Invasion of canine erythrocytes by Bartonella vinsonii subsp. berkhoffii

Bartonella vinsonii subsp. berkhoffii is a recognized cause of endocarditis in dogs and human patients and has been associated with cardiac arrhythmias, myocarditis, granulomatous lymphadenitis, polyarthritis, and granulomatous rhinitis in dogs. Little is known regarding the mode of transmission or cellular localization of this bacteria following infection of a canine host. The aim of the current study was to determine whether erythrocytes may serve as a site of infection by B. vinsonii subsp. berkhoffii. In the study, we successfully demonstrate the invasion of canine erythrocytes by a B. vinsonii subsp. berkhoffii genotype III strain using an in vitro model system. Dog erythrocytes were incubated with B. vinsonii subsp. berkhoffii after which tubes were treated with gentamicin at 12, 24, and 48 h post-inoculation. After gentamicin elimination of extracellular bacteria, there was a gradual increase in intra-erythrocytic bacteria, as assessed by colony forming units per ml, at each collection time point. The largest recovery of intracellular bacteria occurred at 48 h post-infection. These results suggest that canine erythrocytes may serve in the maintenance of bacteremia due to B. vinsonii subsp. berkhoffii within an infected host.     

Isolation or detection of Bartonella vinsonii subspecies berkhoffii and Bartonella rochalimae in the endangered island foxes (Urocyon littoralis)

Bartonella rochalimae (B.r.) and Bartonella vinsonii subsp. berkhoffii (B.v.b.) have been isolated from gray foxes (Urocyon cinereoargenteus) in mainland California and high Bartonella seroprevalence was reported in island foxes (U. litorralis), especially from Santa Cruz and Santa Rosa Islands. As a follow-up study, the objectives were to determine the prevalence of Bartonella bacteremia and seropositivity and to identify the Bartonella species infecting a convenience sample of 51 island foxes living on Santa Rosa Island. Using an immuno-fluorescence antibody test directed against B.v.b and Bartonella clarridgeiae (B.c.), used as a substitute for B.r., the overall antibody prevalence was 62.7% with 16 (31.4%) foxes seropositive for B.c. only, 5 (9.8%) for B.v.b. only, and 11 (21.6%) for both antigens. B.v.b. was isolated from 6 (11.8%) foxes using blood culture medium. An additional seropositive fox tested PCR positive for B.v.b. and 3 other seropositive foxes tested PCR positive for B. rochalimae. All of the isolated B.v.b. colonies and the B.v.b. PCR positive sample belonged to type III, the same type found to infect mainland gray foxes. Therefore, Bartonella infection is widespread within this island fox population with evidence for B.v.b. type III reservoir host-specificity. Presence of B. rochalimae in the Channel Islands has been detected for the first time using PCR.     

The immunologic response of dogs to Bartonella vinsonii subspecies berkhoffii antigens: as assessed by Western immunoblot analysis.

