Is canine leptospirosis underdiagnosed in southern Ontario? A case report and serological survey

An eight-year-old city-dwelling Cairn Terrier was presented to a veterinary hospital in acute renal failure with evidence of hepatic insufficiency. The dog was treated symptomatically over three days, during which time vomiting was largely controlled, but it became jaundiced as hepatic insufficiency worsened. Leptospira pomona was demonstrated in large numbers by immunofluorescent staining of urinary sediment. It was isolated and its identity confirmed as L. pomona genotype kennewicki. The source of the infection was thought to be raccoons.

Sera from 474 blood samples submitted for diagnostic purposes to two clinical pathology laboratories in southern Ontario were examined with the microscopic agglutination test for antibodies to selected leptospiral serovars. Of the sera tested, 39.2% reacted at titers ≥1:100 with one or more serovars, the majority of all sera (26.2%) reacting at low titers to canicola or icterohaemorrhagiae, or both. These reactions likely resulted from vaccination. A smaller proportion reacted to other serovars tested: autumnalis (3.8%), bratislava (8.2%), grippotyphosa (1.9%), hardjo (3.0%), and pomona (3.2%). Among dogs reacting to these latter serovars (other than bratislava), many had broadly cross-reacting and relatively high titers. One dog with a titer of 1:800 to pomona had had a disease typical of leptospirosis two years previously. Three other dogs with high titers to autumnalis, bratislava, or mixed serovars had clinical histories compatible with leptospirosis.

We suggest that leptospiral bacterins for dogs in Ontario be broadened to include at least serovars autumnalis and pomona.

     

Epidemiology of leptospirosis in North-Central Italy: Fifteen years of serological data (2002–2016)

Leptospirosis is a re-emerging bacterial zoonosis. North-Central Italy is characterized by a geographic area that promote Leptospira circulation. Data on sero-epidemiological survey carried out from 2002 to 2016 in North-Central Italy were reported and discussed. Overall, 709 out of the 8488 (8.35%) tested sera were positive for Leptospira at the cut-off titer (1:100) and 218 (2.57%) at higher titer (≥1:400). The highest percentages of positivity was recorded for coypus (22.86%), swine (19.74%) and bovine (13.03%). Pomona and Australis resulted the serogroup more often detected, followed by Sejroe and Icterohaemorrhagiae; while, a low number of positive sera was detected for serogroups Ballum, Canicola and Tarassovi. Percentage of positive sera for each year slightly decreased from 2002 to 2008 and rose from 2009. High percentages of positive reactions were recorded in 2014 (17.23%), 2015 (19.61%) and 2016 (38.05%). In conclusion, the results of this investigation reported an increase of leptospirosis in North-Central Italy. Furthermore, several animals resulted infected as accidental hosts by unusual Leptospira serovars. These data could suggest a change in host range for some serovars, that may promote the adaptation to new hosts.     

An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G₁ that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay.

At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation

     

Leptospirosis in sheep in Western Canada

A survey of 930 ovine sera and kidneys from 33 sheep was conducted to assess the rate of leptospiral infection in sheep slaughtered in Alberta. Sera were tested for the presence of agglutinins to indigenous serovars of Leptospira interrogans. Kidneys with gross lesions were examined for the presence of leptospires by means of an indirect fluorescent antibody test (FAT) and by culture. Antibodies to serovars pomona and hardjo were present at rates of 1.0% and 0.4%, respectively, in sheep from Saskatchewan, Alberta and British Columbia. Sera from 120 feedlot lambs shipped from Oregon reacted to serovars pomona, hardjo and grippotyphosa at rates of 1.7%, 61.7% and 59.1%, respectively. Fluorescent antibody test detected serovars (presumptively) hardjo in 52% of Oregon feedlot lambs and grippotyphosa in 32% of the same group, a finding supported by the isolation of both these serovars from a pool of two fluorescent antibody test-positive kidneys. The grippotyphosa strain was highly virulent for hamsters, producing intense icterus and death. Leptospires, presumptively serovar grippotyphosa were demonstrated by fluorescent antibody test in one Alberta lamb kidney. The possibility of spreading leptospirosis by movement of breeding stock through public facilities and by assembling lambs in feedlots is discussed.     

