Detection of Anaplasma phagocytophilum in Ixodid Ticks from Equine-Inhabited Sites in the Southeastern United States

Abstract

Anaplasma phagocytophilum is a vector-borne, obligate intracellular bacterium that invades the neutrophils and eosinophils of infected individuals, causing granulocytic anaplasmosis. Equine cases have previously been reported in the United States from California, Florida, and Connecticut, but limited surveillance studies in the Southeast have been conducted. The objective of this study was to determine A. phagocytophilum prevalence in Ixodes scapularis ticks at southeastern U.S. horse-inhabited sites to evaluate the potential risk for equine exposure to A. phagocytophilum-infected ticks in these areas. Samples of I. scapularis were collected from selected barrier islands and Georgia mainland sites where feral and domestic equine populations are present, respectively. Ticks were individually tested for infection by amplification of the A. phagocytophilum ankA gene. The collective prevalence of A. phagocytophilum in I. scapularis ticks was 20% (n=808).

Introduction

C ases of equine granulocytic anaplasmosis in the United States, caused by the rickettsial bacterium Anaplasma phagocytophilum, have predominantly been reported in western states, mainly California (Madigan et al. 1990). Early evidence of granulocytic anaplasmosis in horses from the eastern coast was reported in Florida and New Jersey (Brewer et al 1984; Madigan and Gribble 1987). The first recognized cases in New England were reported as a localized epizootic affecting over 50 horses (Madigan et al. 1996). Since then, A. phagocytophilum seroreactivity and granulocytic cases have been described from Minnesota and Wisconsin (Bullock et al. 2000), Massachusetts (Nolen-Walston et al. 2004), and Virginia (Lewis et al. 2009). Within the Southeast, a single surveillance study estimated the prevalence of the bacterial agent in horse populations in Georgia and Virginia at 9% and 15%, respectively (Magnarelli et al. 2001). Surveillance studies detecting A. phagocytophilum in the Southeast's tick vector, Ixodes scapularis, have also been limited. In 2002, the prevalence of A. phagocytophilum-infected I. scapularis ticks ranged from 0% to 20% at coastal sites in South Carolina, Georgia, and Florida, with an overall prevalence of 1.6% (Fang et al. 2002).

The primary objective of this investigation was to determine the prevalence of A. phagocytophilum in I. scapularis collected at domestic and feral horse habitats in order to evaluate the risk of exposure in horses and other susceptible host populations.

Materials and Methods

The study sites included Cape Lookout National Seashore in North Carolina, Cumberland Island National Seashore in Georgia, and private properties of consenting horse owners in Camden, Glynn, Chatham, and Bulloch counties in Georgia. Cumberland Island was further divided into three areas of the island: northern, central, and southern. Other study areas were not further subdivided.

During the winters of 2004 and 2005, ticks were collected from vegetation using a 1×1-m drag cloth and pooled in the tissue preservative RNAlater (Qiagen, Valencia, CA), in groups of ≤25 ticks. Ticks were individually analyzed for A. phagocytophilum infection. DNA extraction was achieved using a modified version of the Master Pure nucleic acid isolation protocol (Epicenter Biotechnologies, Madison, WI), or a previously described hexadecyltrimethyl-ammonium bromide (CTAB) buffer protocol (Doyle and Doyle, 1990).

A nested polymerase chain reaction was used to amplify the ankA gene of A. phagocytophilum based on previously published protocols using primers AQ2F3 and AQ2R2 for the primary reaction, and AQ2F2 and AQ2R1 for the secondary reaction (Caturegli et al. 2000; Massung et al. 2000; Bjöersdorff et al. 2002). Positive samples were run a second time in order to verify the findings. Those samples that produced identical results were counted as positives (>85%). One randomly chosen positive sample from each site (n=4) was prepared for bidirectional sequencing using the QIAquick PCR purification Kit (Qiagen). Sequencing was performed with an ABI 3100 Automated Sequencer (Clemson University Genomic Institute, Clemson University, Clemson, SC). A BLAST search was administered on GenBank to confirm that the amplified sequences were A. phagocytophilum.

Results

Of the 808 ticks sampled, 158 were counted positive for A. phagocytophilum, with an overall prevalence of 20% (CI=2.76) in the region. Cumberland Island had the highest prevalence of 23% (CI=3.23) (Table 1). Tick populations from mainland sites in Camden, Bulloch, Chatham, and Glynn Counties had prevalences ranging from 17% (CI=15.03) to 0.0%. A summary of the ticks collected from each site can be seen in Table 1.

Table 1.

Prevalence of Anaplasma phagocytophilum in Adult Ixodes scapularis Ticks at Barrier Island and Mainland Study Sites in the Southeastern United States

Collection site	Sex	Sampled	No. infected	% Infected
Cumberland Island, GA	Female	366	74	20.2
	Male	267	67	25.1
	Total	633	141	22
Shackleford Banks, NC	Female	21	0	0
	Male	22	8	36.4
	Total	43	8	19
Bulloch County, GA	Female	22	0	0
	Male	22	1	4.5
	Total	44	1	2
Camden County, GA	Female	26	0	0
	Male	29	4	13.8
	Total	55	4	7
Chatham County, GA	Female	11	1	9.1
	Male	13	3	23.1
	Total	24	4	17
Glynn County, GA	Female	3	0	0
	Male	6	0	0
	Total	9	0	0

A highly significant difference in tick infection rate (α≤0.05) was found between the combined numbers of infected ticks on island versus mainland sites (G=19.768; df=1; p=<0.0001). However, there was no significant difference between the two island sites (G=0.328; df=1; p=0.5670) or the four mainland sites (G=5.861; df=3; p=0.1186). At the Cumberland Island study site, a significant difference was found between the three regions: north (36/222), south (65/223), and central (40/188) (G=10.855; df=2; p=0.0044).

The results of the BLAST search confirmed that the randomly chosen amplicons were A. phagocytophilum. Samples from Chatham County and Cumberland Island had the greatest sequence similarity to human and tick samples from the northeastern United States, at 85% and 96%, respectively. Shackleford Banks and Camden county isolates had 92% sequence homology with northeastern variants, in addition to A. phagocytophilum in horses from California.

Discussion

The presence of A. phagocytophilum-infected ticks collected from five of the six study sites provides additional evidence that the bacteria are not confined to the west and northeast. Additionally, the high prevalence indicates that the southeastern United States is a potential focus for A. phagocytophilum. In comparison to the one previous survey conducted in the Southeast, for which the overall infection rate was 1.6% (Fang et al. 2002), the observed infection rates in the current study are much higher. Thus the potential for A. phagocytophilum transmission in the region may have previously been underestimated.

It can be inferred that the feral and domestic horse populations in and near the collection sites are at risk for exposure, as are any other mammals in the area. The risk of infection for domestic horses may be lower because of the regular care of the horses. Conversely, the high prevalence rate on the barrier islands suggests that feral horses may have greater exposure to infected ticks. The greatest potential for transmission and maintenance of A. phagocytophilum is on Cumberland Island, based on the samples tested in this survey. The herd at Shackleford Banks is already monitored for equine infectious anemia and reproductive control by the responsible department; additional testing could also be performed to determine anaplasmosis prevalence. The horses on Cumberland Island, however, are not monitored. We hope that our findings will persuade local veterinarians and administration officials managing the islands to allow such sampling and investigations to occur. In the current study we detected A. phagocytophilum from I. scapularis ticks, demonstrating that the pathogen exists in this region. This suggests that horses and potentially other hosts in areas with infected ticks are at risk for exposure. Thus surveillance and monitoring of these host populations would be prudent.

Footnotes

Acknowledgments

The authors thank the National Parks Service staff and private property owners for assistance and cooperation with sampling on the barrier islands and mainland sites, respectively. Additionally, we thank Dr. Chris Saski and individuals at the Clemson University Genomics Institute for assisting with the sequencing reactions.

Author Disclosure Statement

No competing financial interests exist.

Acknowledgments

This study was partially funded by a College of Science and Technology Academic Excellence Grant and Graduate Student Professional Development Fund Grant awarded by Georgia Southern University.

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