Canine ehrlichiosis seropositivity and associated factors in Kenya and Tanzania: a retrospective study.

Canine ehrlichiosis is an important tick-borne disease caused by bacteria in the Ehrlichia genus with species such as E. canis, E. ewingii and E. chaffeensis resulting in a severe dog illness. This study determined the occurrence of canine ehrlichiosis antibodies and its associated factors in Kenya and Tanzania. This was a retrospective study that evaluated laboratory records of 400 samples from Kenya and Tanzania submitted to Pathologists Lancet Kenya for the IDEXX SNAP 4Dx™ Plus test between the years 2016 and 2021. Records of all samples submitted to the Pathologists Lancet Kenya veterinary laboratory for the diagnostic tests were retrieved, examined, and compiled. Descriptive statistics and univariable and multivariable logistic regression were considered during analysis. The overall proportion of samples that tested positive for canine ehrlichiosis was 23% (92/400). Samples from Kenya accounted for 61% (245/400) of samples, and the percent positive was 31% (29/245). The samples from Tanzania accounted for 39% (155/400), and the percent positive was 69% (63/155). In the final model, the odds of a sample testing positive was 1.7 times for those submitted from July to December compared with those submitted from January to June. Blood samples of dogs from Tanzania had 5.31 times the odds of testing positive on the SNAP test when compared with those from Kenya. This study reports high percent positive in samples originating from Tanzania and those received during the year's second half.

Comparative evaluation of Borrelia burgdorferi antibody detection between the VetScan Flex4 and SNAP 4Dx Plus.

Two studies were developed to compare Borrelia burgdorferi antibody detection between the VetScan Flex4 and SNAP 4Dx Plus tests. The objective of the first study was to evaluate the diagnostic sensitivity (Se) and specificity (Sp) of VetScan Flex4 and SNAP 4Dx Plus B. burgdorferi results using field sourced samples compared to a Western Blot reference method. The sensitivity and specificity of VetScan Flex4 were 81.9 % (95 % CI: 71.9 %-89.5 %) and 89.3 % (95 % CI: 85.2 %-92.9 %) respectively, and SNAP 4Dx Plus's sensitivity and specificity were 80.7 % (95 % CI: 70.6 %-88.6 %) and 92.8 % (95 % CI: 89.1 %-95.5 %) respectively. When comparing VetScan Flex4 and Snap 4Dx Plus, the Simple Kappa Coefficient estimate was 0.76 (95 % CI: 0.69-0.84) indicating substantial agreement between the two methods. McNemar's Test revealed concordance between the two methods was not statistically significant (P = 0.05). The objective of the second study was to evaluate whether VetScan Flex4 differentiates between B. burgdorferi antibodies derived from infection versus vaccination with commonly used canine Lyme vaccines. The sensitivity and specificity of the VetScan Flex4 in differentiating canine Lyme vaccination from infection with Borrelia burgdorferi were 100 % (Se 95 % CI: 78.2 %-100 %; Sp 95 % CI: 91.2 %-100 %). In conclusion, the VetScan Flex4 is a reliably sensitive and specific point-of-care test that is similar to Snap 4Dx Plus, can differentiate between infection and Lyme vaccination, and can be utilized by veterinarians for Lyme disease diagnosis and surveillance of B. burgdorferi exposure.

Prevalence of Dirofilaria immitis in dogs in Shenyang, Northeastern China.

In the present study, we first report the seroprevalence of Dirofilaria immitis in dogs in Shenyang, northeastern China. Sera from 528 randomly selected dogs were examined for D. immitis antigen using SNAP®4Dx test kit; 12.7% tested showed seropositive. No significant difference of infection was observed in different genders and breeds (P>0.05), but the difference was significant in different age groups and rearing conditions (P<0.05). The result suggested that the risk of exposure to D. immitis in dogs is high in Shenyang, and should be given attention.

Detection of zoonotic vector-borne pathogens in domestic dogs in Giza, Egypt.