Survey of leptospiral agglutinins in the sera of swine of southeastern Alabama

The occurrence of leptospirosis in swine of southeastern Alabama was determined. A total of 627 sera were tested, using the microscopic agglutination method, with live antigens of 12 serovars. Of the sera tested, 121 (19.3%) had a titer of 1:100 or greater to the serovars employed. The percentage distribution of sera with titers of greater than or equal to 1:100 among serovars most commonly reported was as follows: Leptospira interrogans serovars pomona, 3.8%; icterohaemorrhagiae, 3.3%; canicola, 1.6%; hardjo, 0.7%; and grippotyphosa, 0.16%. Of the less commonly recognized leptospiral serovars, the percentages reacting were as follows: ballum, 4.9%; autumnalis, 3.2%; pyrogenes, 1.1%; and bataviae, 0.4%. None of the sera reacted with antigen of serovars australis, tarassovi, or wolffi.     

Prevalence of antibodies against Leptospira sp. in snakes,lizards and turtles in Slovenia

Background

Leptospiral infections in poikilothermic (cold blooded) animals have received very little attention and the literature concerning natural infections of these animals is limited. The aim of this study was to determine the prevalence of leptospiral antibodies in reptiles, imported into Slovenia and intended to be pets in close contact with humans. A total of 297 reptiles (22 snakes, 210 lizards and 65 turtles) were tested for specific antibodies against serovars of Leptospira interrogans sensu stricto using the microscopic agglutination test (MAT). Live cultures of different serovars were used as antigens. MAT was performed according to standard procedures and the degree of reaction was interpreted by estimating the percentage of agglutinated leptospires. Samples showing titres of ≥ 50 against one or more serovars were considered as positive.

Results

Antibodies against seven pathogenic serovars of L. interrogans sensu stricto were detected in 46 of 297 reptiles. Among 22 snakes, specific antibodies against pathogenic serovars of three Leptospira species (L. interrogans, L. kirschneri and L. borgpetersenii) at titre levels from 1:50 to 1:400 were detected in 6 snakes. In 31 of 210 lizards, specific antibodies were found in titres from 1:50 to 1:1000 and, finally, among 65 turtles (terrapins and tortoises), 9 had specific antibodies at titre levels between 1:50 and 1:1600. Animals imported from non-EU countries showed significantly higher prevalence (25.0%; 95 confidence interval: 16.7–33.3%) than animals from EU member states (10.4%; confidence interval: 6.1–14.7%).

Conclusions

Reptiles may be considered as potential reservoirs of L. interrogans sensu stricto. Origin of the animals is a risk factor for presence of leptospiral antibodies, especially in lizards. Special attention should be focused on animals from non-EU member states.     

A serological survey of leptospirosis in cats

A serological survey of 225 cats from different parts of the NorthIsland was conducted using 11 leptospiral serovar antigens. Twenty (8.8%) of the sera gave titres to the microagglutination test.The prevalence of titres to the serovars were as follows: five (2.22%) to copenhageni, five (2.22%) to hardjo, four (1.77%) to ballum, four (1.77%) to pomona, one (0.44%) to balanica and one (0.44%) to canicola; no titres to tarassovi, bataviae, pyrogenes and australis were recorded. It is hypothesised that prey-predator transmission between cats and rodents is responsible for infections with serovars copenhageni and ballum. Infections with the other serovars are presumed to result from contact with infected animals or a contaminated environment.     

