Seroprevalence and hematological abnormalities associated with Ehrlichia canis in dogs referred to a veterinary teaching hospital in central-western Brazil / Soroprevalência e alterações hematológicas associadas com Ehrlichia canis em cães encaminhados a um hospital veterinário-escola no centro-oeste do Brasil

Ticks are significant parasites of dogs in the tropics, where tick-borne pathogens are highly prevalent, especially in areas where tick control measures are frequently neglected. This study investigated the seroprevalence and hematological abnormalities associated with Ehrlichia canis in dogs referred to a veterinary teaching hospital in Central-western Brazil. Out of 264 dogs tested for anti-Ehrlichia canis antibodies by an indirect immunofluorescence assay (IFA), 59.1% (156/264) were positive. Seropositivity was significantly associated to anemia and thrombocytopenia, alone or in combination, and to leukopenia. Conversely, there were no differences in terms of seroprevalence according to sex, breed and age. This study demonstrated that dogs referred to a veterinary teaching hospital in Central-western Brazil are highly exposed to E. canis and that seropositive dogs are more likely to present hematological abnormalities, particularly anemia, thrombocytopenia and leukopenia. To our knowledge, this is the first study on detection of anti-E. canis antibodies by means of IFA among dogs in the state of Goiás. These findings highlighted the need for increasing awareness among dog owners regarding tick control measures in Central-western Brazil, ultimately to reduce the risk of exposure to E. canis and other tick-borne pathogens.

Carrapatos são importantes parasitos de cães nos trópicos, onde patógenos transmitidos por carrapatos são altamente prevalentes, especialmente em áreas onde as medidas de controle de carrapatos são frequentemente negligenciadas. O estudo investigou a soroprevalência e as anormalidades hematológicas associadas à Ehrlichia canis em cães encaminhados para um hospital veterinário-escola no Centro-oeste do Brasil. Dos 264 cães testados para anticorpos anti-Ehrlichia canis por meio da reação de imunofluorescência indireta (RIFI), 59.1% (156/264) foram positivos. A soropositividade foi associada significativamente à anemia e trombocitopenia, isoladamente ou em combinação, e à leucopenia. Por outro lado, não houve diferenças quanto à soroprevalência segundo sexo, raça e idade. Este estudo demonstrou que cães encaminhados a um hospital veterinário-escola na região Centro-oeste do Brasil são altamente expostos à E. canis, e que cães soropositivos têm maior probabilidade de apresentar alterações hematológicas, principalmente anemia, trombocitopenia e leucopenia. Para o nosso conhecimento, este é o primeiro estudo sobre a detecção de anticorpos anti-E. canis por meio da RIFI em cães do estado de Goiás. Essas descobertas destacam a necessidade de aumentar a conscientização entre os proprietários de cães em relação às medidas de controle do carrapato no Centro-oeste do Brasil, em última análise, para reduzir o risco de exposição ao E. canis e outros patógenos transmitidos por carrapatos.

Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum.

In canine leishmaniosis caused by the protozoan Leishmania infantum, little is known about how co-infections with or co-seropositivities for other pathogens can influence aggravation of this disease. Therefore, the objectives of this study were to evaluate the frequency of co-infections with or co-seropositivities for certain pathogens in dogs seropositive for L. infantum and their relationship with clinical signs, histological changes and L. infantum load. Sixty-six L. infantum-seropositive dogs were submitted to clinical examination, collection of blood and bone marrow, culling, and necropsy. Antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and Toxoplasma gondii and Dirofilaria immitis antigens were investigated in serum. Samples from different tissues were submitted to histopathology and immunohistochemistry for the detection of Leishmania spp. and T. gondii. Quantitative real-time PCR was used to assess the L. infantum load in spleen samples. For detection of Coxiella burnetii, conventional PCR and nested PCR were performed using bone marrow samples. All 66 dogs tested positive for L. infantum by qPCR and/or culture. Fifty dogs (76%) were co-seropositive for at least one pathogen: T. gondii (59%), Ehrlichia spp., (41%), and Anaplasma spp. (18%). Clinical signs were observed in 15 (94%) dogs monoinfected with L. infantum and in 45 (90%) dogs co-seropositive for certain pathogens. The L. infantum load in spleen and skin did not differ significantly between monoinfected and co-seropositive dogs. The number of inflammatory cells was higher in the spleen, lung and mammary gland of co-seropositive dogs and in the mitral valve of monoinfected dogs. These results suggest that dogs infected with L. infantum and co-seropositive for certain pathogens are common in the region studied. However, co-seropositivities for certain pathogens did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.