The public health implications of zoonotic vector-borne pathogens are numerous because domestic animals, such as dogs, live in close proximity to humans. Blood was collected from 116 domestic dogs in Cairo, Egypt from three different settings at the human-animal interface. The three settings the dogs came from were: privately owned animals seeking care at the Cairo University Faculty of Veterinary Medicine Clinic, non-laboratory reared research dogs maintained at the Cairo University Faculty of Veterinary Medicine, and an urban private animal rescue in Shabramont, Giza, Egypt. Enrolled animals were visually inspected for presence of flea or tick ectoparasites, Rhipicephalus sanguineus sensu lato ticks were recovered from 56 enrolled animals and a flea identified as Ctenocephalides felis was recovered from one animal. To test for past and/or current infection with vector-borne pathogens, conventional PCR and IDEXX SNAP® 4Dx® Plus were performed on whole blood. Pathogen targets included: Anaplasma spp., Ehrlichia spp., Babesia spp., Borrelia spp., Bartonella spp., Dirofilaria spp., and Rickettsia spp. Among dogs sampled across all locations, one dog was positive for Babesia sp. infection and one dog was positive for Anaplasma sp. infection as detected by PCR and confirmed by Sanger sequencing. Three additional dogs were positive for infection but had incomplete sequences obtained: two for Ehrlichia sp. and one for Borrelia sp. The SNAP® test results for all sampled dogs included: eight dogs positive for Anaplasma spp., 14 dogs positive for Ehrlichia spp., and five additional dogs positive for both Anaplasma spp. and Ehrlichia spp. SNAP® test results by sampling location showed that 66% of the dogs at the animal rescue were positive for Anaplasma spp. and/or Ehrlichia spp., 17% of the privately owned dogs at the Faculty of Veterinary medicine were positive for Anaplasma spp. and/or Ehrlichia spp., and none of the research dogs were positive for any of the targets on the SNAP® test. This high proportion of seropositivity in the animals sampled indicates a vector population which is not well controlled and a need for continued owner education and promotion of consistent use of preventive medications and the risk for zoonotic transmission.

Evaluation of the SNAP® 4Dx® plus test for the detection of Dirofilaria immitis antigen and characterization of exposure to tick-borne pathogens in wild canids in southern Ontario.

Population-level surveys for Dirofilaria immitis in wild canids typically rely on identification of the parasite at necropsy. More recently, some studies have employed the use of the SNAP® 4Dx® Plus test. However, since the assay was designed for use with domestic dogs it needs to be validated for use with wild canids for accurate interpretation of results. We therefore evaluated the performance of the SNAP® 4Dx® Plus test for detection of D. immitis in wild canids in southern Ontario. Overall, 199 wild canid carcasses were collected from across the region and assessed for the presence of D. immitis parasites at necropsy; ten were infected. Lung tissue extract (LE) and thoracic fluid filter paper extract (TFE) prepared from each wild canid were tested via the SNAP® 4Dx® Plus test, which simultaneously tests for the presence of D. immitis antigen and antibodies to Borrelia burgdorferi, Anaplasma spp., and Ehrlichia spp. The prevalence adjusted bias adjusted kappa (PABAK) and Gwet's first-order-agreement coefficient (AC1) were used to assess the level of agreement between sample pairs. The PABAK and AC1 between LF and TFE applied to the SNAP® 4Dx® Plus test and the necropsy-confirmed D. immitis status indicated a very high level of agreement for all sample pairs. Compared to necropsy-confirmed D. immitis status, the estimated sensitivity and specificity of the SNAP® 4Dx® Plus test for D. immitis antigen in LE was 80 % (95 % CI 44.4-97.5%) and 98.9 % (95 % CI 96.2-99.9%), respectively. For the TFE, the sensitivity was 70 % (95 % CI 34.8-93.3%) and the specificity was 97.9 % (95 % CI 94.7-99.4%). With respect to the tick-borne pathogen components, 1.5 % (3/199; 95 % CI 0.3-4.5%) of wild canids tested positive for B. burgdorferi antibody; 1.0 % (2/199; 95 % CI 0-3.8%) of LE samples were positive and 0.5 % (1/199; 95 % CI 0-3.1%) of TFE samples were positive. No samples tested positive for antibody to Anaplasma spp. (95 % CI 0-2.3%) or Ehrlichia spp. (95 % CI 0-2.3%). Collectively, the results suggest that the SNAP® 4Dx® Plus test may be a suitable test for use with LE and TFE for the detection of D. immitis antigen in wild canids from southern Ontario.