Salmonella enterica subsp. enterica isolated from chicken carcasses and environment at slaughter in Reunion Island: prevalence, genetic characterization and antibiotic susceptibility

Salmonella contamination of 71 chicken broiler flocks was investigated at the slaughterhouse in Reunion Island between October 2007 and January 2009. Samples were collected from live broiler chickens and chicken carcasses as well as the slaughterhouse environment. Salmonella spp. was isolated from 40 of 71 (56 % with a confidence interval 5 % [45–67]) broiler chicken flocks at slaughter. The most prominent serovars were Blockley (31 %), Typhimurium and Brancaster (14 %), Hadar (10 %), Salmonella multidrug resistant clinical organisms serotypes 1,4,[5],12:i:-, and Virchow (8 %) and Livingstone, St. Paul, Seftenberg, Llandoff, Infantis and Indiana. At the farm, 27 % of the broiler chicken flocks tested positive for Salmonella spp. Salmonella spp. was isolated from 124 of 497 environmental samples (25 %). In most cases, there was no relationship between pulsed field gel electrophoresis (PFGE) pattern and antibiotic resistance pattern. The predominant Salmonella serovars were susceptible to most of the tested antibiotic drugs, but S. Hadar exhibited multidrug resistance. This study highlighted the primary source of Salmonella was the farm of origin and downstream stages in processing could not remedy to but amplify this Salmonella contamination.     

Identification of novel immunogenic proteins in pathogenic Haemophilus parasuis based on genome sequence analysis

Haemophilus parasuis causes contagious porcine Glässer's disease, which is occurring worldwide and leads to severe losses in the pig industry. To identify novel antigen candidates against this disease, 22 surface-exposed or secreted proteins were selected from the annotated H. parasuis genome by reverse vaccinology strategy. Expression of these proteins in Escherichia coli was attempted. Immunogenicity of the expressed candidates was assessed using Western blot analysis with mouse-derived antiserum prepared with whole bacteria of H. parasuis serovar 4 or 5. Three ABC-type transporters (OppA, YfeA and PlpA) and 1 curli protein assembly (CsgG) were identified as potent immunogenic proteins. The proteins show cross-reactions when tested with sera raised against serovars 4 and 5 of H. parasuis.     

Epidemiological investigation of Chlamydophila psittaci in pigeons and free-living birds in Croatia

During 2003, 278 adult pigeons (Columba livia) and 54 birds of 11 other free-living species were caught in the various locations in the City of Zagreb, Croatia. Sera from 182 pigeons were tested for the presence of antibodies against Chlamydophila (C.) psittaci by ELISA test and 174 of them (95.6%) were found positive. Because of the high positivity rate in sera, cloacal swabs of 278 pigeons as well as 54 other species of free-living birds were tested for the presence of C. psittaci antigen. Fourty-four of the 278 pigeons (15.83%) were antigen positive, whereas all 54 of the wild birds were negative. Antigen-positive pigeons were euthanised and examined pathomorphologically and cytologically. Findings of specific antibodies and antigen of C. psittaci confirmed the high rate of infection among urban pigeons in the City of Zagreb, fortunately not among other free-living birds. Although the pigeon serovars of C. psittaci are considered to be of moderate pathogenicity for humans, the identification of 15.8% antigen-positive birds represents a potential source of infection to humans, especially for elderly people and immunodeficient patients, as well as for poultry in the Zagreb city area.     

Immunological and molecular characterization of Leptospira interrogans isolated from a bovine foetus

Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine and can transmit the pathogen from animal to animal or animal to human. Thus, surveillance and monitoring systems for detection of new Leptospira serovars are important for the control of leptospirosis. Here, we report the isolation of a spirochete from a stillborn bovine foetus and its characterization by immunological and molecular techniques. A variable number tandem repeat profile using seven discriminatory primers identified the spirochete as belonging to species Leptospira interrogans serogroup Australis serovar Muenchen. A phenotypic analysis using monoclonal antibodies (mAbs) against leptospiral membrane-associated proteins confirmed the expression of important virulence and pathogenicity factors (LipL32 and LigBrep). Out of 120 reference sera tested, 22 positive (36.66%) and 9 negative (15%) also reacted with the new isolate. Furthermore, the serovar Muenchen isolate was virulent in hamster model. The animal inoculated developed acute lethal infection characterized by hepatic, pulmonary and renal lesions. Local isolates exhibited unique characteristics that differed from those of reference strains; therefore, isolation of leptospires is useful in the surveillance of local pathogenic serovars. In conclusion, the data obtained from this study can contribute to the epidemiological understanding and control of leptospirosis in southern Brazil.     

