Ramaswamy Chandrashekar

Serological and Molecular Prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia Species in Dogs from Minnesota

A population of 731 naturally exposed pet dogs examined at a private practice in Baxter, Minnesota, an area endemic for Lyme disease and anaplasmosis, was tested by serological and molecular methods for evidence of exposure to or infection with selected vector-borne pathogens. Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and for Dirofilaria immitis antigen. Blood samples from 273 dogs were also analyzed by polymerase chain reaction (PCR) for Anaplasma and Ehrlichia species DNA. Based on the owner history and the attending veterinarians physical examination findings, dogs exhibiting illness compatible with anaplasmosis or borreliosis were considered clinical cases, and their results were compared to the healthy dog population. Antibodies to only A. phagocytophilum were detected in 217 (29%) dogs; to only B. burgdorferi, in 80 (11%) dogs; and seroreactivity to both organisms, in 188 (25%) dogs. Of 89 suspected cases of canine anaplasmosis or borreliosis, A. phagocytophilum or B. burgdorferi antibodies were detected in 22 dogs (25%) and 8 dogs (9%) respectively, whereas antibodies to both organisms were found in 38 dogs (43%). Ehrlichia canis antibodies and D. immitis antigen were each detected in 11 (1.5%) dogs. Anaplasma phagocytophilum DNA was amplified from 7 of 222 (3%) healthy dogs and 19 of 51 (37%) clinical cases. Seroreactivity to both A. phagocytophilum and B. burgdorferi was detected more frequently in suspected cases of anaplasmosis and/or borreliosis than seroreactivity to either organism alone. Based on PCR testing, A. phagocytophilum DNA was more prevalent in suspected cases of anaplasmosis or borreliosis than in healthy dogs from the same region.

Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis : hematologic, serologic and molecular findings

BackgroundRhipicephalus sanguineus is a ubiquitous tick responsible for transmitting Ehrlichia canis and most likely Anaplasma platys to dogs, as either single or co-infections. The objective of this study was to assess the effects of either simultaneous or sequential experimental infections with E. canis and A. platys on hematological and serological parameters, duration of infection, and efficacy of doxycycline therapy in dogs infected with one or both organisms. Six dogs per group were either uninfected, A. platys infected, E. canis infected, A. platys and E. canis co-infected, A. platys infected and E. canis challenged or E. canis infected and A. platys challenged at day 112 post-infection (PI). Doxycycline treatment was initiated at 211 days PI, followed by dexamethasone immunosuppression beginning 410 days PI.ResultsInitially, transient decreases in hematocrit occurred in all groups infected with E. canis, but the mean hematocrit was significantly lower in the A. platys and E. canis co-infected group. All dogs except the controls developed marked thrombocytopenia after initial infection followed by gradually increased platelet counts by 112 days PI in groups with the single infections, while platelet counts remained significantly lower in the A. platys and E. canis co-infected group. Both sequential and simultaneous infections of A. platys and E. canis produced an enhanced humoral immune response to A. platys when compared to infection with A. platys alone. Likewise, co-infection with E. canis and A. platys resulted in a more persistent A. platys infection compared to dogs infected with A. platys only, but nearly all A. platys infected dogs became A. platy s PCR negative prior to doxycycline treatment. E. canis infected dogs, whether single or co-infected, remained thrombocytopenic and E. canis PCR positive in blood for 420 days. When treated with doxycycline, all E. canis infected dogs became E. canis PCR negative and the thrombocytopenia resolved. Despite immunosuppression, neither A. platys nor E. canis DNA was PCR amplified from doxycycline-treated dogs.ConclusionsThe results of this study demonstrate that simultaneous or sequential infection with A. platys and E. canis can alter various pathophysiological parameters in experimentally infected dogs, and because natural exposure to multiple tick-borne pathogens occurs frequently in dogs, awareness of co-infection is important in clinical practice.

