Prevalence of Babesia canis, Borrelia afzelii, and Anaplasma phagocytophilum infection in hard ticks removed from dogs in Warsaw (central Poland)

The purposes of this study were to specify the occurrence and prevalence of Babesia canis, Borrelia burgdorferi sensu lato, and Anaplasma phagocytophilum in ticks removed from dogs in Warsaw, and to determine the Borrelia species occurring in Ixodes ricinus ticks. Among 590 collected ticks, 209 were identified as I. ricinus, and 381 as Dermacentor reticulatus. DNA of B. canis was detected in 11% of D. reticulatus ticks. We found that 6.2% of I. ricinus ticks harbored B. burgdorferi s.l. specific DNA and 2.9% harbored A. phagocytophilum DNA. In these samples sequencing of the detected Borrelia amplicon confirmed infection with Borrelia afzelii genospecies. New sequences were submitted to the GenBank database (accession no. EU152128, EU152127, EU152126). This work is the first detection of B. afzelii and A. phagocytophilum in ticks from Warsaw, and the first survey for the prevalence of B. canis, B. afzelii, and A. phagocytophilum in ticks in central Poland.     

Presence and distribution of Borrelia burgdorferi sensu lato species in internal organs and skin of naturally infected symptomatic and asymptomatic dogs, as detected by polymerase chain reaction.

Tissues from Dutch family dogs symptomatic for borreliosis according to established criteria and from infected but asymptomatic dogs were tested for Borrelia burgdorferi sensu lato DNA using a polymerase chain reaction. Subsequently, B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana were identified by hybridization. Symptomatic dogs showed a higher prevalence of Borrelia in liver samples (9 of 15) than asymptomatic dogs (9 of 43) p = 0.0049. Overall, B. garinii was the most prevalent species and occurred together with up to three other species in on liver sample. B. burgdorferi sensu stricto however, was predominantly detected in samples of synovial membranes, skin, cerebrospinal fluid, bladder, heart, and bone marrow. Nine out of 10 symptomatic dogs with a very high antibody titre were positive for Borrelia DNA by PCR in one or more of these tissues. We conclude that dissemination in naturally infected European dogs occurs and that the two most prevalent species, B. burgdorferi sensu stricto and B. garinii, differ in their tropism.     

Borrelia burgdorferi sensu lato and Rickettsia spp. infections in hard ticks (Ixodes ricinus) in the region of Hanover (Germany)

In a total of 605 Ixodes (I.) ricinus ticks collected in the spring-months March, April and May 2005, quantitative real time PCR (qPCR) revealed 26.6% Borrelia (B.) burgdorferi sensu lato (sl)-positive ticks, i. e. divided by sex and stage into 31.9% positive adults (34.8% females and 29.0% males) and 18.5% positive nymphs. Mono-infections with genospecies from the B. burgdorferi sl-complex were found in over two thirds of the positive individuals, whereas almost one third showed double- or even triple-infections. Genospecies-specific conventional PCR determined B. afzelii as the most frequent genospecies followed by B. garinii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). Rickettsia spp. were found in 34.2% of the collected ticks, divided into 37.6% adults (42.5% females and 32.8% males) and 29.0% nymphs. Co-infections of Rickettsia-positive ticks with B. burgdorferi sl spirochaetes were present in 10.1% of the ticks. Thereby, adult ticks exhibited a co-infection rate of 13.4% (15.5% females and 11.3% males) and nymphs of 5.0%. Independently of the above mentioned study, 3939 Ixodes ticks, sent in between 2006 and 2010 for B. burgdorferi sl-diagnostic, were examined by qPCR exclusively for B. burgdorferi sl. The resulting B. burgdorferi sl prevalence was 23.1% and 24.4% in 2006 and 2007, respectively, followed by a continuous decrease to 12.8% in 2010. To analyse whether this observed decrease in infection frequency is due to sampling bias, in a current study randomly sampled ticks collected from defined sites equally distributed over the city of Hanover are investigated in a statistically relevant sample size.     