Prevalence of Leptospira interrogans antibodies in free-ranging Tayassu pecari of the Southern Pantanal, Brazil, an ecosystem where wildlife and cattle interact

We surveyed a wild population of white-lipped peccaries (Tayassu pecari) in the Brazilian Pantanal for evidence of Leptospira interrogans. Serum samples from 71 free-ranging T. pecari were obtained between 2003 and 2005 in the southern Pantanal of Mato Grosso do Sul state. We used microscopic microagglutination to test for antibodies against 14 L. interrogans serovars (antibody titers ≥1:100 were considered seropositive). Seventy percent of captured animals tested positive for leptospirosis antibodies. Antibodies against icterohaemorrhagiae and autumnalis serovars were the most prevalent. We used log-linear analyses to test for associations among seropositivity, age class, and sex of captured animals. Seropositivity was strongly associated with animal age class, but independent of sex. Forty-six percent of animals less than 2 years old, 63% of adults during peak reproductive years, and 100% of the oldest age class were seropositive. A nonparametric multivariate procedure (MRPP) showed that the composition of serovar antibody types changed with age, and ANOVA models demonstrated that antibody titers increased with age, suggesting long-term exposure to a greater number and variety (i.e., serovar types) of L. interrogans infections. This study presents the first quantitative survey of antibodies against L. interrogans serovars in a T. pecari population of the Pantanal. The high prevalence of leptospirosis antibodies in free-ranging white-lipped peccaries and the potential impacts on reproduction and population dynamics emphasize the need for further studies investigating the roles of Pantanal wildlife and livestock in the transmission and maintenance of L. interrogans in the environment.     

Serological survey of pre-weaned New Zealand fur seals (Arctocephalus forsteri) for brucellosis and leptospirosis

AIM: To conduct a longitudinal serological survey for evidence of Brucella spp and Leptospira spp infection of pre-weaned New Zealand fur seals in a colony on the Otago Peninsula.

METHODS: Seal pups were repeatedly captured on a monthly basis from February through to July 2001. Pups were tagged at first capture and a blood sample was taken at each capture event. A total of 163 sera were collected from 118 seal pups. Where sufficient volume was collected, the sera were tested for leptospirosis using the microscopic agglutination test (MAT), and for brucellosis using the competitive enzyme-linked immunosorbent assay (ELISA) for Brucella abortus.

RESULTS: None of 128 sera from 101 seals tested positive to the ELISA for B. abortus. All tests for Leptospira interrogans serovars Grippotyphosa, Copenhageni, Bratislava and Leptospira borgpetersenii serovar Ballum were negative at a cut-off of <1/100 dilution. Positive or suspicious titres were found to L. interrogans serovars Canicola and Pomona and L. borgpetersenii serovar Hardjo. The highest titres (12,800) were found to serovar Pomona. The titre to serovar Pomona in one seal rose from <1/50 in March to 12,800 in April and was <1/50 when re-sampled in July. The titre to serovar Pomona in another seal dropped from 12,800 in May to <1/50 in June. These seals also had titres to serovar Hardjo, which rose or fell in the same manner. All suspicious or positive titres occurred in late April and early May, when the pups were approximately 4–5 months old. In June and July, all seals tested were negative.

CONCLUSIONS: There was no serological evidence of Brucella infection in the pre-weaned fur seals at the colony. Positive titres to serovars Pomona, Hardjo, or Canicola suggest that a Leptospira species was present at the colony, however isolation or visualisation of the organism is required to confirm this. Care should be exercised when handling New Zealand fur seals to prevent human infection or inadvertent transfer of leptospirosis to another marine mammal species.     

Diagnostic specificity, sensitivity and cross-reactivity of an enzyme-linked immunosorbent assay for the detection of antibody against Leptospira interrogans serovars pomona, sejroe and hardjo in cattle.

