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We evaluated spatial-temporal risk for Lyme disease in northwestern North Carolina, USA, by using individual-level canine Borrelia burgdorferi seroprevalence data collected during 2017-2021 at routine veterinary screenings for tickborne diseases. Seroprevalence in dogs increased from 2.2% (47/2,130) in 2017 to 11.2% (339/3,033) in 2021. The percentage of incident seropositivity increased from 2.1% (45/2,130) in 2017 to 7.6% (231/3,033) in 2021. Exploratory geographic analyses found canine seroprevalence shifted from clustered (2017, Moran's I = 0.30) to dispersed (2021, Moran's I = -0.20). Elevation, slope, aspect, and forest land cover density were associated with canine seroprevalence within various household buffer regions in 2017. Slope was associated with seroprevalence at the household level in 2021. Results support the use of individual-level canine seroprevalence data for monitoring human risk for Lyme disease. Establishing sentinel veterinary clinics within Lyme disease-emergent communities might promote prevention and control efforts and provide opportunities for educational and behavioral interventions.

Background: An increased risk for glomerulonephritis and a higher prevalence of antibodies to Borrelia (B.) burgdorferi sensu lato have been reported in Bernese mountain dogs (BMDs). The aim of the study was to determine the prevalence of laboratory abnormalities suggestive of kidney disease in clinically healthy BMDs compared to a control population and to investigate if there is a correlation with the occurrence of antibodies to B. burgdorferi sensu lato, Ehrlichia canis, and Anaplasma (A.) spp. and with the occurrence of Dirofilaria (D.) immitis antigen. A total of 197 BMDs and 57 control dogs were included in the study. Laboratory evidence of kidney disease was defined as renal azotemia and/or proteinuria with a urine protein creatinine ration of more than 0.5 in an inactive urine sediment. A SNAP®4Dx® ELISA (IDEXX, Laboratories, Inc., Westbrook, ME, USA) was used to detect antibodies to B. burgdorferi sensu lato, E. canis and Anaplasma spp. and antigen of D. immitis., Results: Laboratory evidence of kidney disease was significantly more common in BMDs than in control dogs (17.8% versus 1.8%) (p = 0.005). The proportion of BMDs with anti-B. burgdorferi sensu latu antibodies and anti-A. phagocytophilum antibodies was significantly higher in BMDs (p <  0.001). However, an association between these findings could not be identified., Conclusion: BMDs are more often affected by kidney disease and have a higher prevalence of antibodies to bacterial pathogens transmitted by Ixodes ticks than control dogs. However, a causal relationship between these two variables could not be established due to a lack of association between these two findings.

Lyme disease (LD) is a tick-transmitted disease caused by Borreliella burgdorferi (Bb). Temporal studies of maternal antibody (Ab) profiles in Bb infected pregnant dogs and their pups have not been conducted. In this study, Ab profiles of a client-owned Bb C6 Ab positive Rottweiler and her nine pups were assessed. The dam presented with lameness 12 days prior to parturition and was C6 Ab positive with a Quant C6 Ab concentration of 237U/mL. Treatment with amoxicillin was initiated and 11 days later nine pups were delivered. Screening of the sera from the dam and pups against Bb cell lysates and a panel of antigens revealed similar immunoreactivity profiles. While antigen-specific IgG and IgM reactivity persisted in the dam for at least 7 months, a rapid decline in IgG specific for BBA36, BBK53, BB0238, BBA73 and outer surface protein (Osp) E in the pups occurred between days 29 and 52 post-parturition. In contrast, Ab specific for DbpA and the diagnostic antigens VlsE (C6) and OspF, remained elevated in the pups. Sera from the dam displayed potent complement-dependent bactericidal activity against Bb. Sera from the pups was also bactericidal but primarily through a complement-independent mechanism. Lastly, single dose vaccination of the dam at day 51 post-parturition with a LD subunit vaccine consisting of OspA and an OspC chimeritope triggered a broad anti-OspC Ab response indicative of an anamnestic response. Although this study focused on a single case, these findings add to our knowledge of maternal Ab profiles and will aid the interpretation of serological assays in pups delivered by a Bb C6 Ab positive dog., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The Cumberland Gap Region (CGR) of the United States is a natural corridor between the southeastern, northeastern, and midwestern regions of the country. CGR has also many species of ticks and mosquitos that serve as competent vectors for important animal and human pathogens. In this study, we tested dogs from six different animal shelters in the CGR for Rocky Mountain spotted fever (RMSF), anaplasmosis, Lyme disease, canine ehrlichiosis and canine heartworm disease., Results: Sera from 157 shelter dogs were tested for antibodies to RMSF agent, Rickettsia rickettsii, using an indirect immunofluorescence assay. Sixty-six dogs (42.0%) were positive for either IgM or IgG, or both IgM and IgG antibodies to R. rickettsii. Moreover, the same set of sera (n = 157) plus an and additional sera (n = 75) from resident dogs at the same shelters were tested using the SNAP 4Dx Plus. Of 232 dogs tested, two (0.9%) were positive for antibodies to Anaplasma phagocytophilum/A. platys, nine (3.9%) were positive for antibodies to Borrelia burgdorferi, 23 (9.9%) for positive for antibodies to Ehrlichia canis/E. ewingii, and 13 (5.6%) were positive for Dirofilaria immitis antigen. Co-infection with two or more etiologic agents was detected in five animals. Three dogs had antibodies to both B. burgdorferi and E. canis/E. ewingii, and two dogs were positive for D. immitis antigen and antibodies to B. burgdorferi and E. canis/E. ewingii., Conclusions: Shelter dogs in the CGR are exposed to a number of important vector-borne pathogens. Further studies are required to ascertain the roles these animals play in maintenance and transmission of these pathogens.

We characterized the antibody response to decorin-binding protein A (DbpA) or DbpB from immune serum samples collected from 27 dogs infected with Borrelia burgdorferi by Ixodes scapularis ticks. Immunoglobulin M (IgM) antibodies to DbpA or DbpB were rarely detected, but high levels of IgG antibodies to DbpA were detected in 16 of 27 of the immune sera collected 1 mo after infection, 20 of 25 of the sera collected after 2 mo, and each of the 23, 17, or 11 serum samples evaluated after 3, 4, or 5 mo, respectively. In addition, IgG antibodies to DbpB were detected in 22 of 27 ( p  = 0.005) tested dogs after 1 mo, and the frequency of detecting the antibodies thereafter closely mimicked the antibody responses to DbpA. Moreover, antibodies to DbpA or DbpB were not produced by dogs vaccinated with a whole-cell B. burgdorferi bacterin; removing the antibodies to DbpA by adsorption to recombinant DbpA (rDbpA) did not affect the reactivity detected by a rDbpB ELISA. Therefore, the findings from our preliminary study showed that antigenically distinct antibodies to DbpA or DbpB are produced reliably during canine infection with B. burgdorferi , and the response is not confounded by vaccination with a Lyme disease bacterin. Larger studies are warranted to more critically evaluate whether detecting the antibody responses can improve serodiagnostic confirmation of canine Lyme disease.

The capability of imidacloprid 10% + flumethrin 4.5% (Seresto®) collars to prevent transmission of Borrelia burgdorferi sensu lato (Bbsl) and Anaplasma phagocytophilum (Ap) by naturally infected ticks was evaluated in two studies with 44 dogs. In each study, one group served as non-treated control, whereas the other groups were treated with the Seresto® collar. All dogs were exposed to naturally Bbsl- and Ap-infected hard ticks (Ixodes ricinus, Ixodes scapularis). In study 1, tick infestation was performed on study day (SD) 63 (2 months post-treatment [p.t.]); in study 2, it was performed on SD 32 (one month p.t.) respectively SD 219 (seven months p.t.). In situ tick counts were performed 2 days after infestation. Tick counts and removals followed 6 (study 1) or 5 days (study 2) later. Blood sampling was performed for the detection of specific Bbsl and Ap antibodies and, in study 1, for the documentation of Ap DNA by PCR. Skin biopsies were examined for Bbsl by PCR and culture (only study 1). The efficacy against Ixodes spp. was 100% at all time points. In study 1, two of six non-treated dogs became infected with Bbsl, and four of six tested positive for Ap; none of the treated dogs tested positive for Bbsl or Ap. In study 2, ten of ten non-treated dogs became infected with Bbsl and Ap; none of the treated dogs tested positive for Bbsl or Ap; 100% acaricidal efficacy was shown in both studies. Transmission of Bbsl and Ap was successfully blocked for up to 7 months.

