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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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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 (T m ) 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 10 3 copies/μl, while the LOD for H. canis was 10 4 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., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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.

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.

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: 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., (Copyright © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

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 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.

The objective of this study was to evaluate and compare the antioxidant response and the products of oxidative damage analysed by various assays in clinical and subclinical canine monocytic ehrlichiosis (CME). For this purpose, four assays to measure the total serum antioxidant capacity (TAC), such as the cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), trolox equivalent antioxidant capacity (TEAC) using acidic medium (TEAC A ), and the TEAC using the horseradish peroxidase (TEAC H ) were used. In addition, the serum thiol concentrations were analysed. Reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), and ferrous oxidation-xylenol orange (FOX) were measured to determine the concentrations of free radical and the products of oxidative damage as result of the disease. All antioxidant markers were significantly lower in the dogs on clinical ehrlichiosis when compared with healthy dogs; however only the CUPRAC, FRAP and thiol were significantly lower in subclinical CME compared with healthy dogs. TBARS and FOX showed no significant differences between dogs with CME and healthy dogs; however, a significant increased ROS concentration was observed in dogs with clinical and subclinical CME when compared with healthy dogs. Results showed that in CME there is a state of oxidative stress with significant changes in markers of antioxidant defence and in concentrations of free radicals. However, the detection of these changes would depend of the assay used., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: To further clarify the causes of pancytopoenia and to investigate whether underlying cause or severity were associated with survival in an area endemic for vector-borne pathogens., Methods: Retrospective review of medical records of 119 dogs with and 238 dogs without pancytopoenia., Results: Mixed-breed dogs and dogs younger than one year had higher odds of being pancytopoenic. The most common diagnoses included monocytic ehrlichiosis (n=42), leishmaniasis (n=28) and parvoviral enteritis (n=19). The mean white blood cell counts were lower in dogs with ehrlichiosis and parvoviral enteritis compared to dogs with leishmaniasis, while platelet counts were lower in ehrlichiosis compared to leishmaniasis or parvoviral enteritis. Total protein concentrations were lower in dogs with parvoviral enteritis compared to ehrlichiosis and leishmaniasis. Higher haematocrit, platelet and white cell counts were associated with better odds of survival., Clinical Significance: Infectious diseases appear to be the leading causes of canine pancytopoenia in endemic areas; severe leukopoenia (ehrlichiosis, parvoviral enteritis), thrombocytopoenia (ehrlichiosis) and hypoproteinaemia (parvoviral enteritis), represented potentially useful disease-specific diagnostic determinants. The severity of pancytopoenia significantly affects the clinical outcome., (© 2017 British Small Animal Veterinary Association.)

Background: Canine monocytic ehrlichiosis (CME) is one of the most important tick-borne diseases worldwide. Cytopenias have been observed in both acute (nonmyelosuppressive) and chronic (myelosuppressive) CME; however, leukocyte abnormalities and indices have been incompletely described in dogs with acute CME., Objectives: The aims of this study were to analyze temporal changes in differential leukocyte counts, leukocyte morphology, myeloperoxidase index (MPXI), and lobularity index (LI) in dogs with experimental and naturally occurring acute CME., Methods: Differential leukocyte counts and morphology were evaluated in archived blood smears from 13 Beagle dogs experimentally infected with Ehrlichia canis and evaluated weekly for 42 days postinfection (DPI); 20 dogs with naturally occurring acute CME also were evaluated. MPXI and LI were obtained from ADVIA reports. Wilcoxon tests were used to assess changes over time; leukogram results in natural cases were assessed in comparison with reference intervals., Results: In experimental dogs, significant decreases in neutrophil, monocyte, lymphocyte, and eosinophil counts, and a mild left shift occurred within 14 DPI. The MPXI decreased significantly between 14 and 21 DPI and remained low, while LI increased from 14 to 35 DPI. Lymphocyte counts rebounded at 21 DPI, normalizing total WBC counts. Neutrophil toxicity was seen rarely, but reactive lymphocytes were observed frequently. Dogs with natural infection had variable patterns of leukocyte changes., Conclusions: Acute CME is associated with several discrete quantitative and qualitative leukogram changes indicative of concurrent inflammation, antigenic stimulation, and stress. Changes in MPXI and LI warrant further investigation in dogs with CME and other diseases., (© 2016 American Society for Veterinary Clinical Pathology.)