Performance of a commercially available in-clinic ELISA for the detection of antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis antigen in dogs

OBJECTIVE To evaluate the sensitivity and specificity of a commercially available in-clinic ELISA for detection of heartworm infection and tick-borne diseases in dogs. SAMPLE POPULATION 846 serum, plasma, or blood samples obtained from dogs. PROCEDURES Samples were evaluated via the in-clinic ELISA to detect antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis (heartworm) antigen. True infection or immunologic status of samples was assessed by use of results of necropsy, an antigen assay for D immitis, and immunofluorescence assay or western blot analysis for antibodies against B burgdorferi, E canis, and A phagocytophilum. RESULTS Sensitivity and specificity of the in-clinic ELISA for detection of heartworm antigen (99.2% and 100%, respectively), antibodies against B burgdorferi (98.8% and 100%, respectively), and antibodies against E canis (96.2% and 100%, respectively) were similar to results for a similar commercial ELISA. In samples obtained from dogs in the northeast and upper Midwest of the United States, sensitivity and specificity of the in-clinic ELISA for antibodies against Anaplasma spp were 99.1% and 100%, respectively, compared with results for an immunofluorescence assay. Samples from 2 dogs experimentally infected with the NY18 strain of A phagocytophilum were tested by use of the in-clinic ELISA, and antibodies against A phagocytophilum were detected by 8 days after inoculation. Antibodies against Anaplasma platys in experimentally infected dogs cross-reacted with the A phagocytophilum analyte. Coinfections were identified in several of the canine serum samples. CONCLUSIONS AND CLINICAL RELEVANCE The commercially available in-clinic ELISA could be used by veterinarians to screen dogs for heartworm infection and for exposure to tick-borne pathogens.

Validation of a Leishmania infantum ELISA rapid test for serological diagnosis of Leishmania chagasi in dogs

Canine visceral leishmaniasis (CVL) is caused by Leishmania donovani complex parasites including L. donovani, Leishmania infantum and Leishmania chagasi. As some studies suggest that L. chagasi and L. infantum may be very similar or even the same species, the aim of the present study was to evaluate a commercial rapid ELISA test, originally designed for L. infantum, in the diagnosis of CVL in dogs naturally infected by L. chagasi. A total of 400 serum canine samples, including 283 positive dogs for CVL from an endemic area, 86 clinically healthy dogs from a non-endemic area and 31 dogs seropositive for confounding infectious agents (Trypanosoma cruzi, Toxoplasma gondii, Neospora caninum, Babesia canis and Ehrlichia canis) were used for test validation. An overall sensitivity of 94.7% (95% CI=91.41-97.01%) and specificity of 90.6% (95% CI=83.80-95.21%) was found, with a high degree of agreement (k=0.8445) to the indirect ELISA. When confounding infectious diseases were excluded, specificity increased to 100% (95% CI=95.8-100%), with a higher degree of agreement (k=0.8928). In conclusion, the commercial kit designed for L. infantum was a highly sensitive and specific device for detection of L. chagasi infection in dogs, which indicates high immunoreactivity similarities between L. infantum and L. chagasi.

High Prevalence of Tick-Borne Pathogens in Dogs from an Indian Reservation in Northeastern Arizona*

We evaluated the serological and molecular prevalence of selected organisms in 145 dogs during late spring (May/June) of 2005 and in 88 dogs during winter (February) of 2007 from the Hopi Indian reservation. Additionally, in 2005, 442 ticks attached to dogs were collected and identified as Rhipicephalus sanguineus. Infection with or exposure to at least one organism was detected in 69% and 66% of the dogs in May/June 2005 and February 2007, respectively. Exposure to spotted fever group (SFG) rickettsiae was detected in 66.4% (2005) and 53.4% (2007) of dogs, but rickettsial DNA was not detected using polymerase chain reaction. Active Ehrlichia canis infection (by polymerase chain reaction) was identified in 36.6% (2005) and 36.3% (2007) of the dogs. E. canis infection was associated with SFG rickettsiae seroreactivity (p < 0.001). Anaplasma platys DNA was detected in 8.3% (2005) and 4.5% (2007) of the dogs. Babesia canis and Bartonella vinsonii berkhoffii seroprevalences were 6.7% and 1% in 2005, whereas in 2007 prevalences were 0% and 1.1%, respectively. No Bartonella spp., Ehrlichia chaffeensis, or Ehrlichia ewingii DNA was detected. Dogs on this Hopi Indian reservation were most frequently infected with E. canis or A. platys; however, more than half of the dogs were exposed to a SFG-Rickettsia species.