Seroprevalence and Genotypic Analysis of Ehrlichia canis Infection in Dogs and Humans in Cauca, Colombia.

Ehrlichia canis infections have been reported in humans in Venezuela and Costa Rica. In this study, 506 healthy residents and 114 dogs from four municipalities (Cauca, Colombia) were surveyed and blood samples collected. Antibodies to E. canis in human and canine sera were evaluated using the Tandem repeat protein 19 (TRP19) peptide ELISA and indirect immunofluorescence assay (IFA). Ehrlichia canis TRP19 antibodies were detected in only 1/506 human sera, but the single positive sample was negative by IFA. The majority (75/114; 66%) of dogs surveyed had antibodies to the E. canis TRP19 peptide by ELISA, and eight randomly selected sera were further confirmed by E. canis IFA. Genomic DNA samples obtained from 73 E. canis TRP19 ELISA-positive dog blood samples were examined by PCR targeting the 16S ribosomal ribonucleic acid (rRNA) gene. Ehrlichia canis 16S rRNA was amplified in 30 (41%) of the dogs, and 16 amplicons were selected for DNA sequencing, which confirmed that all were E. canis. A second PCR was performed on the 16 confirmed E. canis 16S rRNA PCR-positive samples to determine the TRP36 genotype by amplifying the trp36 gene. TRP36 PCR amplicon sequencing identified nine dogs infected with the U.S. E. canis TRP36 genotype (56%), one dog with the Brazilian genotype (6%), and six dogs with the Costa Rican genotype (38%). Moreover, these molecular genotype signatures were consistent with serologic analysis using TRP36 genotype-specific peptides. Notably, there was no serologic evidence of E. canis infection in humans, suggesting that E. canis infection in dogs in Cauca is not associated with zoonotic human infection.

Perfil clínico e laboratorial de cadelas sororeativas para erliquiose tratadas em um Hospital Veterinário Universitário em Niterói, Estado do Rio de Janeiro, Brasil / Clinical and laboratory profile of dogs seroreactive to ehrlichiosis treated at the Veterinary Medical Teaching Hospital in Niterói, State of Rio de Janeiro, Brazil

Ehrlichiosis is a tick-borne disease highly prevalent in Brazil, and is relevant in canine clinical practice due to its high morbidity and mortality. Its clinical signs are nonspecific and its phases are acute, lasting 2 to 4 weeks; subclinical, i.e., asymptomatic; and chronic, resembling an autoimmune disease. The purpose of this study was to identify the occurrence of reactivity to Ehrlichia canis of bitches treated at the Veterinary Medical Teaching Hospital of the Universidade Federal Fluminense (UFF) - Niterói, RJ, Brazil, based on serological examination by iELISA, and to compare the hematological, biochemical, urinary protein-creatinine and urinary density profiles of reactive and non-reactive animals. This study involved solely bitches, regardless of breed, starting at 1 year of age. One hundred and thirty bitches, 1 to 16 year-old (mean age 7.02 ± 4.00), weighing 1.5 to 50 kg (mean weight 12.12 ± 10.65) were subjected to clinical examination and abdominal ultrasound. Complete blood count, biochemical measurements, urinalysis and serology for E. canis were also performed. The serum was used in the iELISA to identify immunoglobulin G (IgG), using a canine Ehrlichia Imunotest® diagnostic kit (Imunodot®, Jaboticabal, SP, Brazil) according to the manufacturer's instructions. Sixty animals (46.20%) were reactive to E. canis. According to their owners, only 5 (8.3%) of the 60 seroreactive animals had a history of tick-borne disease. The most common profile was that of mixed breed animals living with their owners, older than 7 years, who had not been treated preventatively with specific drugs against ectoparasites. Laboratory tests showed significant differences between groups in terms of total protein (TP), and calcium and urinary protein-creatinine ratio (UPC). TP and UPC were elevated in the non-reactive group, while the only significant change in the reactive group was mild hypocalcemia. In this study, 30% (18/60) of the bitches were seroreactive to E. canis and had hypocalcemia. Of these, 50% (9/18) had a UPC above 0.5. Furthermore, 66.7% (12/18) of this group with hypocalcemia also showed urine density (UD) of less than 1024. Among these 18 bitches, 5 had both alterations, i.e., UPC > 0.5 and UD < 1024. In this study, a high prevalence of bitches seroreactive to Ehrlichia canis was observed, despite the absence of clinical and/or laboratory signs indicative of the disease. In the investigation of IgG class antibodies, it is not possible to determine the exact time of infection, and titers may remain high for a period of more than 11 months, even after treatment and elimination of the bacterium. The fact that most seroreactive bitches showed no symptoms compatible with the disease either before or during the study suggests that they were in the subclinical phase of ehrlichiosis. The main reason for calcium metabolism disorders is a phosphorus imbalance, a condition that occurs in kidney diseases. Isosthenuria reflects the kidney's inability to concentrate urine. This finding may be one of the first clinical manifestations of chronic kidney disease (CKD), especially in dogs. On the other hand, the UPC ratio may increase with the progression of CKD. The presence of hypocalcemia, isosthenuria and increased UPC associated with seroreactivity suggests that infection by E. canis may be associated with the onset of CKD. Veterinarians should keep in mind the complexity of the pathophysiology of ehrlichiosis to ensure the disease is not underdiagnosed in any of its phases, thereby ensuring the correct treatment is provided. Such awareness is expected to reduce the chronicity of the disease and underlying sequelae among dogs.(AU)