There is currently lack of information on the changes of acute phase proteins (APP) and antioxidant markers and their clinical relevance as treatment response indicators in canine monocytic ehrlichiosis (CME). The objective of this study was to investigate the patterns of C-reactive protein (CRP), haptoglobin (Hp), ferritin and paraoxonase-1 (PON-1) during treatment of dogs with acute CME with rifampicin. Blood serum samples from ten Beagle dogs with experimental acute CME were retrospectively examined. Five dogs (Group A) were treated with rifampicin (10mg/Kg/24h), per os, for 3 weeks and 5 dogs (Group B) received no treatment (infected controls). Two Beagle dogs served as uninfected controls. Blood serum samples were serially examined prior to Ehrlichia canis inoculation and on post-inoculation days 14, 21, 28, 35 and 42. Significant changes of CRP, Hp, ferritin and PON-1 values were found in the majority of infected dogs. However, their concentrations did not differ between the two groups during the treatment observation period. The results of this study indicate that although several APP and PON-1 tend to significantly change in the majority of dogs with acute CME, they were of limited clinical relevance as treatment response indicators in this experimental setting., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The canine monocytic ehrlichiosis, caused by Ehrlichia canis, is endemic in several regions of Brazil. Some serological diagnostic techniques using immunodominant proteins of E. canis as antigens are available, but their specificities and sensitivities are questionable. Based on this, the objective of this study was to test the antigenic potential of the recombinant gp19 protein (rGP19) for subsequent use in diagnostic tests. The rGP19 expressed in the Escherichia coli strain BL21 (DE3) C41 was recognized in the sera from experimentally infected dogs using ELISA and Western blotting. Thus, it was possible to obtain a promising antigen with the ability to differentiate between E. canis-positive and -negative animals, even 1 week after infection.

Ehrlichiosis is a zoonotic disease that is caused by bacteria of the genus Ehrlichia. The aims of this study were to detect the presence of Ehrlichia spp. in the blood of dogs in Ituberá, Bahia, and to compare the sensitivities and specificities of blood smear, serological, and molecular examinations. Furthermore, this study identified factors associated with exposure to the agent in dogs in this locality. Blood samples were collected from 379 dogs and submitted for indirect immunofluorescent assay and polymerase chain reaction testing for the detection of Ehrlichia spp. antibodies and DNA, respectively. Additionally, a peripheral blood smear was obtained from the ear tip for parasite identification. Of the 379 animals, 12.4%, 32.7%, and 25.6% were identified as positive on the blood smear, serological, and molecular tests, respectively. The dogs positive in one of the three techniques were considered exposed (46.9%). Younger dogs and rural habitat were protective factors and presence of ticks and contact with other dogs were the risk factors associated with exposure to the agent. It was concluded that dogs of Ituberá have high positivity for Ehrlichia spp. and that the diagnostic methods used for detection are complementary.

This study assessed the occurrence of Ehrlichia spp., Babesia spp. and Hepatozoon spp. infections in 100 tick-harboring dogs from a semiarid region of the State of Paraíba, Northeastern Brazil. Blood samples and ticks were collected from the animals, and a questionnaire was submitted to dog owners to obtain general data. Blood samples were used to perform hemogram, direct blood smear and immunological and molecular hemoparasite detection. The 1,151 ticks collected were identified as Rhipicephalus sanguineus; direct smears revealed E. canis-like morulae in the monocytes of 4% (4/100) of the non-vaccinated female dogs, and 34% and 25% of the dogs tested positive for Ehrlichia canis by indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR), respectively. Blood smear examination revealed Babesia-suggestive merozoites in the erythrocytes of 2% (2/100) of the animals. Babesia vogeli was detected by PCR in ten animals (10%) and was correlated with young age (p = 0.007) and thrombocytopenia (p = 0.01). None of the animals showed Hepatozoon spp. positivity. These results indicate that E. canis is the main tick-borne canine pathogen in the study area and provide the first report of B. vogeli infection in dogs from Paraiba State.

Background: Frequent exposure of Grenadian dogs to Rhipicephalus sanguineus results in Anaplasma platys, and Ehrlichia canis seroreactivity. During elective surgeries, substantial intraoperative hemorrhage occurs in some seroreactive dogs., Objectives: To assess hemostatic parameters and bleeding tendencies as well as prevalence of PCR positivity in apparently healthy A. platys and E. canis seroreactive and seronegative free-roaming dogs from Grenada., Animals: Forty-seven elective surgery dogs allocated to 4 groups: Seronegative control (n = 12), A. platys (n = 10), E. canis (n = 14) and A. platys, and E. canis (n = 11) seroreactive., Methods: Preoperatively, hemostasis was assessed by platelet count, prothrombin time, activated partial thromboplastin time, and buccal mucosal bleeding time. Intra- and postoperative bleeding scores were subjectively assigned. Blood, spleen, bone marrow, and lymph node aspirates were tested by PCR., Results: Bleeding scores in dogs coseroreactive for A. platys and E. canis were higher (P = .015) than those of seronegative dogs. A. platys DNA was amplified from 7/21 (33%) A. platys seroreactive dogs and from 1 E. canis seroreactive dog; E. canis DNA was amplified from 21/25 (84%) E. canis seroreactive dogs. E. canis DNA was amplified most often from blood, whereas A. platys DNA was amplified most often from bone marrow., Conclusions and Clinical Importance: Apparently healthy, free-roaming dogs coseropositive for A. platys and E. canis may have increased intraoperative bleeding tendencies despite normal hemostatic parameters. Future investigations should explore the potential for vascular injury as a cause for bleeding in these dogs. Improved tick control is needed for dogs in Grenada., (Copyright © 2014 by the American College of Veterinary Internal Medicine.)

