Cattle and the Natural Historyof Rickettsia parkeri in Mississippi

Kristine T. Edwards,1 Jerome Goddard,1 Tara L. Jones,2

Christopher D. Paddock,2 and Andrea S. Varela-Stokes3

Abstract

Cattle have been recognized as hosts for Amblyomma maculatum, the Gulf Coast tick, for over 100 years. Fornearly as long, A. maculatum have been known to harbor the spotted fever group Rickettsia (SFGR), now knownas Rickettsia parkeri. However, human infection with R. parkeri was not documented until 2004. Results presentedherein describe a laboratory and a field study evaluating cattle and the natural history of A. maculatum andR. parkeri in Mississippi. In the laboratory study, seroconversion to R. parkeri antigen occurred in calves exposedto R. parkeri by injection or by feeding R. parkeri–infected A. maculatum, and two out of six animals weretransiently rickettsemic. All calves remained clinically normal during the study, except for gotch ear-like lesionsin all tick-infested calves, regardless of infection status of ticks, suggesting that R. parkeri is not involved in thecondition. In the field study, A. maculatum (n¼ 34) removed from Mississippi sale barn cattle (n¼ 183) and thecattle hosts were tested for R. parkeri. Cattle were not rickettsemic by polymerase chain reaction, but 49.7%demonstrated low titers to R. parkeri antigen when tested by indirect fluorescent antibody for SFGR. Of ticksremoved from cattle, 11.8% were hemolymph positive and 8.7% were indirect fluorescent antibody positive.Approximately 22% (5/23) and 4% (1/23) of harvested tick extracts were positive for R. parkeri by polymerasechain reaction of the 17 kDa antigen gene and ompA gene, respectively. An amplicon for the ompA gene from onetick was successfully sequenced and showed 100% similarity with the homologous sequence of R. parkeri. Thus,cattle may harbor R. parkeri–infected A. maculatum and produce antibodies to SFGR. Cattle may play a role in thenatural history of R. parkeri infection by expanding populations of A. maculatum and transporting R. parkeri–infected ticks to various locations, rather than as a reservoir for R. parkeri.

Key Words: Cattle—Ecology—Natural history—Rickettsia—Rickettsia parkeri.

Introduction

The Gulf Coast tick, Amblyomma maculatum [Acari: Ix-odidae], is a large, Nearctic and Neotropical, three-host

tick (Sonenshine 1991, 1993, Estrada-Pena et al. 2005) thatparasitizes a wide range of vertebrate animals in its variouslife stages. Cattle were first identified as hosts for A. macula-tum in the early 1900s (Hooker et al. 1912). In 1937, Dr. R.R.Parker identified a spotted fever group Rickettsia sp. (SFGR) inA. maculatum from cattle, similar to, but distinct from, Rick-ettsia rickettsii, the etiologic agent of Rocky Mountain spottedfever (Parker et al. 1939). Parker speculated that this rickettsiamight cause a Rocky Mountain spotted fever–like illness,which he referred to as ‘‘maculatum infection’’ (Parker 1940).

Eventually, the agent was named Rickettsia parkeri (Lackmanet al. 1965), and a case of R. parkeri rickettsiosis was confirmedin 2002 in a Virginia man who had a spotted-fever-like illnesswith eschars following tick bite (Paddock et al. 2004). Sincethen, a total of 12 suspected or confirmed cases of rickettsiosisdue to R. parkeri have occurred, including 4 in Mississippiresidents (Paddock et al. 2008).

The distribution, abundance, and occurrence of A. macula-tum on wild and domestic mammals in north-central Okla-homa, and the geographical distribution of A. maculatum inMississippi, have been well documented (Semtner and Hair1973, Barker et al. 2004, Goddard and Paddock 2005); how-ever, the potential role of cattle in the natural history of R.parkeri is unknown. Adult ticks prefer feeding on cattle ears,

1Department of Entomology & Plant Pathology, Mississippi State University, Mississippi State, Mississippi.2Centers for Disease Control and Prevention, Atlanta, Georgia.3Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi.

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and when infestations involve sufficient numbers, the earsmay become thickened and curled, causing a condition calledgotch ear (Wright and Barker 2007). Little is known about thepathogenesis or epidemiology of gotch ear except that itusually involves A. maculatum (Gladney 1976, Williams et al.1978, Byford et al. 1992, Mock 2000, Broce and Dryden 2005,Highfill 2006, Wright and Barker 2007, Wright et al. 2007).

