Bartonella henselae Infections In An Owner and Two Papillon Dogs Exposed to Tropical Rat Mites ( Ornithonyssus bacoti )

Abstract

After raccoons were trapped and removed from under a house in New York, the owner and her two Papillon dogs became infested with numerous rat mites (Ornithonyssus bacoti). Two weeks later, both dogs developed pruritus, progressively severe vesicular lesions, focal areas of skin exfoliation, swelling of the vulva or prepuce, abdominal pain, and behavioral changes. Two months after the mite infestation, the owner was hospitalized because of lethargy, fatigue, uncontrollable panic attacks, depression, headaches, chills, swollen neck lymph nodes, and vesicular lesions at the mite bite sites. Due to ongoing illness, 3 months after the mite infestation, alcohol-stored mites and blood and serum from both dogs and the owner were submitted for Bartonella serology and Bartonella alpha Proteobacteria growth medium (BAPGM) enrichment blood culture/PCR. Bartonella henselae DNA was amplified and sequenced from blood or culture specimens derived from both dogs, the owner, and pooled rat mites. Following repeated treatments with doxycycline, both dogs eventually became B. henselae seronegative and blood culture negative and clinical signs resolved. In contrast, the woman was never B. henselae seroreactive, but was again PCR positive for B. henselae 20 months after the mite infestation, despite prior treatment with doxycycline. Clinicians and vector biologists should consider the possibility that rat mites may play a role in Bartonella spp. transmission.

Introduction

B artonella (formerly known as Rochalimaea) comprises a genus of fastidious, small, Gram-negative, slow-growing, intracellular bacteria that can induce a longstanding bacteremia in animals and people. The genus was named in honor of Alberto Leonardo Barton Thompson, a Peruvian scientist born in Argentina, who studied B. bacilliformis. At least 11 Bartonella species are known to infect humans, with B. henselae being the most frequent infection found in nonimmunocompromised individuals (Chomel and Kasten 2010). Presumably, Bartonella spp. have been infecting humans for thousands of years, as demonstrated by amplification of B. quintana DNA from a 4000-year-old human tooth (Drancourt et al. 2005). Bartonella spp. are known to be transmitted by arthropod vectors, including fleas, lice, and sand flies (Bouhsira et al. 2013).

Recently, vector competence for Ixodes ricinus transmission of B. birtlesii has been demonstrated in a laboratory study (Reis et al. 2011). On the basis of epidemiological findings, keds (wingless flies) are thought to be responsible for transmission of Candidatus B. melophagi to sheep (Maggi et al. 2009) and B. schoenbuchensis to deer in North America and Europe (Matsumoto et al. 2008). Cat fleas, Ctenocephalides felis, are thought to be the sole or primary vector for B. henselae transmission to pets and potentially people (Bouhsira et al. 2013). To date, Bh DNA has been PCR amplified from the blood of cats, dogs, feral swine, cattle, horses, sea turtles, wild cats, and several marine mammal species (Harms et al. 2008, Maggi et al. 2008, Cherry et al. 2009, Chomel and Kasten 2010, Psaroulaki et al. 2010, Beard et al. 2011, Cherry et al. 2012, Girard et al. 2012).

In this study, evidence is presented that the tropical rat mite may be capable of transmitting B. henselae. On the basis of the chronology of events, B. henselae infection may have resulted in the gradual onset of illness in both dogs and illness in the owner.

Materials and Methods

Historical and clinical presentations for the dogs and owner

On July 13, 2011, 7 days after raccoons were trapped and removed from under her house located in Trumansburg, NY, an owner and her two Papillon dogs, a 5-year-old spayed female (dog 1) and 3.5-year-old castrated male (dog 2), became infested with numerous biting arthropods. The owner reported observing mites on herself and her dogs, and they all developed cutaneous blister-like lesions at the mite bite sites. Over the next 2 weeks, as depicted in the timeline (Fig. 1), both previously healthy dogs became ill with clinical signs that the attending veterinarian temporally associated with the rat mite infestation. During the initial physical examinations, the attending veterinarian noted large numbers of mites infesting each of the dogs. In addition, two Ixodes scapularis larvae were removed from one of the dogs during mite collection. Their owner reported no prior tick exposure history for her dogs and routinely administered a heartworm preventive and applied a topical acaracide. The owner also reported that she was not aware of previous flea exposures in her dogs. The owner (a registered nurse) had a prior diagnosis of fibromyalgia in 1998 and she had been treated for Lyme disease in 2005 in association with the development of arthritis.