A 5-year-old neutered male Mastiff and an 8-year-old spayed female Labrador Retriever were presented to the University of Minnesota Veterinary Medical Center. The Mastiff was presented for evaluation of lameness and pyoderma one month prior in Missouri, where he tested positive for Ehrlichia canis by serum ELISA test, treated with doxycycline. PCR for Ehrlichia sp, Anaplasma sp, Babesia sp, and Bartonella sp, and PCR for antigen receptor rearrangement were negative, serum protein electrophoresis (SPE) revealed polyclonal gammopathy, and mildly reactive lymphoid cells were seen cytologically. The Labrador presented with a proliferative rostral mandibular gingival mass and lipomas for further presurgical evaluation of cold agglutinin activity documented by a commercial laboratory 2 years earlier prior to removal of a grade II mast cell tumor. This dog had a negative SNAP4Dx, normal SPE, and persistently increased serum ALP activity and polyuria/polydipsia suggestive for hyperadrenocorticism. Both dogs had markedly agglutinated RBC in the EDTA samples that dispersed with warming, and normal plasma color. Cold agglutinin activity was demonstrated by direct saline agglutination testing using whole blood and washed erythrocytes demonstrating agglutination at 30°C, 25°C, 15°C, and 4°C, but not at 37°C. CBC results (ADVIA 2120i) from the Mastiff revealed no significant differences in the RBC results obtained at room temperature (RT) and at 37°C; however, the RT run demonstrated negative bias in neutrophil and platelet concentrations attributed to rapid RBC settling. This uncommon hematologic condition may cause artifacts on the automated leukogram and platelet count, and may be subclinical for long periods., (© 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.)

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.

Ehrlichia canis is the most common cause of monocytotropic ehrlichiosis in dogs around the world. The purpose of the present study was to validate a new automated fluorescence system (Accuplex4™ BioCD system; Antech Diagnostics, Lake Success, New York) to detect antibodies against the E. canis immunodominant glycoprotein 36 (gp36). Sera and blood samples (ethylenediamine tetra-acetic acid) were collected from mixed sex beagles ( n = 8) on days 0, 3, 7, 10, 14, 17, 21, 28, 42, 49, 56, 63, 70, 77, 84, and 98 after intravenous inoculation with culture-derived E. canis. Sera were assayed using the Accuplex4 BioCD system (Accuplex4), an E. canis indirect fluorescent antibody test (IFAT), and a commercially available kit. A complete blood cell count and a proprietary E. canis polymerase chain reaction (PCR) were performed on each blood sample. On the day thrombocytopenia was first detected for each dog, E. canis DNA was amplified from blood of all dogs. At those times, E. canis antibodies were detected in 7 of 8 dogs by the Accuplex4, 1 of 8 dogs by the commercial kit, and 4 of 8 dogs by IFAT. Ehrlichia canis DNA was amplified from blood before seroconversion in any antibody assay for 6 dogs. Antibodies against gp36 were detected by Accuplex4 within 3 days of PCR-positive test results and were detected up to 25 days sooner than the commercial kit. After starting doxycycline treatment, E. canis DNA was no longer amplified by PCR assay, but serum antibodies remained detectable by all assays.

Ehrlichia canis infection causes multisystemic disease in dogs (canine monocytic ehrlichiosis, CME) which is associated with variable morbidity and mortality. Atypical clinical manifestations, including gastrointestinal signs, may occasionally occur in CME and approximately 10-15% of dogs are presented with historical or clinical evidence of vomiting, diarrhea, and/or abdominal discomfort. The objective of this study was to investigate if there are any alterations in serum canine pancreatic lipase immunoreactivity (cPLI) in dogs with experimentally induced or naturally occurring monocytic ehrlichiosis. Serum samples from 10 Beagle dogs experimentally infected with E. canis and two healthy uninfected Beagles were serially examined; samples from 20 naturally infected dogs (10 with non-myelosuppressive [NME] and 10 with myelosuppressive [ME] ehrlichiosis) were also examined at a given point in time (cross-sectional sampling). None of the experimentally infected Beagles showed gastrointestinal signs or increased cPLI concentrations prior to or following the artificial infection. Three naturally infected dogs with NME and one with ME demonstrated serum cPLI concentrations in the diagnostic range for pancreatitis (>400 μg/L) without showing gastrointestinal signs. The results of the present study indicated that 4/20 (20%) of dogs naturally infected with E. canis demonstrated increased serum cPLI concentrations consistent with mild and clinically inapparent pancreatitis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The general aim of this study, which was conducted for the first time in Iran, was to evaluate the seroprevalence and geographical distribution of Ehrlichia canis in a dog population in Iran, followed by molecular confirmation using PCR and sequencing. Blood samples were collected from 240 dogs in different areas of Alborz and Tehran Provinces and initially analyzed using the immunofluorescent antibody (IFA) test to detect anti-Ehrlichia canis IgG antibodies. Subsequently, nested PCR was performed based on a fragment of the 16S rRNA gene of E. canis on serologically positive samples. The results showed that 40/240 dogs (16.6%) presented anti-Ehrlichia canis IgG antibodies and that nine of the blood samples from the 40 seropositive dogs (22.5%) contained E. canis DNA, which was confirmed by sequencing. The seroprevalence of E. canis tended to be higher in purebred, one to three-year-old male dogs living in the Plain zone, in rural areas; however, this difference was not statistically significant.

