A case was defined as a child ≤18 years of age with an epidemiological link by identification of Salmonella enterica in cultures from both the affected child and the exposed reptile. We identified a total of 177 otherwise healthy children (median age 1.0 years, range 2 days to 17.0 years). RAS manifested mainly with gastrointestinal disease, but 15% presented with invasive RAS, including septicemia, meningitis, and bone and joint infection. The children with invasive RAS were significantly younger than children with noninvasive disease (median age 0.17 and 2.0 years, p<0.0001). RAS is most frequently seen after exposure to turtles (42%). However, children with invasive RAS had been exposed more often (p≤0.001) to reptiles other than turtles, including iguanas, bearded dragons, snakes, chameleons, and geckos. Children exposed to those latter reptiles usually kept indoors were younger than children exposed to turtles mostly kept outdoors (p<0.0001). RAS in children is significantly associated with invasive disease at young age, in particular infants <6 months of age. Exposure to reptiles, other than turtles, kept indoors is associated with RAS at younger age and more invasive disease. This finding is helpful for recognizing or even preventing invasive RAS in young infants that are at highest risk.

Differences in the antigen structures of Corynebacterium pseudotuberculosis and the induced humoral immune response in sheep and goats.

Caseous lymphadenitis (CLA), a disease affecting sheep and goats, is caused by Corynebacterium pseudotuberculosis (Cp). Eradication programs are based on the serological identification of Cp infected animals. However, available diagnostic ELISAs are not similarly suitable for sheep and goats. In the present study the comparison of antigens revealed major species specific differences between sheep and goat derived Cp field isolates as well as between field isolates and the Cp ATCC reference strains. Furthermore, we found species-specific differences in the anti-Cp humoral immune response between sheep and goats. The analysis of band frequency was able to distinguish between immunodominant and non-immunodominant protein bands. The 150 kDa, 74 kDa, 48 kDa, and 30 kDa antigens were immunodominant in both, sheep and goats. Interestingly, the most commonly used diagnostic antigen, i.e. the 30 kDa phospholipase D (PLD), was recognized by 100% of the Cp positive goats but only by 70% of the Cp positive sheep. Furthermore, analysis of field sera revealed that there were a particular percentage of Cp positive sera which reacted negative with the PLD. In conclusion our results clearly showed that (1) the application of a combination of further defined immunodominant Cp antigens - in addition to the PLD antigen - and (2) consideration of species-specific differences in the anti-Cp immune response will substantially contribute to the improvement of Cp serological diagnostics and to effective eradication programs in both sheep and goats.

Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis or M. caprae has recently (re-) emerged in livestock and wildlife in all countries bordering Switzerland (CH) and the Principality of Liechtenstein (FL). Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa) and red deer (Cervus elaphus elaphus), act as bTB reservoirs elsewhere in continental Europe. Our aims were (1) to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2) to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC). Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar (3.6%; 95% CI: 1.4-7.8) but none of the 269 red deer (0%; 0-1.4). M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.

ESBL-producing uropathogenic Escherichia coli isolated from dogs and cats in Switzerland.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia (E.) coli have emerged in human and veterinary medicine. The aim of this study was to investigate the presence of ESBL-producers among uropathogenic E. coli isolated from dogs and cats and to characterize detected ESBL-producing isolates by antibiotic susceptibility testing, identification of ESBL genes, multi-locus sequence typing (MLST), detection of putative virulence genes, and analysis of E. coli phylogroups. Among the 107 E. coli isolates derived from urinary samples (59 from dogs, 40 from cats), eight isolates from four different animals (two dogs, two cats) were found to be ESBL-producers. These isolates were of ST533/CTX-M-15/TEM/phylogroup B1 (four strains from one dog), ST410/CTX-M-15/TEM/phylogroup A (three strains, one from a dog and two from a cat), and ST648/CTX-M-15/phylogroup D (one strain from a cat). In terms of putative virulence factors, all isolates harbored lpfA, sat, and tsh, whereas iss was only detected in strains of ST533. Thus, ESBL-producers were detected among uropathogenic E. coli from Swiss companion animals and the eight CTX-M-15-producing isolates belonged to three sequence types (ST410, ST533, ST648) and three E. coli phylogroups (A, B1, D). For the first time, E. coli of ST533 carrying bla(CTX-M-15) were thereby detected in a dog.

Nanotransformation of the haemotrophic Mycoplasma suis during in vitro cultivation attempts using modified cell free Mycoplasma media.