FIG. 1.

Timeline of events starting with removal of raccoons from under the woman's house through the next 6 months following the tropical rat mite infestation.

At the time of initial examination, the owner provided her veterinarian with a container of mites collected at the onset of the infestation from the dogs' bedding, directly from the dogs or from the premises for parasitological identification. Over 20 mites submitted to Cornell University were all identified as tropical rat mites (Ornithonyssus bacoti) by Dr. Susan E. Wade, Director of Parasitology at the Animal Health Diagnostic Center. When Dr. Wade's report became available on August 6, 2011, a professional exterminator treated the house and premises for the tropical rat mite infestation. Due to an ongoing infestation, the premises were again treated 5 days later.

Twenty-six days after the mite infestation was first noted by the owner, the female Papillon (dog 1) was hospitalized due to pruritus, progressively severe vesicular lesions at the sites of the mite bites, a swollen vulva, and focal areas of skin exfoliation on the abdomen and vulva in areas surrounding the mite bites. The dog was painful on palpation of the abdomen and back, refused to walk, and exhibited fearful, hiding behavior. Complete blood count (CBC) and serum biochemical profile values, obtained when dog 1 was hospitalized, were within normal reference intervals. Abdominal radiographs revealed mild splenomegaly. Three days later, the male Papillon (dog 2) was hospitalized, due to inflamed vesicular lesions at mite bite sites, a swollen prepuce, a painful abdomen, reluctance to move, and hematochezia. The male dog would reportedly chew at his prepuce and also exhibited fearful hiding behavior. Neither dog was febrile at the time of admission or during hospitalization. Due to the suspicion that both dogs had been infected systemically with a mite-borne pathogen, the attending veterinarian initiated systemic antibiotic treatment and prescribed a topical medication to decrease the pruritus and to combat potential secondary skin infection and applied an acaricide (Revolution, Zoeitis Animal Health, New York). Both dogs were treated with doxycycline, 25 mg (approximately 6 mg/kg) every 12 h for 21 days. A topical ointment containing nystatin, neomycin sulfate, thiostrepton, and triamcinolone acetonide (Animax, Dechra Veterinary Products, Overland Park, KS) was applied to the vulva, or prepuce, respectively, and to the bite lesions.

Two weeks later, dog 1 was again hospitalized due to anorexia, vomiting, and hematochezia. Subcutaneous fluids, an antiemetic (maropitant citrate tablets, Cerenia™, Pfizer Animal Health, New York), and metronidazole were administered. During September, both dogs remained pruritic, and the female dog had severe skin exfoliation involving the regions around previous mite bites.

Thirty-one days after the rat mite infestation was noted, the owner reported an acute onset of lethargy and fatigue that persisted for the next 3 weeks. On September 11, 2011, she was hospitalized for 4 days due to panic attacks, depression, headaches, chest pain, and chills, a constellation of symptoms that she had not experienced in association with her prior illnesses. At admission, she still had vesicular lesions at the previous mite bite sites and swollen lymph nodes in her neck.

Due to the potential that the rat mites had simultaneously transmitted an infectious agent to the dogs and the owner, the attending veterinarian contacted the Intracellular Pathogens Research Laboratory (IPRL) in September, 2011, seeking diagnostic assistance. In October, 2011, mites collected in July and aseptically collected blood and serum from both dogs and the owner were submitted to the IPRL for testing. Collection and analysis of the woman's data were conducted in conjunction with North Carolina State University Institutional Review Board approval (IRB #1960) and with her permission. A commercial enzyme-linked immunosorbent assay (ELISA) (SNAP 4DX™, IDEXX Laboratories, Westbrook, ME) that detects Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and Dirofilaria immitis antigen was negative for both dogs. Indirect fluorescent antibody assays for Babesia canis, Babesia gibsoni, Ehrlichia canis, and Rickettsia rickettsii were negative.

Using the Bartonella alpha Proteobacteria growth medium (BAPGM) enrichment blood culture/PCR platform, the woman and both dogs were tested for DNA evidence of Bartonella spp. infections. At the time of blood collection, the woman had been treated by her physician with doxycycline 100 mg every 12 h for approximately 1 month. Antibiotics were discontinued for 2 weeks prior to collecting blood for BAPGM enrichment blood culture, after which daily administration of doxycycline (100 mg every 12 h) was continued for an additional 3 weeks followed by 100 mg b.i.d. for 2 days on and 4 days off for an additional month.

