Novel Rickettsia Species Infecting Dogs, United States.

In 2018 and 2019, spotted fever was suspected in 3 dogs in 3 US states. The dogs had fever and hematological abnormalities; blood samples were Rickettsia seroreactive. Identical Rickettsia DNA sequences were amplified from the samples. Multilocus phylogenetic analysis showed the dogs were infected with a novel Rickettsia species related to human Rickettsia pathogens.

MOLECULAR PREVALENCE OF SELECTED VECTOR-BORNE ORGANISMS IN CAPTIVE RED WOLVES (CANIS RUFUS).

The red wolf (Canis rufus) is a critically endangered North American canid, with surviving conspecifics divided between a captive breeding population and a reintroduced free-ranging population. The goal of this study was to assess the prevalence of selected vector-borne pathogens in captive red wolves. Whole blood samples were collected from 35 captive red wolves. Quantitative polymerase chain reaction (PCR) assays were performed on extracted DNA to identify infection by Trypanosoma cruzi and vector-borne organisms within the following genera: Anaplasma, Babesia, Bartonella, Ehrlichia, Mycoplasma, Neoehrlichia, Neorickettsia, and Rickettsia. All red wolves sampled were PCR-negative for all tested organisms. These pathogens are unlikely to constitute threats to red wolf conservation and breeding efforts under current captive management conditions. The results of this study establish a baseline that may facilitate ongoing disease monitoring in this species.

Potentially novel Ehrlichia species in horses, Nicaragua.

Ehrlichia sp. DNA was amplified from 4 Ehrlichia-seroreactive horses from Mérida, Nicaragua. Sequencing of 16S rDNA, sodB, and groEL genes indicated that the bacterium is most likely a novel Ehrlichia species. The tick vector and the potential for canine and human infection remain unknown.

Development and validation of a sensitive and specific sodB-based quantitative PCR assay for molecular detection of Ehrlichia species.

We developed a sensitive and specific sodB-based quantitative PCR assay to detect Ehrlichia spp. The assay's limit of detection was 5 copies/reaction, and it did not amplify nonspecific DNA. Compared with a 16S rRNA gene PCR target, the sodB target may offer an improved molecular diagnostic assay to detect Ehrlichia spp.

Detection of Anaplasma phagocytophilum in an inflammatory pericardial effusion of a dog.

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.

Evaluation of the Veterinary IDEXX SNAP 4Dx Plus Test for the Diagnosis of Lyme Disease in Humans.

Background: Lyme disease, caused by infection with Borrelia burgdorferi, is the most common vector-borne disease in the United States. The standard two-tier testing (STTT) algorithm suffers from low sensitivity, misinterpretation, and long turnaround time, preventing timely detection and treatment. To address these challenges, we hypothesized that the canine point-of-care (PoC) SNAP 4Dx Plus test used to detect Borrelia burgdorferi antibodies could be employed for human diagnosis. Materials and Methods: The SNAP 4Dx Plus testing was conducted in accordance with the manufacturer's instructions, with results read by manual inspection. All analyses were conducted using R version 4.3.1, and agreement between the PoC assay and the STTT was assessed using kappa statistics with GraphPad software. Results: We included 102 previously-tested human serum samples, of which 19 samples (18.6%) were STTT positive. Compared to the STTT, the SNAP 4Dx Plus test demonstrated a low sensitivity of 0.16 (95% CI 0.03 to 0.40). Conclusion: Overall, our results do not support the use of the SNAP 4Dx Plus LD assay for the diagnosis of human Lyme disease. Differences in antibody concentrations between human and canine samples may partly explain our findings.

First reported case of leishmaniasis in a cat in Trinidad and Tobago.

A 3-year-old, female, domestic shorthair cat, was presented to the Veterinary Teaching Hospital at the School of Veterinary Medicine (SVM), Trinidad and Tobago for a swollen nose, and multiple, variably sized small masses on both ears. The initial diagnostic tests included a CBC, serum biochemistry profile, cytological evaluation of masses on the ear and nose, and FeLV/FIV testing. The CBC and biochemistry results were unremarkable except for a hyperproteinaemia and hyperglobulinemia. Cytology of the nose and ear lesions revealed mixed inflammation and high numbers of intracellular and extracellular organisms consistent with Leishmania amastigotes. The cat was FeLV/FIV negative. Histopathology and Leishmania IFA and PCR analysis were subsequently performed, confirming the Leishmania diagnosis. The PCR, DNA sequencing and phylogenetic tree analyses identified L. amazonensis. This is the first reported case of L. amazonensis infection in a domestic animal in Trinidad with molecular characterization indicating it exists in the region and is likely being transmitted by sandflies.

