Vector‐borne disease and its relationship to hematologic abnormalities and microalbuminuria in retired racing and show‐bred greyhounds

Abstract 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 <4000/μL (4/28 AKC; 5/30 NGA, P > .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.


| INTRODUCTION
Greyhounds are popular companion animals in the United States.
Many are retired racing greyhounds registered with the National Greyhound Association (NGA), (https://www.ngagreyhounds.com/ Home). Show-bred greyhounds registered with the American Kennel Club (AKC) have pedigrees that certify they have not raced (http:// www.greyhound-data.com). Therefore, these 2 groups have distinct genetic and environmental backgrounds.
We hypothesized that VBD is more prevalent in retired racing greyhounds than in show-bred greyounds, that organisms in addition to B. vogeli and E. canis are common in retired racing greyhounds, and that occult infection contributes to "breed-related" thrombocytopenia, leukopenia and microalbuminuria.
Our primary objectives were to:

| Statistical analysis
Power calculations for the outcomes of detecting differences in prevalence of VBD, thrombocytopenia and microalbuminuria between groups were calculated using epitools.ausvet.com.au (power = 0.8, Based on previous studies, the seroprevalence of B. vogeli in NGA dogs and AKC dogs was assumed to be 50% and 0%, respectively. 36,37 To detect a difference in VBD prevalence between groups the required sample size was estimated to be n = 15 in each group. Approximately 50% of greyhounds are thrombocytopenic according to most reference intervals established for other breeds (<170 000 platelets/μL). 7,9,10 Assuming 50% of NGA and 0% of AKC greyhounds are thrombocytopenic, the required sample size again was estimated to be n = 15 in each group. Fifty-three percent of urine samples collected from clinically healthy retired racing greyhounds by cystocentesis are positive for microalbuminuria whereas up to 15% of voided urine samples from clinically healthy dogs of other breeds exhibit microalbuminuria. 13,67 We assumed the prevalence of microabluminuria to be at least 53% in voided urine of NGA and 15% in AKC greyhounds, requiring a sample size of n = 29 in each group.  Based upon WBC counts of ≤4000/μL, 9 dogs were leukopenic.
Leukopenia was not more common in the 8 dogs with VBD exposure or infection (1/8) compared to those without VBD exposure or infection (8/50; P = .99). Leukopenia was not more common in AKC (4/28) than NGA dogs (5/30; P > .99). Total WBC, neutrophil, lymphocyte and monocyte counts were lower and eosinophil counts were higher in AKC dogs compared to NGA dogs ( Figure 1B-F).
More AKC show-bred greyhounds (10/21) had microalbuminuria than did retired racers (5/26), but the difference was not significant (P = .06). The magnitude of microalbuminuria was higher in AKC

| DISCUSSION
The overall prevalence of at least 1 positive test for VBD in an individual retired racing greyhound was not significantly higher in retired racing greyhounds compared to show-bred greyhounds. However, the retired racing greyhounds tested positive to more VBD agents than did show-bred greyhounds, and coinfection or coexposure to ≥1 agent was common.
Previous studies have suggested that racing greyhounds are at increased risk for exposure to or infection with B. canis (vogeli) and possibly E. canis because of risk of exposure to R. sanguineus in racing kennels. 36,37,58,59 Interestingly, in our study, the seroprevalence of Unexpectedly, thrombocytopenia was not found in either the retired racing greyhounds or the show-bred greyhounds. Previous studies to establish normal reference ranges for platelets in greyhounds have led to the conclusion that greyhounds have lower platelet counts than other breeds, and that platelet counts between 100 000 and 170 000/μL should not be considered abnormal in this breed. 3,7,[9][10][11] However, a high rate of exposure to R. sanguineus, other ticks, and potentially fleas in some geographic locales may have contributed to the low platelet counts historically reported in greyhounds as a breed. Indeed, the retired racing greyhounds in our study had serologic evidence of exposure to Bartonella, Babesia, and Ehrlichia species, all of which are associated with thrombocytopenia. However, it is important to note that no dog tested PCR positive for these organisms, and titers were low. Therefore, a lack of active infection may explain the uniformly normal platelet counts observed in our study. Although 3 dogs were actively infected with (PCR+) M. haemocanis, infection with this organism is not usually associated with thrombocytopenia or other laboratory abnormalities in healthy dogs. 43,70,71 Therefore, it is not surprising that even dogs in the VBD group in our study were not thrombocytopenic. Larger, contemporary studies re-exploring reference intervals in greyhounds comprehensively screened for VBD agents are warranted.
Like thrombocytopenia, leukopenia and leukocytosis are variably associated with VBD. Some studies have shown that WBC counts are lower in greyhound dogs than in other breeds. 3,8,11 We explored whether occult VBD may account for leukopenia in some greyhounds.
We did not find that leukopenia occurred more commonly in greyhounds testing positive for VBD compared to those testing negative.
Surprisingly, we found that total WBC counts (including neutrophils, lymphocytes, and monocytes) were lower and eosinophils were higher in the show-bred dogs compared to the retired racing dogs. In addition to infection, other factors such as stress, age, sex, and neuter status can affect WBC counts in dogs. [72][73][74] Whether these or other factors account for differences requires further investigation.
Proteinuria is also common in both greyhound dogs and in dogs with VBD. 13,[16][17][18][19][20][21][22]25,34 In our study, microalbuminuria was common in both retired racing greyhounds and in show-bred greyhounds and was not significantly more frequent in dogs with evidence of VBD exposure. This finding may be explained by a lack of active infection with VBD agents such as Babesia and Ehrlichia spp. that cause microalbuminuria. In addition, other mechanisms such as breed-related vascular dysfunction likely contribute to proteinuria in this breed. 2 There were more intact males in the AKC than in the NGA group which, in addition to genetic differences, may have contributed to the higher magnitude of microalbuminuria in the AKC group. 75 One limitation of our study was that platelet clumping precluded the use of quantitative platelet counts for some analyses. Although venipuncture was atraumatic and samples were kept refrigerated during the collection process, platelet clumping was very common. Greyhound platelets are more reactive than those of other breeds, potentially contributing to the propensity for clumping despite atraumatic venipuncture. 3,10 Notably, the prevalence of clumping seen in the greyhounds in our study was similar to a previous study of greyhound platelets. 10 In addition, a delay of several hours occurred from when blood was placed into EDTA tubes and when automated platelet counts were performed at the laboratory because of the field conditions of our study. It is possible that the semiquantitative platelet estimates overestimated platelet counts. However, the formula used in our study would tend to underestimate rather than overestimate the platelet count. 76 In addition, previous reports have shown the magnitude of thrombocytopenia is <150 000/μL in 5% to 53% of retired racing greyhounds. 7,9,10 Decreases of this magnitude should be detected using blood smear-based estimates.
Another limitation was the unexpectedly low prevalence of exposure to VBD agents compared to older studies of B. vogeli exposure in retired racing greyhounds. 36,37 This, coupled with the normal platelet counts in all dogs, precluded us from determining if VBD has any role in contributing to lower platelet counts historically considered normal for the breed, and our ability to detect whether a difference was present in overall prevalence of VBD between groups with our chosen sample size.
Despite this limitation, our results suggest reference intervals for greyhounds should be further assessed by larger scale studies using comprehensive screening for VBD as exclusionary criteria. In addition, like other breeds, retired racing greyhounds with clinically relevant thrombocytopenia should be screened for vector-borne and other diseases.

| CONCLUSIONS
We found retired racing greyhounds are exposed to a variety of VBD

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Informed signed consent from the owner was required for participation. This study was approved by the Western University of Health Sciences IACUC committee, protocol # R16IACUC043.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.