In November, 2011, after the Bartonella enrichment blood culture/PCR results became available, both dogs were again treated with doxycycline, with gradual return to a preinfestation health status. The male dog had suspected clinical relapses in January and September of 2012, each characterized by prepucial swelling, shivering, abdominal pain, and hiding behavior, and each episode was reportedly responsive to repeat doxycycline administration. Unfortunately, this dog was not retested prior to administration of antibiotics at the time of the suspected relapses. The owner and both dogs were retested for serological and BAPGM enrichment blood culture/PCR evidence of Bartonella spp. infection in April of 2013.

Serological analysis

B. canis, B. gibsoni, E. canis, R. rickettsii, Bartonella vinsonii subsp. berkhoffii genotypes I, II, III (Bvb I, II, III), B. henselae Houston 1(BhH1), B. henselae San Antonio2 (BhSA2), and Bartonella koehlerae (Bk) antibodies were determined in the IPRL following traditional immunofluorescence antibody assay (IFA) practices with fluorescein-conjugated goat anti-human immunoglobulin G (IgG; Pierce Biotechnology Rockford, IL). Bartonella organisms of feline isolates of Bk (NCSU 09FO-01) and BhH1 (NCSU 93FO-23), BhSA2 (NCSU 95FO-099), and canine isolates of Bvb genotype I (NCSU 93CO-01), II (NCSU 95CO-08) and III (NCSU 06CO-01) were passed from agar-grown cultures into cell cultures to prepare antigens. Heavily infected cell cultures were spotted onto 30-well Teflon-coated slides (Cel-Line/Thermo Scientific), air dried, acetone fixed, and stored frozen. Serum samples were diluted in phosphate-buffered saline (PBS) solution containing normal goat serum, Tween-20, and powdered nonfat dry milk to block nonspecific antigen binding sites. A dog (Goat Anti-Canine IgG Heavy and Light Chain, MP Biomedicals, ICN Cappel, Costa Mesa, CA) or human (Fluorescein-Conjugated Goat Anti-Human IgG Heavy and Light Chain, Thermo Scientific, Pierce Antibody, Rockford, IL) conjugate was applied as appropriate, and slides were read on a fluorescence microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY). Sera were screened at dilutions of 1:16 to 1:8192. A cutoff titer of ≥1:64 was used to define a seroreactive titer.

DNA extraction, PCR DNA sequencing, and BAPGM enrichment blood culture

The three-part BAPGM diagnostic platform incorporates PCR amplification of Bartonella spp. DNA following extraction from patient blood and serum samples, PCR amplification following BAPGM enrichment blood culture for 7 and 14 days, and PCR from isolates obtained following BAPGM subculture onto trypticase soy agar with 10% rabbit blood. Agar plates were incubated for 4 weeks and checked weekly for evidence of bacterial growth. To assess for potential laboratory contamination, an uninoculated BAPGM culture flask was processed simultaneously and in an identical manner with each batch of patient blood and serum samples tested. The standard operating procedure followed in our laboratory states that that negative-control liquid culture flask is the last flask to which BAPGM medium is added, after which the flask is not closed (capped) until all diagnostic samples are processed. Methods used for testing sample cultures, including DNA extraction, PCR amplification targeting the Bartonella 16S–23S intergenic spacer region (ITS), and sequencing procedures were performed using previously described methods (Maggi et al. 2005, Breitschwerdt et al. 2007, Diniz et al. 2007, Duncan et al. 2007, Maggi et al. 2011).

Following the standard operating procedures in the IPRL, sample preparation including BAPGM cultures and agar plate subinoculation, DNA extraction, PCR preparation, and PCR amplification and analysis were performed in separate laboratory rooms to avoid culture as well as DNA contamination. In addition, negative and positive Bartonella DNA test control samples, consisting of bacteria-free blood DNA and DNA spiked with B. henselae genomic DNA at 0.5 genome copies per microliter, respectively, were used for each batch of DNA tested. For all results reported in this study, PCR products consistent in size with a Bartonella spp. (400–600 bp amplicon size) were sequenced to confirm the species and ITS genotype. Sequences were aligned and compared with GenBank sequences using AlignX software (Vector NTI Suite 6.0, InforMax, Inc.). DNA was automatically extracted from 200 μL of EDTA anti-coagulated blood and serum and from 200 μL of BAPGM using a BioRobot Symphony Workstation and MagAttract DNA blood kit (Qiagen, Valencia, CA). The BioRobot was cleaned and decontaminated monthly. All DNA extraction controls, tested with each sample batch processed in the robot, remained PCR negative for Bartonella sp. DNA throughout the study.