A second-generation, point-of-care immunoassay provided improved detection of Anaplasma and Ehrlichia antibodies in PCR-positive dogs naturally infected with Anaplasma or Ehrlichia species.

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.

Evaluation of Anaplasma spp. seroprevalence in dogs and association with incidence of human anaplasmosis.

Point-of-care (POC) ELISA tests are routinely used in US veterinary practices to screen canine patients for antibodies to tick-transmitted pathogens. Results are also used to monitor spatial and temporal trends in canine seroprevalence, and these data can build awareness of the risk to humans of tick-transmitted diseases such as Lyme disease and anaplasmosis. This study utilized a second-generation test that has incorporated additional Anaplasma-specific peptides into a commercial POC ELISA test to allow detection of Anaplasma spp. antibodies earlier post-infection. A convenience population consisting of 19,894 canine samples from a US commercial diagnostic laboratory were tested using the second-generation POC ELISA test to describe regional Anaplasma spp. canine seroprevalence and assess correlation to anaplasmosis cases reported to Centers for Disease Control and Prevention by state. Antibodies to Anaplasma spp. were detected in 1646 samples (8.3%) with the Northeast and Midwest US census regions having the highest proportion of positive samples. At the state level, a significant correlation was found between canine Anaplasma spp. seroprevalence and human anaplasmosis incidence (r2 = 0.64). Although estimates of canine Anaplasma spp. seroprevalence presented here using the second-generation POC ELISA are generally increased, especially in the Northeast and Midwest, the regional distribution of canine samples testing positive for Anaplasma spp. antibodies is consistent with previous reports. The observed correlation with human anaplasmosis incidence indicates that results from the second-generation POC ELISA will continue to add value in epidemiological assessment of human anaplasmosis risk.

Rickettsia typhi infection in a clinically-ill dog from Houston, Texas.

In 2020, Rickettsia typhi was diagnosed in a dog from Houston, Texas, USA based upon R. typhi IFA seroreactivity in both acute and convalescent sera, and PCR with DNA sequencing of 4 different gene regions, all of which were 100% identical to R. typhi. The dog was clinically ill with intermittent fever, lethargy, inappetence, and lymphadenopathy. Clinicopathological abnormalities included a mild nonregenerative anemia, neutrophilia, lymphopenia, thrombocytopenia, hypoalbuminemia, and elevated ALP. The dog rapidly recovered with doxycycline administration.

An Improved Point-of-Care ELISA for the Diagnosis of Anaplasmosis and Ehrlichiosis During the Acute Phase of Tick-Borne Infections in Dogs.

Veterinarians often test for serologic evidence of vector-borne infections in sick dogs presenting with clinical signs or to screen for subclinical chronic infections. Additional peptide targets for the detection of antibodies to Anaplasma phagocytophilum, Anaplasma platys, and Ehrlichia canis were added to an existing point-of-care (POC) ELISA test (SNAP 4Dx Plus Test, IDEXX Laboratories, Westbrook, ME). This second-generation, multi-analyte test detects Dirofilaria immitis antigen and antibodies to Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp. The second-generation test is expected to better meet the needs of practicing veterinarians and their patients. To assess this expectation, the second-generation POC test was evaluated with serum samples from experimentally infected dogs and a broader field population of dogs. Compared to the first-generation test, most dogs experimentally infected with A phagocytophilum (n = 7/8), A platys (n = 4/6), or E canis (n = 4/6) had detectable antibody responses 3-22 days earlier post-infection; these results demonstrated better alignment with polymerase chain reaction (PCR) amplification results and the onset of clinical signs. Using a convenience sample set of 510 sera from both academic and commercial veterinary diagnostic laboratories, the second-generation test had sensitivities greater than 90% for Anaplasma spp. (94.1%), B burgdorferi (95.5%), Ehrlichia spp. (93.4%) and D immitis (98.0%). Specificity ranged from 96.8% - 100% across the four assays. Results from this study demonstrate that the second-generation POC ELISA had an improved ability to detect serologic responses during the acute phase of A phagocytophilum, A platys, and E canis experimental infections. The results from the broader field samples support overall high sensitivity and specificity, consistent with the historical performance of the first-generation POC ELISA test.