Vaccines against Borrelia burgdorferi are administered frequently to dogs in areas endemic for the infection. These vaccines produce an antibody response to spirochetal proteins that cross-react in many antibody tests, including immunofluorescence assay, Western blot, and whole cell ELISA used to document exposure to B. burgdorferi. The purpose of this study was to determine whether the in-clinic C6 ELISA assay (SNAP® 4Dx® Plus; IDEXX Laboratories, Inc., Westbrook, Maine) and the quantitative format C6 ELISA (Lyme Quant C6® Antibody Test, IDEXX Laboratories, Inc., Westbrook, ME) react to sera from dogs that have been vaccinated with 1 of 4 different commercially available B. burgdorferi vaccines. Four groups of 3 dogs each were each administered one of the 4 vaccines and sera evaluated over time by indirect fluorescent antibody assay, western blot immunoassay, the in-clinic C6 ELISA assay, and the quantitative format C6 ELISA. While all dogs developed B. burgdorferi antibodies detectable by indirect fluorescent antibody assay and western blot immunoassay after vaccination, none of the samples were positive in either of the C 6 peptide-based assays. Based on these results, positive anti-C 6 antibody results in client-owned dogs are likely to reflect exposure to B. burgdorferi rather than vaccination., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Borrelia burgdorferi and Anaplasma phagocytophilum are tick-borne infections transmitted by Ixodes scapularis in the eastern USA; both agents cause disease in dogs and people. To characterize changes in seroprevalence over time, Cochran Armitage trend tests were used to evaluate percent positive test results for antibodies to B. burgdorferi and Anaplasma spp. in approximately 20 million canine tests from 2010-2017 in 25 states and 905 counties in the eastern USA., Results: A significant decreasing trend in seroprevalence to B. burgdorferi was evident in eight states along the mid-Atlantic coast from Virginia to New Hampshire, and in Wisconsin. In contrast, a continued increasing trend was evident in five northeastern and Midwestern states where Lyme borreliosis is endemic or emerging, as well as in three southern states where endemicity has not yet been widely established. Similarly, seroprevalence to Anaplasma spp. showed a significant, although smaller, decreasing trend in five states along the mid-Atlantic coast from Virginia to Connecticut and Rhode Island, as well as in Minnesota and Wisconsin in the Midwest; despite the fact that those trends were significant they were weak. However, a strong increasing trend was evident in Massachusetts and three states in northern New England as well as in Pennsylvania., Conclusions: As expected, seroprevalence continued to increase in regions where Lyme borreliosis and anaplasmosis are more newly endemic. However, the declining seroprevalence evident in other areas was not anticipated. Although the reasons for the decreasing trends are not clear, our finding may reflect shifting ecologic factors that have resulted in decreased infection risk or the combined positive influence of canine vaccination, tick control, and routine testing of dogs in regions where these infections have long been endemic. Analysis of trends in canine test results for tick-borne infections continues to be a valuable tool to understand relative geographical and temporal risk for these zoonotic agents.

Tick-borne relapsing fever (TBRF) is caused by several Borrelia spp. (including Borrelia turicatae ), which are primarily transmitted by Ornithodoros ticks. Relapsing fever group species are found worldwide, except for Antarctica. Approximately 500 human cases were reported between 1990 and 2011 in the United States (likely an underestimate), while cases in domestic and wild dogs were reported from Florida, Texas, and Washington. TBRF spirochetes are related to Borrelia burgdorferi , the agent of Lyme borreliosis. Dogs are routinely screened for B. burgdorferi , but it is unknown if infection with TBRF agents produces antibodies cross-reactive with B. burgdorferi assays. These data are critical for accurate surveillance of TBRF and Lyme borreliosis in dogs. In this study, B. burgdorferi -negative dogs were inoculated with B. turicatae , and seroconversion was confirmed by the rBipA (recombinant Borrelia immunogenic protein A) Western blot. Seropositive samples were tested with commercial and veterinary diagnostic laboratory B. burgdorferi -based tests. Borrelia turicatae -seroreactive samples cross-reacted with a whole-cell indirect fluorescent antibody (IFA) test and two multiantigen tests, but not with single-antigen tests using C6. Cross-reactivity with TBRF can confound epidemiology and surveillance efforts and confuse recommendations made by veterinarians for prevention and control. These findings demonstrate the need to critically evaluate results from B. burgdorferi diagnostic tests in the context of the assay type and the animal's geographical location and history of travel, as well as highlighting the need for commercially available specific diagnostic tests for TBRF spirochetes., (Copyright © 2019 American Society for Microbiology.)

Background: Ehrlichia ewingii is the most seroprevalent Ehrlichia-infecting dogs in the southern and mid-western United States. Fever, lameness, and polyarthritis are commonly reported findings in dogs naturally infected with E. ewingii., Objectives: To evaluate clinicopathologic findings in a population of dogs naturally infected with E. ewingii., Animals: Forty-one dogs PCR positive for E. ewingii and PCR negative for other targeted vector-borne organisms., Methods: Retrospective study. Clinical and clinicopathologic data including physical examination findings, CBC, serum biochemistry, urinalysis (UA), symmetric dimethylarginine (SDMA), and vector-borne disease diagnostic results were reviewed., Results: Frequent clinical diagnoses other than ehrlichiosis (28/41; 68.3%) were renal disease (7/41; 17.1%) and immune-mediated hemolytic anemia (IMHA) (6/41; 14.6%). The most frequent physical examination finding was joint pain (14/41; 34.1%). Prominent hematologic and biochemical abnormalities included abnormal lymphocyte counts (22/36; 61.1%); neutrophilia (21/37; 56.8%); increased alkaline phosphatase (20/35; 57.1%) and alanine transaminase (14/35; 40%) activities; and increased SDMA concentration (11/34; 32.4%). Urinalysis abnormalities included proteinuria (20/27; 74%), most with inactive sediments (16/20; 80%). Dogs were seroreactive by Ehrlichia canis immunofluorescence assay (IFA; 17/39; 43.6%) and Ehrlichia ELISA (34/41; 82.9%). Seroreactivity by IFA for other vector-borne pathogens included Bartonella (1/39; 2.6%), Rickettsia rickettsii (spotted-fever group rickettsiae) (12/39; 30.8%), and Borrelia burgdorferi by ELISA (1/41; 2.4%)., Conclusions and Clinical Importance: Renal disease, IMHA, proteinuria, neutrophilia, abnormal lymphocytes, and increased liver enzyme activities were common in this group of E. ewingii-infected dogs. Studies are needed to determine if E. ewingii contributes to comorbidities or is a precipitating factor in clinical syndromes in persistently infected dogs., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Vector-borne pathogens are emerging concerns in multiple regions of Canada. Determining regional prevalence of canine vector-borne pathogens and documenting change will improve clinician awareness, enable targeted prevention, enhance diagnosis and ideally reduce the risk of disease. Study objectives were to: (i) estimate the prevalence of positive canine vector-borne test results from samples submitted in Canada; (ii) assess change in prevalence over time, from baseline (2008) to 2015; and (iii) estimate the prevalence of pathogen co-infections., Methods: This repeat cross-sectional study evaluated 753,468 test results for D. immitis antigen and B. burgdorferi, Ehrlichia canis/ewingii/muris serology, and 753,208 test results for Anaplasma phagocytophilum/platys serology using the SNAP® 4Dx®Test and SNAP 4Dx® Plus Test., Results: Based on all submitted samples from Canada (2008-2015), the period seroprevalence of B. burgdorferi, Ehrlichia spp., Anaplasma spp. and D. immitis antigen were 2.0%, 0.5%, 0.4% and 0.2%, respectively. Over the 7 years (2008 compared to 2015) we observed a significant increase in seroprevalence for B. burgdorferi (144.4%) and Ehrlichia spp. (150%). Co-infections (positive for two or more pathogens on a single 4 pathogen test kit) were estimated at 5.4% (1162/21,612) of total positive tests., Conclusions: The temporal rise and geographical differences in prevalence detected for these pathogens (notably B. burgdorferi) are consistent with anecdotal information on canine illness related to tick-borne pathogen exposure in multiple regions of Canada, particularly canine Lyme disease.