Prior to extraction of DNA from the tropical rat mites, pooled specimens were washed twice using 2 mL of dH₂O followed by a single wash with 95% ethanol. For DNA extraction, the entire pool of the arthropods was pulverized to a fine powder by bead beating using stainless steel beads. DNA from mites was manually extracted using DNeasy Blood & Tissue Mini Kit following manufacturer's instructions (Qiagen, Valencia, CA). Bartonella DNA was amplified using conventional Bartonella genus PCR primers targeting the 16S–23S ITS using primers 425s (5′-CCGGGGAAGGTTTTCCGGTTTATCC-3′), 325s (5′-CCTCAGATGATGATCCCAAGCCTTTTGGCG-3′), and 1000as (5′-CTGAGCTACGGCCCCTAAATCAGG-3′), as previously described (Maggi et al. 2011). Bartonella sp. sequences were compared using Basic Local Alignment Search Tool (BLAST version 2.0) and AlignX (Vector NTI Advance, Invitrogen) software.

Results

Bartonella spp. serology and BAPGM enrichment PCR results

Bartonella serology and PCR results for the owner and both dogs are summarized in Table 1. When initially tested by the IPRL in October, 2011, the owner was seronegative to Bvb genotypes I, II, and III, BhH1, BhSA2, or Bk antigens. Bh DNA was PCR amplifed and sequenced from two of the woman's blood samples, one serum sample and from one of three BAPGM enrichment blood cultures (Table 1). Sequence analysis revealed 368/383 (96%) identity with B. henselae strain San Antonio 2 (GenBank accession no. AF369529) and 383/383-bp identity (100%) with a previously described B. henselae strain obtained from a cat in Australia (GenBank accession no. AJ441256). When retested in February, 2012, and April, 2013, the woman remained seronegative to all Bartonella spp. test antigens used in this study; however, Bh DNA was again amplified and sequenced from an April, 2013, blood sample. Amplified DNA sequences obtained in October, 2011, and April, 2013, were identical, with the exception of a single base pair difference.

Table 1.

Bartonella spp. Serology, PCR, and BAPGM Enrichment Blood Culture/PCR Results for the Owner and Her Two Papillon Dogs

						Bartonella IFA reciprocal titers
Patient	Collection Date	Sample	PCR original sample	PCR culture	PCR Isolate	Bvb I	Bvb II	Bvb III	Bh H1	Bh SA2	Bk
Owner	10/17/2011	Serum	Bh	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
	10/19/2011	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Bh	Bh	Neg
	10/21/2011	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Bh	Neg	Neg
	2/27/2012	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
	2/29/2012	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
	3/2/2012	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
	4/3/2013	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
	4/5/2013	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
	4/7/2013	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Bh	Neg	Neg
Dog 1	10/10/2011	Serum	N/D	N/D	N/D	<16	<16	<16	<16	64	<16
		Blood	Bh	Neg	Neg
	4/8/2013	Serum	N/D	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
Dog 2	10/10/2011	Serum	N/D	N/D	N/D	<16	<16	<16	<16	64	<16
		Blood	Neg	Neg	Bh
	10/1/2012	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
	10/3/2012	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
	10/5/2012	Serum	Neg	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg
		Urine	Neg	Neg	Neg
	4/8/2013	Serum	N/D	N/D	N/D	<16	<16	<16	<16	<16	<16
		Blood	Neg	Neg	Neg

BAPGM, Bartonella alpha Proteobacteria growth medium; IFA, immunofluorescent antibody assay; Bh, Bartonella henselae, Bh SA2, Bh strain San Antonio; Bh H1, Bh strain Houston 1; Bvb I, II, and III, Bartonella vinsonii subsp. berkhoffii genotypes I, II, and III; Bk, Bartonella koehlerae; N/D, not done; Neg. negative.

When initially tested in the IPRL in October of 2011, both dogs were seroreactive to B. henselae SA2 antigens at titers of 1:64, but were seronegative to Bvb genotypes I, II, and III and BhH1 and Bk antigens (Table 1). On the basis of analysis of the DNA sequences amplified from the October, 2011, blood sample from dog 1 and the BAPGM enrichment subculture isolate from dog 2, respectively, both dogs were infected with B. henselae strains that also shared 100% identity (323/323 bp) with GenBank accession no. AJ441256. The difference between the Bh SA2 sequence (AF369529) and the strain obtained from the woman and both dogs is related to a 15-bp insertion, located at position 296. When retested in April, 2013, both dogs were seronegative to all six test antigens and PCR negative by BAPGM enrichment blood culture (representing three independent PCR results for each dog, i.e., from blood, 7-day, and 14-day BAPGM enrichment blood cultures).