Identification of Borrelia burgdorferi genospecies inducing Lyme disease in dogs from Western Poland

Canine Lyme borreliosis may be caused by three Borrelia burgdorferi sensu lato genospecies. The prevalence of infection by Borrelia species was determined by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with the enzyme Fsp4H I in the blood of dogs naturally infested by ticks in an endemic region of Poland. Blood samples were collected from 98 dogs of various breeds, delivered to the Veterinary Clinic in Szczecin (northwestern Poland) for various reasons. Nested PCR revealed the presence of DNA characteristic of only 1 genospecies, i.e. B. burgdorferi sensu stricto (s.s.), in all PCR-positive samples. Digestion of PCR products from a fragment of the fla gene amplified with primers FLA1 and FLA2 gave only one band pattern consistent with the pattern obtained from sequence analysis of the fla gene from a reference isolate of B. burgdorferi s.s. GeHo (X15660) from GenBank.     

Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.     

Evaluation of different media and a BGM cell culture assay for isolation of Borrelia burgdorferi sensu lato from ticks and dogs

A co-culture assay for isolation of Borrelia burgdorferi sensu lato (sl.) from naturally infected ticks and dogs suspected of Lyme borreliosis (LB) was evaluated using buffalo-green-monkey (BGM) cells as the mammalian component. Four different media were tested for their ability to provide sufficient growth conditions for spirochetes and BGM cells. A total of 176 Ixodes ricinus ticks and 268 specimens from 98 dogs were used to compare cell-free culture with the BGM co-culture. A 1:1 mixture of Barbour-Stoenner-Kelly medium (BSK) and Eagle's minimum essential medium (EMEM) supported the growth of the two test strains, B. burgdorferi sensu stricto B31 and B. valaisiana VS116 to the same extent as BSK medium and the growth as well as the viability of BGM cells in this medium were the same as in EMEM. Using the 1:1 mixture of BSK and EMEM, borrelial growth measured in co-culture with BGM cells did not differ significantly from corresponding values obtained in cell-free cultures. In cell-free culture the isolation rate of B. burgdorferi sl. from ticks was significantly higher in BSK/EMEM 1:1 than in BSK medium (P < 0.01). Co-culture with BGM cells had no significant influence on the isolation rate of borreliae from ticks. However, a significant amount of isolates were obtained by one of the procedures only. Analysing canine specimens accordingly, spirochetes were grown from the blood of one dog after four weeks in BGM cell co-culture. The isolate was classified as B. afzelii by PCR-coupled restriction fragment length polymorphism analysis.     

Serologic cross-reactivity of antibodies against Borrelia theileri, Borrelia burgdorferi, and Borrelia coriaceae in cattle.

OBJECTIVE: To evaluate the immune response induced by Borrelia theileri infection and to determine whether B theileri induces cross-reacting antibodies to other bovine borreliae. ANIMALS: Two 3-month-old calves, 1 of which was splenectomized. PROCEDURE: Calves were exposed to Boophilus microplus infected with B theileri. Rectal temperature, PCV, bacteremia, and clinical signs of infection were monitored. Serum was obtained weekly and used to evaluate the humoral response to homologous antigen and B burgdorferi and B coriaceae, using an indirect fluorescent antibody (IFA) test, and to B burgdorferi, using a commercially available ELISA. The identity of cross-reacting antigens was explored, using monoclonal antibodies to genus- and species-specific antigens in an IFA test. RESULTS: B theileri-infected calves produced antibodies that cross-reacted with B burgdorferi and B coriaceae whole-cell antigens. Borrelia theileri whole-cell antigen was recognized by genus-specific monoclonal antibody H9724 but not by species-specific antibody H5332. False-positive reactions were not observed when serum from B theileri-infected calves was tested by use of the ELISA for B burgdorferi. CONCLUSIONS: B theileri induces humoral responses in infected cattle that can be confused with those of other borrelial infections. Care must be taken to definitively distinguish between the various borreliae that may cause disease in cattle. CLINICAL RELEVANCE: Serologic cross-reactivity must be taken into account when making a serodiagnosis of Lyme borreliosis or epizootic abortion in epidemiologic studies involving cattle.     