The aim of this study was to evaluate adenosine deaminase activity and purines levels in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups with five animals each: healthy animals and animals infected by E. canis. The concentration of purines (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, inosine, hypoxanthine, xanthine and uric acid), and adenosine deaminase (E-ADA) activity in sera were evaluated. Samples were collected on days 12 and 30 post-infection (PI). The E-ADA activity showed a significant reduction on day 12 PI, and increased on day 30 PI in dogs infected with E. canis. On day 12, an increase in seric concentration of ATP, ADP and adenosine was verified, and different levels of hypoxanthine, xanthine and uric acid had a drastic reduction in infected compared healthy dogs (P<0.05). However, on day 30 PI, the levels of seric ADP and AMP decreased, unlike the concentration of xanthine and uric acid that increased significantly in infected dogs (P<0.05). Therefore, the activity of E-ADA and purine levels are altered in experimental canine ehrlichiosis, probably with the purpose of modulating the pathogenesis of the disease related to immune response, oxidative stress and coagulation disorders in acute phase., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Tick-borne pathogens affect a wide range of vertebrate hosts. To identify tick-borne pathogens among dogs from Campo Grande, MS, Brazil testing seropositive for Leishmania infantum (syn. L. chagasi), a serological and molecular study was conducted to detect Ehrlichia canis, Anaplasma platys and Babesia vogeli in 60 serum and spleen samples. A confirmatory diagnosis of L. infantum based on serological and molecular assays was also performed, as was sequence alignment and phylogenetic analysis to assess the identity of the parasite species infecting these animals. IgG antibodies to Ehrlichia spp., B. vogeli and L. infantum were found, respectively, in 39 (65%), 49 (81.6%) and 60 (100%) of the sampled dogs. Twenty-seven (45%), fifty-four (90%), fifty-three (88.3%), two (3.3%) and one (1.6%) dog were positive, respectively, for E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. and Anaplasma sp. in PCR assays. After sequencing, the amplicons showed 99% of identity with E. canis, B. vogeli, A. platys and Leishmania chagasi isolates. The findings of this study indicate that L. infantum-seropositive dogs from Campo Grande are exposed to multiple tick-borne pathogens, which should therefore be included in the differential diagnosis of dogs with clinical suspicion of leishmaniasis.

This study investigated infection by Ehrlichia spp. agents by PCR, immunofluorescence assay test (IFAT), and by Giemsa-stained blood smears in 108 dogs from a semiarid region of the state of Paraíba, Northeastern Brazil. Seventy-five (69.4%) of the 108 dogs were found to be seropositive to Ehrlichia canis, while only four dogs (3.7%) were positive in real-time PCR for E. canis. In six dogs (5.6%) E. canis-like morulae were observed in monocytes. Animals that stayed in environment whose floor was dried dirt, and dogs whose owners reported low frequency of cleaning the dog environment had higher (P<0.05) PCR positivity for E. canis. Increasing seropositivity was found in older dogs (P=0.012). This study provides the first molecular detection of E. canis in the semiarid region of Northeastern Brazil., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Canine ehrlichiosis and babesiosis are the most prevalent tick-borne diseases in Brazilian dogs. Few studies have focused attention in surveying tick-borne diseases in the Brazilian Amazon region. A total of 129 blood samples were collected from dogs living in the Brazilian eastern Amazon. Seventy-two samples from dogs from rural areas of 19 municipalities and 57 samples from urban stray dogs from Santarém municipality were collected. Serum samples were submitted to Indirect Immunofluorescence Assay (IFA) with antigens of Babesia canis vogeli, Ehrlichia canis, and six Rickettsia species. The frequency of dogs containing anti-B. canis vogeli, anti-E. canis, and anti-Rickettsia spp. antibodies was 42.6%, 16.2%, and 31.7%, respectively. Anti-B. canis vogeli antibodies were detected in 59.6% of the urban dogs, and in 29.1% of the rural dogs (P < 0.05). For E. canis, seroprevalence was similar among urban (15.7%) and rural (16.6%) dogs. For Rickettsia spp., rural dogs presented significantly higher (P < 0.05) prevalence (40.3%) than urban animals (21.1%). This first study on tick-borne pathogens in dogs from the Brazilian eastern Amazon indicates that dogs are exposed to several agents, such as Babesia organisms, mostly in the urban area; Spotted Fever group Rickettsia organisms, mostly in the rural area; and Ehrlichia organisms, in dogs from both areas studied.