Canine vector-borne pathogens (VBPs) are a group of globally distributed and rapidly spreading microorganisms transmitted by arthropods. In the present survey, we investigated Anaplasma spp., Ehrlichia canis, Dirofilaria immitis and Borrelia burgdorferi sensu lato seroprevalence between three groups of dogs (asymptomatic, suspected and deceased) from continental and coastal regions of Croatia with the commercial point-of-care SNAP®4Dx®Plus for VBPs. The overall prevalence for tested pathogens in dogs was 6.1% with the highest prevalence detected for Anaplasma spp. (4.5%), while the remaining pathogens were found at a prevalence of less than 1% (E. canis 0.6%, B. burgdorferi s.l. 0.4%, D. immitis 0.6%). No statistically significant differences in VBP detection between dog cohorts could be found with the exception of D. immitis in the deceased group. Interestingly, no evidence of D. immitis could be found in the hearts of dogs in this group at necropsy, however. This study provides the first data on the seroprevalence of selected VBPs between dogs of different health statuses in Croatia. The results demonstrate that serological evidence of VBPs alone or in combination with co-infections were found just as frequently in asymptomatic dogs as those with suspected or confirmed evidence of VBP disease, raising questions about the pathogenic potential of these organisms in domesticated dogs.

Background: Lyme borreliosis, a tick-borne disease, is endemic to some parts of North America and is an emerging disease in other parts of the world. Vaccination is an increasingly common, although controversial, method used in the prevention of Lyme disease in dogs; the reported efficacies of Borrelia burgdorferi vaccines in dogs are highly variable, ranging from 50% to 100%., Objectives: To determine the efficacy of vaccines for prevention of Lyme disease in dogs in North America., Methods: Experimental and observational study designs were eligible for inclusion. The outcome of interest was the reduction of incidence of clinical illness after exposure to B. burgdorferi. Electronic databases searched were MEDLINE, Web of Science, and Centre for Agricultural Biosciences Abstracts. Clinical signs were extracted as dichotomous outcomes: lameness, anorexia, pyrexia, depression, and lymphadenopathy. Study quality was assessed using tools from the Cochrane collaboration., Results: In total, 3 observational studies and 13 challenge trials were included. None of the challenge trials assessed lymphadenopathy, but for each of the remaining 4 clinical signs, a meta-analysis was performed. Compared to unvaccinated dogs, vaccinated dogs had a reduced odds of developing lameness, depression, pyrexia, and anorexia (odds ratio: 0.15-0.23)., Conclusions and Clinical Importance: Based on the quantitative synthesis of results from challenge studies, vaccinated dogs are less likely to develop clinical signs after exposure to B. burgdorferi compared to unvaccinated dogs. These results should be interpreted with caution, however, as several shortcomings related to quality and study design were identified. Future studies should focus on larger sample sizes in field conditions., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Vector-borne agents comprise medically important infections affecting dogs throughout much of the world. Sensitive detection of antibodies directed at tick-borne disease-causing organisms in dogs is diagnostically important for veterinarians, pets and their owners, and epidemiologically important for public health surveillance. The SNAP 4Dx Plus Test (IDEXX Laboratories, Inc., Westbrook, ME) identifies antibodies to or infection with multiple tick-borne pathogens and canine heartworm antigen in a single assay. Recently, VetScan FLEX4 Rapid Test (Abaxis, Inc., Union City, CA) was launched as a new assay to detect tick-borne pathogen antibodies and heartworm antigen. In the present study, we evaluated the comparative performance of SNAP 4Dx Plus (SNAP) and FLEX4 Rapid Test (FLEX4) using samples selected based on geographic distributions for canine vector borne diseases, including Borrelia burgdorferi (n = 105), Anaplasma phagocytophilum (160), Anaplasma platys (115), Ehrlichia canis (154), Ehrlichia ewingii (163), Ehrlichia chaffeensis (151) and Dirofilaria immitis (105). Canine vector borne diseases infection status was established for each sample by a combination of reference methods that included necropsy (D. immitis, heartworm disease), Western immunoblotting (B. burgdorferi), immunofluorescence assays (A. phagocytophilum and E. canis) and species-specific ELISAs (A. platys, E. canis, E. ewingii and E. chaffeensis). For comparisons among the 2 assays, samples were evaluated per the manufacturers' instructions for each test kit. By testing each same sample set compared to the defined reference results, sensitivities differed substantially between SNAP and FLEX4, at 95.5 vs. 40.9%, respectively for B. burgdorferi, 97.1% vs. 61.4% for E. canis, 98.2% vs. 59.3% for E. ewingii, 64.3% vs. 35.7% for E. chaffeensis, 84.5% vs. 12.7% for A. phagocytophilum, 83.3% vs. 33.3% for A. platys, and 94.1% vs. 88.2% for D. immitis. Specificities for both rapid assay tests ranged from 98% to 100%. Based upon the comparative results derived from this study, the SNAP test was more sensitive than the FLEX4 test for detection of antibodies to all tick-borne pathogens and heartworm disease (Dirofilaria immitis) antigen in dogs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Vector-borne diseases constitute a major problem for veterinary and public health, especially in tropical regions like Central America. Domestic dogs may be infected with several vector-borne pathogens of zoonotic relevance, which may also severely compromise canine health., Methods: To assess the prevalence of canine vector-borne diseases in Nicaragua, 329 dogs from seven cities, which were presented to the veterinarian for various reasons, were included in this study. Dogs were examined clinically and diagnostic blood samples were taken for analysis of packed cell volume (PCV) and presence of microfilariae as well as antigen of Dirofilaria immitis and antibodies to Ehrlichia spp., Anaplasma spp. and Borrelia burgdorferi (sensu lato) by use of a commercially available rapid ELISA. To detect current infections, specific PCRs for the detection of E. canis, A. platys and A. phagocytophilum were carried out on blood samples of the respective seropositive dogs. Microfilaremic blood samples, as well as D. immitis antigen positive samples were further subjected to PCR and subsequent sequencing for filarial species identification., Results: Antibodies against Ehrlichia spp. were present in 62.9% of dogs, while Anaplasma spp. seroprevalence was 28.6%. Antibodies against species of both genera were detected in 24.9% of dogs. Borrelia burgdorferi (s.l.) antibodies were not detected. Dirofilaria immitis antigen was present in six animals (1.8%), two of which also showed D. immitis microfilariae in buffy coat. In addition to D. immitis, Acanthocheilonema reconditum was identified by PCR and sequencing in two of four additional microfilaremic blood samples, which were tested negative for D. immitis antigen. Current E. canis infections as defined by DNA detection were present in 58.5% of Ehrlichia-seropositive dogs, while 5.3% of Anaplasma-seropositive dogs were PCR-positive for A. platys, 2.2% for A. phagocytophilum and 16.0% for both Anaplasma species. Current E. canis infection had a statistically significant negative impact on PCV, whereas no relationship between infection status and clinical signs of disease could be observed., Conclusions: These results indicate that canine vector-borne diseases are widespread in Nicaragua and that dogs may constitute a reservoir for human infection with E. canis, A. phagocytophilum and D. immitis. Thus, the use of repellents or acaricides to protect dogs from vector-borne diseases is strongly recommended.

Background: Prevention of Lyme disease in dogs in North America depends on effective vaccination against infection by the tick vector-born spirochete Borrelia burgdorferi. Most vaccines effectively prevent spirochete transmission to dogs during tick feeding based on immunization with the outer-surface lipoprotein A (OspA) of B. burgdorferi. More recently, vaccines containing additional OspC protein moieties have been introduced. These are designed to enhance protection by forming a second line of defense within the vertebrate host, where OspC expression replaces OspA as the dominant surface antigen. However, supportive data for demonstration of OspC mediated protection is still lacking. Since OspA immunogenicity is of paramount importance to protection against spirochete transmission; this study was designed to compare the immunogenicity of two commercially available vaccines against the Borrelia burgdorferi OspA. We further characterized OspA antigen fractions of these vaccines with respect to their biochemical and biophysical properties., Results: Two groups of beagle dogs (n = 9) were administered either: (1) a nonadjuvanted/monovalent, recombinant OspA vaccine (Recombitek® Lyme) or (2) an adjuvanted, recombinant OspA /OspC chimeric fusion vaccine (Vanguard® crLyme). The onset of the anti-OspA antibody response elicited by the nonadjuvanted/monovalent OspA vaccine was significantly earlier than that for the bivalent OspA /OspC vaccine and serum borreliacidal activity was significantly greater at all post-vaccination time points. As expected, only dogs inoculated with the bivalent OspA/OspC vaccine mounted a humoral anti-OspC response. However, only three out of nine dogs in that group had a positive response. Comparison of the OspA vaccine structures revealed that the OspA in the nonadjuvanted/monovalent vaccine was primarily in the lipidated form, eluting (SEC-HPLC) at a high molecular weight, suggestive of micelle formation. Conversely, the OspA moiety of the OspA/OspC vaccine was found to be nonlipidated and eluted as the monomeric protein., Conclusions: We hypothesize that these structural differences may account for the superior immunogenicity of the nonadjuvanted monovalent recombinant OspA vaccine in dogs over the adjuvanted OspA fraction of the OspA/OspC vaccine.