Serosurvey of arthropod-borne diseases among shelter dogs in the Cumberland Gap Region of the United States.

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.

Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis.

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

Simultaneous differential detection of canine blood parasites: Multiplex high-resolution melting analysis (mHRM).

Recently, the incidence of canine infection by the tick-borne parasites Babesia spp., Hepatozoon canis, Ehrlichia canis and Anaplasma platys has been increasing globally. We have developed a multiplex high-resolution melting analysis (mHRM) technique to reduce the time demands and costs associated with detecting haemoparasites in canine blood, while increasing the degree of reliability of this method of analysis. We have designed primers that are specific for protozoans (B. vogeli and H. canis) and Rickettsia-like bacteria (E. canis and A. platys) based on the 18S or 16S rDNA sequences, respectively. Two primer pairs (Protz18S-C and Bact16S-A) were found to be suitable for detecting these agents since their melting temperatures (Tm) exhibited discernible differences among the four haemoparasites, A. platys, B. vogeli, E. canis and H. canis (83.10 °C, 82.41 °C, 80.37 °C and 78.56 °C, respectively). The sequences acquired from these PCR products were >94 % identical to those of A. platys, B. vogeli, E. canis and H. canis in GenBank. The limit of detection (LOD) for B. vogeli, E. canis and A. platys was 103 copies/µl, while the LOD for H. canis was 104 copies/µl. Of the 68 dogs tested, 28 (41 %) were infected with these agents. The most commonly occurring infection involved E. canis, followed by B. vogeli, A. platys and H. canis, with infection percentages of 26 %, 13 %, 7 % and 6 %, respectively. These results demonstrate that mHRM can serve as a rapid, economical and reliable tool for the detection of parasitic diseases in canine blood for diagnosis and epidemiology.

Laboratory Blood-Based Testing for Non-Lyme Disease Tick-Borne Infections at a National Reference Laboratory.

OBJECTIVES: We evaluated trends in non-Lyme disease tick-borne disease (NLTBI) testing at a national reference laboratory. METHODS: Testing data performed at Quest Diagnostics during 2010 to 2016 were analyzed nationally and at the state level. RESULTS: Testing and positivity for most NLTBIs increased dramatically from 2010 through 2016 based on testing from a large reference laboratory. The number of positive cases, though not as stringent as criteria for public health reporting, generally exceeds that reported by the Centers for Disease Control and Prevention. The frequency of NLTBI in the US is seasonal but testing activity and positive test results are observed throughout all months of the year. Positive results for NLTBI testing mostly originated from a limited number of states, indicating the geographic concentration and distribution of NLTBIs reported in this study. CONCLUSIONS: This report provides an important complementary source of data to best understand trends in and spread of NLTBI.

Characterization of serum protein fractions of dogs naturally infected with Ehrlichia canis or Anaplasma platys associated with uveitis.