Canine infection with Dirofilaria immitis, Borrelia burgdorferi, Anaplasma spp., and Ehrlichia spp. in the United States, 2010–2012

BackgroundThe geographic distribution of canine infection with vector-borne disease agents in the United States appears to be expanding.MethodsTo provide an updated assessment of geographic trends in canine infection with Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia spp., and Anaplasma spp., we evaluated results from an average of 3,588,477 dogs tested annually by veterinarians throughout the United States from 2010 – 2012.ResultsAs in an earlier summary report, the percent positive test results varied by agent and region, with antigen of D. immitis and antibody to Ehrlichia spp. most commonly identified in the Southeast (2.9% and 3.2%, respectively) and antibody to both B. burgdorferi and Anaplasma spp. most commonly identified in the Northeast (13.3% and 7.1%, respectively) and upper Midwest (4.4% and 3.9%, respectively). Percent positive test results for D. immitis antigen were lower in every region considered, including in the Southeast, than previously reported. Percent positive test results for antibodies to B. burgdorferi and Ehrlichia spp. were higher nationally than previously reported, and, for antibodies to Anaplasma spp., were higher in the Northeast but lower in the Midwest and West, than in the initial report. Annual reports of human cases of Lyme disease, ehrlichiosis, and anaplasmosis were associated with percent positive canine test results by state for each respective tick-borne disease agent (R2 = 0.701, 0.457, and 0.314, respectively). Within endemic areas, percent positive test results for all three tick-borne agents demonstrated evidence of geographic expansion.ConclusionsContinued national monitoring of canine test results for vector-borne zoonotic agents is an important tool for accurately mapping the geographic distribution of these agents, and greatly aids our understanding of the veterinary and public health threats they pose.

Performance of a commercially available in-clinic ELISA for detection of antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii and Dirofilaria immitis antigen in dogs

OBJECTIVE To evaluate the performance of an in-clinic ELISA designed for detection of heartworm antigen and antibodies against 5 tick-borne pathogens. DESIGN Validation study. SAMPLE 1,601 serum or matched serum, plasma, and blood samples from dogs. PROCEDURES Samples were tested for Dirofilaria immitis (heartworm) antigen and antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii by means of an in-clinic ELISA. Evaluation of assay sensitivity and specificity, agreement of results among sample types, and cross-reactivity of E canis antigens in the assay with anti-Ehrlichia chaffeensis antibodies in stored samples from experimentally infected dogs were performed at a reference laboratory. Field tests of the in-clinic ELISA were performed at 6 veterinary facilities. Results were compared with confirmatory test results. RESULTS Sensitivity and specificity of the in-clinic ELISA were > 89% for detection of antibodies against A phagocytophilum (93.2% and 99.2%, respectively), A platys (89.2% and 99.2%, respectively), B burgdorferi (96.7% and 98.8%, respectively), E canis (97.8% and 92.3%, respectively), and E ewingii (96.5% and 93.9%, respectively). Sensitivity of the assay for detection of D immitis was 98.9%, with 99.3% specificity. The in-clinic ELISA identified exposure to > 1 vector-borne pathogen in 354 of 1,195 samples. Cross-reactivity of E canis antigens with anti-E chaffeensis antibodies was confirmed. Results of field evaluations confirmed that the in-clinic ELISA could be reliably used under typical clinical conditions to identify dogs exposed to the pathogens of interest. CONCLUSIONS AND CLINICAL RELEVANCE The in-clinic ELISA provided a comprehensive in-house serologic screening test for all vector-borne pathogens evaluated.