PCR testing of tropical rat mites

Bh DNA was amplified and sequenced from pooled rat mites collected from the dogs and premises by the owner in July, 2011. The Bh DNA sequence obtained from the rat mites shared 99.7% identity (294/295 bp) with GenBank accession no. AJ441256. The overlapping Bh DNA from the woman and two dogs shared 100% sequence identity in this region and 99.7% identity to the rat mite sequence.

Discussion

As a result of this rat mite infestation investigation, the same Bh ITS genotype was amplified from blood or culture specimens obtained from the owner and both dogs In addition, Bh DNA was successfully amplified and sequenced from pooled rat mites, which were obtained from the dogs and the premises at the time of the initial infestation. The detection of the same Bh genotype in the mites as was found in the owner and her dogs could be due to DNA contamination of the mites with blood from infected animals (dogs, human, raccoons), or alternatively infected mites could have transmitted the bacteria to the dogs and woman. Because both dogs were healthy prior to the mite exposure, and because both dogs developed a similar and near simultaneous onset of illness following mite exposure, B. henselae transmission from the mites to the dogs seems more likely to have occurred. However, because the woman had a historical diagnosis of Lyme disease and because Ixodes ticks have been implicated in the transmission of Bartonella spp. (Reis et al. 2011), there is the potential that the woman and dogs were previously infected with B. henselae genotype SA2 by tick transmission.

C. felis, the common cat flea, is a documented arthropod vector for transmission of B. henselae to both cats and dogs. Cats are considered the primary reservoir host for B. henselae throughout the world, with bacteremic prevalences as high as 50–75% in feral cat populations (Chomel and Kasten 2010). The owner did not own cats, routinely applied acaracide products to her dogs, denied flea infestations on her dogs, and rarely observed stray cats around her house, making flea transmission from a cat reservoir to the owner and her dogs less likely. I. scapularis larvae were found on one of the dogs at the time of mite collection, thus it is also possible that both dogs were infected via tick bites. C. felis, is known to infest raccoons, opossums, foxes, coyotes, and other small mammals that inhabit the urban, suburban, and rural environments (Gracia et al. 2008, Abramowicz et al. 2012). Interestingly, a recent study using PCR amplification from raccoon and cat blood specimens reported B. henselae bacteremia in 32% of raccoons and 35% of feral cats on St. Simons Island, Georgia, suggesting that raccoons that comingle with cats and their fleas may serve as wildlife reservoir hosts for B. henselae (Hwang and Gottdenker 2013). On the basis of the historical sequence of events, we hypothesize that the rat mites became infected with B. henselae when obtaining a blood meal from the raccoons.

Females rat mites (O. bacoti) can live for up to 6 months without a blood meal (Beck and Folster-Holst 2009), and after a single blood meal can lay 90–120 eggs within a 2- to 3-day period. In the environment, mites occupy various types of nests and are infrequently found on the skin surface of the host. During the night, mites search for their preferential hosts (wild and domestic rodents) to obtain a blood meal and are generally not active during daylight hours. If suitable preferential hosts are unavailable, the mites occasionally infest alternative hosts, such as dogs and humans (Charlesworth and Clegern 1977, Theis et al., 1981, Lopatina et al. 1992, Chung et al. 1998, Skirnisson 2001, Beck and Pfister 2004). The bite of these mites often causes tiny, clear blisters that are accompanied by a rash. There are multiple articles from around the world that describe human infestation with O. bacoti, (Charlesworth and Clegern 1977, Beck and Pfister 2004), in Moscow (Lopatina et al. 1992), in Iceland (Skirnisson 2001), and in America (Chung et al. 1998); however, only recently have investigators described the potential for disease transmission by Dermanyssus spp. mites.