Bartonella vinsonii subspecies berkhoffii is a recently recognized zoonotic pathogen that causes endocarditis, granulomatous rhinitis, and granulomatous lymphadenitis in dogs. Isolation of B. vinsonii (berkhoffii) from blood or tissue samples is frequently unsuccessful; therefore, diagnosis is primarily dependent on serologic or molecular testing modalities. Because previous canine serologic studies have used an indirect immunofluorescence assay (IFA), without Western immunoblot (WI) confirmation, the overall objective of this study was to examine the diagnostic use of WI for confirmation of B. vinsonii (berkhoffii) infection in dogs. To confirm that agar-grown and cell culture-grown organisms yielded similar patterns of WI antigenic protein recognition, the 2 preparations were compared using IFA-reactive sera obtained from dogs experimentally infected with B. vinsonii (berkhoffii). Temporal changes in the pattern of antigenic protein recognition were characterized using sera obtained from dogs at various time points after experimental B. vinsonii (berkhoffii) infection. The specificity of B. vinsonii (berkhoffii) WI was examined by testing canine sera that were reactive to B. henselae, B. clarridgeiae, Ehrlichia canis, Rickettsia rickettsii, Babesia canis, Anaplasma phagocytophilum (previously E. equi), or Brucella canis antigens. Clinical accessions including serum samples obtained from B. vinsonii (berkhoffii) culture-positive dogs and B. vinsonii (berkhoffii) culture-negative dogs that were IFA seroreactive to B. vinsonii (berkhoffii) antigens were examined by WI. The results of this study indicate that WI using agar-grown or cell culture-grown B. vinsonii (berkhoffii) antigens produce identical patterns of antigenic protein recognition. After experimental infection, there is a progressive increase in the number of antigenic proteins that are recognized by WI, with the 33-kD antigen representing the first and the most persistent antigen recognized by B. vinsonii (berkhoffii)-infected dogs. Regarding specificity, sera from dogs that were reactive to various heterologous antigens did not recognize B. vinsonii (berkhoffii) antigens by IFA or WI, and sera from dogs experimentally infected with B. henselae did not recognize B. vinsonii (berkhoffii) antigens by WI. Regarding clinical accessions, there was good agreement between B. vinsonii (berkhoffii) IFA test results and WI analysis. Western immunoblot analysis can be used to detect or confirm exposure to B. vinsonii (berkhoffii) in dogs.     

Seroepidemiology of Bartonella vinsonii subsp berkhoffii exposure among healthy dogs

OBJECTIVE: To determine seroprevalence of antibodies to Bartonella vinsonii subsp berkhoffii and risk factors for seropositivity among working dogs owned by the US government. DESIGN: Cross-sectional study. ANIMALS: 1,872 dogs. PROCEDURE: An ELISA was used to detect antibodies to B vinsonii subsp berkhoffii. RESULTS: Antibodies to B vinsonii subsp berkhoffii were detected in 162 dogs (8.7%; 95% confidence interval, 7.4 to 10.0%). Dogs living in the southeast, plains states, southwest, and south-central were significantly more likely to be seropositive than were dogs living in other regions of the United States. German Shepherd-type dogs were significantly less likely to be seropositive than were dogs of other breeds, and dogs entering training programs or that had been rejected from a training program were significantly more likely to be seropositive than were dogs used for narcotics detection and dogs trained to patrol or detect explosives. Dogs used by the border patrol or Federal Aviation Administration were more likely to be seropositive than were dogs used by the Department of Defense or customs service. Odds that dogs would be seropositive were significantly higher for dogs stationed in the southern United States, the northeastern United States, or a foreign country, compared with dogs stationed in all other regions of the United States. CONCLUSIONS AND CLINICAL RELEVANCE: Overall, 8.7% of this diverse group of healthy dogs was found to be seropositive for antibodies to B vinsonii subsp berkhoffii, and seropositivity rates were associated with location, suggesting either that there are multiple vectors for the organism or that the major vector for the organism depends on geographic and environmental factors.     

Immunopathology of Bartonella vinsonii (berkhoffii) in experimentally infected dogs.

Following natural infection with Bartonella, dogs and humans develop comparable disease manifestations including endocarditis, peliosis hepatis, and granulomatous disease. As the immunologic response to infection in these hosts has not been clearly established, data presented here was derived from the experimental infection of six specific pathogen free (SPF) beagles with a known pathogenic strain of Bartonella. Six dogs were inoculated intravenously with 10(9)cfu of B. vinsonii ssp. berkhoffii and six control dogs were injected intravenously with an equivalent volume of sterile saline. Despite production of substantial levels of specific antibody, blood culture and molecular analyses indicated that Bartonella established chronic infection in these dogs. Flow cytometric analysis of monocytes indicated impaired bacterial phagocytosis during chronic Bartonella infection. There was also a sustained decrease in the percentage of CD8+ lymphocytes in the peripheral blood. Moreover, modulation of adhesion molecule expression (downregulation of L-selectin, VLA-4, and LFA-1) on CD8+ lymphocytes suggested quantitative and qualitative impairment of this cell subset in Bartonella-infected dogs. When compared with control dogs, flow cytometric analysis of lymph node (LN) cells from B. vinsonii infected dogs revealed an expanded population of CD4+ T cells with an apparent na?ve phenotype (CD45RA+/CD62L+/CD49D(dim)). However, fewer B cells from infected dogs expressed cell-surface MHC II, implicating impaired antigen presentation to helper T cells within LN. Taken together, results from this study indicate that B. vinsonii establishes chronic infection in dogs which may result in immune suppression characterized by defects in monocytic phagocytosis, an impaired subset of CD8+ T lymphocytes, and impaired antigen presentation within LN.     