Canine borreliosis: a laboratory diagnostic trial

The aim of this study was to investigate samples from dogs suggestive of active canine borreliosis (group A) by culture and PCR and the detection of antibodies against Borrelia burgdorferi sensu lato in order to confirm a presumptive clinical diagnosis of canine borreliosis by laboratory results. Criteria for such a diagnosis were: history of tick exposure, lameness, neurological signs, nephropathy, lethargy, anorexia, and fever. A total of 302 samples comprising EDTA blood, urine, synovial fluid, cerebrospinal fluid, and tissue (skin, synovial membrane, kidney) from 98 dogs (26 with arthritis, 46 with neurological signs, 21 with nephropathy, 5 with non-specific symptoms) were collected and examined. Moreover, 55 healthy dogs (group B) and 236 dogs with symptoms or injuries unlikely to be associated with borreliosis (group C) were included in this study. Blood serum samples collected from all individuals (n=389) were analysed by ELISA. Twenty-one (21%) out of 98 dogs from group A, 4 (7%) out of 55 from group B and 15 (6%) out of 236 dogs from group C were positive for antibodies against B. burgdorferi sensu lato. The seroprevalences between groups A, B and C differed significantly. None of the corresponding samples investigated by PCR and culture were positive for spirochetal DNA or viable spirochetes. Borrelia afzelii was grown from one EDTA-blood sample but the corresponding blood serum sample remained antibody-negative. Consequently, the etiologic role of B. afzelii in this case is unclear. In approximately 40% of the presumptive canine borreliosis cases, other lesions have been found to be responsible for clinical signs. This study affirms that a definitive diagnosis of canine borreliosis cannot be made by clinical symptoms and serology based on a single consultation. Moreover, this study clearly revealed that the diagnostic sensitivity is enhanced by a thorough consideration and exclusion of other diseases.     

Diagnosis of Lyme disease in two cows by the detection of Borrelia burgdorferi DNA

Two cows from different herds in a district of Switzerland known to harbour ixodid ticks had erythematous lesions on the hairless skin of the udder, were in poor general condition with a poor appetite and decreased milk production, and had a stiff gait and swollen joints. Borrelia burgdorferi sensu strictu DNA was detected in samples of synovial fluid and milk from one of the cows and Borrelia afzelii DNA was detected in synovial fluid from the other by means of a real-time PCR.     

Efficacy of an amitraz-impregnated collar in preventing transmission of Borrelia burgdorferi by adult Ixodes scapularis to dogs

OBJECTIVE: To determine whether an amitraz-impregnated collar could prevent transmission of Borrelia burgdorferi by Ixodes scapularis to dogs. DESIGN: Laboratory trial. ANIMALS: 8 specific-pathogen-free Beagles. PROCEDURE: On days -15 and -1, all dogs had negative ELISA results for serum antibodies against B. burgdorferi. On day 0, 4 dogs were each fitted with an amitraz-impregnated (9%) collar, and 4 dogs served as untreated controls. On day 7, all dogs were infested with 100/scapularis (approx 50 females and 50 males) with a known B. burgdorferi infectivity rate of 39.4%. On days 21, 28, 35, 42, 56, 70, and 84, each dog was tested for serum antibodies against B. burgdorferi via ELISA and a western blot technique. Additional ELISA were also performed for serum antibodies against antigenically similar organisms. RESULTS: By day 70, all control dogs had developed serum ELISA responses ranging from 328 to 510 kinetics-ELISA units (equivalent to end-point titers of approx 43,500 to 60,000), whereas treated dogs remained seronegative throughout the study. Western blot assays performed on all serum samples confirmed that antibodies detected in control dogs reflected responses to specific antigens of B. burgdorferi, whereas treated dogs had no such antibodies. Additional serologic analyses confirmed that antibody responses observed in control dogs were not attributable to antigenically similar organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Amitraz-impregnated collars prevented transmission of B. burgdorferi in 4 of 4 treated dogs and may be a useful management tool for prevention of borreliosis in dogs.     

