Barbara A. Qurollo

Spotted Fever Group Rickettsia in Dogs

In mammals, detection of Wolbachia bacteria can be used to diagnose filarial infection, while antibiotic treatment to eliminate Wolbachia can assist in eliminating filarial infections. Because Wolbachia are necessary for survival of several filarioids and closely related to Anaplasma and Ehrlichia , we analyzed Wolbachia DNA amplification by Anaplasma/Ehrlichia qPCR, from 39,526 domestic and wildlife animal blood samples submitted to a diagnostic laboratory between 2017 and 2023. Filarial infection was confirmed by 28S gene amplification, followed by phylogenetic analysis utilizing filarial cytochrome oxidase subunit 1 ( cox1 ), myosin heavy chain ( myoHC ), and 70 kilodalton heat shock protein ( hsp70 ) gene sequencing. Wolbachia DNA was detected in 57 domestic dogs ( Canis familiaris ) and three raccoons ( Procyon lotor ) from 23 states and Puerto Rico. A majority of the Wolbachia sequences from dogs were Dirofilaria immitis ‐associated (89%, 51/57), whereas DNA from other Wolbachia were associated with insects (9%, 5/57) or Dirofilaria repens (2%, 1/57). D. immitis infection was confirmed by 28S filarial PCR for all samples with D. immitis ‐associated Wolbachia available for testing ( n = 41). D. repens infection was confirmed by 28S and cox1 PCR in the dog infected with D. repens ‐associated Wolbachia . This dog was originally imported from Slovakia. The Wolbachia DNA amplified from raccoons most closely aligned with Wolbachia from Mansonella ozzardi (98.9%). 28S filarial, cox1 , myoHC , and hsp70 sequencing did not align with currently available GenBank sequences but did align with Mansonella . Morphologically, microfilariae from additional raccoons were consistent with Mansonella llewellyni . Molecular surveillance for Wolbachia in wildlife and domestic animals has the potential to identify novel filarial species in the United States, including zoonotic species.

An 11-year-old female spayed German Wirehaired Pointer with a 1-week history of lethargy, hyporexia, diarrhea, and coughing presented with pericardial effusion causing cardiac tamponade. An echocardiogram revealed no structural cause for pericardial effusion. The pericardial effusion was an exudate with mixed macrophagic and neutrophilic inflammation. Morulae occasionally were found within neutrophils. The pericardial fluid and blood were qPCR and cPCR positive for Anaplasma phagocytophilum (NC State University, Vector-borne Disease Diagnostic Laboratory, Raleigh, NC). The dog's blood was negative by ELISA (Vetscan Flex4 Rapid Test, Zoetis, Parsippany, NJ) for A. phagocytophilum antibodies at initial presentation and subsequently positive (SNAP4DxPlus, IDEXX, Westbrook, ME) 7 days later. After pericardiocentesis and administration of doxycycline (5 mg/kg PO q12h for 14 days), a repeat echocardiogram performed 1 month later showed no recurrence of pericardial effusion.

A validated second-generation SNAP 4Dx Plus (Idexx) incorporates new peptides for improved detection of antibodies against Anaplasma and Ehrlichia tick-borne pathogens in dogs. We compared the first- and second-generation SNAP 4Dx Plus using dogs naturally infected with Anaplasma or Ehrlichia species, or dogs seroreactive by an E. canis indirect fluorescent antibody test (IFAT). The second-generation immunoassay was more sensitive than the first-generation for dogs infected with A. phagocytophilum (51.1% and 29.2%, respectively), A. platys (63.6% and 35.3%, respectively), E. canis (96.2% and 88.3%, respectively), or E. ewingii (73.7% and 70.8%, respectively), and for dogs seroreactive by E. canis IFAT (87.3% and 83.9%, respectively). The second-generation immunoassay detected significantly more Anaplasma- or Ehrlichia-infected dogs that were Anaplasma ( p < 0.001) or Ehrlichia ( p = 0.031) seroreactive, respectively, than did the first-generation test. When Ehrlichia seroreactivity by E. canis IFAT and both immunoassays was compared, significantly more E. canis–infected dogs were seroreactive by E. canis IFAT than the first-generation ( p = 0.006) but not the second-generation ( p = 0.125) immunoassay. Significantly more E. ewingii–infected dogs were seroreactive by the first- ( p = 0.011) and second-generation ( p = 0.049) immunoassays than the E. canis IFAT. Medical records available for 7 dogs that were Anaplasma seroreactive by the second-generation but not the first-generation immunoassay revealed case management decisions that might have been different with an immediate anaplasmosis diagnosis, including earlier doxycycline therapy and less hospitalization. The second-generation SNAP 4Dx Plus test offered improved serologic detection of Anaplasma and Ehrlichia in naturally infected dogs.