Cyclic CD8+ lymphopenia in dogs experimentally infected with Bartonella vinsonii subsp. berkhoffii

Until recently, it was presumed that Bartonella vinsonii only infected voles, a species of North American rodents. In April of 1993, however, our laboratory isolated a novel subspecies of B. vinsonii (B. vinsonii subsp. berkhoffii) from the blood of a dog diagnosed with vegetative valvular endocarditis. Subsequently, based on a seroepidemiologic survey of dogs from North Carolina and Virginia presenting for a variety of medical problems, we found evidence supporting a potentially important association between B. vinsonii and Ehrlichia canis co-infection in dogs. In the following study, eight dogs were infected with B. vinsonii: four specific pathogen free dogs and four dogs that had previously been infected with E. canis. Flow cytometric analysis of peripheral blood lymphocytes revealed a cyclic elevation of the CD4/CD8 T-cell ratio that correlated with cyclic CD8+ lymphopenia in all dogs infected with B. vinsonii, regardless of prior exposure to E. canis.     

Aortic valvular endocarditis caused by Bartonella vinsonii subsp. berkhoffii in 2 dogs presenting for fulminant pulmonary edema

Objective: To describe the clinical course of 2 dogs that presented with fulminant cardiogenic pulmonary edema due to aortic valvular endocarditis caused by Bartonella vinsonii subsp. berkhoffii. Case series summary: Two dogs that were presented for respiratory distress had severe pulmonary infiltrates. Mechanical ventilation was required to support the dogs while the cause of the infiltrates was determined. The diagnosis of cardiogenic edema was made based on echocardiographic findings of aortic valve vegetation and severe aortic valvular regurgitation. Values obtained from pulmonary artery catheterization supported this diagnosis. Both dogs were euthanized, one within 24 hours of admission due to severe aortic regurgitation thought to be untreatable, and the other 9 days after admission due to the development of acute renal failure. Histological evaluation of the aortic valves, serology, and polymerase chain reaction confirmed Bartonella vinsonii subsp. berkhoffii as the cause of the aortic endocarditis. New or unique information provided: In medium‐to‐large breed dogs presenting with fulminant pulmonary edema, aortic valvular endocarditis due to Bartonella spp. should be considered as a causal agent.     

Canine bartonellosis: serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii

The purpose of this study was to determine the serological and molecular prevalence of Bartonella spp. infection in a sick dog population from Brazil. At the S?o Paulo State University Veterinary Teaching Hospital in Botucatu, 198 consecutive dogs with clinicopathological abnormalities consistent with tick-borne infections were sampled. Antibodies to Bartonella henselae and Bartonella vinsonii subsp. berkhoffii were detected in 2.0% (4/197) and 1.5% (3/197) of the dogs, respectively. Using 16S-23S rRNA intergenic transcribed spacer (ITS) primers, Bartonella DNA was amplified from only 1/198 blood samples. Bartonella seroreactive and/or PCR positive blood samples (n=8) were inoculated into a liquid pre-enrichment growth medium (BAPGM) and subsequently sub-inoculated onto BAPGM/blood-agar plates. PCR targeting the ITS region, pap31 and rpoB genes amplified B. henselae from the blood and/or isolates of the PCR positive dog (ITS: DQ346666; pap31 gene: DQ351240; rpoB: EF196806). B. henselae and B. vinsonii subsp. berkhoffii (pap31: DQ906160; rpoB: EF196805) co-infection was found in one of the B. vinsonii subsp. berkhoffii seroreactive dogs. We conclude that dogs in this study population were infrequently exposed to or infected with a Bartonella species. The B. henselae and B. vinsonii subsp. berkhoffii strains identified in this study are genetically similar to strains isolated from septicemic cats, dogs, coyotes and human beings from other parts of the world. To our knowledge, these isolates provide the first Brazilian DNA sequences from these Bartonella species and the first evidence of Bartonella co-infection in dogs.     