Trends in canine seroprevalence to Borrelia burgdorferi and Anaplasma spp. in the eastern USA, 2010-2017.

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.

Detection of Dirofilaria immitis antigen and antibodies against Anaplasma phagocytophilum, Borrelia burgdorferi and Ehrlichia canis in dogs from ten provinces of China.

Despite the fact vector-borne diseases (VBDs) have been increasingly reported in dogs worldwide, there are only limited reports on VBDs in dogs in China with most being based on molecular detection of active infections. To provide further data on the exposure of dogs in China to VBD agents, we used commercial immunochromatographic assays to test plasma from 637 apparently healthy indoor and breeding colony dogs from 21 veterinary clinics in 10 provinces in China and a commercial dog breeding facility for circulating antigen of Dirofilaria immitis, and for circulating antibodies against Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. Overall, we found only low levels of exposure to Ehrlichia spp. (4.7%; 30/637), Anaplasma spp. (1.4%; 9/637), B. burgdorferi (0.9%; 6/637) and D. immitis (0.2%; 1/637) with most of the positive animals coming from the commercial breeding colony (26/103; 25.2%) where ectoparasites were most commonly noted. At least one vector-borne agent was found in dogs from 6 of the 10 provinces investigated. Our results confirm exposure of dogs from around China to a variety of VBDs, even indoor pets seldom observed to harbor ectoparasites.

Canine and human infection with Borrelia burgdorferi in the New York City metropolitan area.

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 (R2 = 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 (R2 = 0.90) but was less accurate at predicting human case reports (R2 = 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.

Comparative Evaluation of 2 In-Clinic Assays for Vector-Borne Disease Testing in Dogs.

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.

Serological detection of antibodies to Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen in dogs from Costa Rica.

In a study in Costa Rica 314 serum samples from dogs throughout all seven provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis, and of circulating antigen of Dirofilaria immitis. A total of 6.4% (20/314) and 38.2% (120/314) were positive for Anaplasma spp. (An) and E. canis (Ec) antibodies. Overall, 8.0% (25/314) were positive for D. immitis (Di) antigen. One single dog reacted positive with B. burgdorferi s.l. (Bb) antigen (0.3%, 1/314). E. canis positive dogs were detected in all provinces (highest percentages in Guanacaste, Puntarenas [both significantly different compared to the overall] and Limón). Guanacaste and Puntarenas also showed the highest prevalences of Anaplasma spp. (both significantly different compared to the overall). The highest prevalence of D. immitis was detected in Puntarenas (significantly different compared to the overall). Double pathogen exposure (Ec plus An; Ec plus Di; Ec plus Bb) were recorded in 8.9% (28/314). Two dogs showed a triple pathogen exposure (0.6%, 2/314; An, Ec and Di). There was a significant difference between male (11.5%, 18/156) and female (4.4%, 7/158) animals for D. immitis positive results. There was also a significant difference between breed and no breed dogs regarding the characteristics of a general positive test, as well as seropositivity to the single pathogens of Anaplasma spp., E. canis and D. immitis. Finally there was a significant difference in the presence of clinical signs again regarding the characteristics of a general positive test, as well as seropositivity to Anaplasma spp., E. canis and D. immitis. Practitioners in Costa Rica should be aware of the canine vector-borne diseases mentioned as dogs are at risk of becoming infected. Concerning the positive B. burgdorferi s.l. dog, an autochthonous occurrence cannot be confirmed due to a history of adoption and an unusual tattoo number. Veterinary advice to protect dogs and limit transmission of vector-borne pathogens, also to humans, by using prophylactic measures is strongly recommended.