Comparative Strain Analysis of Anaplasma phagocytophilum Infection and Clinical Outcomes in a Canine Model of Granulocytic Anaplasmosis

A pilot study was conducted to determine whether existing human or canine strains of Anaplasma phagocytophilum would reproduce clinical disease in experimentally inoculated dogs similar to dogs with naturally acquired granulocytic anaplasmosis. Six hounds were inoculated intravenously with one human and two canine strains of A. phagocytophilum that were propagated in vitro in HL-60 cells or in infected autologous neutrophils. Infected dogs were monitored for lethargy, anorexia, petechiae, lymphadenopathy, and fever. Dogs were assessed for complete blood count (CBC), serum chemistry, and serology (IFA and SNAP® 4Dx®); for A. phagocytophilum blood load by quantitative polymerase chain reaction; and for cytokine production. Prominent clinical signs were generalized lymphadenopathy and scleral injection; only one dog developed fever lasting 4 days. Notable laboratory alterations included sustained leukopenia and thrombocytopenia in all dogs. A. phagocytophilum morulae were noted in blood between days 10 and 11, although all dogs retained A. phagocytophilum DNA in blood through day 60. All dogs seroconverted by days 10-15 by IFA, and by days 17-30 by SNAP 4Dx; cytokine analyses revealed 10-fold increases in interleukin-2 and interleukin-18 in the neutrophil-propagated 98E4 strain-infected dog. All A. phagocytophilum strains produced infection, although canine 98E4 strain reproduced clinical signs, hematologic changes, and inflammatory cytokine elevations most consistent with granulocytic anaplasmosis when recognized clinically. Therefore, this strain should be considered for use in future studies of A. phagocytophilum canine infection models.

A serological survey of tick-borne pathogens in dogs in North America and the Caribbean as assessed by Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeensis, E. ewingii , and Borrelia burgdorferi species-specific peptides

Introduction Tick-borne pathogens cause a spectrum of disease manifestations in both dogs and humans. Recognizing regional and temporal shifts in exposure are important as tick distributions change. To better delineate regional exposure to canine tick-borne pathogens, an expanded set of species-specific peptides were used to detect Anaplasma phagocytophilum (Aph), Anaplasma platys (Apl), Ehrlichia canis (Ec), Ehrlichia chaffeensis (Ech), Ehrlichia ewingii (Eew), and Borrelia burgdorferi (Bb) antibodies in canine serum. Methods Archived canine serum samples (n=6,582) collected during 2008–2010 and in 2012 from the US, Canada, and the Caribbean were retrospectively screened for antibodies against Ehrlichia and Anaplasma species-specific peptides. Overall, regional and temporal seroprevalence rates were determined. Results Overall Bb and Eew were the most seroprevalent pathogens. During 2008–2010, seroprevalence rates increased overall for Aph and Ech, and regionally, Bb and Aph seroprevalence rates increased in the South. Canada had unexpectedly high seroprevalence rates for Ec and Apl. The most common co-exposures were Eew+Ech, followed by Aph+Bb and Eew+Bb. Conclusions This study demonstrated significant shifts in canine vector-borne disease seroprevalence rates. The use of specific peptides facilitated improved geographic delineation of tick-borne pathogen distributions among dogs, which may enhance epidemiological surveillance of vector-borne pathogens shared by dogs and humans.

Ehrlichia ewingii infection and exposure rates in dogs from the southcentral United States.

We used PCR and a novel serologic assay to determine infection and exposure rates to Ehrlichia ewingii in dogs from an area of northeast Oklahoma and northwest Arkansas where Amblyomma americanum ticks are abundant. Of 143 dogs assayed, 13 (9.1%) harbored E. ewingii by PCR and 64 (44.8%) had antibodies to E. ewingii detected using a peptide-based microtiter plate ELISA. Dogs were more likely (P=0.001) to be positive by PCR if sampled in August (30.8%) but no association was found between seropositive status and month of collection of sample (P>0.05). Additional testing revealed PCR evidence of Ehrlichia chaffeensis (4/143; 2.8%) and Anaplasma platys (5/143; 3.5%) as well as antibodies reactive to E. chaffeensis (25/143; 17.5%), Ehrlichia canis (2/143; 1.4%), and Anaplasma spp. (8/143; 5.6%). Testing of another 200 dogs from the area revealed additional PCR and/or serologic evidence of E. ewingii, E. canis, E. chaffeensis, and A. platys. None of the 343 dogs evaluated had evidence of Borrelia burgdorferi exposure. These data support the interpretation that E. ewingii may be the primary agent of canine ehrlichiosis in this region, and suggest that diagnostic evaluation of dogs suspected to have a tick-borne disease should include assays targeting this organism.