Spatial analysis of Yersinia pestis and Bartonella vinsonii subsp. berkhoffii seroprevalence in California coyotes (Canis latrans)

Zoonotic transmission of sylvatic plague caused by Yersinia pestis occurs in California, USA. Human infections with various Bartonella species have been reported recently. Coyotes (Canis latrans) are ubiquitous throughout California and can become infected with both bacterial agents, making the species useful for surveillance purposes. This study examined the geographic distribution of 863 coyotes tested for Y. pestis and Bartonella vinsonii subsp. berkhoffii serologic status to gain insight into the natural history of B. vinsonii subsp. berkhoffii and to characterize the spatial distribution of the two agents.

We found 11.7% of specimens positive to Y. pestis and 35.5% positive to B. vinsonii subsp. berkhoffii. The two pathogens had distinct spatial clusters: Y. pestis was more prevalent in eastern portions of the state and B. vinsonii subsp. berkhoffii in coastal regions. Prevalence of Y. pestis increased with increasing elevation, whereas prevalence of B. vinsonii subsp. berkhoffii decreased with increasing elevation. There were differences in the proportions of positive animals on a yearly basis to both pathogens.     

Movement of Rhipicephalus sanguineus adults between co-housed dogs during active feeding

Adult male ticks have been shown capable of experimental acquisition and transmission of tick-borne pathogens without requiring a molt. To determine the ecological relevance of this intrastadial transmission route, we evaluated the extent to which actively feeding male Rhipicephalus sanguineus move naturally between co-housed infested dogs. Dogs (n=4) were infested with single color-coded ticks, individually housed in tick-confinement cages for 48 h while infestations established, and then each dog examined and the ticks present counted. Dogs were then co-housed in a large, group pen for an additional 7 (study 1) or 5 (study 2) days. In the first study, sex ratios were adjusted to encourage migration, with two dogs receiving predominantly female ticks and two dogs receiving all male ticks. In the second study, each dog received a ratio of ticks that parallels that found in natural infestations (4:1 male to female). Results showed that ticks readily migrated between infested, co-housed dogs. Rates of immigration, defined as the percentage of ticks previously attached to one dog that moved onto another dog, ranged from 0 to 46% (mean=31.1% study 1; 9.4% study 2). Emigration rates, defined as the number of ticks initially infesting one dog that moved to another dog, averaged 35.2% in study 1 and 10.8% in study 2 (3.6-67.6%). Movement of adult ticks between dogs represents a naturally occurring form of interrupted feeding, a strategy which has been shown to shorten the feeding time necessary to allow transmission of pathogens. In ticks that readily detach from one host and reattach to a second host to resume feeding, replication of any pathogens present has already been initiated and therefore the same delay in transmission seen in ticks attached to a host for the first time may not occur.     