Total protein concentration and diagnostic test results for gray wolf (Canis lupus) serum using Nobuto filter paper strips.

Nobuto filter paper strips are widely used for storing blood-serum samples, but the recovery of proteins from these strips following rehydration is unknown. Poor recovery of proteins could reduce the concentration of antibodies and antigens and reduce the sensitivity of diagnostic assays. We compared the protein concentration, and its association with test sensitivity, of eluted Nobuto strip samples with paired sera. We collected and froze serum from five gray wolves (Canis lupus) for 8 mo. When thawed, we used a spectrophotometer (absorbance 280 nm) to determine the serum protein concentration for paired sera and Nobuto eluates for each animal in 2-fold serial dilutions. Total protein concentration was similar for both sample storage methods (Nobuto eluates and control sera), except for the undiluted samples in which Nobuto eluates had higher total protein concentrations. Both sample storage methods appear to produce similar results using the SNAP® 4Dx® Test to detect antibodies against pathogens causing Lyme disease, anaplasmosis, and ehrlichiosis as well as antigen for canine heartworm disease.

SNAP Assay Technology.

The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

Comparison of different diagnostic tools for the detection of Anaplasma phagocytophilum in dogs.

BACKGROUND: Anaplasma phagocytophilum is common in dogs, but the best way to diagnose an infection is still not determined. Antibody detection assays are frequently used in veterinary practice. Additionally, polymerase chain reaction (PCR) is available for detection of A phagocytophilum DNA. It is still unknown, how well different diagnostic methods correlate. OBJECTIVES: The aim of the study was to compare 2 antibody detection assays, an immunofluorescence assay (IFA) and the ELISA SNAP4Dx, and to determine the correlation of these assays by evaluating the sensitivity and specificity compared with PCR as a direct detection method of the organism. METHODS: Sera of 200 prospectively included dogs were tested for antibodies to A phagocytophilum using IFA and SNAP4Dx. DNA of the organism was detected by PCR on whole blood. Sensitivity, specificity, and predictive values, including their 95% confidence intervals, were calculated for IFA and SNAP4Dx in relation to PCR. RESULTS: Four of 200 animals were PCR-positive. Sensitivity of IFA and SNAP4Dx was 100% (95% CI 51.01-100). Specificity of IFA was 52.9% (95% CI 50.42-64.17) and that of SNAP4Dx, 57.4% (95% CI 45.83-59.70). Agreement of the 2 antibody tests was fair (κ 0.334). CONCLUSIONS: Immunofluorescence assay and SNAP4Dx were very sensitive and therefore can be useful as screening tests for A phagocytophilum infection. However, the specificity was low, and agreement between both antibody tests was insufficient. This could be due to either false-positive antibody test results, or false-negative PCR results in dogs that were actually infected.

Comparison of different diagnostic assays for the detection of Borrelia burgdorferi-specific antibodies in dogs.

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.

Babesia canis vogeli, Ehrlichia canis, and Anaplasma platys infection in a dog.

A 12-month-old male neutered mixed breed dog was presented with a history of diarrhea, lethargy, emaciation, polydypsia, and sniffling. Physical examination findings included pale mucous membranes, increased heart and respiratory rates, and normal rectal temperature (38°C). Hematologic abnormalities included anemia and thrombocytopenia. Biochemical abnormalities included hypoalbuminemia, hyperbilirubinemia, and elevated ALP and ALT activities. A SNAP 4Dx test result was positive for Ehrlichia canis. Babesia canis vogeli organisms were found in the peripheral blood films, while morulae of E canis were not seen. Real-time polymerase chain reaction testing confirmed the presence of both B c vogeli and E canis organisms, and also was positive for Anaplasma platys infection. The dog recovered following treatment with doxycycline and imidocarb dipropionate, with normal hematology and biochemical profiles.