Estimating Lyme disease risk using pet dogs as sentinels

The reported number of cases of Lyme disease, Borrelia burgdorferi sensu lato, is thought to have increased in the UK over the past decade, but consistent surveillance data are lacking. Here the prevalence of B. burgdorferi in ticks attached to pet dogs was examined - using them as sentinels for human disease risk. Dogs give a good indication of the exposure of their human owners to infected ticks, since they largely share the same environment and visit the same outdoor areas. PCR was used to test 739 tick samples collected from 3534 dogs selected at random as they visited veterinary practices over a period of six months. Overall, the prevalence of infected ticks on all dogs was 0.5% giving an estimated 481 infected ticks per 100,000 dogs. The data suggest that the prevalence of Borrelia in the UK tick population is considerably higher than most recent estimates indicate.     

Shared flagellar epitopes of Borrelia burgdorferi and Borrelia anserina

Antigenic cross-reactivity between Borrelia burgdorferi and Borrelia anserina was studied using mouse immune sera and monoclonal antibodies. With immune sera, significant cross-reactivity between B. burgdorferi and B. anserina was demonstrated by indirect immunofluorescent assay. In immunoblots, most of the cross-reactivity was shown to be associated with the periplasmic flagella. Using monoclonal antibodies in immunoblots, it was shown that B. burgdorferi and B. anserina shared at least two flagellar epitopes, one of which was not shared with Borrelia hermsii or Borrelia coriaceae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole cell lysates and the use of a species-specific monoclonal antibody (H5332) which reacts with a major outer surface protein (Osp A) of B. burgdorferi readily differentiated the two species at the molecular level.     

A fluorescent bead-based multiplex assay for the simultaneous detection of antibodies to B. burgdorferi outer surface proteins in canine serum

Lyme disease is a zoonotic, vector-borne disease affecting humans, dogs, horses and other species. It is caused by infection with spirochetes of the Borrelia burgdorferi sensu lato group which are transmitted to the mammalian host by infected ticks (Ixodes). Exposure to B. burgdorferi is commonly diagnosed by serological testing. The gold standard for the detection of antibodies to B. burgdorferi is a two-step procedure of an ELISA followed by confirmatory Western blotting (WB). Here, we developed and validated a new bead-based multiplex assay for the detection of antibodies to B. burgdorferi in canine serum which combined the testing by ELISA and WB in a single quantitative test. B. burgdorferi outer surface protein A (OspA), OspC and OspF were expressed in E. coli. The recombinant proteins were coupled to fluorescent beads providing the matrix of the assay. Two sets of canine sera were used for validation of the multiplex assay. First, sera from 79 dogs with known ELISA and WB results were used to establish the conditions of the assay. These samples were selected to provide similar numbers of pre-tested sera ranging from negative to high positive results and included sera from vaccinated and/or naturally infected dogs. A high correlation was observed for detection of antibodies to B. burgdorferi in the single and multiplex assays (n=79). Spearman's rank correlations were 0.93, 0.88 and 0.96 for OspA, OspC and OspF, respectively. Second, a total of 188 canine serum samples that were not tested previously were used for further multiplex assay validation. All samples were also blindly analyzed for antibodies to B. burgdorferi antigens by WB. The WB results provided a 'relative gold standard' for each antigen and were used to perform a receiver operating curve analysis. The areas under the curves were 0.93 for OspA, 0.82 for OspC, and 0.89 for OspF. Multiplex assay interpretation ranges for antibodies to all three B. burgdorferi antigens in canine serum were established by likelihood analysis. The diagnostic sensitivities of the individual OspA, OspC and OspF bead-based assays were 83%, 62% and 82%, respectively, and the diagnostic specificities were 90%, 89% and 86%, respectively. The new multiplex assay provides a sensitive and fully quantitative platform for the simultaneous evaluation of antibodies to B. burgdorferi OspA, OspC and OspF antigens and distinguishes between antibodies that originated from vaccination or natural exposure to B. burgdorferi.     