Whole blood samples were collected from 515 dogs in the practice region surrounding Saskatoon, Saskatchewan, Canada between 2008 and 2010 and evaluated for seroprevalence of vector-borne diseases. Of 515 samples, 12 (2.3%) were positive, with 7 (1.4%) positive for antibodies to Borrelia burgdorferi. These prevalences are higher than those previously reported for this region., Competing Interests: Disclosure statement IDEXX Labs Canada provided the SNAP 4Dx tests. There are no competing financial interests.

Background: Autochthonous transmission of Borrelia burgdorferi, the primary agent of Lyme disease in dogs and people in North America, commonly occurs in the northeastern United States, including the New York City metropolitan area, a region with a large human and pet population and broadly diverse demographics and habitats., Methods: We evaluated results from a specific, C6-based serologic assay performed on 234,633 canine samples to compare evidence of past or current infection with B. burgdorferi (sensu stricto) in dogs to county-wide social and environmental factors, as well as to reported cases of Lyme disease in people., Results: The data revealed a wide range of county level percent positive canine test results (1.2-27.3%) and human case reports (0.5-438.7 case reports/100,000 people). Dogs from highly (> 50%) forested areas and counties with lower population density had the highest percent positive test results, at 21.1% and 17.9%, respectively. Canine percent positive tests correlated with population-adjusted human case reports (R 2 = 0.48, P < 0.0001), as well as population density, development intensity, temperature, normalized difference vegetation index, and habitat type. Subsequent multiple regression allowed an accurate prediction of infection risk in dogs (R 2 = 0.90) but was less accurate at predicting human case reports (R 2 = 0.74)., Conclusion: In areas where Lyme disease is endemic, canine serology continues to provide insight into risk factors for transmission to both dogs and people although some differences in geographic patterns of canine infection and human disease reports are evident.

We attempted to determine if Leptospira spp. antibodies induced by vaccination would cross-react with Borrelia burgdorferi antigens used in a commercial automated immunofluorescent assay (AccuPlex 4 BioCD; Antech). Staff- and student-owned dogs ( n = 31) were recruited at a veterinary teaching hospital in a B. burgdorferi nonendemic area. The dogs were randomized and administered 1 of 4 commercial Leptospira spp. vaccines that contained serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona, then booster vaccinated 3 wk later. Blood was collected on weeks 0, 3, 4, 8, and 12. After confirming that maximal Leptospira spp. titers occurred on week 4, aliquots of sera from week 4 were shipped frozen for analysis of B. burgdorferi antibodies against OspA, OspC, OspF, P39, and SLP with the AccuPlex system. Week 4 sera from all 31 dogs had a titer of 1:100 for at least 1 Leptospira spp. serovar. Titers of 1:800 or greater were detected against multiple serovars in 27 dogs. None of the samples contained antibodies against the B. burgdorferi OspA, OspC, OspF, P39, and SLP peptides used in the commercial assay. The B. burgdorferi peptides used in the AccuPlex system do not recognize naturally occurring Leptospira spp. antibodies or those induced by the commercial Leptospira spp. vaccines administered in our study.

Objective: The study aimed to investigate the distribution of specific immune responses (IgG) to Borrelia burgdorferi using a line immunoassay with recombinant VlsE (variable major protein-like sequence, expressed) protein and synthetic C peptide among other antigens. We compared the immune responses to VlsE protein and C 6 peptide, because both antigens have been considered specific for a Borrelia infection., Materials and Methods: A total of 1355 blood samples from dogs suspected of Borrelia infection were analysed. The line immunoassay employed nine antigens., Results: A total of 64.4% of all samples tested negative, 16.4% were positive for an infection and 17.4% were positive for vaccination. Band patterns specific for both infection and vaccination were observed in 1.2% of the dogs. The bands that most frequently tested positive were p100 (24.3%), p31/OspA (18.5%), C 6 (16.3%) and VlsE (13.9%). A total of 236 dogs (17.4% of the population) had antibodies to VlsE and/or C 6 peptide. In 73.3% of these dogs, results for VlsE and C 6 peptide were consistent, whereas this was not the case for 26.7% of these animals., Conclusion and Clinical Relevance: Testing using a line immunoassay allows for qualitative analyses of different immune responses to various antigens used as probes. In our study, >  26% of the dogs displayed discrepant results with regard to VlsE and C 6 , the two antigens considered specific for Borrelia burgdorferi infection. To confirm or rule out infection, the results of several band patterns, thought to be specific for infection, need to be taken into consideration.

This survey was conducted in four Brazilian cities, from three federative units (Pernambuco, Minas Gerais, and Federal District), representing different socioeconomic settings, to determine the presence of antibodies to or antigens and DNA of selected pathogens in privately owned dogs. From January to April 2015, blood and serum samples were collected and assayed using different tests. In particular, antibodies to Anaplasma spp., Borrelia burgdorferi sensu lato, and Ehrlichia spp., and antigens of Dirofilaria immitis were detected using a rapid enzyme-linked immunosorbent assay, whereas antibodies to Babesia spp. were detected by an immunofluorescence antibody assay. Moreover, the presence of Leishmania DNA in blood samples was assessed by real-time polymerase chain reaction. Overall, 208 (69.3%) out of 300 dogs were positive for at least one tested pathogen (intended here as antibodies, antigen or DNA, as abovementioned), with 139 (66.8%) of them being positive to two or more pathogens. No dog presented antibodies to B. burgdorferi s.l., and D. immitis antigens were detected exclusively in dogs from Pernambuco. Among positive dogs, the most common clinical signs were lymphadenomegaly (45.2%), onychogryphosis (41.3%), dermatitis (34.1%), pale mucous membranes (19.7%), weight loss (14.9%), fever (12.5%), alopecia (11.1%), and lethargy (4.8%). Tick and flea infestations were recorded in 41.7% and 29.3% of the dogs, respectively, with 49 (16.3%) dogs being co-infested by both ticks and fleas. Most of the tick- and flea-infested dogs presented high level of infestation (>10 ticks and >20 fleas). The level of tick infestation varied significantly among federative units, being highest in Minas Gerais (68.0%), followed by Pernambuco (36.0%) and Federal District (21.0%). On the other hand, the level of flea infestation was higher in Pernambuco (50.0%), followed by Minas Gerais (29.0%) and Federal District (9.0%). The number of dog owners reporting the use of ectoparasiticides (on dogs and/or in the environment) varied significantly, ranging from 6.0% in Pernambuco to 60.0% in Federal District. This study reveals disparate results in terms of dog exposure to fleas and ticks in the studied communities, which may be related to factors such as the owners' capability/willingness to afford the use of ectoparasiticides on their dogs and the dog's level of restriction. Further research is needed to establish the relationship between dog owners' socioeconomic situation and the level of exposure to ectoparasites and their transmitted pathogens., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Anaplasma phagocytophilum and Borrelia burgdorferi are both transmitted by Ixodes spp. and are associated with clinical illness in some infected dogs. This study evaluated canine antibody responses to the A. phagocytophilum p44 peptides APH-1 and APH-4 as well as the B. burgdorferi C6 peptide before and after doxycycline treatment. A total of eight dogs were infested with wild-caught I. scapularis for 1 week. Blood was collected prior to tick attachment and from Days 3-77 to 218-302 with doxycycline treatment beginning on Day 218. Blood was assayed for A. phagocytophilum DNA by PCR assay. Sera was assessed for antibodies by immunofluorescent antibody (IFA) test and ELISA. Anaplasma phagocytophilum DNA was amplified from blood of all dogs by Day 7. Antibodies to APH-4 were detected in serum as early as 14days after tick exposure and six dogs had APH-4 antibodies detected 3-7 days before antibodies against APH-1. All dogs were seropositive for A. phagocytophilum from Days 218 to 302. Antibodies to B. burgdorferi were detected in 6/8 dogs beginning 21days after I. scapularis infestation. Among the five dogs that remained seropositive at Day 218, C6 antibody levels declined on average 81% within 84days of initiating treatment. The results suggest that the APH-4 peptide may be more useful than APH-1 for detecting antibodies earlier in the course of an A. phagocytophilum infection. After doxycycline administration, C6 antibody levels but not APH-1 or APH-4 antibody levels decreased, suggesting a treatment effect on C6 antibody production., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Canine test results generated by veterinarians throughout Canada from 2013-2014 were evaluated to assess the geographical distribution of canine infection with Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia spp., and Anaplasma spp., Methods: The percent positive test results of 115,636 SNAP® 4Dx® Plus tests from dogs tested were collated by province and municipality to determine the distribution of these vector-borne infections in Canada., Results: A total of 2,844/115,636 (2.5%) dogs tested positive for antibody to B. burgdorferi. In contrast, positive test results for D. immitis antigen and antibodies to Ehrlichia spp. and Anaplasma spp. were low, with less than 0.5% of dogs testing positive for any one of these three agents nationwide. Provincial seroprevalence for antibodies to B. burgdorferi ranged from 0.5% (Saskatchewan)-15.7% (Nova Scotia); the areas of highest percent positive test results were in proximity to regions in the USA considered endemic for Lyme borreliosis, including Nova Scotia (15.7%) and Eastern Ontario (5.1%). These high endemic foci, which had significantly higher percent positive test results than the rest of the nation (P < 0.0001), were surrounded by areas of moderate to low seroprevalence in New Brunswick (3.7%), Quebec (2.8%), and the rest of Ontario (0.9%), as well as northward and westward through Manitoba (2.4%) and Saskatchewan (0.5%). Insufficient results were available from the westernmost provinces, including Alberta and British Columbia, to allow analysis., Conclusion: Increased surveillance of these vector-borne disease agents, especially B. burgdorferi, is important as climate, vector range, and habitat continues to change throughout Canada. Using dogs as sentinels for these pathogens can aid in recognition of the public and veterinary health threat that each pose.