Mycoplasma suis belongs to haemotrophic mycoplasmas (HMs) which cause infectious anaemia in a large variety of mammals. To date, no in vitro cultivation system for M. suis or other HMs has been established. We hypothesised that M. suis could grow in classical Mycoplasma media supplemented with nutrients (e.g. glucose, iron-binding proteins) which are naturally available from its host environment, the porcine blood. Blood from experimentally M. suis-infected pigs was used to inoculate either standard SP-4 Mycoplasma medium supplemented with iron-binding proteins (transferrin, haemin, and haemoglobin) or glucose-enriched Hayflick Mycoplasma medium. A quantitative M. suis-specific real-time PCR assay was applied to determine and quantify M. suis loads weekly during 12 week-incubation. The first 2 weeks after inoculation M. suis loads decreased remarkably and then persisted at a stationary level over the observation time of 12 weeks in iron-binding protein- or glucose supplemented media variants. Scanning electron microscopic analysis of liquid M. suis sub-cultures on Hayflick agar showed small, densely-packed microcolonies of irregular M. suis cells of reduced size (0.2-0.6µm) indicating nanotransformation. The partial 16S rDNA sequence of these cultured M. suis nanocells was 99.9% identical to M. suis. M. suis cells derived from liquid cultures interact in vitro with porcine erythrocytes by fibril-like structures. We conclude, that the modified Mycoplasma media used for M. suis cultivation are obviously unfavourable for growth but lead to culture persistence. M. suis adapt to inappropriate culture conditions by alteration into nanoforms.

The surface-localised α-enolase of Mycoplasma suis is an adhesion protein.

Mycoplasma suis belongs to the haemotrophic mycoplasmas which colonise red blood cells of a wide range of vertebrates. Adhesion to red blood cells is the crucial step in the unique lifecycle of M. suis. Due to the lack of a cultivation system, identification of adhesion structures has been difficult. So far, only one adhesion protein, i.e. MSG1 was identified. In order to determine further adhesion molecules of M. suis, we screened genomic M. suis libraries and performed Southern blot hybridisation analyses of genomic M. suis DNA. The α-enolase of M. suis was identified and analysed genetically and functionally. The encoding gene has 1623 bp in size. The deduced amino acid sequence showed an overall identity of 59.6-65.1% to α-enolases of other pathogenic mycoplasmas. The 540 aa M. suis α-enolase displays a size extension of about 90 aa in comparison to α-enolases of other mycoplasmas. Recombinant α-enolase expressed in Escherichia coli demonstrated immunogenicity in experimentally infected pigs. Immunoblot, confocal laser scanning microscopy and immune electron microscopy analysis using antibodies against recombinant α-enolase, indicate the membrane and surface localisation of native α-enolase in M. suis, though no typical signal sequences exist. Furthermore, we showed that recombinant α-enolase binds to porcine erythrocyte lysate in a dose-dependent manner. E. coli transformants which express α-enolase on their surface acquire the ability to adhere to porcine red blood cells. In conclusion, our observations indicate that α-enolase could be involved in the adhesion of M. suis to porcine red blood cells.

Complete genome sequence of the hemotrophic Mycoplasma suis strain KI3806.

Mycoplasma suis, a member of the hemotrophic mycoplasma (HM) group, parasitize erythrocytes of pigs. Increasing evidence suggests that M. suis is also a zoonotic agent. Highly pathogenic strains of M. suis (e.g., M. suis KI3806) have been demonstrated to invade erythrocytes. This complete sequenced and manually annotated genome of M. suis KI3806 is the first available from this species and from the HM group. The DNA was isolated from blood samples of experimentally infected pigs due to the lack of an in vitro cultivation system. The small circular chromosome of 709,270 bp, encoding an unexpectedly high number of hypothetical proteins and limited transport and metabolic capacities, could reflect the unique lifestyle of HM on the surface of erythrocytes.

Detection of Candidatus Mycoplasma haemobos in cattle with anaemia.

Haemotrophic mycoplasmas are unculturable eperythrocytic pathogens that are found in a wide range of domestic and wild animals. In this study an outbreak of haemotrophic mycoplasmosis in cattle herds in Northern Germany is reported. Affected animals exhibited anaemia and depression and infection was confirmed following microscopic examination of blood smears and on PCR. Sequence analysis indicated that in addition to infection with Mycoplasma wenyonii, animals were infected with a novel bovine haemotrophic mycoplasma Candidatus Mycoplasma haemobos.

The effect of Chlamydophila pneumoniae Major Outer Membrane Protein (MOMP) on macrophage and T cell-mediated immune responses.