Anaplasma phagocytophilum is an intracellular tick-borne rickettsial pathogen, which causes granulocytic anaplasmosis in various species of livestock and companion animals and also in humans. Previously A. phagocytophilum has been isolated and propagated in cell lines derived from the tick Ixodes scapularis and in the human promyelocytic cell line HL60. In this study we used the Ixodes ricinus-derived cell line IRE/CTVM20 to isolate and propagate two new canine strains of A. phagocytophilum. Blood samples were collected by veterinarians from two dogs, one from Germany and the other from Austria. Suspicion of clinical canine granulocytic anaplasmosis was raised by the treating veterinarians and after confirmation of A. phagocytophilum infection by real-time PCR, buffy coat cells were isolated and co-cultivated with IRE/CTVM20 cells maintained at 28 °C in L15/L15B medium. In the tick cells, rickettsial inclusions were first recognised after 86 days of incubation. Electron microscopic examination of tick cells infected with one of the isolates revealed cytoplasmic vacuoles containing pleomorphic organisms with individual bacteria enveloped by a bilayer membrane. Sequencing of 16S rRNA genes confirmed the isolation of A. phagocytophilum and showed the highest identity to the A. phagocytophilum human HZ strain. The two A. phagocytophilum isolates were passaged several times in IRE/CTVM20 cells and transferred to the I. scapularis cell line ISE6. This confirms for the first time the successful establishment and continuous cultivation of this pathogen in I. ricinus cells as well as infectivity of these canine strains for I. scapularis cells., (Copyright © 2012. Published by Elsevier B.V.)

The present study evaluated the presence of Ehrlichia DNA in the blood samples of 320 dogs from the urban and rural areas of the municipality of Poconé, Pantanal region, Mato Grosso state, by Polymerase Chain Reaction (PCR), targeting the ehrlichial dsb gene. Risk factors for infection in dogs were also evaluated. Forty-eight (15%, 95% CI: 11.4-19.5%) dogs were positive: 25 (15.6%, 95% CI: 10.4-22.2%) from the urban area and 23 (14.4%, 95% CI: 9.3-20.8%) from the rural area (P > 0.05). Partial DNA sequence obtained from PCR products of 18 samples from the urban area and 16 samples from the rural area were 100% identical to E. canis from Brazil and the USA. This study reports the first E. canis molecular detection in dogs from the northern Pantanal region.

The aim of this study was to determine whether serum cardiac troponin I (cTnI) concentration measured on admission was associated with the clinical severity of canine monocytic ehrlichiosis and was predictive of clinical outcome (death or survival) in dogs naturally infected with Ehrlichia canis. Serum cTnI concentration was compared among 22 dogs with non-myelosuppressive ehrlichiosis (NME), 22 dogs with myelosuppressive ehrlichiosis (ME) and 10 healthy dogs. Unlike healthy dogs, 45.5% NME and 59.1% ME dogs had increased cTnI concentrations. There was no difference in the frequency of cTnI increase or mean cTnI concentrations between the NME and ME groups, whereas mean cTnI concentration was significantly lower in healthy dogs. No association was established between cTnI concentration on admission and clinical outcome., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In view of the fact that stray dogs are a reservoir for many diseases, this study was undertaken to determine the prevalence of Ehrlichia canis in stray dogs in North Trinidad and to evaluate the diagnostic implications of haematological alterations associated with seropositivity. Overall, 41 (44.6%) of 92 stray dogs were seropositive to E. canis by the indirect immunofluorescent antibody test. Dogs, one year of age and older (59.7%) were more likely to be seropositive than dogs less than one year old (13.3%) (p<0.001). No significant differences in seropositivity between females and males were found. The odds ratios showed that seropositive dogs were 3.34 (CI 95%; 1.33-8.59) and 5.17 (CI 95%; 0.19-1.26) times more likely to have low platelet counts and elevated total serum protein concentrations (p=0.014 and p<0.001, respectively) than seronegative dogs. Lower mean platelet counts and a higher mean total protein concentration were associated with seropositivity (p<0.01). Mean eosinophil and segmented neutrophil counts were elevated in dogs that tested negative for E. canis antibodies (p=0.002 and p<0.005, respectively). Other haematological parameters were not different between the 2 groups. The high percentage of stray dogs infected with E. canis should alert veterinarians to the potential risk of transmission of the disease. A comprehensive study possibly using molecular methods such as nested PCR should be undertaken to determine how co-infection with other pathogens may alter haematological profiles. In general, control of ticks and stray dog populations may help to control the spread of tick-borne diseases., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Previous research suggested that clinical manifestations, histopathological lesions, and even infection maintenance in the course of canine monocytic ehrlichiosis (CME) are directly related to the immune response developed by the host. In the present study, blood lymphocyte subsets were analyzed by multiparametric flow cytometry in 37 dogs with naturally occurring CME and 47 healthy dogs used as controls. T, T helper (Th), T cytotoxic (Tc), B, non-T, non-B lymphocytes and those that express MHC class II were characterized in every dog. Animals with CME showed higher relative values of T and Tc cells and a higher absolute number of Tc cells in peripheral blood. The percentage of Th cells and the absolute and relative values of B cells were higher in healthy animals than in CME-affected dogs. The significance of these changes on the pathogenesis of natural Ehrlichia canis infection in dogs needs further evaluation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Early diagnosis of canine ehrlichiosis favors prompt institution of treatment and improves the prognosis for the animal, since this disease causes mortality among dogs. Studies have shown that determining the concentration of acute-phase proteins (APPs) may contribute towards early detection of disease and aid in predicting the prognosis. This study aimed to evaluate the APP profile in dogs experimentally infected with Ehrlichia canis, at the start of the infection and after treatment. It also investigated whether any correlation between APP levels and the clinical and laboratory alterations over the course of the disease would be possible. The results obtained showed abnormal levels of all the APPs on the third day after infection (D3), with the highest levels being reached on D18, with the exception of ceruloplasmin and acid glycoprotein, which presented their peaks on D6 and D12 respectively. We concluded that assessment of APP levels could contribute towards establishing an early diagnosis of canine ehrlichiosis, particularly regarding acid glycoprotein and ceruloplasmin, since these proteins were detected at increased levels even before the onset of clinical and laboratory findings of the disease.