Because infection with R. parkeri in human hosts produces anecrotic lesion (Paddock et al. 2008), we hypothesized thatgotch ear might also be attributable to this infection. Further,because tick infection rates with R. parkeri appear to be higherthan those with other SFGR (Sumner et al. 2007), exposure ofcattle to this pathogen seems likely (Bishopp and Hixon 1936,Bishopp and Trembley 1945, Williams et al. 1978, Byford et al.1992, Mock 2000, Barker et al. 2004, Broce and Dryden 2005,Ketchum et al. 2005, Highfill 2006, Wright et al. 2007). The aimof this study was to evaluate the role of cattle in the naturalhistory of R. parkeri.

Materials and Methods

Clinical definition of gotch ear

We defined gotch ear as a condition of the ear in cattle as-sociated with tick infestation, predominantly A. maculatum,exhibiting variable degrees of edema, and lesions at tick at-tachment sites on the outer or inner pinnae that includedcrusting, alopecia, erythema, and excoriation, with or withoutcurling of the tip of the pinnae and a loss of a portion of the ear.

Experimental study in calves

All studies were approved by the Institutional Animal Careand Use Committee at Mississippi State University. Eighthealthy Holstein bull-calves from the Mississippi State Uni-versity dairy (age range: 3 to 6 months) with no detectableantibodies to SFGR by indirect fluorescent antibody (IFA) ordetectable circulating SFGR by polymerase chain reaction(PCR) to the ompA and 17 kDa antigen genes were maintainedin a tick-free environment for the entire course of the study.These were kept in biosecurity level-2 rooms located in astand-alone building at the Mississippi State University Col-lege of Veterinary Medicine. Four calves were chosen at ran-dom for the injection group; three were inoculated by threedifferent routes with 0.3 mL of R. parkeri–infected Vero cellsuspension (Tate’s Hell isolate): intradermally over the tricepsmuscle of the left shoulder, intravenously in the left jugularvein, and subcutaneously near the other injection sites.Growth parameters for the rickettsiae in Vero cells were asfollows: inocula for all experiments were from two- to six-passage infected Vero cell suspensions with an approximatedose of 1.8�106 Vero cells in 1 mL for the negative control and*2.63�106 Vero cells infected with R. parkeri in 1 mL (75%–80% infected). The fourth calf served as a negative control,and was injected with 0.3 mL of an uninfected Vero cell sus-pension at each of the same locations.

To establish an infected tick colony, 285 engorged nymphalticks obtained from an Oklahoma State University colonywere demonstrated R. parkeri negative by hemolymph test(Gimenez 1964, Burgdorfer 1970), PCR for the ompA gene, andIFA techniques. These ticks were injected percutaneouslywith either R. parkeri–infected cell suspensions (n¼ 210 ticks)or sterile phosphate-buffered saline (PBS; pH 7.4; n¼ 75 ticks)

using a 30-gauge needle as previously described (Goddard2003).

All ticks were kept in vials in humidity chambers and ex-amined daily to determine whether they had molted. In theinjected tick colony, 95% (199/210) of the R. parkeri–injectedticks molted and 96% (72/75) of the PBS-injected ticks molted.The mean days to molt for the 199 ticks were 24.46 and 25.28for the R. parkeri–injected and PBS-injected ticks, respec-tively. After molting to the adult stage, a subsample of 15/18(83.3%) R. parkeri–injected ticks were positive when tested bynested PCR for the ompA gene, whereas all PBS-injected tickswere negative for this gene. The same subsample was 86%(16/18) hemolymph positive.

For the IFA test, tick hemolymph was spotted onto thewells of slides and stored at �208C until ready to stain. Aseparate antigen-coated slide was used as a positive controland hemolymph-negative ticks were included as negativecontrols. Each sample and control field was loaded with 40mLof anti–R. rickettsii human serum (kindly provided by Dr.William Nicholson, Centers for Disease Control and Preven-tion) diluted 1/100 in PBS and incubated at 378C for 35 min.Slides were washed three times for 5 min each, twice in PBSand once in dH2O, and allowed to air-dry. Forty microliters of1:20 fluorescein-isothiocyanate-conjugated anti-human im-munoglobulin G (IgG) was pipetted onto each field and theslides were incubated again at 378C for 35 min. The washeswere repeated and the last dH2O wash was counterstainedwith Eriochrome black T. After drying, the slides were cov-ered with two drops VECTASHIELD� (Vector Laboratories)and cover slips were placed over them. Sera showing titers�32 were titrated by twofold dilutions to their endpoints.