Immunogenicity and efficacy study of a commercial Borrelia burgdorferi bacterin.

The immunogenicity and efficacy of a commercial Borrelia burgdorferi bacterin was evaluated for stimulation of the host immune response and protection against clinical disease associated with experimentally induced borreliosis in dogs. A total of 30 vaccinated and 24 control dogs were used in 3 separate studies. The vaccine was given IM as two 1-ml doses separated by a 3-week interval. Two weeks or 5 months following the last vaccination, the dogs were challenge inoculated with 7 daily doses of a virulent preparation of a B burgdorferi field isolate through intraperitoneal, subcutaneous, and intradermal routes with or without glucocorticoid administration at the same time. The development of B burgdorferi spirochetemia and clinical disease in the dogs after challenge exposure was studied. Serum samples were obtained from the dogs at various times during the study for serum neutralizing antibody determination and protein immunoblot antibody assay against various geographic isolates of B burgdorferi. Challenge exposure induced limb/joint disorder, fever, anorexia, signs of depression, and B burgdorferi spirochetemia in the nonvaccinated control dogs. The vaccine was found to elicit cross-reactive serum neutralizing and protein immunoblot antibody responses in dogs to various isolates of B burgdorferi and to protect the vaccinated dogs against experimentally induced borreliosis.     

Prevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in questing Ixodes ricinus ticks in relation to the density of wild cervids

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum have been considered as pathogens in animals and humans. The role of wild cervids in the epidemiology is not clear. We analyzed questing Ixodes ricinus ticks collected in spring for these pathogens from sites with high (Fjelløyvær and Strøm) and low density (Tjore, Hinnebu and Jomfruland) of wild cervids to study the spread of the pathogens in questing ticks.

Methods

For detection of Anaplasma phagocytophilum a 77-bp fragment in the msp2 gene was used. Detection of Borrelia burgdorferi sensu lato was performed using the FL6 and FL7 primers according to sequences of conserved regions of the fla gene. The OspA gene located on the linear 49-kb plasmid was used as target in multiplex PCR for genotyping. Genospecies-specific primers were used in the PCR for Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii.

Results

Infection rates with Borrelia spp. were significantly lower at Fjelløyvær and Strøm compared to Tjore and Hinnebu; Fjelløyvær vs. Tjore (χ² = 20.27, p < 0.0001); Fjelløyvær vs. Hinnebu (χ² = 24.04, p < 0.0001); Strøm vs. Tjore (χ² = 11.47, p = 0.0007) and Strøm vs. Hinnebu (χ² = 16.63, p < 0.0001). The Borrelia genospecies were dominated by. B. afzelii (82%) followed by B. garinii (9.7%) and B. burgdorferi sensu stricto (6.9%). B. burgdorferi s.s. was only found on the island of Jomfruland. The infection rate of Anaplasma phagocytophilum showed the following figures; Fjelløyvær vs Hinnebu (χ² = 16.27, p = 0.0001); Strøm vs. Tjore (χ² = 13.16, p = 0.0003); Strøm vs. Hinnebu (χ² = 34.71, p < 0.0001); Fjelløyvær vs. Tjore (χ² = 3.19, p = 0.0742) and Fjelløyvær vs. Støm (χ² = 5.06, p = 0.0245). Wild cervids may serve as a reservoir for A. phagocytophilum. Jomfruland, with no wild cervids but high levels of migrating birds and rodents, harboured both B. burgdorferi s.l. and A. phagocytophilum in questing I. ricinus ticks. Birds and rodents may play an important role in maintaining the pathogens on Jomfruland.