Ehrlichiosis and babesiosis are tick-borne diseases, caused mainly by Ehrlichia canis and Babesia canis, respectively, with a worldwide occurrence in dogs, whose main vector is the brown-dog tick, Rhipicephalus sanguineus. The present work aimed to detect the presence of E. canis and Babesia sp. in 91 dog blood samples in Colombia, by molecular and serological techniques. We also performed sequence alignment to indicate the identity of the parasite species infecting these animals. The present work shows the first molecular detection of E. canis and B. vogeli in dogs from Colombia. Immunoglobulin-G (IgG) antibodies to E. canis and Babesia vogeli were found in 75 (82.4%) and 47 (51.6%) sampled dogs, respectively. Thirty-seven (40.6%) and 5 (5.5%) dogs were positive in PCR for E. canis and Babesia sp., respectively. After sequencing, amplicons showed 99% of identity with isolates of E. canis and B. vogeli. The phylogenetic trees based on 16S rRNA-Anaplasmataceae sequences and 18S rRNA-piroplasmid sequences supported the identity of the found E. canis and B. vogeli DNAs, respectively. The present work shows the first molecular detection of E. canis and B. vogeli in dogs in Colombia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Thirty naturally infected dogs were divided into three groups (10 animals each): group I (canine leishmaniosis), group II (canine ehrlichiosis) and group III (dogs co-infected by both infectious agents). Ten clinically healthy dogs were used as controls. A haematological profile was obtained and dog sera were assessed for the presence of platelet-bound IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. Eight of the naturally co-infected dogs (80%), five of the leishmaniotic dogs (50%) and six of the ehrlichiotic dogs (60%) had detectable levels of platelet-bound antibodies (PBIg). No PBIg were observed in the control group. The hypothesis that Leishmania and Ehrlichia co-infection could more severely affect the immune response of infected dogs is discussed., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Ehrlichiosis is the multiorgan infectious disease caused by small, intracellular rickettsias from the genus Ehrlichia. These microorganisms are known as an etiologic factor of infections world wide in humans and in different species of animals. Dog ehrlichiosis can be caused by several species of Ehrlichia attacking different groups of blood cells, but most often an infection by Ehrlichia canis is diagnosed with special relation to monocytes. A vector for E. canis are Rhipicephalus sanguineus and Ixodes ricinus, commonly occurring in Poland. Disease caused by E. canis is known as Canine Monocytic Ehrlichiosis (CME). The disease most often has an asymptomatic course which can, in favourable circumstances, run into acute or chronic forms. The acute form of CME proceeds usually with fever, apathy, weakness and accompanying respiratory symptoms, lameness and disturbances in blood coagulation. In laboratory examinations thrombocytopenia, anemia and leucopenia are ascertained. The chronic form of CME proceeds among gentle, unspecific symptoms which may last even 5 years. The CME diagnosis is difficult and often demands parallel different diagnostic methods. A medicines of choice in the ehrlichiosis treatment are antibiotics from the group of tetracyclines, given at least for 28 days. They are largely efficient during treatment of the acute CME, causing the quick improvement. Instead, in the case of chronic form, answer for treatment can be weak, and cases of resistance to antibiotics ave known.