A second group of four calves were selected at random forthe tick infestation portion of the study. Fifteen to 20 R. par-keri–infected adult A. maculatum (*50% male) ticks wereplaced on the right ear of each of three calves. Similarly, 20PBS-injected ticks were placed on the right ear of the controlcalf. The tick-infested ear of each calf was covered with a sock,which was adhered to the ear base with tissue glue. All tickshad either fed to repletion and remained attached, or hadalready dropped off by the seventh day after placement.Calves with ticks were sedated with xylazine and ticks fromtheir ears were removed. Tick attachment sites were biopsiedafter local anesthesia had been applied while the calves werestill under sedation from the zylazine.

At least 3 mL of whole blood in EDTA tubes and 3 mL ofblood in serum separator tubes was collected from all calvesonce before the experiment and three times weekly afterplacement of ticks or injection, by jugular venipuncture forhematologic, molecular, and serologic tests. The calves weregiven a physical examination including monitoring of tem-perature, appetite, and attitude on each sampling day for 30days. The calves’ tick attachment sites were also visually in-spected for signs of gotch ear. Calf sera in both groups werescreened for antibodies to SFGR using R. parkeri–coated slidesfor IFA to detect IgG antibodies at a 1:32 dilution. Fluorescein-isothiocyanate-labeled goat anti-bovine IgG (Kirkegaard andPerry Laboratories) was used to determine serologic responseto infection. Sera showing titers �32 were titrated by twofolddilutions to their endpoints. Anti–R. rickettsii antibodies inhuman serum were used for a positive control and cattleserum negative by PCR of the ompA gene and determinedseronegative before the study was used as a negative control.

486 EDWARDS ET AL.

Skin biopsy specimens collected using an 8-mm punchwere obtained from the site of tick attachment from the fourtick-infested calves 7 days after exposure to R. parkeri–infectedticks as described above. Skin biopsy specimens were col-lected in a similar fashion (local anesthesia) from the fourinjected calves. Samples from all eight calves were fixed in10% formalin and processed for histopathologic evaluation.Sections (3 mm thick) were cut from formalin-fixed, paraffin-embedded tissues and tested using an immunoalkalinephosphatase staining method for SFGR, as described in Pad-dock et al. (2004). DNA was extracted from blood using anIllustra Blood Genomic Prep Mini Spin kit (GE Healthcare) forall sampled days postinfection (DPI) for each calf, and theextracts were evaluated using a nested PCR assay designed toamplify a segment of the ompA gene based on a publishedprotocol (Sumner et al. 2007). Extracts were also evaluatedusing a nested PCR assay designed to amplify a segment of the17 kDa antigen gene based on a published protocol (Paddocket al. 2004). DNA extracted from cultivated Tate’s HellR. parkeri in Vero cells (CDC) was used for a positive controland water was used for a negative control. We observed theprimary product by electrophoresis in 2% agarose gels con-taining ethidium bromide. DNA extractions, primary andsecondary PCR assays, as well as gel electrophoresis wereperformed in separate designated areas of the lab to minimizechance of DNA contamination.

Observational study in sale barn cattle

From July through October 2008, A. maculatum ticks andwhole-blood samples were collected from cattle (Table 1) at

cattle auctions in six Mississippi counties while accompany-ing the veterinarian designated for these sales and in accor-dance with the Mississippi Department of Agricultureguidelines (Fig. 1). Cattle were sampled randomly, withoutregard to age, gender, or breed. Blood specimens were ob-tained from all animals regardless of the presence of ticks.DNA was extracted as described above from whole bloodand samples were evaluated using nested PCR assays de-signed to amplify segments of the 17 kDa antigen gene and theompA genes.

Ticks collected from cattle were deposited in labeled vialsfor transport to the lab, where they were held in a humiditychamber until testing. Hemolymph tests (Gimenez 1964), IFA,and PCR methods were used to assay for SFGR as describedabove. For PCR testing, the ticks were minced using a scalpelblade and DNA was extracted using an Illustra Tissue andCells Genomic Prep Mini Spin kit (GE Healthcare) and testedby PCR assays designed to amplify segments of the 17 kDaantigen and ompA genes. PCR products were sequencedthrough MWG Biotech and analyzed using ClustalX2 andthe BLAST programs to identify the product (version 2.0;National Center for Biotechnology Information).