Background: Lyme borreliosis is a common tick-borne disease of the northern hemisphere that is caused by bacterial spirochaetes of the Borrelia burgdorferi (sensu lato) (Bbsl) complex. To date, there has been no convincing evidence for locally-acquired Lyme borreliosis on the Australian continent and there is currently a national debate concerning the nature and distributions of zoonotic tick-transmitted infectious disease in Australia. In studies conducted in Europe and the United States, dogs have been used as sentinels for tick-associated illness in people since they readily contact ticks that may harbour zoonotic pathogens. Applying this principle, we used a combination of serological assays to test dogs living in tick 'hot spots' and exposed to the Australian paralysis tick, Ixodes holocyclus, for evidence of exposure to B. burgdorferi (s.l.) antigens and other vector-borne pathogens., Results: Altogether, 555 dogs from four demographic groups were recruited into this study. One dog had evidence of exposure to Anaplasma spp. but no other dog was positive in screening tests. A total of 122 dogs (22.0%) had a kinetic ELISA (KELA) unit value > 100, and one dog with a high titre (399.9 KELA units) had been vaccinated against B. burgdorferi (sensu stricto) before travelling to Australia. Older dogs and those with a history of tick paralysis were significantly more likely to have a KELA unit value > 100. Line immunoassay analysis revealed moderate-to-weak (equivocal) bands in 27 (4.9%) dogs., Conclusions: Except for a single dog presumed to have been exposed to Anaplasma platys, infection with Anaplasma spp. B. burgdorferi (s.l.), Ehrlichia spp., and Dirofilaria immitis, was not detected in the cohort of Australian dogs evaluated in this study. These results provide further evidence that Lyme borreliosis does not exist in Australia but that cross-reacting antibodies (false positive results) are common and may be caused by the transmission of other tick-associated organisms.

A randomized, blinded, negative controlled study was conducted to determine whether treatment with afoxolaner (NexGard ® , Merial, Inc.) would prevent the transmission of Borrelia burgdorferi to dogs by wild caught Ixodes scapularis ticks. Twenty healthy dogs were randomly assigned to two groups of ten dogs each. Ten dogs were treated orally on Day 0 at a dose near the minimum recommended dose of afoxolaner of 2.5mg/kg (actual doses 2.5-3.1mg/kg) and ten control dogs were not treated. On Day 28, each dog was infested with approximately 50 adult unfed wild caught I. scapularis that had a 67% B. burgdorferi infection rate (determined by polymerase chain reaction). On Day 33, live ticks were counted and removed. No ticks were found on treated dogs while control dogs had an average of 21.4 ticks. To detect infection, the B. burgdorferi-specific C6 antibody SNAP ® 4Dx ® test (IDEXX) was performed on serum collected before infestation (all dogs seronegative on Days -6 and 27) and on Days 48, 63, 77 and 92. The ten treated dogs remained seronegative through the end of the study (Day 92), while nine out of the ten control dogs were infected, as demonstrated by their seroconversion to being positive for the presence of the B. burgdorferi-specific C6 antibody starting on Day 48. In this study, all dogs treated with NexGard ® 28days prior to challenge with wild caught I. scapularis ticks were protected from B. burgdorferi infection, while nine out of the ten untreated control dogs were infected., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

OspC performs essential functions during the enzootic cycle of the Lyme disease (LD) spirochetes. In this study, the specificity of antibody (Ab) responses to OspC was profiled to define the antigenic determinants during infection and after vaccination. Several OspC variants or 'types' were screened with serum from SNAP4Dx C6 positive dogs and with serum from rabbits hyperimmunized with OspC proteins. The OspC type-specific nature of the Ab response revealed that variable domains of OspC are immunodominant during infection and upon vaccination. To assess the potential of OspC to elicit Ab in the context of a bacterin vaccine, OspC production in strains cultivated in vitro was assessed. Immunoblot and indirect immunofluorescent antibody analyses demonstrated that production is low and that only a subset of cells actively produces OspC in vitro, raising questions about the potential of bacterin vaccines to stimulate significant anti-OspC Ab responses. The specificity of the OspC Ab response in experimentally infected mice over time was assessed to determine if domains shielded in the OspC homodimer become accessible and stimulate Ab production as infection progresses. The results demonstrate that the OspC Ab response remains focused on surface exposed variable regions of the protein throughout infection. In contrast to some earlier studies, it is concluded that conserved domains of OspC, including the C7 or C10 domain, do not elicit significant Ab responses during infection or upon vaccination. Collectively, the results indicate that OspC diversity must be considered in vaccine design and in the interpretation of diagnostic assays that employ OspC as a diagnostic antigen., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lyme disease in dogs can be effectively prevented by vaccination against antigens expressed by the spirochete Borrelia burgdorferi during transmission by the tick vector Ixodes sp. Lyme vaccine efficacy has traditionally been based on indicators of infection following wild-caught tick challenge whereas most other types of vaccine are required to demonstrate protection from clinical signs of disease. In this vaccination-challenge study we sought to demonstrate the ability of a nonadjuvanted, outer surface protein A (OspA) vaccine to protect from infection and to prevent synovial lesions consistent with Borreliosis. Thirty, purpose-bred beagles were randomly divided into vaccinated and unvaccinated groups. The vaccinated group was administered two subcutaneous doses of a nonadjuvanted, purified, Borrelia burgdorferi OspA vaccine at a 21- day interval. Dogs were challenged by wild-caught, B. burgdorferi-infected ticks (Ixodes scapularis). Clinical signs, serology, Borrelia isolation and PCR evaluated antemortem vaccine efficacy. Postmortem histopathological analysis of synovial tissue was compared to antemortem infection status. Borreliosis was demonstrated by Borrelia isolation from skin biopsies in 13 out of 15 unvaccinated dogs. All unvaccinated dogs' Western blot profiles were consistent with infection. Two of 15 vaccinated dogs had at least one positive spirochete culture which cleared 91days post-challenge, and Western blot profiles were consistent with vaccination alone. No dogs, vaccinated or unvaccinated, exhibited clinical signs consistent with borreliosis. Based on a histopathological cumulative joint scoring system (CJS), all unvaccinated dogs had synovial lesions indicative of Lyme disease. Only one of the vaccinated dogs had a CJS that was greater than the statistical cut off score for the absence of synovial lesions. There was high correlation between clinical histopathology and spirochete isolation. Infection with B burgdorferi may produce inconsistent clinical signs of lameness. Histopathological changes in joints from infected dogs are reliable indicators of borreliosis and correlate well with other indicators of infection. This model provides support that vaccination with a nonadjuvanted, purified OspA vaccine offers protection from Borrelia infection and the resulting synovial lesions that can lead to clinical signs of lameness., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study assessed the seroprevalence of anti-Borrelia burgdorferi antibodies in New Brunswick dogs. Testing of 699 serum samples from dogs across the province revealed a 6% province-wide seropositivity, more than 6 times higher than that found in 2008. The rapid increase in seropositivity indicates increased Lyme disease risk to both canine and human health.

