Typical and atypical manifestations of Anaplasma phagocytophilum infection in dogs.

Eighteen clinically ill dogs, naturally infected with Anaplasma phagocytophilum, were examined at a veterinary practice in Baxter, Minnesota. A clinical examination, complete blood cell count, enzyme- linked immunosorbent assay (ELISA) for A phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and Dirofilaria immitis antigen, and a polymerase chain reaction test for A phagocytophilum DNA were obtained for all dogs. Physical examination findings included fever, arthropathy, lymphadenopathy, epistaxis, acute gastritis, cervical hyperpathia, and central nervous system dysfunction. Complete blood cell count abnormalities included thrombocytopenia, morulae in neutrophils, anemia, leukopenia, eosinopenia, lymphopenia, and monocytosis. Seroreactivity to A phagocytophilum was found in 61%, B burgdorferi antibodies in 17%, and D immitis antigen in 5% of the dogs. Fever, arthropathy, neurologic dysfunction, and epistaxis are clinical syndromes that can be associated with A phagocytophilum infection. Treatment with doxycycline resulted in rapid resolution of clinical signs in all dogs.

Comparison of Anaplasma and Ehrlichia species-specific peptide ELISAs with whole organism-based immunofluorescent assays for serologic diagnosis of anaplasmosis and ehrlichiosis in dogs.

OBJECTIVE: To compare the performance of 5 synthetic peptide-based ELISAs with that of 3 commercially available immunofluorescent assays (IFAs) for serologic diagnosis of anaplasmosis and ehrlichiosis in dogs. SAMPLE: A convenience set of 109 serum samples obtained before and at various times after inoculation for 23 dogs that were experimentally infected with Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Ehrlichia chaffeensis, or Ehrlichia ewingii and 1 uninfected control dog in previous studies. PROCEDURES: All serum samples were assessed with 5 synthetic peptide-based ELISAs designed to detect antibodies against A phagocytophilum, A platys, E canis, E chaffeensis, and E ewingii and 3 whole organism-based IFAs designed to detect antibodies against A phagocytophilum, E canis, and E chaffeensis. The species-specific seroreactivity, cross-reactivity with the other tick-borne pathogens (TBPs), and diagnostic sensitivity and specificity were calculated for each assay and compared among assays. RESULTS: All serum samples obtained from dogs experimentally infected with a TBP yielded positive results on a serologic assay specific for that pathogen. In general, sensitivity was comparable between ELISAs and IFAs and tended to increase with duration after inoculation. Compared with the IFAs, the corresponding ELISAs were highly specific and rarely cross-reacted with antibodies against other TBPs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that peptide-based ELISAs had enhanced specificity relative to whole organism-based IFAs for detection of antibodies against Anaplasma and Ehrlichia spp, which should facilitate accurate diagnosis and may help detect dogs coinfected with multiple TBPs.

Evaluation of peptide- and recombinant protein-based assays for detection of anti-Ehrlichia ewingii antibodies in experimentally and naturally infected dogs. [corrected]

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.

Borrelia burgdorferi Antibody Test Results in Dogs Administered 4 Different Vaccines.

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

Nitrocellulose: a tried and true polymer finds utility as a post-genomic substrate.

High-throughput techniques for genomics and proteomics differ greatly from traditional bio-molecular research techniques in the amount of data that can be obtained from a given experiment. However, many of these novel techniques rely heavily on the traditional concepts of molecular immobilization followed by hybridization, binding or analysis. These concepts, which predate even traditional blotting techniques, have become so widely used that the complexity of their fundamental precepts is often overlooked. In this review, we explore the history and use of one of the most common immobilization surfaces, nitrocellulose. This unique polymer has been used as a surface in biological research for more than 6 decades, and continues to find utility in the post-genomic era with high-throughput techniques.

Cloning and expression of the rabbit prostaglandin EP2 receptor.