Results

Experimental study in calves

Calves exposed to R. parkeri remained alert, nonfebrile, andmaintained normal appetites during the course of the study.Both negative control calves were negative for R. parkeri in-fection by PCR throughout the study. All calves (negative

Table 1. Gulf Coast Ticks (Amblyomma maculatum) Infected with Spotted Fever Group Rickettsiae,

Mississippi Sale Barn Cattle, 2008

Salebarn County

Datecollected

No. ofticks Sex

Hemolymphpositive

PCR-positive17 kDa

PCR-positiveompA

IFApositive Cattle breed Age

1 Lee 9 July 02 Clay 15 July 1 Male 0 0 0 0 Brahma Unknown3 Noxubee 28 July 1 Female 0 0 0 0 Hereford 3

3 Male 0 0 0 010 Female 1 2 1a 1 Hereford 21 Male 1 2 0 0

4 Walthall 5 August 1 Male 0 0 0 1 Angus cross Unknown1 Male 0 0 0 0 Black and Whiteb Unknown1 Female 1 0 0 0

5 Adams 19 August 1 Nymph c c c c Charolais cross 51 Female 1 c c c Charolais cross 57 Male 0 c c c Angus cross 53 Female c c c c

2 Male 0 c c c Angus cross Unknown1 Female 0 c c c

6 Lauderdale 6 October 2 Female 0 0 0 0 Angus cross 101 Female 0 1 0 0 Angus 71 Female 0 0 0 0 Angus 01 Female 0 0 0 0 Angus 6

Total 39 4 5 1 2 13

Total tested 34 23 23 23

aSequence analysis for ompA amplicons showed 100% similarity with the homologous sequence of R. parkeri (GenBank accession no.U43802).

bCommon name for any unknown black and white cattle breed.cTicks lost to follow-up: 5 died before hemolymph testing; another 11 died before PCR or IFA testing.IFA, indirect fluorescent antibody; PCR, polymerase chain reaction.

Rickettsia parkeri IN CATTLE 487

control calves and R. parkeri–exposed calves) were negativefor R. parkeri using nested PCR of the ompA gene. However,two of six calves exposed to R. parkeri were transiently posi-tive to SFGR by PCR of the 17 kDa antigen gene (one tick-infected calf on DPI-23 and one inoculum-infected calf onDPI-11 and DPI-14). Each of the six calves, exposed to R.parkeri by needle inoculation or by feeding of infected ticks,developed low IgG titers (�64) to R. parkeri (Fig. 2) and bothnegative control calves remained seronegative. Attachmentsites from all four calves receiving ticks showed edema,crusting and erythematous lesions on the outer pinna, andcurling of the tip of the ear (Fig. 3A) and, in some cases, severeexcoriations (Fig. 3B). These lesions were present in the rightear of all tick-infested calves, regardless of whether ticks were

R. parkeri infected. Indurated swellings ranging from *2 to6 cm developed at injection sites in all R. parkeri–injectedcalves as well as the control calf. Immunohistochemicalstaining of tissue samples collected from ears of three calvesexposed to R. parkeri via tick bite and from skin at the injectionsite on the neck of three calves exposed via needle inoculationrevealed SFGR in the inflammatory cell infiltrates (Fig. 4).

Observational study in sale barn cattle

Blood samples were collected from 183 cattle at six salebarns, representing 25% of all sale barns in the state. Thirty-nine A. maculatum were removed from 13 (7.1%) cattle at 5 salebarns (Table 1). Cattle comprised a variety of beef breeds and

FIG. 1. Geographic distribution of Amblyomma maculatum collections in Mississippi by county, 2005, distribution of salebarns, and distribution of sale barns visited in this study, 2008 (map constructed by Joe MacGown).

488 EDWARDS ET AL.

ranged in age from 1 to 14 years (mean¼ 5); 176 (96%) of theseanimals were female. All cattle were negative for R. parkeri byPCR of both the 17 kDa antigen gene and the ompA gene.Ninety-one (50%) of cattle sera showed an IFA titer of 32 whenreacted with R. parkeri antigen; however, when titrated, allsamples were negative at a 1:64 dilution.