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.

Genetic diversity of Anaplasma and Ehrlichia bacteria found in Dermacentor and Ixodes ticks in Mongolia.

Anaplasma and Ehrlichia are tick-borne bacterial pathogens that cause human granulocytic anaplasmosis, human monocytic ehrlichiosis, and are severe threats to livestock economies like Mongolia. In this study, ticks were collected, identified, and pooled (n = 299) from three distinct environments across central Mongolia. Each pool was initially tested for Anaplasma/Ehrlichia using a 16S rRNA PCR assay that detects both genera, and specific PCR testing was done to identify those positive samples. Maximum likelihood estimation (MLE) of infection rates of ticks collected from the environment in Selenge aimag (province) found infection rates of Ixodes persulcatus ticks to be 2.0% (95% CI: 0.7, 4.3%) for A. phagocytophilum and 0.8% (95% CI: 0.1, 2.5%) for both nonspecific Ehrlichia and Anaplasma. Ehrlichia muris was only detected in I. persulcatus ticks collected from the Selenge aimag, where the MLE was 1.2% (95% CI: 0.1, 2.5%). The calculated MLE infection rate of Anaplasma spp. in questing Dermacentor nuttalli ticks ranged from 1.9% (95% CI: 1.1, 9.1%) in the Tov aimag to 2.3% (95% CI: 1.3, 10.8%) in the Selenge aimag. However, when examining MLE in ticks removed from livestock, estimates increase substantially, ranging from 7.8% (95% CI: 4.2, 13.3%) in Dornogovi to 22.5% (95% CI: 14.3, 34.3%) in Selenge, suggesting that livestock play a key role in disease maintenance. Considering the collective economic losses that can result from these pathogens and the potential for illness in nomadic herdsmen, these results highlight the need for enhanced TBD surveillance and prevention measures within Mongolia.

Prevalence of Babesia spp. and clinical characteristics of Babesia vulpes infections in North American dogs.

BACKGROUND: Babesiosis is an important cause of thrombocytopenia and hemolytic anemia in dogs. Babesia vulpes, reported in European dogs and North American foxes, rarely has been reported in domestic North American dogs. Newly optimized polymerase chain reaction (PCR) primers facilitate more sensitive amplification of B. vulpes DNA. OBJECTIVES: To determine the prevalence of Babesia sp. infections in dogs being tested for Babesia infection, and to describe co-infections and clinicopathologic abnormalities in B. vulpes positive dogs. ANIMALS: Dog blood or tissue samples (n = 9367) submitted to a diagnostic laboratory between June 2015 and June 2018 were tested using an optimized Babesia PCR assay. METHODS: Comprehensive canine vector-borne disease diagnostic testing was performed on convenience samples. RESULTS: Babesia sp. DNA was amplified from 269/9367 (2.9%) North American dogs. Babesia sp. infections included B. gibsoni monoinfection (157; 1.7%), B. vulpes monoinfection (19; 0.20%), and B. gibsoni and B. vulpes coinfection (29; 0.31%). Forty-three of the 48 total B. vulpes-infected dogs were American Pit Bull Terrier-type breeds, of which 36 historically were involved with dog fights. Coinfections with Mycoplasma, Dirofilaria immitis, or Wolbachia and coexposures to Bartonella, Ehrlichia, and Rickettsia spp. were documented in B. vulpes-infected dogs. Clinicopathologic data in B. vulpes-infected dogs both with and without coinfections included anemia, thrombocytopenia, hyperglobulinemia, hypoalbuminemia, and proteinuria. CONCLUSIONS AND CLINICAL IMPORTANCE: Babesia vulpes infection in domestic North American dogs is commonly found in conjunction with other coinfections, including B. gibsoni and hemotropic Mycoplasma. Similar to B. gibsoni, dog-to-dog transmission of B. vulpes may be a frequent mode of transmission.