Outer sheath antigen was prepared from Leptospira interrogans serovars pomona, sejroe and hardjo by treating the organisms with 1.0M NaC1 followed by 0.04% sodium dodecyl sulfate (SDS). Sodium dodecyl sulfate was removed from the SDS-protein complexes by the extraction of dodecyl sulfate anions as ion pairs with triethylammonium cations into an organic solvent. The outer sheath antigen was recovered from the organic solvent as a precipitate and used as the source of leptospiral enzyme-linked immunosorbent assay (ELISA) antigen. Utilizing this antigen, ELISA was adapted to detect bovine serum antibody to L. interrogans serovars pomona, sejroe and hardjo. The specificity of this assay in 344 bovine sera, which were negative in the microscopic agglutination test (MAT) for seven serovars, was 99.4%. In sera from 37 and 87 cattle which revealed MAT titers greater than or equal to 1:50 for L. interrogans serovars pomona and sejroe, the relative sensitivity of the test was 100%. The ELISA also showed a considerable degree of low level cross-reactivity with other serovars. Sixty-six (75.9%) out of 87 bovine sera which were MAT-positive (MAT titer of greater than or equal to 1:50) with serovars sejroe and hardjo only were ELISA positive with heterologous pomona antigen; 16 (43.2%) and six 16.2%) out of 37 bovine sera which were MAT positive MAT titer of greater than or equal to 1:50) with serovar pomona only were ELISA positive with heterologous sejroe and hardjo ELISA antigen respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimicrobial susceptibility and multi-drug resistance of Salmonella enterica subspecies enterica serovars in Sudan

This study was undertaken to determine the antimicrobial resistance patterns of Salmonella enterica subspecies enterica recovered from human, food, water, and animal samples collected in Khartoum State, Sudan. A total of 64 Salmonella isolates belonging to 28 different serovars were tested for their susceptibility to 13 antimicrobial agents. The majority of isolates (98.4 %) were resistant to at least one antimicrobial agent. Isolates were frequently resistant to ampicillin (90.6 %), cephalexin (50.0 %), nalidixic acid (25.0 %), streptomycin (21.9 %), kanamycin (18.8 %), gentamicin (17.2 %), and co-trimoxazole and trimethoprim (12.5 %). The most common pattern of multiple drug resistance included resistance to ampicillin and cephalexin. Most isolates were sensitive to chloramphenicol (98.4 %), ciprofloxacin (93.8 %), and norfloxacin (90.6 %). Two chicken- and the two human-origin S. Kentucky isolates were resistant to both ciprofloxacin and norfloxacin. All S. Kentucky isolates and the one S. Rissen isolate demonstrated multi-drug resistance. The results indicate the significance of multi-drug-resistant Salmonella serovars isolated from chickens and other animals and foods as sources for multi-drug-resistant Salmonella in humans in Sudan.     

Epidemiological Aspects in the Leptospira spp. and Toxoplasma gondii Infection in Horses from Botucatu,São Paulo,Brazil

Leptospirosis and toxoplasmosis are zoonoses with high importance because of the economic and public health impact. This study was aimed to determine the seroprevalence of leptospirosis and toxoplamosis in 714 serum samples of horses from different farms from Botucatu, São Paulo, Brazil. The samples were researched for Toxoplasma gondii antibodies by indirect immunofluorescence antibody test (IFAT) and for Leptospira spp. antibodies by microscopic agglutination test. Of 714 serum samples, 128 (17.9%; 95% CI: 15.3%-20.9%) were positive for one or more serovars of Leptospira spp., with icterohaemorrhagiae, canicola, and castellonis as the most prevalent serovars, whereas 42 (5.9%; 95% CI: 4.4%-7.9%) were positive for T gondii, of which 33 samples (78.57%; 95% CI: 64.0%-88.2%) presented a titer of 16, 7 (16.7%; 95% CI: 8.4%-30.7%) a titer of 64, and 1 (2.38%; 95% CI: 0.6%-12.3%) a titer of 256. No significant difference was found among the results obtained and the associated variables such as age and sex.     