The standard for detecting vector-borne pathogens is real-time PCR (rtPCR). However, this requires many individual tests to obtain an accurate diagnosis. The purpose of this study was to develop and validate a targeted next-generation sequencing (NGS) assay for vector-borne pathogens. Pathogen target regions were amplified via PCR using two primer pools that were developed in conjunction with ThermoFisher Scientific, and barcoded DNA libraries were prepared and sequenced with the Ion Torrent S5 system. Data were assembled using SPAdes and mapped to a reference file containing sequences from the pathogens. The raw reads were analyzed to confirm the results. Test feasibility and analytical specificity were evaluated with type strains or validated positive clinical samples from dogs. The analytical sensitivity of the method was compared to Ct values obtained by rtPCR testing. Diagnostic sensitivity and specificity were assessed with a set of known positive and negative clinical samples based on rtPCR testing. Positive and negative percent agreements and Cohen’s kappa were calculated. The primer sets were specific for the intended targets, based on sequence analysis of the amplified products, and the method detected 17 different pathogens. Analytical sensitivity was equivalent to an rtPCR Ct value of approximately 35–36. The positive percent agreement was 92%, and the negative percent agreement was 88%. Cohen’s kappa was 0.804, which indicates almost perfect agreement between the rtPCR assays and the targeted NGS assay. Using a targeted method reduces the costs associated with NGS sequencing and allows for a 2–3 day turn-around time, making this a viable method for detection of vector-borne pathogens in canine whole blood samples.

Background: Reference intervals for platelets and white blood cell (WBCs) counts are lower in greyhounds than other breeds. Proteinuria is common. Vector-borne diseases (VBD) cause thrombocytopenia, leukopenia, and proteinuria. Racing greyhounds are commonly exposed to vectors that carry multiple organisms capable of chronically infecting clinically healthy dogs. Hypothesis/Objectives: Vector-borne disease prevalence is higher in retired racing greyhounds than in show-bred greyhounds. Occult infection contributes to breed-related laboratory abnormalities. Animals: Thirty National Greyhound Association (NGA) retired racing and 28 American Kennel Club (AKC) show-bred greyhounds. Methods: Peripheral blood was tested for Anaplasma, Babesia, Bartonella, Ehrlichia, hemotropic Mycoplasma, and Rickettsia species using PCR. Antibodies to Anaplasma, Babesia, Bartonella, Ehrlichia, and Rickettsia species and Borrelia burgdorferi were detected using immunofluorescence and ELISA assays. Complete blood counts, semiquantitative platelet estimates, and microalbuminuria concentration were determined. Results: Seven of 30 NGA and 1/28 AKC greyhounds tested positive for ≥1 VBD (P = .05). More positive tests were documented in NGA (10/630) than in AKC dogs (1/588; P = .02). Exposure to Bartonella species (3/30), Babesia vogeli (2/30), Ehrlichia canis (1/30), and infection with Mycoplasma hemocanis (3/30) occurred in NGA dogs. Platelet counts or estimates were >170 000/μL. White blood cell counts .99; 1/8 VBD positive; 8/51 VBD negative, P = .99) and microalbuminuria (10/21 AKC; 5/26 NGA, P = .06; 1/8 VBD positive; 14/25 VBD negative, P = .41) were not associated with VBD. Conclusions and Clinical Importance: The prevalence of thrombocytopenia and B. vogeli exposure was lower than previously documented. Larger studies investigating the health impact of multiple VBD organisms are warranted.