Molecular identification of vector-borne organisms in Ehrlichia seropositive Nicaraguan horses and first report of Rickettsia felis infection in the horse.

Certain vector-borne organisms serve as etiological agents of equine disease. After previously identifying a new Ehrlichia species in horses from Mérida, we aimed to determine the infection frequency and screen for a wide range of vector-borne organisms from 93 tick-exposed, Ehrlichia seropositive horses in this region. PCR assays were performed to identify infection by organisms within the following genera: Anaplasma, Babesia, Bartonella, Ehrlichia, Leishmania, Mycoplasma, Neorickettsia, Rickettsia and Theileria. Overall, 90/93 horses (96.8%) were infected with one or more vector-borne organisms. Ninety (96.8%) horses were infected with Theileria equi and 21 (26.8%) with Babesia caballi. Nine (9.7%) horses were infected with the novel Ehrlichia species previously designated H7, reported in horses from Nicaragua and Brazil. Two horses (2.2%) were infected with Rickettsia felis. Anaplasma, Bartonella, Leishmania, Mycoplasma, or Neorickettsia species DNA was not amplified from any horse. Ticks collected from horses infected with vector-borne organisms were identified as Amblyomma cajennense sensu lato and Dermacentor nitens. Horses in Mérida are infected by a range of vector-borne organisms, including B. caballi, T. equi, Ehrlichia species H7, and R. felis. To the authors' knowledge, this constitutes the first report of molecular detection of R. felis in horses.

Clinicopathological findings in 41 dogs (2008-2018) naturally infected with Ehrlichia ewingii.

BACKGROUND: Ehrlichia ewingii is the most seroprevalent Ehrlichia-infecting dogs in the southern and mid-western United States. Fever, lameness, and polyarthritis are commonly reported findings in dogs naturally infected with E. ewingii. OBJECTIVES: To evaluate clinicopathologic findings in a population of dogs naturally infected with E. ewingii. ANIMALS: Forty-one dogs PCR positive for E. ewingii and PCR negative for other targeted vector-borne organisms. METHODS: Retrospective study. Clinical and clinicopathologic data including physical examination findings, CBC, serum biochemistry, urinalysis (UA), symmetric dimethylarginine (SDMA), and vector-borne disease diagnostic results were reviewed. RESULTS: Frequent clinical diagnoses other than ehrlichiosis (28/41; 68.3%) were renal disease (7/41; 17.1%) and immune-mediated hemolytic anemia (IMHA) (6/41; 14.6%). The most frequent physical examination finding was joint pain (14/41; 34.1%). Prominent hematologic and biochemical abnormalities included abnormal lymphocyte counts (22/36; 61.1%); neutrophilia (21/37; 56.8%); increased alkaline phosphatase (20/35; 57.1%) and alanine transaminase (14/35; 40%) activities; and increased SDMA concentration (11/34; 32.4%). Urinalysis abnormalities included proteinuria (20/27; 74%), most with inactive sediments (16/20; 80%). Dogs were seroreactive by Ehrlichia canis immunofluorescence assay (IFA; 17/39; 43.6%) and Ehrlichia ELISA (34/41; 82.9%). Seroreactivity by IFA for other vector-borne pathogens included Bartonella (1/39; 2.6%), Rickettsia rickettsii (spotted-fever group rickettsiae) (12/39; 30.8%), and Borrelia burgdorferi by ELISA (1/41; 2.4%). CONCLUSIONS AND CLINICAL IMPORTANCE: Renal disease, IMHA, proteinuria, neutrophilia, abnormal lymphocytes, and increased liver enzyme activities were common in this group of E. ewingii-infected dogs. Studies are needed to determine if E. ewingii contributes to comorbidities or is a precipitating factor in clinical syndromes in persistently infected dogs.

Babesia gibsoni cytochrome b mutations in canine blood samples submitted to a US veterinary diagnostic laboratory.