The aim of the study was to establish the prevalence of Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi in dogs in eastern Poland and to determine the factors associated with exposure (seroposity) or infection (PCR). Anti-A. phagocytophilum, anti-B. burgdorferi and anti-E. canis antibodies were determined in 400 dogs, using the SNAP 4Dx ® test (IDEXX Laboratories). In addition, PCRs were performed for the detection of E. canis, A. phagocytophilum and B. burgdorferi DNA. In reference to the risk factor analysis, a regression logistic model was determined for each aetiological agent. The overall seroprevalence was highest for B. burgdorferi (11.0 %), followed by A. phagocytophilum (8.0 %) and E. canis (1.5 %). Eleven healthy dogs were found to be infected with A. phagocytophilum, as determined by PCR, while the remainder were seronegative. For B. burgdorferi, the DNA of the spirochetes was detected in the blood of 20 dogs, while the presence of anti-B. burgdorferi IgG was detected in the sera of ten of these. For E. canis, none of the dogs tested positive by PCR. Tick control was included as a protective factor for A. phagocytophilum and B. burgdorferi, while the origin (rural) was included as a risk factor for B. burgdorferi and A. phagocytophilum infection. In addition, breed (pure) was a risk factor for B. burgdorferi infection, and sex (female) was a risk factor for E. canis.

Objective: Bernese Mountain Dogs (BMD) have a higher prevalence of Borrelia burgdorferi sensu lato (Bbsl) antibodies than other breeds, but it is not known whether this is the case for other pathogens. Therefore, the aim of the study was to determine the frequency and level of specific antibodies against members of the Bbsl group, Anaplasma phagocytophilum (Ap), and Leptospira (L.) interrogans serovars in BMD and compare the results with those found in dogs of other breeds., Materials and Methods: A total of 171 healthy BMD and 57 healthy control dogs of other breeds were included in the study. Controls were large dogs (> 30 kg) with long, dark hair coats. A two-tiered testing method consisting of computerized kinetic enzyme-linked immunosorbent assay (KELA) and Western blotting was used for detection of antibodies against Bbsl, an immunofluorescence assay (IFA) was used for detection of antibodies against Ap, and microscopic agglutination test (MAT) for antibodies to 18 different serovars of L. interrogans., Results: The prevalence of anti-Bbsl antibodies was significantly higher in BMD (43.3%) than in controls (17.5%) (p < 0.001). Antibodies to Bbsl attributable to vaccination were excluded from the calculation of prevalence. Antibodies to Ap were found in 50.3% of BMD, whereas only 24.6% of the controls dogs were tested positive for Ap (p < 0.001). Antibody titers of the 18 different serovars of L. interrogans antibodies did not differ significantly between BMD and control dogs except for L. copenhageni antibody titers which were higher in BMD. Significantly higher antibody titers to L. canicola (p = 0.003), L. copenhageni (p = 0.005), L. grippothyphosa (p = 0.029) and L. vanderhoedoni (p = 0.035) were seen in BMD compared to control dogs., Conclusion and Clinical Relevance: BMD had a higher prevalence of anti-Bbsl, anti-L. copenhageni and anti-Ap antibodies than control dogs. Significantly higher antibody titers against L. canicola (p = 0.003), L. copenhageni (p = 0.005), L. grippothyphosa (p = 0.029) and L. vanderhoedoni (p = 0.035) were seen in BMD compared with control dogs, but the reason for this and potential clinical implications are not known.

The primary objective of our study was to optimize detection of serum antibodies to Borrelia burgdorferi using a new commercial automated fluorescence system (Accuplex4 BioCD system, Antech Diagnostics, Lake Success, New York). The system used multiple natural and artificial peptides-outer surface proteins (OspA, OspC, OspF), an outer membrane protein (P39), and a proprietary synthetic peptide (small Lyme peptide [SLP])-and the results were compared with a commercially available enzyme-linked immunosorbent assay that uses a proprietary peptide (C6). Sera from 4 groups were evaluated: dogs vaccinated with 1 of 3 commercially available vaccines (n = 18); dogs infested with adult Ixodes scapularis (black-legged tick; n = 18); dogs previously vaccinated and then infested with I. scapularis (n = 18); and dogs with B. burgdorferi infection that were then vaccinated (n = 14). All of the vaccines evaluated induced OspA responses. However, antibodies against OspF or C6 were not induced in any of the vaccinated dogs. Additionally, the OspF antibodies had 100% sensitivity and specificity when compared to antibodies against C6 peptide. In B. burgdorferi-infected dogs, antibodies against OspC and SLP were detected in serum sooner than antibodies against the other targets. Low levels of antibodies against OspA developed in 6 of 14 B. burgdorferi-infected, unvaccinated dogs and had the shortest duration compared to the other antibodies. Detection of antibody responses to multiple B. burgdorferi targets with this system can be used to help differentiate vaccinated dogs from exposed dogs as well as acute infection from chronic infection., (© 2015 The Author(s).)

Beagles received placebo or ospA- and ospB-negative Borrelia burgdorferi before a tick challenge. A total of 28 (41%) ticks and skin biopsy specimens from each control dog (n = 10) contained B. burgdorferi. In contrast, 12 (19%) ticks recovered from the vaccine recipients (n = 10) were infected (P = 0.0077), and 5 dogs yielded spirochetes from the skin biopsy specimens (P = 0.0325). In addition, 9 (90%) placebo recipients and 4 (40%) vaccine recipients developed joint abnormalities (P = 0.0573). Therefore, vaccination with the ospA- and ospB-negative spirochete provided significant protection against Lyme disease., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Antibody levels to outer surface proteins C and F (OspC and OspF, respectively) in sera collected from laboratory Beagle dogs at 1, 2, and 4 months after challenge with infected black-legged ticks (Ixodes scapularis) were determined. Each dog was confirmed by culture to harbor Borrelia burgdorferi in the skin (n = 10) or the skin and joints (n = 14). Significant levels of immunoglobulin M (Ig)M or IgG anti-OspC antibodies were detected in single serum samples from only 3 (13%) dogs. Similarly, IgM anti-OspF antibodies were detected in only 1 (4%) serum sample collected from a dog with B. burgdorferi in the skin and joints. In contrast, 4 (29%) dogs with skin and joint infections produced IgG anti-OspF antibodies after 2 months, and the response expanded to include 2 (20%) dogs with skin infection and 4 additional dogs with skin and joint infections (overall sensitivity = 62%) after 4 months. The findings failed to support the utility of OspC-based antibody tests for diagnosing canine Lyme disease, but demonstrated that dogs with B. burgdorferi colonizing joint tissue most often produced significant levels of IgG anti-OspF antibodies. Therefore, additional studies to more thoroughly evaluate the clinical utility of OspF-based antibody tests are warranted., (© 2015 The Author(s).)

Background: In North America, Borrelia burgdorferi is the causative bacterial agent of canine Lyme borreliosis and is transmitted following prolonged attachment and feeding of vector ticks, Ixodes scapularis or Ixodes pacificus. Its prevention is predicated upon tick-avoidance, effective on-animal tick control and effective immunization strategies. The purpose of this study is to characterize dogs that are newly seropositive for Borrelia burgdorferi infection in relation to compliant use of a recombinant OspA canine Lyme borreliosis vaccine. Specifically, Preventive Fractions (PF) and Risk Ratios (RR) associated with the degree of vaccine compliancy (complete versus incomplete) are determined., Methods: 6,202 dogs were tested over a five year period in a single veterinary hospital utilizing a non-adjuvanted, recombinant OspA vaccine according to a 0, 1, 6 month (then yearly) protocol. Rates of newly acquired "Lyme-positive" antibody test results were compared between protocol compliant and poorly compliant (incompletely and/or non-vaccinated) dogs., Results: Over the five-year span, one percent (range 0.39 - 1.3) of protocol compliant vaccinated, previously antibody negative dogs became seropositive for infection. Approximately twenty-one percent (range 16.8 - 33.3) of incompletely vaccinated dogs became positive for infection-specific antibodies. The Preventative Fraction for testing positive for antibodies specific for infection with Borrelia burgdorferi in any given year based on optimal vaccine compliance was, on average, 95.3% (range 93.29 - 98.08). The Risk Ratio for becoming infected with Borrelia burgdorferi antibodies in any given year if vaccine non-compliant was 21.41 (range 14.9 - 52.1). There was a high statistically significant relationship (p = <0.0001) in the observed data in terms of vaccination protocol compliance and the probability of Borrelia burgdorferi infection in each of the five years under study., Conclusions: The recombinant outer surface protein A (rOspA) vaccine for dogs is highly effective in preventing new seropositive cases of Borrelia burgdoferi infection over a five-year period in dogs living in an endemic area. Dogs that were vaccine protocol-compliant were significantly less likely to become infected (as indirectly assessed by antibody) with the agent of canine Lyme borreliosis as measured by Preventive Fraction and Risk Ratio calculations.