Bacteria stained by the Gimenez technique were detected inthe hemolymph from 4 (11.8%) of 34 A. maculatum removedfrom cattle, and 2 of these also stained by IFA assay, includingticks from cattle at one sale barn in Walthall County whereA. maculatum had not previously been reported (Goddard and

Paddock 2005). Rickettsial DNA was detected using the17 kDa PCR assay in 5 (22%) of 23 ticks removed from cattle atNoxubee and Lauderdale county sale barns. A 540-bp seg-ment of the ompA gene was amplified from one of these ticksand showed 100% identity with the homologous sequence ofR. parkeri (GenBank accession no. U43802).

Discussion

In this investigation, A. maculatum successfully fed to re-pletion on the ears of calves, and exposed calves showed a

FIG. 2. Tick-infected calves and Rickettsia parkeri–injected calves seroconverted at a 1:32 dilution on days postinfection(DPI)-2; most were transiently infected and then remained positive for the duration of the study.

FIG. 3. Gotch ear in calf infested with R. parkeri–infected A. maculatum ticks (A) and gotch ear of calf infested withA. maculatum ticks without organism (B).

FIG. 4. Immunohistochemistry of biopsied ear in calf infested with R. parkeri–infected A. maculatum ticks revealing spottedfever group rickettsiae in the inflammatory cell infiltrates (A) and immunohistochemistry of biopsied injection site in calfexposed to R. parkeri by needle injection revealing spotted fever group rickettsiae in the inflammatory cell infiltrates (arrow) (B).

Rickettsia parkeri IN CATTLE 489

transient rickettsemia and antibody production. A cutaneousinfection was identified by immunohistochemical stain insome of these calves; however, none of the R. parkeri–infectedanimals developed signs of systemic illness, and gotch earoccurred in all animals exposed to ticks, regardless of infec-tion with R. parkeri.

We found no evidence of rickettsemia by PCR in any cattle,despite the relatively frequent occurrence of A. maculatum oncattle from sale barns in Mississippi, and the identification oflow antibody titers to SFGR in *50% of these animals.However, this assessment took place at one point in time, sosera were only evaluated as either positive or negative for aparticular dilution.

Using the 1:32 dilution, some experimentally infectedcalves (from both injected and tick-infested groups) showedan antibody response as early as DPI-2. It is difficult to explainthese results, but it is noteworthy that both negative controlcalves were negative by IFA at both the 1:32 and the 1:64dilutions in a blinded evaluation. A study of related rickett-siae (Rickettsia conorii) in cattle showed that the rate of sero-conversion is dose related (Kelly et al. 1991). In that study, allof the cattle were inoculated with either 3000 organisms (lowdose) or 100,000 organisms (high dose). The high-dose cattleshowed an IgG response (titer �1:40) by DPI-7 and low-dosecattle by DPI-15. The dose used in our study was higher thantheir higher dose by a factor of nearly 30.

These data show that R. parkeri–infected A. maculatum arecapable of transmitting this SFGR to naı̈ve calves and that R.parkeri–infected ticks can be found on cattle in nature. In ad-dition, at least some sale barn cattle demonstrated low titers ofSFGR antibodies, suggesting past exposure to SFGR, possiblythrough the bite of infected A. maculatum.

Although there was no evidence of persistent systemic in-fection in cattle by R. parkeri and thus cattle do not likely serveas competent reservoir hosts for R. parkeri, we believe thatcattle may play an important role in the maintenance of therickettsia by providing a blood meal to large numbers of ticksand increasing tick populations and distribution in thesoutheastern United States. This may ultimately increase ex-posure of wildlife and people to R. parkeri–infected ticks.These results may have important implications in the devel-opment, prevention, and management approaches for gotchear with emphasis placed on tick control rather than antibiotictreatment. Additional studies are underway in our lab to as-sess the role of wildlife as potential reservoir hosts for R.parkeri.

Acknowledgments

The authors would like to thank LARAC personnel for theirkind attention to animal care throughout this study. The au-thors would also like to thank Jesse Carter for his skilled as-sistance in handling the calves. This article has been approvedfor publication as Journal Article No J-11653 of the MississippiAgricultural and Forestry Experiment Station, MississippiState University.

Disclosure Statement

None of the authors have any commercial associations thatmight create a conflict of interest in connection with this ar-ticle. No competing financial interests exist. The findings and

conclusions described in this article are those of the authorsand do not necessarily represent the opinions of the U.S.Department of Health and Human Services.

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Address correspondence to:Kristine T. Edwards

Department of Entomology & Plant PathologyMississippi State University

100 Twelve LaneMississippi State, MS 39762

E-mail: kt20@msstate.edu

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