BACKGROUND: Babesiosis caused by Babesia gibsoni is recognized throughout the world and can be difficult to treat. Resistance to atovaquone is associated with mutations in the B. gibsoni mitochondrial genome, specifically the M128 position of cytochrome b (cytb). The prevalence of cytb mutations in North America has not been reported. HYPOTHESIS/OBJECTIVES: The objective of our study was to describe the prevalence of cytb M128 mutations in B. gibsoni in canine blood samples submitted to a US veterinary diagnostic laboratory. A secondary objective was to determine whether or not some dogs had wild-type cytb in our initial samples then had M128 mutations detected in follow-up samples. ANIMALS: One-Hundred seventy-four dogs that tested positive for the presence of B. gibsoni between 2012 and 2017. METHODS: Case series of consecutive samples submitted to a veterinary diagnostic laboratory. Partial B. gibsoni cytb genes were amplified by polymerase chain reaction and screened for the presence of mutations at the M128 position. RESULTS: The overall prevalence of M128 mutants was 3.5% (6/173 dogs) in the initial samples. The incidence of new cytb mutants in dogs that tested positive for B. gibsoni, which then had follow-up testing, was 12.1% (5/41). Conclusions and Clinic Importance: Our study reaffirms that B. gibsoni infection is widespread and most commonly detected in American Staffordshire Terrier/American Pit Bull Terrier dogs (128/174, 74% of the infected dogs in our study). The prevalence of cytb mutations does not warrant pretreatment genotyping.

Molecular surveillance of novel tick-borne organisms in Madagascar's lemurs.

The discovery and characterization of emerging tick-borne organisms are critical for global health initiatives to improve animal and human welfare (One Health). It is possible that unknown tick-borne organisms underlie a subset of undiagnosed illness in wildlife, domesticated species, and humans. Our study lends support to the One Health concept by highlighting the prevalence of three blood-borne organisms in wild lemurs living in close proximity to domesticated species and humans. Previously, our team identified three novel, presumably tick-borne, intravascular organisms, belonging to the genera Babesia, Borrelia, and Neoehrlichia, circulating in two of Madagascar's lemur species. Here, we extend our previous observation by developing a targeted molecular surveillance approach aimed at determining the prevalence of these organisms in lemurs. Using quantitative PCR, we provide Babesia, Borrelia, and Neoehrlichia prevalence data for 76 individuals comprising four lemur species located in eastern Madagascar. Our results indicate a high prevalence (96%) of Babesia across sampled individuals with lower prevalences for Neoehrlichia (36%) and Borrelia (14.5%). In light of our results, we recommend additional studies of these tick-borne organisms to determine pathogenicity and assess zoonotic potency to other animals and humans in Madagascar.

Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA.

BACKGROUND: Babesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. Commonly used approaches to diagnose babesiosis include microscopic examination of peripheral blood smears, detection of circulating antibodies and PCR. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene. These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. However, differences in the 18S rRNA gene sequence of distantly related clades can make it difficult to design assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. By targeting Babesia mitochondrial genome (mtDNA), we designed a novel three primer qPCR with greater sensitivity and broader screening capabilities to diagnose and differentiate Babesia spp. METHODS: Using 13 Babesia mtDNA sequences, a region spanning two large subunit rRNA gene fragments (lsu5-lsu4) was aligned to design three primers for use in a qPCR assay (LSU qPCR) capable of amplifying a wide range of Babesia spp. Plasmid clones were generated and used as standards to determine efficiency, linear dynamic range and analytical sensitivity. Animals naturally infected with vector-borne pathogens were tested retrospectively and prospectively to determine relative clinical sensitivity and specificity by comparing the LSU qPCR to an established 18S rDNA qPCR. RESULTS: The LSU qPCR efficiencies ranged between 92 and 100% with the limit of detection at five copies/reaction. The assay did not amplify mammalian host or other vector-borne pathogen gDNA except Cytauxzoon felis (a feline protozoal pathogen). The LSU qPCR assay amplified 12 different Babesia. sp. and C. felis from 31/31 (100%) archived samples, whereas the 18S qPCR amplified only 26/31 (83.9%). By prospective analysis, 19/394 diagnostic accessions (4.8%) were LSU qPCR positive, compared to 11/394 (2.8%) 18S rDNA qPCR positive. CONCLUSIONS: We have developed a more sensitive qPCR assay with a more expansive range of Babesia spp. detection by targeting a highly conserved region of mtDNA, when compared to an established 18S qPCR.