To evaluate the possible use of desmopressin acetate (DDAVP) in haemorrhagic disorders consequent to canine monocytic ehrlichiosis (CME), three dogs infected by Ehrlichia canis, with a history of thrombocytopenia and recent bleeding, were studied. The dogs were administered desmopressin (1 μg/kg b.w. s.c.) every 24 h on three occasions. Blood samples were collected immediately before, and after 2 and 48 h the first DDAVP administration, to assess haematological, clinical chemistry and clotting time parameters. Spontaneous bleeding stopped within 1 h after the first DDAVP injection. Buccal mucosa bleeding time (BMBT) was shortened from 9.6 to 2.3 min within 2 h after the treatment. A statistically significant increase in platelet PLT count and fibrinogen, and a statistically significant decrease of PT and aPTT were observed after DDAVP administration. The haemorrhagic disorders caused by CME appear to be quickly corrected by DDAVP administration, giving the clinician the time necessary to administer appropriate chemotherapy.

Ehrlichia canis and Babesia canis vogeli are two tick-borne canine pathogens with a worldwide importance. Both pathogens are transmitted by Rhipicephalus sanguineus, the brown dog tick, which has an increasing global distribution. A multiplex quantitative real-time PCR (qPCR) assay for the simultaneous detection of the tick-borne pathogens E. canis and B. canis vogeli was developed using dual-labeled probes. The target genes were the 16S rRNA of E. canis and the heat shock protein 70 (hsp70) of B. canis vogeli. The canine beta actin (ACTB) gene was used as an internal control gene. The assay was conducted without using any pre-amplification steps such as nested reactions. The sensitivity of each reaction in the multiplex qPCR assay was tested in the presence of high template concentrations of the other amplified genes in the same tube and in the presence of canine DNA. The detection threshold of the multiplex assay was 1-10 copies/μl in all channels and the amplifications of the B. canis hsp70 and ACTB were not affected by the other simultaneous reactions, while minor interference was observed in the amplification of the E. canis 16S rRNA gene. This assay would be useful for diagnostic laboratories and may save time, labor and costs., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objective: To evaluate microtiter-plate format ELISAs constructed by use of different diagnostic targets derived from the Ehrlichia ewingii p28 outer membrane protein for detection of E ewingii antibodies in experimentally and naturally infected dogs., Sample Population: Serum samples from 87 kenneled dogs, 9 dogs experimentally infected with anti-E ewingii, and 180 potentially naturally exposed dogs from Missouri., Procedures: The capacities of the synthetic peptide and truncated recombinant protein to function as detection reagents in ELISAs were compared by use of PCR assay, western blot analysis, and a full-length recombinant protein ELISA. Diagnostic targets included an E ewingii synthetic peptide (EESP) and 2 recombinant proteins: a full-length E ewingii outer membrane protein (EEp28) and a truncated E ewingii outer membrane protein (EETp28), Results: A subset of Ehrlichia canis-positive samples cross-reacted in the EEp28 ELISA; none were reactive in the EESP and EETp28 ELISAs. The EESP- and EETp28-based ELISAs detected E ewingii seroconversion at approximately the same time after infection as the EEp28 ELISAs. In afield population, each of the ELISAs identified the same 35 samples as reactive and 27 samples as nonreactive. Anaplasma and E can is peptides used in a commercially available ELISA platform did not detect anti-E ewingii antibodies in experimentally infected dogs., Conclusions and Clinical Relevance: The EESP and EETp28 ELISAs were suitable for specifically detecting anti-E ewingii antibodies in experimentally and naturally infected dogs.

Canine ehrlichiosis is a disease transmitted by ticks Rhipicephalus sanguineus and caused by Ehrlichia canis, obligatory intracellular bacteria. The present study examined the prevalence of anti-E. canis in 254 dogs from four administrative regions of Cuiabá, Mato Grosso, by indirect immunofluorescence assay. There was a prevalence of 42.5% (108/254) without significant difference between the studied regions. The variables age, breed, sex, habitat, access to rural and ticks were analyzed. The antibody titers ranged from 1:40 to 1:2,560. Only 32 (29.63%) seropositive dogs were infested with ticks, all R. sanguineus. The results confirm that do not have breed, sex or age predisposition to ehrlichiosis due E. canis, while the lowest occurrence of reactive dogs indoors probably related to low tick infestation, although no significant difference between dogs with or without infestation with the tick vector.