In 2012, Melter and colleagues described B. quintana transmission from a mite to a family of high socioeconomic status in the Czech Republic (Melter et al. 2012). After the mites migrated from the attic into a top-floor apartment, the parents, two grandparents, two children, and the epidemiologist investigating the outbreak experienced papular rashes and pruritic vesicular lesions on the body and legs and recurrent fevers. Dermanyssus spp. mites, a parasite of synanthropic pigeons, collected in the apartment contained B. quintana strain Toulouse, as did a day-4 blood sample from the father. In addition, all family members, except the grandfather, seroconverted to B. quintana antigens. Reeves and colleagues described a unique groEL Bartonella sp. DNA sequence from pooled O. bacoti collected from rats (Rattus norvegicus) in Egypt (Reeves et al. 2006) and in Steatonyssus spp. mites from the superfamily Dermanyssoidea collected in Tanzania (Reeves et al. 2007). Collectively, these observations suggest that some mite species may be vector competent for the transmission of Bartonella spp.

An interesting feature of the illnesses described in the owner and both of her dogs was an acute onset of behavioral changes, consisting of hiding and fearful behavior in the dogs and agitation and panic attacks in the owner. Although temporally associated with the mite infestation, it is impossible to determine if their behavioral abnormalities were causally related to the B. henselae bacteremic state, to the cutaneous irritation from the mite bites, or was not related to the mites or the B. henselae bacteremia. Previous studies have implicated a role for Bartonella spp. as a cause of behavioral abnormalities (Harvey et al. 1991, Schaller et al. 2007, Breitschwerdt et al. 2011, Maggi et al. 2011). The role of Bartonella spp. as a cause of neurocognitive abnormalities in humans and behavioral abnormalities in animals deserves future research consideration.

From a diagnostic perspective, it is common for B. henselae bacteremic dogs (Perez et al. 2011) and human patients (Maggi et al. 2011, Maggi et al. 2012) to have very low or undetectable IFA antibody titers when tested in the same laboratory by the same technician using the same IFA antigen slides and species-specific conjugate. Although the specificity (Hegarty et al. 2014) of B. henselae serology in dogs appears to be good, the sensitivity (Perez et al. 2011) is extremely poor. Both dogs in this report were not seroreactive to a Bh Houston 1 genotype, whereas both had 1:64 IFA antibody titers to a Bh SA2 genotype, suggesting genotype-specific antibody production. Due to diagnostic limitations associated with serology and direct blood plating methods, a more sensitive detection system combining PCR and culture should be used, and multiple samples from different time points should be tested (Duncan et al 2007, Maggi et al 2011, Perez et al 2011, Maggi et al 2012, Pultorak et al 2013). Even using these sensitive techniques, only one Bartonella isolate was obtained from the multiple samples tested from this owner and her dogs. Despite prior doxycycline administration to both dogs and doxycycline administration to the owner for 1 month prior to initial blood sample collection in October, 2011, PCR/culture evidence of ongoing B. henselae infection was generated.

Conclusions

Our results support the possibility that raccoons (Procyon cancrivorus) may serve as a reservoir host for B. henselae. We conclude that rat mite vector competence studies for B. henselae transmission should be performed in the future and that further research is clearly needed to establish the pathogenicity of Bartonella spp. genotpyes and to more fully define the pathogenesis of bartonellosis in animals and humans.

Footnotes

Acknowledgments

Testing of human blood samples fell under a study that was approved by the North Carolina State University Institutional Review Board (IRB#1960). We would like to thank Dr. Susan E. Wade, Director of Parasitology at Cornell University and the Animal Health Diagnostic Center for providing the mite identification, and Dr. Catherine Hegarty Ross at Colonial Veterinary Hospital, Ithaca, NY, for providing the medical record data for the two dogs in this report. This research was supported in part by the State of North Carolina and an unrestricted donation from Bayer Animal Health.

Author Disclosure Statement

In conjunction with Dr. Sushama Sontakke and North Carolina State University, Dr. Breitschwerdt holds US Patent No. 7,115,385, Media and Methods for Cultivation of Microorganisms, which was issued October 3, 2006. He is the chief scientific officer for Galaxy Diagnostics, a company that provides diagnostic testing for the detection of Bartonella spp. infection in animals and human patients. Dr. Ricardo Maggi has led research efforts to optimize the BAPGM platform and is the Scientific Technical Advisor for Galaxy Diagnostics. For all other authors, no competing financial interests exist.

This work was presented as an abstract (#41) at the 7^th International Conference on Bartonella as Animal and Human Pathogens, April 25–28, 2012, Raleigh NC (www.upch.edu.pe/tropicales/boletines/2012/BOLETIN1_2012/GUYANA/Bartonella%20Book%20final_electronic.pdf).

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