A serosurvey for antibodies to Leptospira in dogs in the lower North Island of New Zealand

AIMS: To investigate the prevalence of antibodies to endemic and exotic Leptospira serovars in samples from a serum bank, collected from dogs in the lower North Island of New Zealand. METHODS: Sera (n=466), which had been collected from apparently healthy dogs, were screened using the microscopic agglutination test (MAT) for antibodies to serovars L. borgpeterseni serovar hardjo, L. interrogans serovars pomona, copenhageni and canicola, and L. kirschneri serovar grippotyphosa. RESULTS: Antibody to Leptospiral antigen was found in 14.2% of dogs tested. The highest level of reactivity was with serovar copenhageni, to which 9.5% (41/433) of sera were positive. Antibodies to serovars grippotyphosa and canicola were not detected in this population of dogs. CONCLUSIONS: Leptospira infection is relatively common in dogs in the lower North Island . CLINICAL RELEVANCE: Vaccination of dogs against leptospirosis should be considered using vaccine containing antigen to serovars hardjo, pomona and copenhageni. The presence of moderate levels of copenhageni antibody in dogs in the lower North Island raises the possibility that this serovar has become established in rodent populations in this region.     

A Serological Survey of Dogs for Brucella canis in Southwestern Ontario

Sera from 2 000 dogs from southwestern Ontario were tested for antibodies to Brucella canis by a rapid slide agglutination test. The 100 positively reacting sera were tested by tube agglutination and immunoprecipitation (gel diffusion) tests. Thirty-one of these sera gave suspicious titres and one a positive titre in the tube agglutination test. Six of the rapid slide test positive sera gave positive reactions in immunoprecipitation tests. One dog was identified which was found to have a history very suggestive of B. canis infection. It was judged that 0.3% of sera tested showed serological evidence of B. canis infection. The complexities of the serological diagnosis of B. canis infection was apparent, in particular the tendency to false-positive results in the rapid slide agglutination test.     

Leptospiral antibodies in cattle in alberta and evidence of an emerging serovar

Antibodies to Leptospira interrogans serovars hardjo and pomona were present in 8.3% and 0.5% of sera respectively, from adult female cattle in Alberta surveyed in 1984-85. Criterion for a positive serum sample was 50% agglutination at 1/100 dilution in the microscopic agglutination test. A positive herd contained one or more cows with positive serum. Prevalences were calculated on sample sizes that would give 80-95% reliability. Hardjo antibody prevalences and hardjo-positive herd prevalences were 0-53.9% and 0-83.3%, respectively, among 65 municipalities surveyed. Pomona prevalences by comparison were 0-3.4% and 0-11.7% respectively. Hardjo had increased significantly since 1980-82, and antibodies were found throughout the province. Pomona occurred mainly in southeastern Alberta, where it was isolated from cattle, swine and skunks. Hardjo was isolated only from cattle and it was found in many areas. Antibodies to icterohaemorrhagiae were present in 0.4% of sera from parts of Alberta surveyed in 1980; evidence of the presence of leptospires related to this serovar in bovine and porcine urinary tracts was obtained by immunofluorescence.     

Seroprevalence of Leptospira spp. in Working Horses Located in the Central Region of Chile

Urban working horses live in close contact with their owners. They are usually kept in periurban areas of big cities and cohabit with other animals under precarious sanitary conditions, whereas army horses are kept under controlled management and work. These characteristics leave urban working horses in higher risk of exposure to Leptospira spp. and could become a zoonotic risk for their owners. The aim of this study was to determine the frequency of seropositive working horses to diverse serovars of Leptospira spp. and compare them to a group of army horses. The microscopic agglutination test was used to assess the serum of 426 horses (160 working horses and 266 army horses) against two serovars of Leptospira borgpetersenii (Hardjo and Ballum) and four of Leptospira interrogans (Pomona, Canicola, Icterohaemorrhagiae, and Autumnalis). In the urban working horses group, 30.63% of horses were positive to at least one serovar at titers above 1:100, whereas 23.31% of the army horses were positive. The most frequent serovar in the working horse group was Ballum followed by Canicola, whereas in the army group was Autumnalis followed by Ballum. The serovars Hardjo, Pomona, and Icterohaemorrhagiae were not present in the army horses, whereas all serovars studied were detected in urban working horses. Although no horses studied presented clinical signs of leptospirosis, the study confirms exposure to Leptospira spp. and the importance of studying in more detail the livelihood conditions in which working horses are kept and possible risk of transmission to their owners.