Comparison of acid- versus heat-treatment for immune complex dissociation and detection of Dirofilaria immitis antigen in canine plasma.

Annual antigen testing is a mainstay for diagnosing infection with Dirofiliaria immitis in dogs; yet, it has been documented that some heartworm-infected dogs and cats test false-negative for antigen due to the presence of antigen-antibody complexes. Several studies have reported the use of heat as a reliable means of immune-complex dissociation (ICD) in recent years; however, the data regarding the use of acid as a reliable method of ICD for D. immitis detection are limited. The objective of this study was to compare an acid-based form of ICD to the more established and evaluated method of heat-based ICD in experimentally infected and non-infected dogs. Plasma from class A dogs experimentally infected ∼4 months prior with D. immitis (infected; n = 24) and dogs reared indoors with no history of exposure to mosquitoes (non-infected; n = 75) were evaluated for presence of D. immitis antigen (DiroCHEK® Heartworm antigen test kit). Each sample was divided into three aliquots for testing: [1] Control plasma (no acid- or heat-treatment), [2] acid-treated plasma (trichloroacetic acid (TCA), incubation, centrifugation for 5 min at 16,000 X g, buffer), and [3] heat-treated plasma (104 °C followed by centrifugation at 16,000 X g). Treatments for each aliquot were performed and tested in triplicate; results were determined both visually (color change) and by spectrophotometric analysis (optical density [OD] value). Of the 24 infected dogs, 0/24 tested positive for antigen in the absence of acid- or heat-treatment. Those same plasma samples following processing by either acid- or heat-treatment yielded 18/24 (75.0%) and 19/24 (79.2%) antigen-positive results, respectively. Of the 75 plasma samples from non-infected dogs, neither acid- nor heat-treatment of plasma caused any false-positive color changes or spectrophotometric values. These results indicate that acid as a means of ICD reliably allowed for the detection of D. immitis antigen in infected plasma while not inducing false-positive results in non-infected plasma samples.

Sarcocystis fayeri-Induced Granulomatous and Eosinophilic Myositis in 2 Related Horses.

This report describes 2 genetically related paint mares, case Nos. 1 and 2, presented to the Oklahoma State University Boren Veterinary Medical Teaching Hospital for chronic weight loss and abnormal gait, respectively. Notable findings in both cases included marked persistent eosinophilia and multiple intramuscular lateral thoracic masses. Histologic examination of masses revealed eosinophilic, centrally necrotic granulomas and marked eosinophilic myositis. Granulomas in case No. 1 also contained intralesional Sarcocystis sp material, and adjacent muscle fibers contained intact protozoal cysts. Case No. 1 developed severe refractory muscle pain and recurrent esophageal dysphagia. At necropsy, disseminated, grossly visible granulomas were present throughout all examined striated muscles. Nested polymerase chain reaction of the 18S rRNA gene revealed >99% homology with Sarcocystis fayeri. Sarcocystis spp are apicomplexan protozoa that infect striated muscle of many omnivorous species, typically without inciting clinical disease. Sarcocystosis should be considered a rare cause of granulomatous eosinophilic myositis and choke in horses.

Persistent Ehrlichia ewingii infection in dogs after natural tick infestation.

BACKGROUND: Ehrlichia ewingii, which causes disease in dogs and people, is the most common Ehrlichia spp. infecting dogs in the United States, but little is known about how long E. ewingii infection persists in dogs. HYPOTHESIS/OBJECTIVES: To evaluate the persistence of natural infection with E. ewingii in dogs. ANIMALS: Four Class A Beagles; no previous exposure to ticks or tick-borne infectious agents. METHODS: Dogs were exposed to ticks by weekly walks through tick habitat in north central Oklahoma; dogs positive for infection with Ehrlichia spp. by sequence-confirmed PCR and peptide-specific serology were evaluated for 733 days (D). Whole blood was collected once weekly for PCR, and serum was collected once monthly for detection of antibodies to Ehrlichia canis (peptide p16), Ehrlichia chaffeensis (indirect fluorescence antibody [IFA] and variable-length PCR target [VLPT]), and E. ewingii (peptide p28). RESULTS: All dogs (4/4) became infected with Ehrlichia spp. as evidenced by seroconversion on IFA to E. chaffeensis (4/4); PCR detection of E. ewingii (4/4) and E. chaffeensis (2/4) DNA using both nested and real-time assays; and presence of specific antibodies to E. ewingii (4/4) and E. chaffeensis (2/4). Infection with E. chaffeensis was not detected after D55. Intermittent E. ewingii rickettsemia persisted in 3 of 4 dogs for as long as 733 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Our data demonstrate that dogs infected with E. ewingii from tick feeding are capable of maintaining infection with this pathogen long-term, and may serve as a reservoir host for the maintenance of E. ewingii in nature.