Conclusion

The high abundance of roe deer and red deer on the Norwegian islands of Fjelløyvær and Strøm may reduce the infection rate of Borrelia burgdorferi sensu lato in host seeking Ixodes ricinus, in contrast to mainland sites at Hinnebu and Tjore with moderate abundance of wild cervids. The infection rate of Anaplasma phagocytophilum showed the opposite result with a high prevalence in questing ticks in localities with a high density of wild cervids compared to localities with lower density.     

Association of urine protein excretion and infection with Borrelia burgdorferi sensu lato in Bernese Mountain dogs

Bernese Mountain dogs (BMDs) are prone to develop a familial glomerulonephropathy and a pathogenic role of Borrelia burgdorferi sensu lato in this disease has been suspected. Glomerular disease in many affected dogs is clinically inapparent and proteinuria is found incidentally. In this study, urine protein excretion was evaluated in 122 clinically healthy BMDs and 55 controls. The seroprevalence of B. burgdorferi in BMDs was 57%, compared to 16% in controls. There were no significant differences in the occurrence of positive dipstick results, microalbuminuria, urine protein-to-urine creatinine ratio or abnormal urine protein pattern (determined by sodium dodecyl sulphate agarose gel electrophoresis) between BMDs and controls and BMDs with and without antibodies against B. burgdorferi. It was concluded that antibodies against B. burgdorferi are not associated with proteinuria as an early sign of renal disease in BMDs.     

Microalbuminuria and comparison of serologic testing for exposure to Borrelia burgdorferi in nonclinical Labrador and Golden Retrievers.

Canine Lyme disease is caused by the spirochete Borrelia burgdorferi after transmission by an Ixodes tick, typically resulting in joint pain, fever and lethargy. Lyme nephritis is a poorly characterized syndrome associated with severe glomerular and tubular renal injury and poor clinical outcome in young to middle-aged dogs positive for exposure to B. burgdorferi. The aims of this study were to identify associations between natural exposure to B. burgdorferi and the presence of microalbuminuria in nonclinical young Labrador and Golden Retrievers and to compare two commonly used serologic tests available to document B. burgdorferi exposure: the Western blot and the commercial point-of-care C6 peptide enzyme-linked immunosorbent assay (ELISA) tests. Microalbuminuria was assessed using a commercial point-of-care ELISA specific for canine albumin. Blood and urine samples from 268 asymptomatic Labrador and Golden Retrievers were included. Of these, 18.7% were positive for B. burgdorferi exposure according to the C6 ELISA; 21.2% were positive for natural exposure to B. burgdorferi and 11.5% for vaccinal antibodies according to the Western blot. The agreement rate was 93% between the two tests (kappa = 0.78, P < 0.0001) for natural exposure. Urine from 6.1% of the dogs was positive for microalbuminuria. There was no association between microalbuminuria and exposure to B. burgdorferi based on results of a Western blot (P = 0.57) or C6 ELISA (P = 0.53). Microalbuminuria is likely not a consequence of B. burgdorferi exposure in young nonclinical Labrador and Golden Retrievers.     

Serologic prevalence of Dirofilaria immitis,Ehrlichia canis,and Borrelia burgdorferi infections in Brazil

Dogs infected with Dirofilaria immitis, Ehrlichia canis, or Borrelia burgdorferi may show nonspecific clinical signs or may be asymptomatic. In Brazil, E. canis and D. immitis infections are frequently diagnosed based on the presence of classical signs; however, serologic tests are seldom performed to confirm the presence of infection. To estimate the seroprevalence of these three canine diseases in Brazil, 2,553 dogs presented at veterinary practices for various tests, routine treatments, or examinations were evaluated by an in-office commercial ELISA test kit (SNAP 3Dx, IDEXX Laboratories). Each dog was examined by the veterinarian, and a whole-blood sample was collected and immediately tested for the simultaneous detection of B. burgdorferi and E. canis antibodies and D. immitis antigen. D. immitis infection was detected in 51 dogs (2.0%) and E. canis antibodies were present in 505 dogs 19.8%). Only one dog tested positive for B. burgdorferi antibodies.     

A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle

Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi.