Veterinary Parasitology 160 (2009) 138–148

Prevalence and geographic distribution of Dirofilaria immitis, Borreliaburgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in theUnited States: Results of a national clinic-based serologic survey

Dwight Bowman a,1, Susan E. Little b,2, Leif Lorentzen c, James Shields c,Michael P. Sullivan c, Ellen P. Carlin a,*a Department of Microbiology and Immunology, Cornell College of Veterinary Medicine, C4-119 Veterinary Medical Center, Ithaca, NY 14853, United Statesb Department of Veterinary Pathobiology, 250 McElroy Hall, Center for Veterinary Health Sciences, Oklahoma State University,

Stillwater, OK 74078-2007, United Statesc IDEXX Laboratories, One IDEXX Drive, Westbrook, ME 04092, United States

A R T I C L E I N F O

Article history:

Received 25 March 2008

Received in revised form 22 September 2008

Accepted 2 October 2008

Keywords:

Anaplasma

Anaplasmosis

Antigen test

Borrelia burgdorferi

Canine

Dirofilaria immitis

Dirofilariasis

Ehrlichia

Ehrlichiosis

ELISA

Heartworm

Lyme borreliosis

A B S T R A C T

We evaluated a comprehensive national database that documents canine infection with, or

exposure to, four vector-borne disease agents, Dirofilaria immitis, Borrelia burgdorferi,

Ehrlichia canis, and Anaplasma phagocytophilum in order to assess geographic trends in rates

of positive tests. While the percent positive test results varied by agent in different regions

of the United States, with D. immitis antigen and antibodies to E. canis more commonly

identified in dogs from the South (3.9% and 1.3%, respectively), and antibody to B.

burgdorferi and A. phagocytophilum found more frequently in dogs from the upper Midwest

and Northeast (4.0–6.7% and 5.5–11.6%, respectively), evidence of at least one agent was

found in dogs from every state considered. Furthermore, each organism also appeared to

occur in endemic foci within larger areas of relatively low prevalence. Relocation of

infected or previously exposed dogs from endemic regions likely accounts for some of the

unexpected geographic distribution seen, although local transmission in previously under-

recognized areas of endemicity could also be occurring. Although data were only available

from the 48 contiguous states (Alaska and Hawaii were not included), taken together, our

results suggest that these disease agents may be present over a wider geographic area, and

thus pose greater animal and public health risks, than is currently recognized. Dogs can

serve as sentinels to identify the presence of vector-borne disease agents of both veterinary

and public health significance.

Published by Elsevier B.V.

Contents lists available at ScienceDirect

Veterinary Parasitology

journa l homepage: www.e lsevier .com/ locate /vetpar

1. Introduction

Concern over vector-borne disease in domestic dogs isevidenced by the common use of tick, mosquito, and

* Corresponding author. Tel.: +1 202 249 1275.

E-mail addresses: ddb3@cornell.edu (D. Bowman),

susan.little@okstate.edu (S.E. Little), carlinvet@gmail.com (E.P. Carlin).1 Tel.: +1 607 253 3406; fax: +1 607 253 4077.2 Tel.: +1 405 744 8523; fax: +1 405 744 5275.

0304-4017/$ – see front matter . Published by Elsevier B.V.

doi:10.1016/j.vetpar.2008.10.093

heartworm preventatives in small animal practice; justover half of all pet owners report administering parasitecontrol products to the pets in their care (APPMA NationalPet Owners Survey, 2003–2004). The vector-borne caninedisease agents of most common concern to the U.S.veterinary community are Dirofilaria immitis, Borrelia

burgdorferi, Ehrlichia spp., and Anaplasma spp. Whileinfection with these agents may be prevented to someextent through vector avoidance or other control mea-sures, morbidity and mortality due to these diseasescontinue to occur in domestic dogs. Indeed, the use of

D. Bowman et al. / Veterinary Parasitology 160 (2009) 138–148 139

acaricides and insecticides alone is an ineffective means ofbreaking the enzootic transmission cycles of these patho-gens. As the roles that these agents play in animal andhuman health have become elucidated over the last fewdecades, the need for further data on the natural historyand prevalence of these infections has become apparent.

Heartworm, a disease of canids caused by infection withthe nematode D. immitis, is perhaps the most importanthelminthic disease of dogs in North America. Microfilariaecirculate in the blood, where they may be ingested by afeeding mosquito. Development into infective third-stagelarvae occurs in the malpighian tubules of the vector, andthe parasites can then infect a new host (or reinfect thesame host) upon subsequent feeding by the mosquito.Ultimately, the immature heartworms migrate to the dog’spulmonary arteries, and within 6–9 months begin produ-cing microfilariae. It is this presence of the adults in thepulmonary arteries that causes right heart and/or pul-monary disease. Although dogs are the natural hosts,infection can also occur in coyotes and ferrets. Infections incats and people often undergo truncated development, butthese infections are sometimes associated with pathogenicmanifestation, sometimes severe (Bowman, 2003; Theis,2005).

Lyme borreliosis is a bacterial disease caused byinfection with the spirochete B. burgdorferi; in dogs,disease is commonly characterized by lameness, fever,anorexia, lethargy, and lymphadenopathy (Kahn, 2005).The most important vector in the eastern U.S. is Ixodes

scapularis, commonly known as the black-legged tick ordeer tick. On the West Coast, I. pacificus serves as the mainvector for B. burgdorferi. The Ixodes spp. involved in B.

burgdorferi transmission are three-host ticks that acquirespirochetes when feeding on rodents as larvae or nymphs,and then can transmit infection as nymphs or adults. Themost important reservoir host is thought to be the white-footed mouse (Peromyscus leucopus), although otherrodents may also serve as a source of spirochetes to infectticks (Schmidt and Ostfeld, 2001; LoGiudice et al., 2003;Brisson et al., 2008). Mean infection rates in I. scapularis

nymphs in endemic areas ranges from about 20% to 40%(Daniels et al., 1998; Tsao et al., 2004; Wang et al., 2003).Infection of the nymphal western vector, I. pacificus, islower, in the range of 0–14% (Eisen et al., 2004).