Uveitis associated with Ehrlichia canis or Anaplasma platys infections were reported in dogs. However, only two E. canis-infected dogs with hypergammaglobulinemia showed acute blindness were reported. There were limited data of the species of Ehrlichia or Anaplasma and the alteration of serum protein fractions in infected dogs. Thus, the species of causative pathogen were investigated and compared the serum protein fractions between infected dogs associated with anterior uveitis and panuveitis in clinical situations. All 103 studied dogs were brought into the ophthalmology clinic which each dog showed signs of unilateral or bilateral uveitis related to ehrlichial infection. Dogs were divided into anterior uveitis and panuveitis groups. The species of Ehrlichia or Anaplasma were identified using nested-PCR based on the 16S rRNA gene and DNA sequencing from blood samples. The serum protein fractions were analyzed using electrophoresis. Fifty-eight dogs (56.31%) were positive of which E. canis and A. platys were detected in 51 and 7 dogs, respectively. The total serum protein and globulin levels were higher in the infected dogs associated with panuveitis than anterior uveitis while the albumin levels were significantly lower in the panuveitis group. The A/G ratios significantly decreased in both groups. Gamma globulin was detected at high levels in both groups while beta globulin significantly increased in the panuveitis group. Hypergammaglobulinemia was detected in 76.92 and 90.90% of infected dogs associated with anterior uveitis and panuveitis, respectively. Most of the infected dogs associated with panuveitis showed significantly levels of hyperproteinemia, hyperbetaglobulinemia and hypergammaglobulinemia compared with anterior uveitis group. E. canis was found as the major pathogen in infected dogs associated with uveitis in this study.

Associations between clinical canine leishmaniosis and multiple vector-borne co-infections: a case-control serological study.

BACKGROUND: Dogs that have clinical leishmaniosis (ClinL), caused by the parasite Leishmania infantum, are commonly co-infected with other pathogens, especially vector-borne pathogens (VBP). A recent PCR-based study found that ClinL dogs are more likely to be additionally infected with the rickettsial bacteria Ehrlichia canis. Further information on co-infections in ClinL cases with VBP, as assessed by serology, is required. The research described in this report determined if dogs with ClinL are at higher risk of exposure to VBP than healthy control dogs using a case-control serology study. RESULTS: Of the 47 dogs with ClinL, anti-E. canis/ Ehrlichia ewingii antibodies were detected in 17 (36.2%), anti-Anaplasma phagocytophilum/Anaplasma platys antibodies in 5 (10.6%) and antigen for Dirofilaria immitis in 2 (4.3%). Of the 87 control dogs, anti-E. canis/E. ewingii antibodies were detected in 14 (16.1%) and anti-A. phagocytophilum/A. platys antibodies in 2 (2.3%). No anti-Borrelia burgdorferi antibody tests were positive. No statistical differences between the ClinL dogs and control dogs regarding lifestyle or use of ectoparasitic prevention, were identified. The ClinL was significantly associated with anti-E. canis/E. ewingii antibodies (odds ratio = 2.9, 95% confidence interval: 1.3-6.7, P = 0.010) compared to controls by both multivariable logistic regression and structural equation modelling. CONCLUSIONS: It was demonstrated that an increased risk for E. canis/E. ewingii seropositivity is present in dogs with ClinL compared to clinically healthy control dogs, despite similar ectoparasitic prevention use and lifestyle. Based on these findings it is suggested that dogs with ClinL should not only be tested for E. canis co-infection using PCR but also serologically for E. canis/E. ewingii.

Seroprevalence of six pathogens transmitted by the Ixodes ricinus ticks in asymptomatic individuals with HIV infection and in blood donors.

The objective of our study was to estimate the seroprevalence of six pathogens transmitted by ticks in HIV-infected persons and blood donors in Poland (B. burgdorferi s.l., A. phagocytophilum, Ehrlichia spp., Babesia spp., Rickettsia spp. Bartonella henselae) to assess the frequency of exposure to such microorganisms in immunocompetent and immunocompromised individuals in endemic regions for I. ricinus ticks. Serum samples were collected from 227 HIV-infected patients and 199 blood donors. All samples were analyzed for antibodies against six tick-borne pathogens and seroprevalence rates were statistically compared between two tested group as well as age, sex and lymphocyte T CD4+ level in HIV infected patients. The seroprevalence of tick-borne infections in HIV-infected patients is higher than that of the healthy population in Poland, although no association between serological status of patients and lymphocyte CD4+ T cell level has been observed. The frequency of tick-borne coinfections and doubtful results of serological tests were significantly higher in HIV-positive individuals. In Poland, the possibility of tick-borne diseases transmission with blood is rather negligible.

Occupational risk for personnel working in veterinary clinics through exposure to vectors of rickettsial pathogens.

Workers at veterinary clinics are exposed to zoonotic pathogens, which, though infrequently, can lead to serious consequences. The aim of the present study was to estimate the seroprevalence for rickettsial pathogens in veterinary clinic personnel in Ciudad Juárez, Chihuahua, Mexico. Voluntary participants included 106 veterinarians, 19 pet groomers, 36 veterinary assistants and 6 administrative workers who supplied blood samples taken by venipuncture. Detection of Rickettsia rickettsii, Ehrlichia spp. and Anaplasma phagocytophilum was conducted by indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR) techniques. To determine risk factors for the seroprevalence, the study considered social, demographic and occupational variables as well as the clinical signs and symptoms of the participants. Logistic regression procedures were performed, and the odds ratio (OR) was calculated with confidence intervals corresponding to a probability of 95%. The results indicate that 54% of the participants had a positive seroreaction to at least one of the pathogens evaluated and 2% were positive to all. The coexposure was as follows: 3% to R. rickettsii and A. phagocytophilum, 9% to Ehrlichia spp. and A. phagocytophilum, and 2% to Ehrlichia spp. and R. rickettsii. It was established that grooming posed the highest risk for R. rickettsii exposure. The implementation of appropriate management practices for zoonotic diseases is recommended to avoid health problems that may be life threatening to personnel in veterinary clinics.

Seroprevalence of vector-borne pathogens in dogs from Croatia.

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.

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 Evidence of Borrelia burgdorferi, Anaplasma phagocytophilum and Ehrlichia Spp. Infections in Horses from Southeastern Bulgaria.

Lyme Borreliosis and granulocytic anaplasmosis are less extensively studied in horses than in dogs and humans. Equine ehrlichiosis is not known in Europe and is in the initial stage of investigation in South, Central, and North America. The aim of this study was to determine the seroprevalence of these infections in Bulgaria. A total of 155 horses were investigated from five regions in Southeastern Bulgaria. Horses were tested for Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia spp. antibodies by a commercial rapid ELISA test. B. burgdorferi and A. phagocytophilum antibodies were found in all five regions (Burgas, Sliven, Stara Zagora, Haskovo, and Kardzhali) at frequencies of 36/155 (23.2%; 95% CI: 16.8-30.7%; ranging by region from 6.4% to 50%) and 31/155 (20%; 95% CI: 14-27.2%; ranging by region from 10% to 30.8%), respectively. Antibodies against Ehrlichia spp. were found in horses from three regions (Burgas, Stara Zagora, and Haskovo) at a rate of 6/155 (3.9%; 95% CI: 1.4-8.2%; ranging by region from 5.7% to 6.4%). The combination of B. burgdorferi/A. phagocytophilum (11/155; 7.1%; 95% CI: 3.6-12.3%) was the most common coexposure observed, followed by B. burgdorferi/Ehrlichia spp. (2/155; 1.3%; 95% CI: 0.2-4.6%) and A. phagocytophilum/Ehrlichia spp. (1/155; 0.6%; 95% CI: 0-3.5%). The study shows that horses in Bulgaria are exposed or coexposed to three tick-transmitted zoonotic bacterial species. Furthermore, it reports Ehrlichia spp. seroreactivity in equines in Europe.

A new TaqMan method for the reliable diagnosis of Ehrlichia spp. in canine whole blood.

BACKGROUND: Ehrlichiosis is an important emerging infectious disease of the canid family and humans worldwide. To date, no extensive evaluation or validation of a molecular diagnostic test for ehrlichiosis has been published. Here, we present data for a newly designed TaqMan assay and compare its performance to a commercial technology (PCRun®). Both of these real-time methods of analysis were evaluated using a comprehensive number of prospective and retrospective samples collected from dogs exhibiting symptoms of ehrlichiosis. RESULTS: Whole blood samples collected from dogs, retrospectively in the United Kingdom and prospectively in Israel, were analysed for the presence of Ehrlichia canis and Ehrlichia minasensis DNA using the TaqMan PCR, developed specifically for this study. The results were compared to those of a real time commercial isothermal amplification method (PCRun® system developed by Biogal Galed Labs ACS, Galed, Israel). The sensitivity and specificity (CI: 95%) of the TaqMan PCR and PCRun® were both determined to be 100% and absolute, for all of the samples tested. Interestingly, both tests were demonstrated to be highly comparable, irrespective of differences in amplification chemistry or sequences targeted. Host differences, incidence of disease and geographical location of the isolates had little impact on the positivity recorded by each of the diagnostic methods. CONCLUSIONS: It was evident that both amplification methods were equally suited for diagnosing canine ehrlichiosis and while the PCRun® clearly amplified all clinically relevant Ehrlichia species known to infect dogs and humans, the TaqMan method was more specific for E. canis and E. minasensis. This work demonstrates that despite good analytical sensitivities and specificities for Ehrlichia spp. neither method could fully account for the clinical diagnosis of thrombocytopenia.

Anaplasma phagocytophilum infection in thrombocytopenic dogs.

Anaplasma and Ehrlichia spp. are tick-transmitted bacteria of clinical relevance in European dogs. The diagnosis of infection is often difficult due to the wide spectrum of disease caused by them. During infection, reduction in platelet count is considered the most common haematological abnormality, frequently representing the sole alteration in asymptomatic dogs. In this study, the presence of bacteria belonging to the genera Anaplasma and Ehrlichia was investigated in Northern Italy in blood samples from 159 thrombocytopenic dogs using a polymerase chain reaction (PCR) assay amplifying a portion of the heat shock gene (groEL). Obtained amplicons were sequenced and analysed. Two dogs were positive for A. phagocytophilum, while A. platys and E. canis were not detected. None of the PCR-positive dogs were diagnosed at the time of hospital admission, even in the presence of clinical signs and clinicopathological abnormalities potentially related to A. phagocytophilum infection. Nucleotide sequence analysis showed that the 2 detected strains belonged to the cluster Europe 1 and were different from each other. This study confirms the presence of A. phagocytophilum infections in dogs of Northern Italy, causing clinical signs and laboratory abnormalities that could not be properly diagnosed and treated.

Characteristics of hemostasis during experimental Ehrlichia canis infection.

BACKGROUND: Ehrlichia canis infection in dogs can cause thrombocytopenia and clinical evidence of bleeding. It is unknown why some dogs show signs of bleeding whereas others do not despite clinically relevant thrombocytopenia. HYPOTHESIS/OBJECTIVES: Activated platelets, decreased fibrinolysis or both mitigate bleeding tendency. Assess standard hemostatic variables, platelet dynamics, and specialized coagulation testing in dogs experimentally infected with E. canis to evaluate this clinical discrepancy. ANIMALS: Four healthy laboratory beagles. METHODS: Dogs were given blood infected with E. canis IV. Platelet indices of activation, platelet aggregometry, antiplatelet antibodies (percent IgG), complete coagulation panel, and thromboelastography (TEG) were measured before inoculation and on weeks 1-8. Dogs were treated with doxycycline at approximately 5 mg/kg PO q12h between weeks 3 and 4 (day 24). For each variable, 1-way repeated measures analysis (1-way ANOVA) with post-hoc analysis was performed with statistical significance set at P < .05. RESULTS: Dogs had significantly lower platelet counts, evidence of activated platelets, and antiplatelet antibodies during E. canis infection. Dogs also appeared hypercoagulable and hypofibrinolytic using TEG as compared with baseline, changes that persisted for variable amounts of time after doxycycline administration. No overt signs of bleeding were noted during the study. CONCLUSIONS AND CLINICAL IMPORTANCE: Activated platelets and a hypercoagulable, hypofibrinolytic state could explain the lack of a bleeding phenotype in some dogs despite clinically relevant thrombocytopenia. Findings from our pilot study indicate that additional studies are warranted.

Graft loss attributed to possible transfusion-transmitted ehrlichiosis following cord blood stem cell transplant.

We present a case of possible transfusion-transmitted Ehrlichia chaffeensis infection in a heavily transfused cord blood transplant recipient, resulting in severe infection and graft loss. Transfusion-transmitted, vector-borne infections in immunocompromised individuals can have severe consequences, and should be considered in hospitalized patients receiving blood products with unexplained fever or sepsis.