Background: Antibody tests are frequently used in the diagnosis of canine Lyme borreliosis, including immunofluorescence assay (IFA) for immunoglobulin G (IgG) and M (IgM) antibodies, kinetic enzyme-linked immunosorbent assay (KELA), and Western blotting (WB). Recently, the SNAP4Dx, an in-house test using C6 technology has become available., Objectives: The aim of this study was to compare IFA, KELA, and SNAP4Dx assay results, and to determine their sensitivity and specificity when compared with the WB, used as gold standard in this study., Methods: Two hundred canine sera were tested for the presence of specific antibodies against Borrelia spp. using the above-mentioned tests., Results: The sensitivity and specificity of IFA-IgG was 76.6% (95% confidence interval [CI] 46.87-86.72) and 87.1% (95% CI 80.06-91.90), and 26.3% (95% CI 11.81-48.79) and 81.0% (95% CI 73.64-86.71) for IFA-IgM, respectively. KELA was 100% (95% CI 83.18-100) sensitive and 75.4% (95% CI 67.02-82.09) specific, and the SNAP4Dx was 84.2% (95% CI 62.43-94.48) sensitive and 98.5% (95% CI 94.83-99.60) specific., Conclusions: Both IFAs had very low sensitivity and specificity and cannot be recommended for screening purposes. In contrast, KELA showed excellent sensitivity, but positive results always need to be confirmed by WB to differentiate the source of antibody formation. The SNAP4Dx had a high sensitivity and specificity, and thus can potentially replace the more labor-intensive WB, at least in untreated dogs., (© 2014 American Society for Veterinary Clinical Pathology.)

Vector-borne disease (VBD) pathogens remain an emerging health concern for animals and humans throughout the world. Surveillance studies of ticks and humans have made substantial contributions to our knowledge of VBD epidemiology trends, but long-term VBD surveillance data of dogs in the United States is limited. This seroreactivity study assessed US temporal and regional trends and co-exposures to Anaplasma, Babesia, Bartonella, Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia spp., and spotted fever group Rickettsia in dogs from 2004-2010. Dog serum samples (N=14,496) were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Disease Diagnostic Laboratory for vector-borne pathogens diagnostic testing using immunofluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) assays. These convenience samples were retrospectively reviewed and analyzed. The largest proportion of samples originated from the South (47.6%), with the highest percent of seroreactive samples observed in the Midatlantic (43.4%), compared to other US regions. The overall seroreactivity of evaluated VBD antigens were Rickettsia rickettsia (10.4%), B. burgdorferi (5.2%), Ehrlichia spp. (4.3%), Bartonella henselae (3.8%), Anaplasma spp. (1.9%), Bartonella vinsonii subsp. berkhoffii (1.5%), Babesia canis (1.1%), and D. immitis (0.8%). Significant regional and annual seroreactivity variation was observed with B. burgdorferi, Ehrlichia, and Rickettsia exposures. Seasonal seroreactivity variation was evident with Rickettsia. Seroreactivity to more than one antigen was present in 16.5% of exposed dogs. Nationally, the most prevalent co-exposure was Rickettsia with Ehrlichia spp. (5.3%), and the highest odds of co-exposure was associated with Anaplasma spp. and B. burgdorferi (odds ratio=6.6; 95% confidence interval 5.0, 8.8). Notable annual and regional seroreactivity variation was observed with certain pathogens over 7 years of study, suggesting canine surveillance studies may have value in contributing to future VBD knowledge.

Canine vector-borne diseases (CVBDs) have increasingly become a focus of attention in the past few years. Nevertheless, in many parts of Europe information on their occurrence is still scarce. In a large study in Poland 3,094 serum samples taken from dogs throughout all 16 Polish provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen. A total of 12.31% (381/3,094; 95% confidence interval [CI]: 11.18-13.52%) and 3.75% (116/3,094; 95% CI: 3.11-4.48%) of the dogs were positive for A. phagocytophilum and B. burgdorferi s.l. antibodies, respectively. Furthermore, 0.26% (8/3,094; 95% CI: 0.11-0.51%) were positive for E. canis antibodies and 0.16% (5/3,094; 95% CI: 0.05-0.38%) for D. immitis antigen. The highest percentages of A. phagocytophilum-positive dogs were noted in Lesser Poland, Silesia and Łódź Provinces. For B. burgdorferi s.l., the highest prevalence was recorded in Łódź Province. Co-infections with A. phagocytophilum and B. burgdorferi s.l. were recorded in 1.71% of all examined dogs (53/3,094; 95% CI: 1.29-2.23%). One dog even had a triple infection, testing positive for E. canis too. Both A. phagocytophilum and B. burgdorferi s.l. have previously been reported in Poland and were confirmed in the present study by positive samples from all 16 provinces. Concerning E. canis and D. immitis travel history or importation cannot be excluded as factors which may have determined the occurrence of these pathogens in the relevant animals. Practitioners in Poland should be aware of the above mentioned CVBDs and of prophylactic measures to protect dogs and their owners.

The first comprehensive study on the prevalence of canine vector-borne pathogens (Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis, and Dirofilaria immitis) was carried in Hungary because, except for babesiosis and dirofilariosis caused by Dirofilaria repens, there were no data on their regional distribution and prevalence. In 2011 and 2012, 1305 blood samples were collected from randomly selected, apparently healthy pet dogs in 167 localities of 19 counties of Hungary. All sera samples from dogs were screened for simultaneous qualitative detection of circulating antibodies to E. canis and B. burgdorferi sensu lato and A. phagocytophilum and D. immitis antigen using SNAP(®) 4Dx (IDEXX Laboratories). Overall, 170 dogs (13.0%; 95% confidence interval [CI] 11-15) were serologically positive to one or more of the tested pathogens. A. phagocytophilum was the most prevalent pathogen detected in 102 dogs by antibody titers (7.9%, 95% CI 6.5-9.5), followed by D. immitis (2.4%, 95% CI 1.0-4.0, n=64) and B. burgdorferi (0.4%, 95% CI 0.0-1.1, n=11) out of 1305 tested dogs. The least prevalent infection was with E. canis, with only two positive dogs (0.16%, 95% CI 0.03-0.6). Co-infection was found in eight dogs (0.61%, 95% CI 0.29-1.21), of which seven were seropositive to two pathogens (five with A. phagocytophilum and D. immitis, two with A. phagocytophilum and B. burgdorferi). One dog was serologically positive to three pathogens (A. phagocytophilum, B. burgdorferi, and D. immitis). Purebred and crossbred animals did not show significantly different levels of seropositivity. There was no significant association between the gender and the results of diagnostic testing. Logistic regression analysis showed a higher chance of seropositivity in the older dogs.

In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the north-east of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n=2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lyme disease, a tick-borne illness caused by Borrelia burgdorferi, infects humans and other species, including dogs. Canine seroprevalence has been suggested as a sentinel marker of human disease risk. A recent publication reported high canine seroprevalence (>5%) in Routt County, Colorado, an area where Lyme disease is generally considered nonendemic. We surveyed veterinarians in Routt County and discovered that 11 of 12 seropositive dogs (>90%) had a documented history of travel to or residence in a Lyme disease endemic area. These findings do not support the presence of an undocumented disease focus and reveal that despite its high sensitivity, there are limitations in the specificity and positive predictive value of elevated canine seroprevalence as a marker of human risk.