Lyme disease is the most common tick-borne infectionamong people in North America and Europe (Wormseret al., 2006), with approximately 20,000 cases reported inthe United States each year (CDC, 2007a). Cases in thesouthern U.S., however, are notably low (CDC, 2007a).Infection of I. scapularis nymphs with B. burgdorferi has notbeen documented in the South (Wormser et al., 2006), andinfection rates in adult I. scapularis are much lower thanthose in the northeastern U.S., at 1.4–4.6% (Clark, 2004;Oliver et al., 2000). Confirmed human infection with B.

burgdorferi is considered rare, if it occurs at all, in statessouth of Maryland and Virginia (Wormser et al., 2006;Dennis, 2005). Disease is characterized in people by anearly set of skin-related and flu-like symptoms, and, in theabsence of treatment, may be followed by arthritic orneurologic complications (Wormser et al., 2006; Steereet al., 2004).

The rickettsial organisms Ehrlichia canis and Anaplasma

phagocytophilum (the latter formerly known as E. phago-

cytophila and E. equi) are both tick-borne obligateintracellular bacteria with a tropism for leukocytes(Rikihisa, 1991). Disease caused by infection with thesepathogens is typically characterized by fever, depression,myalgia, anorexia, and thrombocytopenia. Domestic andwild dogs are the natural hosts of E. canis, which has aworldwide distribution. The primary means of transmis-sion is through the bite of Rhipicephalus sanguineus, thebrown dog tick, although Dermacentor variabilis, theAmerican dog tick, has also been shown to be a capablevector (Groves et al., 1975; Lewis et al., 1977; Johnsonet al., 1998). The host range of A. phagocytophilum issignificantly wider; rodents are considered the primaryreservoir host, but infections also occur in dogs, sheep,cows, horses, and various species of wildlife (Rikihisa,1991; Bown et al., 2003). Transmitted by I. scapularis in theNortheast and upper Midwest, and I. pacificus in theWestern states, A. phagocytophilum is also responsible forhuman granulocytic anaplasmosis (HGA, formerly humangranulocytic ehrlichiosis) (Rikihisa, 2006). Because A.

phagocytophilum shares a vector and reservoir hostsystem with B. burgdorferi, the geographic distributionof cases of HGA parallels that of Lyme borreliosis, and co-infections with the two agents may be seen (Daniels et al.,1998).

Evidence of infection with or exposure to the causativeagents of all four of these diseases can be tested for via asingle-use, in-house diagnostic known as the SNAP1 4Dx1

Test (IDEXX Laboratories, Westbrook, ME). Many veter-inarians are already familiar with this approach as theSNAP1 3Dx1 and SNAP1 4Dx1 test are widely usedthroughout the United States as an annual heartworm plustick-borne disease screening tool. A portion of these resultsare captured through a central reporting system, which hasassembled data from tests performed on several milliondogs since 2001. Access to this comprehensive datasetprovided an excellent opportunity to assess prevalenceand distribution of these four organisms in well-cared fordogs throughout the United States. These results were thencompared with previous assessments of geographic rangeand prevalence. In addition, because multiple agents weretested for simultaneously, the frequency of co-infectionwas evaluated.

2. Materials and methods

2.1. Source of data

The SNAP1 3Dx1 test (IDEXX Laboratories, Westbrook,ME) is an in-clinic ELISA for simultaneous qualitativedetection of canine antibodies to E. canis and B. burgdorferi,and to D. immitis antigen, in canine serum, plasma, orwhole blood. In 2001 the test became available forcommercial use as a replacement option for in-clinic‘‘heartworm only’’ screening protocols. Starting in 2001,IDEXX began offering practice rebates toward the cost ofthe SNAP1 3Dx1 assay in exchange for practices submit-ting a log of all test results. The offer was extended toveterinary practices across the United States; therefore,

D. Bowman et al. / Veterinary Parasitology 160 (2009) 138–148140

the clinic geographic distribution was randomly selected.These practices agreed to use the SNAP1 3Dx1 assayexclusively for all regular canine heartworm screenings,and committed to providing the screening data bycompiling and submitting the data set on an IDEXX-provided data sheet. For the years evaluated, all pathogenswere reported, except for the years 2002 and 2003 whenthe data collection forms did not include D. immitis. Clinicenrollments began in September of each year and typicallycontinued through spring; clinics were required to havetheir data collection forms returned to IDEXX by the end ofAugust each year in order to process the data and the clinicrebates. On average, clinics supplied 120 datapoints(unique test results from individual dogs); in some cases,in excess of 1500 datapoints per practice were supplied.Results were only reported a single time from each dog (norepeat test results reported).

In September of 2006, IDEXX introduced the SNAP1

4Dx1 Test, which included the same analytes as theSNAP1 3Dx1, as well as a fourth analyte for the detectionof A. phagocytophilum antibody. Similar to the studydesign described previously with the SNAP1 3Dx1,customers adopting the use of the SNAP1 4Dx1 wereoffered rebates in exchange for submitting test results. Forthe years 2006 and 2007, therefore, the data set isprimarily comprised of results generated from the SNAP1

4Dx1 assay.Because dogs in the western U.S. are often not

routinely tested by veterinarians in clinic for heartwormantigen or tick-borne disease exposure, additional datapoints were needed for this region. To increase thedistribution and number of data points for the D. immitis

national database, data were retrospectively included forthe years 2001 to 2006 from the IDEXX ReferenceLaboratories network (IDEXX Laboratories, Inc., West-brook, Maine) and added to the data set generated byclinics using the SNAP assays. Reference laboratoryevaluations were performed on the IDEXX PetChek1

microtiter plate that uses antibodies specific to heart-worm antigen. Canine heartworm reference laboratoryresults were analyzed for duplicate test results in a givenyear, and subsequent results for an individual dog withinthe year were excluded from the analysis. This approachincreased the number of results evaluated from dogsfrom western states for heartworm, but not tick-bornedisease agents.

2.2. Heartworm assay

The D. immitis analyte for both the SNAP assays as wellas the microtiter plate is derived from antibodies specificto the heartworm antigen. Test sensitivity in necropsy-categorized samples was 84% (175/208) and ranged from64% to 93% in 1 and 4 worm burden infections, respectively(Atkins, 2003). Test specificity was 97% (30/31) in the samestudy.

2.3. B. burgdorferi assays

The SNAP B. burgdorferi analyte detects antibodiesspecific to the C6 peptide. Test sensitivity was 94.4% (238/

252) when compared to a combination of immunofluor-escence assay (IFA) and Western blot (WB) (O’Connor et al.,2004). (The C6 analyte has been shown, in both humansand canines, not to react to antibodies elicited following B.

burgdorferi vaccination (O’Connor et al., 2004; Marqueset al., 2002).) SNAP specificity was 99.5% when used onfield samples from 987 dogs in North Carolina (Duncanet al., 2004).

2.4. Ehrlichia assay

The SNAP Ehrlichia analyte detects antibody generatedagainst peptides from the p30 and p30-1 proteins. Thesensitivity of the analyte was 95.7% (134/140) whencompared to IFA and/or WB (O’Connor et al., 2002). Testspecificity was 100% as compared to IFA and WB in twoseparate investigations (O’Connor et al., 2004, 2006). Itshould be noted that some strains of E. chaffeensis havehomologous proteins to those of E. canis and as a result, someE. chaffeensis infections will induce cross-reacting antibo-dies on the SNAP E. canis peptide (O’Connor et al., 2004).

2.5. Anaplasma assay

The A. phagocytophilum analyte detects antibodygenerated against a synthetic peptide from the majorsurface protein (p44/MSP2). In a subset population ofsamples, SNAP1 4Dx1 sensitivity and specificity were99.1% and 100%, respectively, relative to the IFA (Chan-drashekar et al., 2007). Preliminary studies indicate thatthe A. phagocytophilum analyte in the SNAP1 4Dx1 cross-reacts with samples from Anaplasma platys infected dogs(unpublished data, source: SNAP1 4Dx1 Test kit insert). Inareas where the Ixodes tick vector is less prevalent orabsent, a positive Anaplasma result could be the result of A.

platys exposure.

2.6. Data analyses

2.6.1. Regional distribution and percent positive test

calculations

Data were collated by county of residence of each dogtested according to postal zip code provided with eachrecord, and then assembled into state and regional groupsas previously described (Blagburn et al., 1996). For ease ofpresentation, only four regional groups (Midwest, North-east, Southeast, and West) were considered (Blagburnet al., 1996). Percent positive test results were calculatedby dividing the number of dogs reported positive for eachagent by the total number of dogs tested.

2.6.2. Population growth

Population growth was analyzed using actual countylevel population data from the U.S. Bureau of the Census.Two sources of data were used for population andmigration statistics. The 2000 Census long form askedrespondents to report the county where they lived in 1995.The data are compiled in the Census ‘‘County to CountyMigration Flow Files.’’ Migration numbers can be mea-sured as a percentage of 1 April 2000 county population fora relative penetration number of migration into a county.

D. Bowman et al. / Veterinary Parasitology 160 (2009) 138–148 141

Population growth data are from 1 April 2000 to 1 July2006 and are compiled from the U.S. Census ‘‘PopulationEstimates Program.’’ This program publishes county levelpopulation estimates between censuses.

2.6.3. Statistical analyses

Differences in the frequency of reported positive testresults between counties, states, and regions were evaluatedfor significance with a t-test using SAS (Windows 9.1) (SASInstitute Inc., Cary, NC) with significance assigned atp < 0.0001.

3. Results

The number of practices that submitted samples totaled2573, consisting of 5,626,926 datapoints from over 3million dogs from all 48 contiguous states. Results ofheartworm testing were available from 3,182,614 dogs:1,039,295 from the Midwest, 707,875 from the Northeast,515,157 from the Southeast, and 920,287 from the West.Results of B. burgdorferi and E. canis testing were availablefrom 982,336 of these dogs: 373,090 from the Midwest,271,070 from the Northeast, 290,636 from the Southeast,and 47,540 from the West. Results of A. phagocytophilum

testing were available from 479,640 of these dogs: 175,829from the Midwest, 188,438 from the Northeast, 101,148from the Southeast, and 14,225 from the West. Evidence ofat least one agent was found in dogs from every stateconsidered (Table 1).

3.1. Heartworm infection

The highest percentage of D. immitis antigen-positivesamples was obtained from the Southeast, at 3.9% (Table 1and Fig. 1). The positive samples tended to occur in clustersof endemic foci, surrounded by areas of relatively lowprevalence (Fig. 1). The prevalence map demonstrates thehighest percent positive rates manifesting in individualcounties, mostly in the Southeast, as well as clusters innorthern California, where more than 9% of dogs tested werepositive in Amador, Lake, Trinity, and Tehema County.

3.2. Lyme borreliosis

Positive titers to B. burgdorferi were most common inthe Northeast, with 11.6% of all samples from this regionreported as positive. In contrast, of the samples that camefrom the Southeast, only 1.0% were positive, the majority ofwhich were from Virginia. Like heartworm, the Lyme-positive samples were often clustered in apparenthyperendemic foci. The prevalence map demonstratesthe most prominent clusters in the coastal Northeast andupper Midwest (Fig. 2), where individual areas/countiesrecorded percent positive results as high as 44.1% (PutnamCounty, NY), 43.0% (Washburn County, WI), and 60.9%(Todd County, Minnesota).

3.3. Ehrlichiosis

Antibodies to Ehrlichia were detected most often in dogsin the Southeast, where 1.3% of samples were reported as

positive (Table 1 and Fig. 3). All other regions weresignificantly below that rate and ranged from 0.3% to 0.6%.Again, endemic foci were seen where local percent positivetest results were more than 10-fold greater than that forthe state or region as a whole.

3.4. Anaplasmosis

The highest prevalence of samples with antibodies to A.

phagocytophilum were reported from the Midwest (6.7%);samples from the Northeast also were frequently found tobe positive (5.5%; Table 1 and Fig. 4). In some individualcounties, such as Washburn County, Wisconsin, NantucketCounty, Massachusetts, Todd County, Minnesota, andLitchfield County, Connecticut, more than 50% of dogswere reported as positive for A. phagocytophilum. Dogstesting positive for both Anaplasma and B. burgdorferi wereseen most commonly in the Midwest (2.0%), followed bythe Northeast (1.4%; Table 1). In one county (Todd County,Minnesota), 40.8% of the dogs were reported to haveantibodies to both agents.

4. Discussion

Using a comprehensive data set we were able todetermine prevalence values of four major vector-bornedisease agents in dogs presenting to veterinary hospitals inthe continental U.S. As expected, heartworm and Ehrlichia-positive samples were more commonly reported in theSouth, and B. burgdorferi- and Anaplasma-positive testswere more frequently found in the Northeast, upperMidwest, and in northern California (Table 1 and Figs. 1–4).However, evidence of current or previous infection with atleast one agent was found in all regions of the U.S. and inevery state considered (Table 1 and Figs. 1–4).

For heartworm, it was not surprising that infectionrates were highest overall in the southern states, as thisarea is well-known by veterinarians to be beset by clinicalheartworm disease. Indeed, the map of percent positivetests for D. immitis generated from our data (Fig. 1)corresponds well with the map of reported cases compiledby the American Heartworm Society (Guerrero et al.,2006), including having the highest prevalence of infec-tions in the coastal states of the southeastern U.S. (SC 5.7%,GA 2.7%, AL 3.4%, MS 7.4%, LA 6.0%, and TX 5.5%) and arelatively low prevalence of infection in pet dogs in Florida(1.8%), where veterinarians and clients may be particularlyconscientious about administering preventatives year-round. Also, as with the AHS map, there is a similarindication that the infection is more common along theMississippi River (AR 6.8%, MO 2.0%, TN 3.6%). In addition,both our analysis and the AHS map show that heartworm ispresent over a wider area than generally acknowledged;positive dogs were commonly reported from areastraditionally considered by many to be largely free ofheartworm, including northern, central, and southernCalifornia (Table 1 and Fig. 1). The results from thewestern states, particularly California and Arizona, may besomewhat unexpected. However, D. immitis infectionshave been documented in these states (Bowman et al.,2007; Corselli and Platzer, 1982; Pensinger, 1986).

Table 1

Percent positive test results (number positive/number tested) in dogs by region and state for antigen of Dirofilaria immitis and antibody to Borrelia

burgdorferi, Anaplasma phagocytophilum, Ehrlichia canis, and co-infection with B. burgdorferi and A. phagocytophilum.

State D. immitis B. burgdorferi Ehrlichia Anaplasma Borrelia & Anaplasma

co-infection

Northeast

CT 0.6% (236/37,650) 18.1% (1,846/10,209) 0.2% (21/10,209) 21.8% (1,499/6,887) 4.6% (317/6,887)

DE 1.6% (79/4,986) 11.2% (516/4,595) 1.0% (48/4,595) 1.1% (48/4,315) 0.4% (17/4,315)

MA 0.7% (1,657/252,281) 19.8% (6,729/33,915) 0.3% (107/33,915) 10.4% (2,803/26,911) 3.0% (811/26,911)

MD 0.8% (221/28,770) 12.6% (2,882/22,945) 0.7% (165/22,945) 1.7% (282/16,307) 0.4% (63/16,307)

ME 0.6% (173/27,247) 11.6% (3,269/28,230) 0.1% (39/28,230) 5.4% (1,341/24,632) 1.1% (271/24,632)

NH 0.8% (168/21,056) 12.9% (2,343/18,122) 0.2% (36/18,122) 4.5% (618/13,743) 1.3% (173/13,743)

NJ 0.3% (384/111,245) 14.2% (2,913/20,575) 0.4% (89/20,575) 9.8% (1,339/13,721) 3.2% (435/13,721)

NY 0.5% (780/158,926) 7.1% (5,781/81,305) 0.2% (179/81,305) 3.6% (1,741/48,201) 0.9% (437/48,201)

PA 0.4% (191/45,815) 9.4% (3,869/40,948) 0.2% (80/40,948) 1.6% (449/27,641) 0.6% (155/27,641)

RI 0.8% (123/16,199) 14.3% (933/6,508) 0.1% (6/6,508) 4.7% (158/3,396) 0.7% (24/3,396)

VT 0.7% (24/3,682) 9.9% (368/3,718) 0.2% (7/3,718) 1.7% (46/2,684) 0.5% (14/2,684)

Regional mean 0.6% (4,036/707,857) 11.6% (31,449/271,070) 0.3% (777/271,070) 5.5% (10,324/188,438) 1.4% (2,717/188,438)

Midwest

IA 0.9% (164/19,097) 0.9% (149/17,390) 0.4% (61/17,390) 0.4% (21/4,840) 0.1% (5/4,840)

IL 0.9% (2,915/337,434) 1.0% (324/31,976) 0.4% (135/31,976) 0.4% (51/11,899) 0.1% (6/11,899)

IN 1.8% (428/24,290) 1.1% (231/20,515) 0.3% (54/20,515) 0.4% (26/7,084) 0.2% (11/7,084)

KS 2.7% (170/6,264) 0.1% (6/5,473) 2.2% (119/5,473) 0.5% (7/1,452) 0.0% (0/1,452)

MI 0.7% (2,031/292,171) 0.6% (431/67,625) 0.1% (34/67,625) 1.2% (190/16,312) 0.4% (68/16,312)

MN 0.4% (332/80,810) 9.5% (7,267/76,610) 0.3% (202/76,610) 9.8% (6,002/61,374) 3.1% (1,928/61,374)

MO 2.0% (457/22,673) 0.2% (59/24,095) 1.9% (462/24,095) 0.3% (14/5,250) 0.0% (0/5,250)

ND 0.5% (25/4,914) 3.0% (136/4,558) 0.0% (1/4,558) 2.4% (40/1,692) 1.2% (21/1,692)

NE 0.8% (34/4,387) 0.1% (5/4,282) 0.3% (13/4,282) – –

OH 0.9% (1,242/136,548) 0.2% (140/61,138) 0.1% (79/61,138) 0.1% (13/14,414) 0.0% (0/14,414)

SD 0.1% (1/962) 0.3% (1/358) 0.0% (0/358) - -

WI 0.6% (616/109,745) 10.2% (6,018/59,070) 0.3% (194/59,070) 10.5% (5,409/51,512) 2.9% (1,510/51,512)

Regional mean 0.8% (8,415/1,039,295) 4.0% (14,767/373,090) 0.4% (1,354/373,090) 6.7% (11,773/175,829) 2.0% (3,550/175,829)

Southeast

AL 3.4% (622/18,388) 0.1% (27/18,998) 0.3% (64/18,998) 0.1% (4/4,331) 0.0% (0/4,331)

AR 6.8% (578/8,526) 0.1% (7/8,391) 3.9% (324/8,391) 0.6% (10/1,743) 0.0% (0/1,743)

FL 1.8% (1,408/80,280) 0.5% (256/54,982) 0.8% (425/54,982) 0.5% (166/31,690) 0.1% (25/31,690)

GA 2.7% (1,373/51,494) 0.3% (77/23,333) 1.9% (444/23,333) 0.2% (15/8,856) 0.0% (2/8,856)

KY 1.1% (227/20,092) 0.2% (45/18,935) 0.8% (152/18,935) 0.1% (5/4,319) 0.0% (0/4,319)

LA 6.0% (871/14,468) 0.1% (9/11,197) 0.2% (18/11,197) 0.1% (1/707) 0.0% (0/707)

MS 7.4% (183/2,459) 0.0% (1/2,198) 3.1% (68/2,198) 0.0% (0/300) 0.0% (0/300)

NC 3.0% (663/22,005) 1.3% (263/20,783) 2.1% (431/20,783) 0.4% (25/6,841) 0.1% (8/6,841)

OK 2.1% (254/11,913) 0.2% (19/11,549) 3.8% (439/11,549) 1.2% (70/5,920) 0.1% (4/5,920)

SC 5.7% (860/15,019) 1.3% (148/11,562) 0.8% (95/11,562) 0.1% (9/6,507) 0.0% (1/6,507)

TN 3.6% (498/13,787) 0.2% (47/18,891) 2.3% (428/18,891) 0.1% (4/4,324) 0.0% (1/4,324)

TX 5.5% (12,160/220,829) 0.2% (91/58,088) 0.8% (441/58,088) 0.6% (90/14,788) 0.1% (16/14,788)

VA 1.1% (331/29,766) 6.7% (1,924/28,787) 1.8% (532/28,787) 0.9% (96/10,195) 0.3% (27/10,195)

WV 0.8% (51/6,131) 0.3% (9/2,942) 0.1% (4/2,942) 0.2% (1/627) 0.0% (0/627)

Regional mean 3.9% (20,079/515,157) 1.0% (2,923/290,636) 1.3% (3,865/290,636) 0.5% (496/101,148) 0.1% (84/101,148)

West

AZ 1.2% (620/53,809) 0.4% (4/992) 3.2% (32/992) 0.7% (4/583) 0.2% (1/583)

CA 1.6% (8,478/530,788) 1.8% (540/29,454) 0.8% (225/29,454) 4.8% (612/12,673) 0.9% (112/12,673)

CO 0.4% (1,028/261,358) 0.4% (49/11,557) 0.2% (19/11,557) 0.0% (0/86) 0.0% (0/86)

ID 0.6% (32/5,748) 0.3% (1/369) 0.0% (0/369) 0.7% (2/298) 0.0% (0/298)

MT 0.6% (16/2,801) – – – –

NM 1.8% (427/23,429) 0.3% (7/2,060) 1.0% (21/2,060) 0.3% (1/289) 0.0% (0/289)

NV 1.2% (74/6,180) – – – –

OR 0.8% (235/29,176) 2.8% (77/2,798) 0.1% (2/2,798) 7.4% (22/296) 0.7% (2/296)

UT 0.6% (11/1,904) 0.0% (0/93) 0.0% (0/93) – –

WA 1.0% (39/4,099) 0.0% (0/33) 0.0% (0/33) – –

WY 1.2% (10/700) 0.0% (0/184) 0.0% (0/184) – –

Regional mean 1.2% (10,970/919,992) 1.4% (678/47,540) 0.6% (299/47,540) 4.5% (641/14,225) 0.8% (115/14,225)

Overall mean 1.4% (43,500/3,182,301) 5.1% (49,817/982,336) 0.6% (6,295/982,336) 4.8% (23,234/479,640) 1.3% (6,466/479,640)

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Endemic transmission is known to occur and is routinelyreported in the foothills of the Coast and Sierra Nevadamountain ranges (Roy et al., 1993). In addition, competentmosquito vectors are found along the lower Colorado River(Corselli and Platzer, 1982). Interestingly, it is in some of

the California counties with high prevalence rates (i.e.,Nevada, Placer, Riverside, and Shasta) in which auto-chthonous cases of human heartworm infections inCalifornia have been reported (Theis et al., 2001). PlacerCounty (Auburn, CA) was found in a fairly recent national

Fig. 1. Evidence of antigen to Dirofilaria immitis in dogs by county, grouped according to percent positive tests. No results (<10) were received from counties

shaded gray, precluding interpretation of the presence of the parasite in these areas. Counties depicted in white had no dogs reported as positive (0%).

Remaining counties were coded as follows: 0.1–2.0% (taupe), 2.1–4.0% (salmon), 4.1–6.0% (red), 6.1–20.0% (brick red). Note: perception of a low heartworm

prevalence in this area leads to lower levels of testing; anecdotally, the SNAP test is not used a lot in the West. Inclusion of laboratory data to create a more

robust dataset may have contributed to this somewhat unexpected appearance.

Fig. 2. Evidence of antibodies to Borrelia burgdorferi in dogs by county, grouped according to percent positive tests. No results (<10) were received from

counties shaded gray, precluding interpretation of the presence of antibodies in dogs from these areas. Counties depicted in white had no dogs reported as

positive (0%). Remaining counties were coded as follows: 0.01–0.49% (pale blue), 0.5–1.0% (eucalyptus), 1.1–5.0% (bright blue), 5.1–40% (slate blue).

D. Bowman et al. / Veterinary Parasitology 160 (2009) 138–148 143

Fig. 3. Evidence of antibodies to Ehrlichia canis in dogs by county, grouped according to percent positive tests. No results (<10) were received from counties

shaded gray, precluding interpretation of the presence of antibodies in dogs from these areas. Counties depicted in white had no dogs reported as positive

(0%). Remaining counties were coded as follows: 0.01–0.49% (pale lavender), 0.5–1.0% (lavender), 1.1–2.0% (magenta), 2.1–15.0% (intense purple).

Fig. 4. Evidence of antibodies to Anaplasma phagocytophilum in dogs by county, grouped according to percent positive tests. No results (<10) were received from

counties shaded gray, precluding interpretation of the presence of antibodies in dogs from these areas. Counties depicted in white had no dogs reported as

positive (0%). Remaining counties were coded as follows: 0.01–0.49% (pale green), 0.5–1.0% (lichen), 1.1–5.0% (kelly green), 5.1–40% (dark moss).

D. Bowman et al. / Veterinary Parasitology 160 (2009) 138–148144

D. Bowman et al. / Veterinary Parasitology 160 (2009) 138–148 145

survey to have the highest prevalence of heartwormantibody-positive cats (33% of 110 of 2190 total sampledcats from 18 states and the District of Columbia) (Milleret al., 2000). In general, a comparison of the map presentedby Theis (2005) (human heartworm diagnosis as well asthe number of canine cases presented by the AHS in 2001)with the map shown here (or that of the AHS 2005) seemshighly suggestive that human infections tend to occur inareas of high canine prevalence. These include the east andsoutheastern coastal states, the midwestern states, andCalifornia where heartworms are highly endemic in thedomestic dog and coyote population (Sacks et al., 2004).

Moreover, when prevalence of infection was consideredon a county level, we found a pattern of apparent endemicfoci of heartworm infections within areas of relativelylower prevalence (Fig. 1). For example, in California,Trinity, Amador, Tehema, and Lake Counties, had dogsreported positive for heartworm (9.1%, 9.4%, 10.1%, and12.5%, respectively) at rates significantly higher than fordogs in the rest of the state (1.6%, p < 0.0001) and thewestern region overall (1.2%, p < 0.0001). This region ofCalifornia has experienced higher than average populationgrowth in recent years. The ‘‘Population EstimatesProgram’’ shows an average growth of these counties at11.3% compared to 7.6% between April, 2000 and July,2006. Positive dogs in these counties may thus representcases of heartworm imported into the region from areasconsidered ‘‘more’’ endemic. In addition, veterinariansmay be more likely to test dogs that have a history of priorresidence in a heartworm-endemic area. However, mos-quitoes capable of transmitting D. immitis are present inthese counties and throughout much of the western region(Corselli and Platzer, 1982; Walters and Lavoipierre, 1982),and it has been well documented in California that naturaltransmission occurs in coyotes in much of the state (Sackset al., 2004). The continued importation of infected dogs,the capable vectors in the region, and the high levels oftransmission occurring in coyotes in California underscorethe need for regular testing and preventive use.

With any test, there is a greater chance of a falsepositive when the population is almost certain to be allnegative (Peregrine, 2005; Peregrine et al., 2007). Thus, in astudy such as this one, where serological tests are beingperformed on a large number of animals in many areaswith a low prevalence, the positive predictive value [PV+]of a test, i.e., the probability that a positive test result iscorrect, must be considered. If calculated for this studyusing the sensitivity and specificity for heartworm given inthe materials and methods, then with a 1% prevalence (thecase in much of the nation), the PV+ is 22.05%, the test islikely to be correct 1 in 5 times; at 3.9% prevalence (thenumbers seen in Florida), the PV+ would be 53.25%, so thetest is likely to be correct about half of the time; and at 6%(the prevalence in some of the counties in California), thePV+ is 64.12% or correct about two-thirds of the time.Therefore, it is important to retest dogs with positiveresults with a second test before treatment with anarsenical. Using the sensitivity (76%) and specificity (97%)calculated for the IDEXX in-house PetChek PF as calculatedby Courtney and Zeng (2001) for dogs with low wormburdens of 1–10 worms (mean 2.3, median 3), the PV+

markedly increases when the results are validated usingthis as the second test. For a prevalence of 1% the PV+improves to 87.67%; for a prevalence of 3.9% it improves to96.51%, and at 6% it improves to 97.95%. Thus, when dogsinitially test positive and appear clinically normal, theyshould be rechecked using this or a similar test forverification of infection status before treatment. Also, theremay be occasions in areas of low prevalence when theclinical status of normal will take precedence over the twotests in series, because even they can be incorrect at a 1%prevalence about 15% of the time. Also, even in levels ofhigh prevalence, it is possible that the diagnosis of anastute clinician will outweigh even the two positivesobtained using these tests, as the tests are not infallible.

As expected, dogs positive for antibodies to B.

burgdorferi were most commonly reported from areaswhere Lyme borreliosis is known to be endemic orhyperendemic, including the Northeast, upper Midwest,and West Coast (Table 1 and Fig. 2). Prevalence ofantibodies in dogs from the northeastern U.S. averaged11.6%, with some hyperendemic localities identified in theNortheast and upper Midwest where rates of positive testsin dogs were greater than 40% (Fig. 2). We also foundconfirmatory evidence of exposure of dogs to the agent ofLyme disease in recognized endemic areas in California(Nevada, Humboldt, and Mendocino County, California:7.7%, 9.2%, and 9.3%, respectively). However, exposure ofdogs to B. burgdorferi was documented over a wider areathan expected, particularly with regard to the southernU.S.

The 2005 national map of reported human Lymeborreliosis cases by county is far sparser in this region(CDC, 2007a), and Lyme disease is considered rare, if itoccurs at all, in states south of Maryland or Virginia(Wormser et al., 2006). Indeed, B. burgdorferi-specificantibodies were reported from only 1% of dogs from theSouth (Table 1), and even this low percent positive ratemay be somewhat inflated by the inclusion of Virginia, astate where Lyme borreliosis is known to occur, into thesoutheastern region (CDC, 2007a). The positive rate forVirginia was 6.7%, whereas that for the rest of thesoutheastern region, omitting the dogs reported fromVirginia, is only 0.36%.

As with the distribution of D. immitis, dogs withantibodies to B. burgdorferi appeared to be reported fromendemic foci within larger areas of relatively lowendemnicity (Fig. 2). For example, in Montgomery County,Texas and Indian River County, Florida, 1.9% and 1.5% ofdogs, respectively, were positive for B. burgdorferi, rateswhich were significantly higher than the prevalence in therest of each state as a whole (TX prevalence 0.2%,p < 0.0001; FL prevalence 0.5%, p < 0.0001) and signifi-cantly higher than that in all southern border statescombined (0.3%, p < 0.0001). As we saw with D. immitis

distribution in unexpected areas, both of these countieshave experienced large increases in population in recentyears. The ‘‘Population Estimates Program’’ demonstratesgrowth of the Montgomery County population at 35.6%compared to 12.7% for all of Texas between April, 2000 andJuly, 2006. For the same time period Indian River Countygrew at 15.2% compared to 13.2% statewide. Montgomery

D. Bowman et al. / Veterinary Parasitology 160 (2009) 138–148146

County, Texas had 1.5% of the population migrate from astate where B. burgdorferi is more endemic. This comparesto 1% for the entire state. Indian River County, Floridashows a 7.5% migration from endemic areas compared to5.4% statewide. These positive dogs may thereforerepresent movement of B. burgdorferi-infected dogs intothe region from areas where transmission of this agentmore commonly occurs.

Our findings of dogs in the deep South with antibodiesto the agent of Lyme disease (Fig. 2) are of particularinterest because a laboratory-confirmed human case ofautochthonus Lyme borreliosis has not been reported fromany state south of Maryland or Virginia (Wormser et al.,2006). Published data are also lacking to support locallyacquired cases of Lyme disease in dogs in the South(Duncan et al., 2004), although some veterinarianspracticing in this region have witnessed seroconversionto C6 in dogs without a travel history to endemic areas(Alleman, personal communication). Infection with B.

burgdorferi sensu stricto has been described in both rodentreservoir hosts and tick vectors in the southern U.S. (Oliveret al., 2000), and the organism is known to be circulating innature, but the relatively low prevalence and distinctnatural history of the tick vector involved is thought tolargely limit transmission to people in this region (Oliveret al., 2003). States that border areas where Lymeborreliosis is known to be endemic, such as North Carolina,do have a higher percent positive rate than those furthersouth (Table 1). We do not have travel histories on the1049 dogs from southeastern and southcentral statessouth of Maryland or Virginia that tested positive for B.

burgdorferi in this study, and thus the data reported herecannot serve to confirm or deny the presence of endemictransmission of B. burgdorferi in the southern U.S. However,these data do suggest that the question of local transmis-sion of B. burgdorferi to both dogs and people in areashistorically thought to be non-endemic for Lyme borre-liosis is one which warrants continued consideration.

In North America, A. phagocytophilum is maintainedlargely in the same tick vector/reservoir host system as B.

burgdorferi, and thus we anticipated the geographicdistribution of these two agents would be similar in ourstudy. As expected, antibodies to A. phagocytophilum weremost commonly reported from dogs in the Midwest andNortheast, where 6.7% and 5.5% of dogs, respectively,tested positive for this agent (Table 1 and Fig. 3). In manylocales, such as Crow Wing and Cass County, Minnesota,and in both the Midwest and Western regions as a whole,the prevalence of A. phagocytophilum in dogs was muchgreater than that of B. burgdorferi, and hyperendemic fociagain appeared to be identified (Fig. 3). For example, inLichtfield County, Connecticut, 50.2% of dogs were positivefor A. phagocytophilum, a prevalence which was signifi-cantly higher than the rest of the state, the northeasternregion, and the country as a whole (21.8%, 5.5% and 4.7%,respectively; p < 0.0001). Foci of infection were alsoidentified in California; 19.4% of dogs in Humboldt County,California were reported as positive for A. phagocytophilum,a rate that is significantly greater than the 4.8% seen in therest of the state, and the 4.5% for the entire West(p < 0.0001). Prevalence rates for A. phagocytophilum were

significantly lower in the South (0.5%; p < 0.0001; Fig. 3), aregion where human infections are also considered lesscommon, although 509 dogs testing positive were reportedfrom this area. Some dogs in the Southeast that werereported as positive to A. phagocytophilum may haverelocated from an area where this agent is endemic or,alternatively, could be the result of infection with A. platys,which generates positives on the assay used. In studiesinvolving dogs infected with a laboratory strain of A. platys,the SNAP1 4Dx1 was reactive with serum from 10 out of10 infected animals (unpublished data, source: SNAP1

4Dx1 Test kit insert). A. platys is thought to be transmittedby R. sanguineus, the brown dog tick, and thus is consideredwidely distributed throughout the U.S. (Harvey, 2006).

As was also expected, evidence of co-infection of dogswith both B. burgdorferi and A. phagocytophilum was seen,particularly in areas where prevalence of both organismswas high. In the Midwest, 2.0% of dogs had evidence ofexposure to both agents, with co-infection rates in ToddCounty, Minnesota reaching a maximum observed level of40.8%. Similarly, in the Northeast, 1.4% of dogs were co-infected with these two pathogens; prevalence of co-infection was as high as 19% in Nantucket County,Massachusetts. B. burgdorferi and A. phagocytophilum sharea common tick vector and reservoir host, and I. scapularis

ticks infected with both pathogens have been reported(Courtney et al., 2003; Adelson et al., 2004; Holman et al.,2004); thus, we were not surprised by this high prevalenceof co-infection. In California, where the overall prevalenceof B. burgdorferi is somewhat lower (1.8%), only 0.9% ofdogs were reported as co-infected in the state as a whole.

The disease agent for which we saw the lowestprevalence nationally and on a regional level was E. canis.Only 0.6% of dogs were positive for E. canis nationwide,with the highest regional rate, 1.3%, reported from thesoutheastern states. However, even with this relatively lowoverall prevalence, a number of foci of infection wereidentified where prevalence rates ranged from 2% to morethan 10%. For some of these areas, such as HumphriesCounty, Tennessee or Caddo County, Oklahoma, wesuspect that cross-reactions with E. chaffeensis, as is knownto occur with the analyte used in the assay (O’Connor et al.,2006) accounted for some of the reported positive results.Several areas where prevalence rates to E. canis werehigher than 2.0% (Fig. 4) correspond to areas of hyper-endemicity of E. chaffeensis identified in other studies(Yabsley et al., 2005), suggesting that some of these dogsmay also be exposed to or infected with E. chaffeensis.

Both R. sanguineus and E. canis are considered morecommon in the South, although they are somewhat widelydistributed across most of the country. We were surprisedby an apparent hyperendemic focus of E. canis infection inImperial County, California, where 11.1% of dogs werereported as positive, compared to 0.8% for the rest of thestate as a whole, particularly because E. chaffeensis in notconsidered common in this area of the country (Foley et al.,1998; Kramer et al., 1999; Lane et al., 2001; Fritz et al.,2005). We were similarly surprised by foci of E. canis

reactivity in dogs in Wisconsin and Minnesota (Fig. 4),another region where E. chaffeensis is not particularlycommon. There may be foci of intense E. canis transmission

D. Bowman et al. / Veterinary Parasitology 160 (2009) 138–148 147

in these particular areas. Alternatively, the test results inthese dogs could represent exposure to a novel Ehrlichia sp.that has yet to be described; novel Ehrlichia and Anaplasma

spp. continue to be discovered in North America(Brandsma et al., 1999; Loftis et al., 2006; Munderlohet al., 2007).

There were several limitations to this study. Firstly, apositive antigen or antibody test is not equivalent to theexistence of an agent in a particular locale; it is evidenceonly of prior exposure at some point and some location in adog’s history. Areas experiencing a population influx likelyare also experiencing an influx of dogs from other regionsof the country, and pets testing positive in these areas maywell have been exposed elsewhere. Furthermore, only adistinct subset of canids was sampled—that is, companiondogs brought to a veterinarian, and whose veterinariansand/or owners opted to test for the agents in question; ourestimates of ‘‘prevalence’’ must be viewed with this bias inmind. In addition, while the overall study looked at 7 yearsof data, A. phagocytophilum results were only available forthe last year, which could make the data vulnerable to anyanomalies affecting that year, such as vector abundance.

All four of these agents can also cause disease inhumans. The three tick-borne diseases (Lyme borreliosis,ehrlichiosis, and anaplasmosis) are nationally reportablewhen diagnosed in people, and all have been documentedin 2007 (CDC, 2007b,c,d). Heartworm infection has beendemonstrated in people in North America both with andwithout a travel history to areas of high endemicity, andpresents a clinical challenge due to the seriousness of thedifferential diagnoses for pulmonary nodules (Theis, 2005;Lagrotteria et al., 2003). The public health implications ofthe study are therefore significant. A physician’s decisionto diagnose a particular infection in a locale or region ispartly influenced by the local prevalence of the agent.Therefore, people living in areas where a disease is notthought to be endemic may go undiagnosed. Conversely,some conditions may be misdiagnosed, followed byinappropriate treatment. The situation with Lyme disease,for instance, is complicated throughout the range of thelone star tick, Amblyomma americanum, which has beenassociated with the development of lesions similar to thecardinal erythema migrans rash characteristic of earlyLyme infection (Dennis, 2005). Further prevalence studiesare indicated, particularly those that focus on these newlyidentified areas of endemicity, and we foresee dogs (or adata set of dog infections) being used as sentinels.

5. Conclusion

These vector-borne disease agents are more widelydistributed than expected. Evidence of at least one agentwas found in every state analyzed, and most states hadevidence of all four organisms, representing all regions ofthe continental U.S. These data will provide veterinarianswith a heightened awareness of the vector-borne diseaseagents common in their practice areas, and elevate theirconsideration of these infections when taking a travelhistory and choosing diagnostics. An important conclusionfor the human population is that people living in areaswhere the disease was not thought to be endemic may also

be at risk for infection. Dogs may serve as sentinels toidentify the presence of vector-borne disease agents ofboth veterinary and public health significance.

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