The Major Outer Membrane Protein (MOMP) belongs to the membrane complex of cysteine-rich proteins of Chlamydophila pneumoniae. Although MOMP can elicit strong immune responses it fails to confer long-term protection against infection in animal models. This effect has been attributed, at least in part, to an inadequate induction of protective Th1-mediated immune responses. In an effort to understand the cellular mechanisms associated to the immunomodulatory properties of MOMP we studied the effect of this protein on mouse macrophages and naïve T-lymphocytes. We found that incubation of mouse macrophages with recombinant MOMP (rMOMP) results in an increased secretion of MMP-9 and a down-regulation of MHC class II, CD86 and CD40. This was accompanied by an increase in IL-10 and IFNγ but not in IL-12 secretion. rMOMP induced a down-regulation of the expression of CD69 and CD154 markers by activated CD4(+) T cells, and enhanced the secretion of IL-2 and IL-10 by these cells. Conversely, rMOMP-treated macrophages up-regulated the expression of CD69 but not CD154, inhibited the synthesis of IL-10 and up-regulated the production of IFNγ by activated CD8(+) T cells. Immunization of mice with MOMP induced the synthesis only of MOMP-specific IgG1 but no differences in cytokine profile were observed compared to controls. Our results provide new evidence on the role of MOMP in modulating T cell-mediated immune responses.

Inorganic pyrophosphatase in uncultivable hemotrophic mycoplasmas: identification and properties of the enzyme from Mycoplasma suis.

BACKGROUND: Mycoplasma suis belongs to a group of highly specialized hemotrophic bacteria that attach to the surface of host erythrocytes. Hemotrophic mycoplasmas are uncultivable and the genomes are not sequenced so far. Therefore, there is a need for the clarification of essential metabolic pathways which could be crucial barriers for the establishment of an in vitro cultivation system for these veterinary significant bacteria.Inorganic pyrophosphatases (PPase) are important enzymes that catalyze the hydrolysis of inorganic pyrophosphate PPi to inorganic phosphate Pi. PPases are essential and ubiquitous metal-dependent enzymes providing a thermodynamic pull for many biosynthetic reactions. Here, we describe the identification, recombinant production and characterization of the soluble (s)PPase of Mycoplasma suis. RESULTS: Screening of genomic M. suis libraries was used to identify a gene encoding the M. suis inorganic pyrophosphatase (sPPase). The M. suis sPPase consists of 164 amino acids with a molecular mass of 20 kDa. The highest identity of 63.7% was found to the M. penetrans sPPase. The typical 13 active site residues as well as the cation binding signature could be also identified in the M. suis sPPase. The activity of the M. suis enzyme was strongly dependent on Mg2+ and significantly lower in the presence of Mn2+ and Zn2+. Addition of Ca2+ and EDTA inhibited the M. suis sPPase activity. These characteristics confirmed the affiliation of the M. suis PPase to family I soluble PPases. The highest activity was determined at pH 9.0. In M. suis the sPPase builds tetramers of 80 kDa which were detected by convalescent sera from experimentally M. suis infected pigs. CONCLUSION: The identification and characterization of the sPPase of M. suis is an additional step towards the clarification of the metabolism of hemotrophic mycoplasmas and, thus, important for the establishment of an in vitro cultivation system. As an antigenic and conserved protein the M. suis sPPase could in future be further analyzed as a diagnostic antigen.

Haemotrophic Mycoplasma infection in horses.

Haemotrophic mycoplasmas (HM) are parasites on the surface of red blood cells and known to infect a wide range of animals. However, there are no previous evidences of HM infections in horses. In this study HM were detected for the first time in the blood of two horses suffering from poor performance, apathy, weight loss, and anaemia. Using a HM specific PCR assay and subsequent sequencing the infective agents isolated from the blood of said horses were confirmed as closely related to the HM species Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos'.

Occurrence of Mycoplasma suis in wild boars (Sus scrofa L.).

Porcine infectious anemia is a well-known disease that occurs worldwide and is caused by the uncultivable hemotrophic bacterium Mycoplasma suis. In this study the occurrence of M. suis in wild boars was investigated by employing a quantitative real-time LightCycler PCR. M. suis infections were detected in 36 out of 359 wild boars (10.03%). Sequencing of the 16S rRNA gene and subsequent phylogenetic analysis revealed the existence of two genetically distinct M. suis subtypes in the wild boar population: one subtype was >99.0% identical to known American and European M. suis isolates, and the second subtype showed the highest homology to known Chinese isolates. In summary, this is the first detection of M. suis in wild boars. The role of M. suis as pathogen in wild boars has yet to be established, but the present findings revealed a possible wildlife reservoir for these bacteria.

Complement C3 in Bernese Mountain dogs.

BACKGROUND: Previous research suggests that low serum concentrations of the third component of complement (C3) are associated with both the susceptibility to infectious agents such as Borrelia burgdorferi and the development of glomerular disease. We hypothesized that low levels of C3 are associated with the coincident occurrence of B. burgdorferi infection and glomerulonephritis in Bernese Mountain dogs. OBJECTIVES: The aims of this study were to evaluate the serum concentration of C3 in Bernese Mountain dogs with and without antibodies against B. burgdorferi and to compare this concentration with that of healthy control dogs. METHODS: Eighty-three clinically healthy Bernese Mountain dogs and 46 control dogs were included. Antibodies against B. burgdorferi were determined using an ELISA with a whole cell sonicate as antigen. Results were confirmed using Western blot. C3 was measured using a single radial immunodiffusion test. Results were reported as the percentage concentration of C3 compared with that in pooled preserved canine serum (100% C3 concentration). RESULTS: Median C3 concentration was 128.5% in Bernese Mountain dogs with antibodies against B. burgdorferi, 133.5% in B. burgdorferi-negative Bernese Mountain dogs, 87.8% in positive control dogs, and 102.2% in negative control dogs. Within Bernese Mountain and control groups, C3 was lower in dogs with antibodies against B. burgdorferi compared with those without. Percentage concentration of C3 was higher in healthy Bernese Mountain dogs compared with control dogs. CONCLUSION: Low C3 concentration is not an explanation for the high prevalence of B. burgdorferi infections and glomerular disease in Bernese Mountain dogs.

In vivo transmission studies of 'Candidatus Mycoplasma turicensis' in the domestic cat.

The natural transmission routes of the three feline haemotropic mycoplasmas--Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum', and 'Candidatus Mycoplasma turicensis' (CMt)--are largely unknown. Since CMt has been detected in the saliva of infected cats using PCR, we hypothesised that direct transmission via social or aggressive contact may occur. The aim of this study was to evaluate this transmission route. CMt-positive saliva and blood samples were obtained from three prednisolonetreated specific pathogen-free (SPF) cats that were infected intraperitoneally with CMt. Five SPF cats were inoculated with CMt-positive saliva or blood subcutaneously to mimic cat bites, and five cats were inoculated orally with blood or oronasally with saliva to mimic social contact. Blood samples were monitored for CMt infection using quantitative real-time PCR and for seroconversion using a novel western blot assay. Neither oronasal nor subcutaneous inoculation with CMt-positive saliva led to CMt infection in the recipient cats, as determined by PCR, independent of prior prednisolone treatment. However, when blood containing the same CMt dose was given subcutaneously, 4 of the 5 cats became PCR-positive, while none of the 5 cats inoculated orally with up to 500 microL of CMt-positive blood became PCR-positive. Subsequently, the latter cats were successfully subcutaneously infected with blood. All 13 CMt-exposed cats seroconverted. In conclusion, CMt transmission by social contact seems less likely than transmission by aggressive interaction. The latter transmission may occur if the recipient cat is exposed to blood from an infected cat.

Follow-up of Bernese Mountain dogs and other dogs with serologically diagnosed Borrelia burgdorferi infection: what happens to seropositive animals?

BACKGROUND: Data on the long-term outcome of B. burgdorferi infections in adult dogs are sparse. The aim of the present study was to investigate whether Bernese Mountain dogs with serological evidence of natural B. burgdorferi infection more often develop signs such as lameness, azotemia or proteinuria during a follow-up period of 2.5 to 3.0 years. Seropositive Bernese Mountain dogs were compared to seronegative Bernese Mountain dogs and to seropositive and seronegative control dogs of other breeds. Dogs included in a previous study on the prevalence of antibodies against B. burgdorferi in Bernese Mountain dogs were re-evaluated. Antibodies against B. burgdorferi were determined using an ELISA with a whole-cell sonicate as antigen and results were confirmed using a Western blot assay. RESULTS: Fifty-three Bernese Mountain dogs and 30 control dogs were re-evaluated. Re-evaluation was performed between 2.5 and 3.0 years (median 2.7 years) after the first assessment.The age of the dogs at the second evaluation ranged from 3 to 11 years (median 6 years). There were no significant differences with regard to poor general condition or lameness between the first and the second evaluation. At the first evaluation 22 (42%) of the Bernese Mountain dogs and 11 (37%) of the control dogs were considered positive for antibodies against B. burgdorferi. At the second evaluation 25 (47%) of the Bernese Mountain dogs and 12 (40%) of the control dogs were considered positive; 69% of the dogs showed the same serological result at both examinations and 31% were seroconverted or seroreverted. During the first examination, azotemia was diagnosed in 6 Bernese Mountain dogs and during the second examination in 11 Bernese Mountain dogs. No control dogs had azotemia in this study. In seropositive dogs there was no increase in lameness or signs of renal disease over time. CONCLUSION: It may be concluded that antibodies against B. burgdorferi determined by whole cell ELISA and confirmed by Western blot were neither associated with the development of lameness nor with signs of renal disease like azotemia or proteinuria in dogs observed over a period of 2.5 to 3.0 years.