Bartonella vinsonii subspecies berkhoffi as a possible cause of anterior uveitis and choroiditis in a dog

A 2-year old, neutered, female spaniel mixed breed was referred to the North Carolina State University Veterinary Teaching Hospital for evaluation of bilateral anterior uveitis. The dog was febrile and, in addition to anterior uveitis, multifocal hyporeflective lesions were present in the tapetal fundus of both eyes. The antibody titer for Bartonella vinsonii subspecies berkhoffi was positive (1 : 512). Aqueous paracentesis was performed for PCR in an attempt to detect B. vinsonii in the eye but was unsuccessful. The ocular manifestations of Bartonella infection in humans are currently expanding as more sensitive serologic and PCR techniques are being developed to identify Bartonella spp. In addition to optic neuritis and neuroretinitis, retinochoroidal lesions are one of the most common manifestations of B. henselae infection, and are frequently accompanied by vitreous or anterior segment inflammation. Diagnosis of a Bartonella infection in humans can be made on serology alone, in conjunction with ocular examination findings. The ultimate proof of B. vinsonii (berkhoffi) as a direct cause of ocular disease would be detection of the infectious agent in the eye. However, it is unknown at this time whether Bartonella causes ocular disease primarily, secondarily via an autoimmune reaction, or both. Due to the difficulties associated with culture of Bartonella spp. and the limitations of PCR, serology is currently the most useful tool for screening dogs for possible Bartonella spp. infection. In the case presented here, even though the PCR was negative, the clinical signs of anterior uveitis and choroiditis might reasonably be associated with B. vinsonii (berkhoffi) seroreactivity, which was repeatable on three separate occasions. Clinical improvement was also accompanied by a post-treatment decrease in B. vinsonii (berkhoffi) seroreactivity, potentially supporting resolution of Bartonella infection in this dog. This is the first reported case of a possible association between uveitis, choroiditis and Bartonella infection in the dog, without clinical manifestations of other organ or tissue involvement. Future studies based on PCR analysis of intraocular fluids may clarify the involvement of B. vinsonii (berkhoffi) in dogs with intraocular inflammatory disease. Furthermore, performing fluorescein angiography in dogs with elevated Bartonella titers may also prove useful in the identification and characterization of lesions.     

Transmission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus).

Two strains of Rhipicephalus sanguineus acquired Ehrlichia canis by feeding as either larvae or nymphs on acutely infected dogs and, in subsequent instars, transmitted the agent to normal dogs. Three strains of R sanguineus transmitted E canis as adults after their larval and nymphal stages fed on infected dogs. More than 400 adult female ticks were fed on infected dogs as larvae or nymphs or both, but none transmitted E canis transovarially.     

Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.     

Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada

To identify the tick-borne pathogens in dogs from Grenada, we conducted a serologic survey for Ehrlichia canis in 2004 (104 dogs) and a comprehensive serologic and molecular survey for a variety of tick-borne pathogens in 2006 (73 dogs). In 2004 and 2006, 44 and 32 dogs (42.3% and 43.8%) were seropositive for E. canis, respectively. In 2006, several tick-borne pathogens were identified by serology and PCR. DNA of E. canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, and Bartonella sp. were identified in 18 (24.7%), 14 (19.2%), 5 (7%), 5 (7%), and 1 (1.4%) dogs, respectively. Six (8.2%) dogs were seropositive for Bartonella vinsonii subsp. berkhoffii. All dogs were seronegative and PCR-negative for Rickettsia spp. Coinfection with two or three pathogens was observed in eight dogs. Partial 16S rRNA E. canis and A. platys sequences were identical to sequences in GenBank. Partial 18S rRNA gene sequences from the Grenadian H. canis were identical to each other and had one possible mismatch (ambiguous base) from H. canis detected from Spain and Brazil. Grenadian B. c. vogeli sequences were identical to B. c. vogeli from Brazil and Japan. All of the detected pathogens are transmitted, or suspected to be transmitted, by Rhipicephalus sanguineus. Results of this study indicate that dogs from Grenada are infected with multiple tick-borne pathogens; therefore, tick-borne diseases should be included as differentials for dogs exhibiting thrombocytopenia, leukopenia, fever, or lethargy. One pathogen, E. canis, is also of potential public health significance.     

Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.