The aim of this study is to present molecular, serologic, and clinical findings for dogs that were naturally infected with Anaplasma phagocytophilum or Borrelia burgdorferi sensu lato (s. l.) in the Czech Republic. This data can provide information relevant to human infection. In total, blood samples from 296 dogs and 118 engorged ticks were examined. Samples were tested for A. phagocytophilum using polymerase chain reaction (PCR) amplification, nested PCR, and direct sequencing of the 16S rDNA, and for B. burgdorferi s. l. using PCR amplification of the 16S rDNA and restriction fragment length polymorphism analysis of the 5S-23S rDNA intergenic spacer. In addition, blood samples were screened for antibodies to these bacteria. Ten (3.4%) dogs were PCR-positive for A. phagocytophilum. Morulae of A. phagocytophilum in granulocytes were found in two of these dogs. Nine of the PCR-positive dogs had clinical signs related to anaplasmosis. Statistically significant differences in the PCR detection rates were found between breeds and between symptomatic and asymptomatic dogs. Infection with Borrelia garinii was detected by PCR in a dog with meningoencephalitis. DNA of A. phagocytophilum and B. burgdorferi s. l. (B. garinii or Borrelia afzelii) was detected in 8.5% and 6.8% of ticks, respectively. Immunoglobulin (Ig) G seropositivity to A. phagocytophilum was 26%. Significant differences were found with respect to breed and gender. IgM and IgG antibodies to B. burgdorferi s. l. were detected in 2.4% and 10.3% of dogs, respectively. Our findings suggest that the exposure to B. burgdorferi s. l. exists in dogs in the Czech Republic, and exposure to A. phagocytophilum is common.

The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates (P<0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs (P=0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available.

Eighty-six dogs were selected based upon Ehrlichia (E.) canis SNAP 3Dx positive results to determine clinical relevance of annual E. canis screening. Immunofluorescence assay showed 72 (84%) of 86 dogs were seroreactive for E. canis. Polymerase chain reaction (PCR) revealed that 12 (14%) of 86 dogs had Ehrlichia deoxyribonucleic acid; seven had E. canis, four had E. ewingii, and one was coinfected with E. chaffeensis and E. ewingii. Thrombocytopenia (<164,000 platelets/microL) was found in 28 (39%) of 72 dogs. In this study, thrombocytopenia was frequently detected in healthy Ehrlichia SNAP 3Dx-positive dogs, whereas active infection was infrequently confirmed by PCR. Therefore, treatment based upon screening results alone is not recommended.

Fifty-five dogs with suspected tickborne disease were tested by immunofluorescence assay and PCR for Anaplasma phagocytophilum infection. Thirty (54.5 per cent) of the dogs were seropositive and five of them fulfilled the serological criteria for an active infection, with either seroconversion or a fourfold increase in antibody titres. Fragments of DNA of the expected size were detected by PCR in two seropositive and three seronegative dogs. However, direct amplicon sequencing failed to identify active A phagocytophilum infections, but revealed the presence of Anaplasma platys DNA in the PCR-positive animals.

Ehrlichia canis is the etiologic agent of canine monocytic ehrlichiosis (CME) and is a useful model for zoonotic tick-borne pathogens, many of which infect dogs. The purpose of this study was to evaluate rifampin and doxycycline regimens for clearance of E. canis infections in addition to alleviation of CME. Beagles were infected with E. canis by intravenous inoculation with carrier blood and treated with either rifampin or doxycycline after the acute phase of CME. Improved hematological values demonstrated that both treatments effectively relieved signs of the disease. Peripheral blood from all dogs became PCR negative after antibiotic treatment, suggesting that these infections were eliminated and that rifampin is an effective alternative chemotherapeutic agent for treatment of CME.

Background: Ehrlichiosis is a multisystemic disease with the potential to cause cardiomyocyte injury in naturally infected dogs., Hypothesis: Myocardial injury occurs in dogs infected with Ehrlichia canis., Animals: One-hundred and ninety-four dogs from Brazil with clinical and laboratory abnormalities indicative of ehrlichiosis. Sixteen healthy dogs served as controls., Methods: Electrocardiogram, echocardiogram, noninvasive blood pressure measurement, and serum cardiac troponin I (cTnI) concentrations were evaluated. Serologic assays and PCR determined the exposure and infection status for E. canis, Anaplasma spp., Babesia canis vogeli, Bartonella spp., Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia chaffeensis, Ehrlichia ewingii, Leishmania chagasi, and spotted-fever group Rickettsia. Dogs were assigned to groups according to PCR status: E. canis infected, infected with other vector-borne organisms, sick dogs lacking PCR evidence for infection, and healthy controls., Results: E. canis-infected dogs had higher serum cTnI concentrations than controls (median: 0.04 ng/dL; range 0.04-9.12 ng/dL; control median: 0.04 ng/dL; range: 0.04-0.10 ng/dL; P= .012), and acute E. canis infection was associated with myocardial injury (odds ratio [OR]: 2.67, confidence interval [CI] 95%: 1.12-6.40, P= .027). Severity of anemia was correlated with increased risk of cardiomyocyte damage (r= 0.84, P< .001). Dogs with clinical signs of systemic inflammatory response syndrome (SIRS) were at higher risk for myocardial injury than were other sick dogs (OR: 2.55, CI 95%: 1.31-4.95, P= .005)., Conclusions and Clinical Importance: Acute infection with E. canis is a risk factor for myocardial injury in naturally infected Brazilian dogs. Severity of anemia and SIRS might contribute to the pathophysiology of myocardial damage.