Background: Controlling canine vector-borne diseases (CVBD) is a major concern, since some of these diseases are serious zoonoses. This study was designed to determine seropositivity rates in Spain for agents causing the following five CVBD: leishmaniosis (Leishmania infantum: Li), heartworm (Dirofilaria immitis: Di), ehrlichiosis (Ehrlichia canis: Ec), anaplasmosis (Anaplasma phagocytophilum/Anaplasma platys: An) and Lyme disease (Borrelia burgdorferi: Bb)., Methods: Anti-An, -Bb, and -Ec antibodies and the Di antigen were determined using the 4DX SNAP® Test (IDEXX Laboratories) and anti-L. infantum (Li) antibodies using the Leishmania SNAP® Test (IDEXX Laboratories) in blood and/or serum samples., Results: Among 1100 dogs examined, overall seropositivity rates were: Li (15.7%), Ec (5%), An (3.1%), Di (1.25%) and Bb (0.4%). While seropositivity towards Bb and Di was similar in all geographic regions, rates were significantly higher in the east of Spain (8.3%) for An, significantly higher in the north (20%) for Ec, and significantly higher in the Southeast (46.6%) and South (27.4%), and significantly lower in the north (0%) for Li.No statistical associations were observed between sex and the CVBD analyzed (p ≥ 0.05) while the following associations with other variables were detected: a higher seropositivity to Ec (40%) and Bb (6.7%) in dogs under one year of age compared with adults (p < 0.05); and a higher seropositivity to An and Li in dogs that lived outdoors versus indoors (p = 0.01; p < 0.001, respectively). Seropositivity rates of 2.1%, 0%, 1.7%, 0.5% and 4.2% were recorded respectively for An, Bb, Ec, Di and Li in dogs with no clinical signs (n = 556) versus 3.8%, 0.6%, 7.5%, 1.8% and 25.9% for those with signs (n = 507) suggestive of a CVBD., Conclusion: The data obtained indicate a risk for dogs in Spain of acquiring any of the five CVBD examined. Veterinarians in the different regions should include these diseases in their differential diagnoses and recommend the use of repellents and other prophylactic measures to prevent disease transmission by arthropod vectors. Public health authorities also need to become more involved in the problem, since some of the CVBD examined here also affect humans.

In this study we endeavored to determine the seroprevalence of tick-borne infections in the military working dog (MWD) population in the Republic of Korea (ROK). Our sample population consisted of 182 serum samples from MWDs for 3 different years (1996, 2002, and 2007). In addition, 63 whole blood samples from 2007 were available for polymerase chain reaction (PCR). Serum samples were evaluated by a commercially available enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA) for Ehrlichia canis and Anaplasma phagocytophilum, and by ELISA only for Borrelia burgdorferi. PCR amplification of DNA was performed to screen for Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum, A. platys, Borrelia burgdorferi, and Rickettsia rickettsii, as well as Babesia and Theileria species using previously published primers and probes. A total of 56 (30.8%) MWDs were positive by at least one serologic test. Seroprevalences for Anaplasma and Ehrlichia were 4.4% and 0.6% based on the ELISA, and 24.7% and 22.5% based on the IFA, respectively. ELISA testing for Borrelia yielded 2 (1.1%) positive results. In parallel testing using both the ELISA and IFA tests, the percentages of dogs with one or more positive results were 34.1%, 25.9%, and 28.4%, for 1996, 2002, and 2007, respectively. There was no significant differences in seroprevalence based on location, year, breed, or sex of the MWD. There was poor agreement between IFA and ELISA test results. No MWD sample had a positive PCR result. MWDs stationed in Korea had serologic evidence of exposure to several tick-borne pathogens, but PCR testing did not identify any active infections.

Tick-borne diseases are of great concern worldwide. Despite this, in Romania there is only limited information regarding the prevalence of vector-borne pathogens in dogs. In all, 1146 serum samples were tested by SNAP(®) 4Dx(®) (IDEXX Laboratories, Inc., Westbrook, ME) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies, and for Dirofilaria immitis antigen. The correlation between positive cases and their geographic distribution, as well as potential risk factors (age, sex, breed, type of dog, habitat, and prophylactic treatments) were evaluated. Overall, 129 dogs (11.3%) were serologically-positive to one or more of the tested pathogens. The seroprevalence for the four infectious agents were: A. phagocytophilum 5.5% (63/1146), D. immitis 3.3% (38/1146), E. canis 2.1% (24/1146), and B. burgdorferi 0.5% (6/1146). Co-infection with E. canis and A. phagocytophilum was registered in 2 dogs (0.2%). The geographical distribution of the seropositive cases suggests clustered foci in southern regions and in the western part of the country for D. immitis, and in the southeastern region (Constanţa County) for E. canis. A. phagocytophilum and B. burgdorferi showed a homogenous distribution, with a tendency for Lyme-positive samples to concentrate in central Romania. For D. immitis, A. phagocytophilum, and E. canis, administering prophylactic treatments was a risk factor associated with infection. Another associated risk factor was the type of dog (stray dogs were at risk being positive for D. immitis, shelter dogs for E. canis, and hunting dogs for B. burgdorferi). The prevalence of D. immitis was significantly higher in males and in dogs older than 2 years. This survey represents the first data detailing A. phagocytophilum and E. canis seroprevalence in Romanian dogs, and the most comprehensive epidemiological study on vector-borne infections in dogs from this country.

Lyme disease in the United States is caused by Borrelia burgdorferi sensu stricto, which is transmitted to mammals by infected ticks. Borrelia spirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses to B. burgdorferi Osp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs.

Objective: To evaluate the Anaplasma phagocytophilum (A. phagocytophilum), Ehrlichia canis (E. canis), Dirofilaria immitis (D. immitis) (canine heartworm), Borrelia burgdorferi (B. burgdorferi) infections in countryside dogs from Yunnan, Hainan and Anhui provinces., Methods: Serum samples were collected from 26 dogs in Yunnan, Hainan and Anhui provinces. The samples were tested using a commercial ELISA rapid diagnostic assay kit (SNAP(®) 4Dx(®); IDEXX Laboratories, Inc. U.S.A.). Meanwhile, indirect immunofluorescence assay (IFA) recommended by WHO was conducted to detect IgG to A. phagocytophilum. Two methods were analyzed and compared., Results: The number of serologically positive dogs for IgG to A. phagocytophilum was only 2 which was from Hainan province and none of the 26 dogs responded positive for E. canis, D. immitis (canine heartworm), and B. burgdorferi by ELISA rapid diagnostic method. The number of serologically positive dogs for IgG to A. phagocytophilum was 13 (50%) by IFA method. Data of the two methods were analyzed by statistical software and the difference was statistically significant (P=0.002)., Conclusions: It can be concluded that IFA method was more sensitive than ELISA rapid diagnostic method. However, we need conduct further and intensive epidemiology survey on tick-born diseases pathogens including A. phagocytophilum, E. canis, D. immitis (canine heartworm), and B. burgdorferi which have public health significance.

Background: In the US little spatially defined information regarding exposure to most vector-borne pathogens in dogs is available for the states of California (CA), Oregon (OR), and Washington (WA)., Objectives: The purpose of the present study was to evaluate the spatial distribution of seroprevalence for 4 vector-borne pathogens, Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis, and Dirofilaria immitis, across the 3 western coastal states of the contiguous United States that extend from the northern Mexican to the southern Canadian border., Methods: A convenience sample, targeting blood from 20 pet dogs per county across CA, OR, and WA, was evaluated using a canine point-of-care ELISA kit. Geographic coordinates of home zip code were displayed using a geographic information system. A total of 2431 dogs from CA, OR, and WA were tested., Results: The overall seroprevalence was highest for A. phagocytophilum (2.4%), followed by B. burgdorferi (1.2%), and E. canis (0.7%). The prevalence of infection with D. immitis was 0.7%. At the individual dog level, there was a significant association between seropositivity to B. burgdorferi and A. phagocytophilum (odds ratio=18.7, 95% confidence interval=6.8-47.1). For most positive results, prevalence tended to decrease with increasing latitude; thus, the highest rates of seropositivity occurred in CA, followed by OR, and then WA; one exception was seropositivity for B. burgdorferi, which was higher in WA (0.38%) than in OR (0.15%), but considerably lower than in CA (2.00%). In WA, dogs that tested positive for A. phagocytophilum, E. canis, and B. burgdorferi were in the southern Puget Sound area. For D. immitis, none of the dogs in WA was positive., Conclusions: Seropositivity for vector-borne pathogens is broadly but patchily distributed in dogs in CA, OR, and WA., (©2011 American Society for Veterinary Clinical Pathology.)

To determine if the range of deer ticks in Maine had expanded, we conducted a multitarget serosurvey of domestic dogs (Canis lupus familiaris) in 2007. An extension of exposure to Borrelia burgdorferi to the northern border and local transmission of Anaplasma phagocytophilum throughout southern areas was found.

The seropositivity of dogs to Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia canis antibodies, and Dirofilaria immitis antigen was assessed in Canada. Borrelia burgdorferi had the highest seroprevalence, while that of Dirofilaria immitis has not changed significantly in the past 20 y. The risk for these vector-borne infectious agents in Canadian dogs is low but widespread with foci of higher prevalence.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)