BACKGROUND: Prostaglandin E2 (PGE2) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP2 receptor in regulating fertility, vascular tone and renal function. RESULTS: The full-length rabbit EP2 receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP2 exhibited specific [3H]PGE2 binding with a Kd of 19.1 +/- 1.7 nM. [3H]PGE2 was displaced by unlabeled ligands in the following order: PGE2>>PGD2=PGF2alpha=iloprost. Binding of [3H]PGE2 was also displaced by EP receptor subtype selective agonists with a rank order of affinity consistent with the EP2 receptor (butaprost>AH13205>misoprostol>sulprostone). Butaprost free acid produced a concentration-dependent increase in cAMP accumulation in rabbit EP2 transfected COS-1 cells with a half-maximal effective concentration of 480 nM. RNase protection assay revealed high expression in the ileum, spleen, and liver with lower expression in the kidney, lung, heart, uterus, adrenal gland and skeletal muscle. In situ hybridization localized EP2 mRNA to the uterine endometrium, but showed no distinct localization in the kidney. EP2 mRNA expression along the nephron was determined by RT-PCR and its expression was present in glomeruli, MCD, tDL and CCD. In cultured cells EP2 receptor was not detected in collecting ducts but was detected in renal interstitial cells and vascular smooth muscle cells. EP2 mRNA was also detected in arteries, veins, and preglomerular vessels of the kidney. CONCLUSION: EP2 expression pattern is consistent with the known functional roles for cAMP coupled PGE2 effects in reproductive and vascular tissues and renal interstitial cells. It remains uncertain whether it is also expressed in renal tubules.

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

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

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

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

Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure.

Dogs exposed to ticks in the southern US may become infected with multiple species of Ehrlichia. To better define infection risk, blood samples collected from 10 dogs infested with ticks via a natural infestation model were evaluated by blood smear examination, PCR, patient-side ELISAs (SNAP® 4Dx® and SNAP® 4Dx® Plus), IFA, and peptide based ELISA for evidence of infection with Ehrlichia canis, E. chaffeensis, and/or E. ewingii. Although morulae were rarely identified in blood smears, every dog (10/10) became infected with Ehrlichia spp. as evidenced by nested PCR detection of E. chaffeensis (7/10) and E. ewingii DNA (10/10); real-time PCR detection of E. chaffeensis (0/10) and E. ewingii (9/10); seroconversion on two different patient-side ELISAs (4/10 or 10/10); seroconversion on IFA to E. canis (10/10, maximum inverse titer=128-4096, GMTMAX=548.7) and E. chaffeensis (10/10, maximum inverse titer=1024-32,768, GMTMAX=4096); and seroconversion on peptide specific ELISA to E. chaffeensis VLPT (7/10) and E. ewingii p28 (9/10). Rickettsemia with E. chaffeensis and E. ewingii, as determined by nested PCR, persisted in dogs for an average of 3.2 or 30.5 days, respectively. Ehrlichia canis was not detected in any dog by any method, and no dogs developed signs of clinical disease. Our data suggest that in areas where ticks are common, dogs are at high risk of infection with Ehrlichia spp., particularly E. ewingii and E. chaffeensis, and can serve as a sentinel for monitoring for the presence of these zoonotic pathogens.

Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America.

BACKGROUND: This study evaluated the exposure of dogs to three different Ehrlichia spp. in the south and central regions of the United States where vector-borne disease prevalence has been previously difficult to ascertain, particularly beyond the metropolitan areas. METHODS: Dog blood samples (n = 8,662) were submitted from 14 veterinary colleges, 6 private veterinary practices and 4 diagnostic laboratories across this region. Samples were tested for E. canis, E. chaffeensis and E. ewingii specific antibodies using peptide microtiter ELISAs. RESULTS: Overall, E. canis, E. chaffeensis and E. ewingii seroprevalence was 0.8%, 2.8%, and 5.1%, respectively. The highest E. canis seroprevalence (2.3%) was found in a region encompassing Arkansas, Louisiana, Oklahoma, Tennessee and Texas. E. chaffeensis seroreactivity was 6.6% in the central region (Arkansas, Kansas, Missouri, and Oklahoma) and 4.6% in the southeast region (Georgia, Maryland, North Carolina, South Carolina, Tennessee and Virginia). Seroreactivity to E. ewingii was also highest in the central region (14.6%) followed by the southeast region (5.9%). The geospatial pattern derived from E. chaffeensis and E. ewingii seropositive samples was similar to previous reports based on E. chaffeensis seroreactivity in white-tailed deer and the distribution of human monocytic ehrlichiosis (HME) cases reported by the CDC. CONCLUSIONS: The results of this study provide the first large scale regional documentation of exposure to E. canis, E. chaffeensis and E. ewingii in pet dogs, highlighting regional differences in seroprevalence and providing the basis for heightened awareness of these emerging vector-borne pathogens by veterinarians and public health agencies.

Comparative strain analysis of Anaplasma phagocytophilum infection and clinical outcomes in a canine model of granulocytic anaplasmosis.

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