Antibodies Reactive to Ehrlichia spp. Are Common in Oklahoma Horses

Abstract

Tick infestations and infection with tick-borne agents are commonly recognized in horses in North America, but equine infection with true Ehrlichia spp. has not been described. To determine the degree to which horses in the south-central United States are naturally exposed to and infected with tick-borne disease agents, serum samples were collected at random (n=240) or from horses with active tick infestations (n=73) and tested by immunofluorescence antibody assay (IFA) and/or enzyme-linked immunosorbent assay (ELISA) for evidence of antibodies reactive to Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. Positive samples were further evaluated by species-specific serology for antibodies reactive to E. canis and E. chaffeensis, and whole blood samples were tested by PCR for evidence of infection with E. canis, E. chaffeensis, E. ewingii, and an E. ruminantium–like organism referred to as the Panola Mountain Ehrlichia. Antibodies reactive to Ehrlichia spp. were identified in 8.75% (21/240) of the randomly acquired samples and 24.7% (18/73) of the serum samples from tick-infested horses, but species-specific ELISA and PCR failed to confirm exposure to or infection with any known Ehrlichia spp. Antibodies to Anaplasma spp. (5/313; 1.6%) and B. burgdorferi (3/313; 1.0%) were uncommon. These data suggest that horses in the south-central United States are likely exposed to a novel Ehrlichia sp. Further research is needed to identify the etiologic agent responsible for the serologic activity seen and to determine the clinical significance, if any, of this finding.

Introduction

Horses in North America are commonly infested with ticks and infected with tick borne disease agents, including Borrelia burgdorferi sensu stricto (s.s.), the causative agent of Lyme borreliosis, and Anaplasma phagocytophilum, which was originally designated as Ehrlichia equi (Bishopp and Trembley 1945, Gribble 1969, Holland et al. 1985, Madigan and Pusterla 2000, Butler et al. 2005); both agents are transmitted by Ixodes spp. ticks, which readily feed on horses. In the southern United States, autochthonous transmission of B. burgdorferi sensu stricto (s.s.) and A. phagocytophilum to people, dogs, and presumably horses is rare, if it occurs at all (Wormser et al. 2006, Bowman et al. 2009). In contrast, infection of dogs and people with Ehrlichia spp. in the South is common, particularly in areas with intense populations of vector ticks and reservoir hosts (Bowman et al. 2009, Salinas et al. 2010, Dahlgren et al. 2011).

In 2001, a comprehensive reorganization of rickettsial organisms resulted in the formation of three main genera: Anaplasma, Ehrlichia, and Neorickettsia (Dumler et al. 2001). Disease agents of horses that were previously in the genus Ehrlichia, including those known to cause Potomac horse fever and equine “ehrlichiosis,” were found to be more closely related phylogenetically to Neorickettsia spp. and Anaplasma spp., respectively. Accordingly, E. risticii was renamed N. risticii, and E. equi was synonymized with E. phagocytophila and the human granulocytic ehrlichiosis (HGE) agent and, collectively, renamed A. phagocytophilum (Dumler et al. 2001, Dumler et al. 2005). Established and novel Ehrlichia organisms described from North America are summarized, together with their predominant vertebrate reservoir hosts and tick vectors, in Table 1. To date, no true Ehrlichia spp., organisms currently considered members of the genus Ehrlichia, have been described from horses in North America.

Table 1.

Ehrlichia spp. Reported from Vertebrate Hosts and Tick Vectors in North America

Ehrlichia spp.	Primary reservoir host	Predominant tick vector	Hosts in which clinical disease develops	References
E. canis	Domestic dogs	Rhipicephalus sanguineus	Dogs	Reviewed in Little 2010
E. chaffeensis	White-tailed deer	Amblyomma americanum	Humans	Lockhart et al. 1996, Dawson et al. 1991
Panola Mountain Ehrlichia sp.	White-tailed deer	Amblyomma americanum	Unknown, goats suspected	Loftis et al. 2006, Yabsley et al. 2008
E. muris–like agent	Unknown, rodents suspected	Ixodes scapularis (not confirmed)	Humans, dogs	Pritt et al. 2011, Hegarty et al. 2012
Ehrlichia sp. of cattle	Unknown	Unknown	Unknown	Gajadhar et al. 2010
E. ewingii	Dogs, white-tailed deer	Amblyomma americanum	Dogs, humans	Anziani et al. 1990, Ewing et al. 1995, Buller et al. 1999, Yabsley et al. 2002

An in-clinic enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. burgdorferi, A. phagocytophilum, and E. canis in dogs is in widespread use in veterinary medicine. This assay has been validated for use in horses to detect antibodies to B. burgdorferi (Johnson et al. 2008) and has been used in equine seroprevalence studies in other areas of the world (Hansen et al. 2010). We used this assay to evaluate over 300 serum samples from horses from Oklahoma, and then further tested a subset of samples for evidence of past or current infection with Ehrlichia spp. using immunofluorescence antibody assay (IFA), species-specific peptide ELISAs, and species-specific and Ehrlichia-wide PCR assays.

Materials and Methods

Equine serum samples (n=240) remaining from routine testing for equine infectious anemia (Coggin tests) were obtained from the Oklahoma Animal Disease and Diagnostic Laboratory (OADDL), Oklahoma State University, Stillwater, Oklahoma. Additionally, whole blood and sera were collected from horses (n=73) with owner-reported tick exposure. Briefly, blood was collected via jugular venipuncture directly into 10-mL collection tubes with no additive and 7-mL collection tubes containing EDTA (Monoject ™ Tyco, Mansfield, MA). All study protocols were approved through the institutional animal care and use committee at Oklahoma State University. Blood collected into tubes with no additive was allowed to clot, and the serum was harvested following centrifugation. Serum was tested immediately using a point-of-care ELISA (described below). Remaining whole blood and serum samples were stored at −20°C until further assayed.

Serum samples from all 313 horses were analyzed for antibodies reactive to Ehrlichia spp., Anaplasma spp., and B. burgdorferi using a commercial ELISA (4DX^® SNAP^®, IDEXX Laboratories, Inc., Westbrook, ME) according to manufacturer's instructions. As previously described, this assay detects antibodies against p30/p30-1 of E. canis, p44/MSP2 of A. phagocytophilum, and the C₆ peptide of B. burgdorferi (O'Connor et al. 2002, 2006). Some strains of E. chaffeensis have homologous proteins to E. canis and therefore cross-reaction can occur (O'Connor et al. 2002, 2006). Similarly, the A. phagocytophilum peptide detects antibodies induced in dogs upon infection with A. platys (unpublished data; SNAP 4Dx Test kit insert; Diniz et al. 2010). The C6 peptide for B. burgdorferi is very specific (Duncan et al. 2004); cross-reactions with other infections are not known to occur.

Serum samples from the horses with known tick exposure (n=73) were also evaluated for antibodies reactive to E. chaffeensis by indirect IFA, as previously described (Dawson et al. 1994, Lockhart et al. 1996) using an initial screening dilution of 1:64 and a 1:50 dilution of fluorescein isothiocyanate (FITC)-labeled goat anti-horse immunoglobulin G (IgG) (Kirkegaard and Perry Laboratories, Gaithersburg, MD) as conjugate. All samples testing positive at 1:64 were then evaluated in serial two-fold dilutions, and titers were reported as the reciprocal of the highest dilution at which specific fluorescence was observed.

A subset (n=16) of serum samples was also analyzed for specific antibodies to E. canis and E. chaffeensis using species-specific peptides in separate microtiter plate-based ELISA format assays (O'Connor et al. 2010, Beall et al. 2012). Synthetic peptides were derived from the E. canis p16 protein and the tandem repeat unit of the E. chaffeensis variable-length PCR target (VLPT) protein. Peptides were coated onto microtiter plates and conjugated to horseradish peroxidase (HRP) as previously described (O'Connor et al. 2010). Test samples and peptide:HRP conjugate were added to peptide-coated microtiter wells and incubated for 30 min. Microtiter wells were aspirated, washed six times, and incubated with a substrate solution containing 3,3′,5,5′-tetramethylbenzidine. Optical density values were determined at 650 nm.

PCR for E. chaffeensis, E. ewingii, and E. canis was performed as previously described (Little et al. 2010) on all of the 73 samples from targeted horses. Total nucleic acid was extracted from EDTA-anticoagulated whole blood using a commercial kit (GFX Genomic Blood Purification, Amersham Pharmacia Biotech, Piscataway, NJ). A fragment of the 16S rRNA gene was amplified in a nested PCR using primers that will detect any Ehrlichia/Anaplasma species in a primary PCR followed by species-targeted primers for secondary PCR (Anderson et al. 1992a, Anderson et al. 1992b, Dawson et al. 1994, Dawson et al. 1996). An additional PCR assay for a gltA gene fragment of an Ehrlichia sp., commonly referred to as the Panola Mountain Ehrlichia (PME), was also performed as previously described (Loftis et al. 2008). Positive controls consisting of previously identified positive dog (E. canis, E. ewingii, E. chaffeensis) or deer (PME) blood samples, and negative (water) controls were included in each PCR assay. A subset (n=20) of samples from horses with high tick infestations and serologic evidence of Ehrlichia spp. infection were submitted to a commercial laboratory for confirmatory real-time PCR assay (IDEXX RealPCR™, IDEXX Laboratories, Sacramento, CA).

Chi-squared analysis with significance assigned at p<0.05 (Cross and Daniel 2013) was used to evaluate the association between presence of antibodies reactive to Ehrlichia spp. and age and gender of horses in a subset of horses (n=190) for which data were available.

Results

The results of all samples evaluated by commercial ELISA and IFA are presented in Table 2. Antibodies reactive to B. burgdorferi and A. phagocytophilum were uncommon and were detected in only three randomly sampled horses, one of which had antibodies reactive to both agents, and four of the horses with a history of tick exposure. However, 8.8% of the randomly sampled horses and more than 20% of the horses with a history of tick infestation had antibodies reactive to Ehrlichia spp. on both commercial ELISA and IFA (Table 2). A subset of these (n=16), nine of which were positive and seven of which were negative to p30/p30-1 on patient-side ELISA, were tested by peptide-specific plate ELISA for antibodies to p16 of E. canis and VLPT of E. chaffeensis; none had antibodies to E. canis p16 and two had antibodies to E. chaffeensis VLPT.

Table 2.

Prevalence of Antibodies Reactive to Ehrlichia spp. in Serum Samples from Random-Source Horses and Those with a History of Tick Exposure

	Assay used to detect antibodies
Sample population	IFA to Ehrlichia chaffeensis	Ehrlichia canis p30/p30-1	Anaplasma phagocytophilum p44/MSP2	Borrelia burgdoferi C6
Random-source from diagnostic laboratory	Not done	21/240 (8.8%)	2/240 (0.8%)	2/240 (0.8%)
Horses with history of tick exposure	18/73 (24.7%)	16/73 (21.9%)	3/73 (4.1%)	1/73 (1.3%)

No agents were amplified by nested PCR of whole blood samples for 16S rRNA (E. chaffeensis, E. ewingii, E. canis) and gltA (PME) from all 73 horses with known tick exposure. Agents were not detected upon additional real-time PCR testing for E. canis, E. ewingii, and E. chaffeensis performed by a commercial laboratory (IDEXX RealPCR™, IDEXX Laboratories, Sacramento, CA) on a subset of horses, all of which had antibodies to Ehrlichia spp. and were heavily infected with ticks.

There was no significant difference in the presence of antibodies between male (16.3% positive) and female (15.3% positive) horses (p>0.05). In the random source sample set, horses with antibodies to Ehrlichia spp. were older (12.9±7.3 years) than horses testing negative (7.1±5.2 years), and horses >10 years of age were more likely to have detectable antibodies to Ehrlichia spp. than horses <10 years of age (p=0.003). However, an association with age with was not seen in horses selected for a history of tick infestations (p>0.05).

Discussion

The present study documents that horses in this area are commonly infected with or exposed to Ehrlichia spp., although these infections may have resolved by the time of testing. The relatively large number of horses testing positive for antibodies to Ehrlichia spp. was unexpected. To our knowledge, no true Ehrlichia sp. has yet been described from horses. Repeated attempts to definitively identify the organism(s) responsible for the antibodies present in these horses have been unsuccessful to date. Although specific antibodies to E. chaffeensis were identified in two of the equine samples, our species-specific antibody results for E. canis and E. chaffeensis failed to implicate either agent as entirely responsible for the findings on the patient-side ELISA. Antibodies to E. ewingii were not detected on the patient-side ELISA used, and so further testing was not conducted (Stillman et al. 2010). However, we cannot exclude the possibility that these horses harbor antibodies reactive to E. ewingii or to the PME agent, the latter of which may be detected on the patient-side ELISA used. Furthermore, known equine rickettsial pathogens in North America, including A. phagocytophilum and N. risticii, both former members of the genus Ehrlichia, do not induce antibodies that react to the p30 protein (Rikihisa 1991) or, in the case of A. phagocytophilum, to the p30 peptide (Stillman et al. 2010).

Similarly, PCR testing for E. canis, E. ewingii, E. chaffeensis, and PME by both conventional, nested assays and commercial real-time assays was negative, as was Ehrlichia-wide PCR. On the basis of rickettsial infection dynamics in other systems, PCR results are most likely to be positive in the early stages of infection, when rickettsemia is greatest (Little et al. 2010, Allison and Little 2013). The infections in the horses in the present study may have resolved prior to collection of samples. However, PCR readily identifies persistent infection in competent hosts for Ehrlichia spp., including white-tailed deer infected with E. chaffeensis and dogs infected with E. ewingii (Lockhart et al. 1996, Little et al. 2010). Exposing naïve horses to wild-caught ticks followed by careful molecular and serologic evaluation may allow identification of the agent(s) responsible for the antibody response seen.

A related study found that antibodies reactive to Ehrlichia spp. were significantly more common in horses with high A. americanum infestations (Duell et al. 2013). Serologic cross-reactivity is common within the genus Ehrlichia, and A. americanum transmits several Ehrlichia spp., including E. chaffeensis, E. ewingii, and a novel Ehrlichia described from Panola Mountain State Park, Georgia, commonly referred to as the PME agent (Anziani et al.1990, Ewing et al. 1995, Yabsley et al. 2008). We were not able to confirm that the antibodies observed in these horses were generated against known Ehrlichia spp. However, specific serologic assays for the PME agent are not available, and other novel, closely related organisms also likely cycle in nature; exposure to the PME agent or a related organism may well be responsible for the antibody reactivity seen. For example, serologic reactivity in dogs in Minnesota and Wisconsin to a p30-based assay was likely due to infection with a novel E. muris–like (EML) agent (Bowman et al. 2009, Hegarty et al. 2012). The EML agent may infect horses as well but is not known to circulate in Oklahoma.

Results of the present study also support the interpretation that exposure to A. phagocytophilum and B. burgdorferi among horses in the south-central United States is uncommon. This finding is consistent with published exposure rates among dogs in the same geographic region (Bowman et al. 2009, Little et al. 2010). Complete travel history was not available for the horses in this study, and it is possible that the few positive horses were infected elsewhere prior to being tested. On the basis of our results, and as is understood in both people and dogs, clinical disease associated with A. phagocytophilum and B. burgdorferi should be considered unlikely in horses in the south-central United States without a history of travel to endemic regions.

Currently, no evidence exists to suggest that clinical disease is associated with exposure to this particular Ehrlichia sp. in horses. Complete blood counts and chemistry screens were performed on a small number (n=6) of seropositive horses and revealed no abnormalities (data not shown). However, documentation of recent infection in naïve horses, while monitoring clinical signs and routine clinicopathologic values, may be warranted to further evaluate the significance of this infection, particularly in light of the fact that the in-clinic assay is often used in horses by veterinarians. The data in the present study indicate that veterinarians in the south-central United States are likely to encounter horses with antibodies to Ehrlichia spp. although the significance of these antibodies for equine health is unclear. We suspect Ehrlichia spp. antibodies would be present in horses throughout much of the eastern United States given the wide distribution of the most likely vector tick, A. americanum (Paddock and Yabsley 2007, Duell et al. 2013), but additional testing of horses from a wider geographic area will be necessary to confirm this supposition.

Footnotes

Acknowledgments

The authors gratefully acknowledge the cooperation of the Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, in providing serum samples from horses and the many participating equine owners and veterinarians who allowed us to collect blood samples from horses.

Author Disclosure Statement

IDEXX Laboratories (Westbrook, ME) donated the 4DX^® SNAP^® assays for use in this study at the request of S.E.L., who has received research support and consulting fees from IDEXX Laboratories in the past 5 years. Two authors (T.O., C.L.) are employees of IDEXX Laboratories. The other authors (R.C., J.D., T.H., and B.H.) have no competing financial interests to disclose.

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