Dynamics of Co-Infection with Bartonella henselae Genotypes I and II in Naturally Infected Cats: Implications for Feline Vaccine Development.

Bartonella henselae is an emerging bacterial pathogen causing cat-scratch disease and potentially fatal bacillary angiomatosis in humans. Bacteremic cats constitute a large reservoir for human infection. Although feline vaccination is a potential strategy to prevent human infection, selection of appropriate B. henselae strains is critical for successful vaccine development. Two distinct genotypes of B. henselae (type I, type II) have been identified and are known to co-infect the feline host, but very little is known about the interaction of these two genotypes during co-infection in vivo. To study the in vivo dynamics of type I and type II co-infection, we evaluated three kittens that were naturally flea-infected with both B. henselae type I and type II. Fifty individual bloodstream isolates from each of the cats over multiple time points were molecularly typed (by 16S rRNA gene sequencing), to determine the prevalence of the two genotypes over 2 years of persistent infection. We found that both B. henselae genotypes were transmitted simultaneously to each cat via natural flea infestation, resulting in mixed infection with both genotypes. Although the initial infection was predominately type I, after the first 2 months, the isolated genotype shifted to exclusively type II, which then persisted with a relapsing pattern. Understanding the parameters of protection against both genotypes of B. henselae, and the competitive dynamics in vivo between the two genotypes, will be critical in the development of a successful feline vaccine that can ultimately prevent B. henselae transmission to human contacts.

Bartonella Infection among Cats Adopted from a San Francisco Shelter, Revisited.

Bartonella infection among cats from shelters can pose a health risk to adopters. Bartonella henselae is the most common species, with B. clarridgeiae and B. koehlerae being less common. The lower rates of infection by the latter species may reflect their rarity or an inefficiency of culture techniques. To assess the incidence of infection, blood cultures, serology, and PCR testing were performed on 193 kittens (6 to 17 weeks old) and 158 young adult cats (5 to 12 months old) from a modern regional shelter. Classical B. henselae culture medium was compared to a medium supplemented with insect cell growth factors. Bartonella colonies were isolated from 115 (32.8%) animals, including 50 (25.9%) kittens and 65 (41.1%) young adults. Therefore, young adults were twice as likely to be culture positive as kittens. Enhanced culture methods did not improve either the isolation rate or species profile. B. henselae was isolated from 40 kittens and 55 young adults, while B. clarridgeiae was cultured from 10 animals in each group. B. koehlerae was detected in one young adult by PCR only. B. henselae genotype II was more commonly isolated from young adults, and genotype I was more frequently isolated from kittens. Kittens were 4.7 times more likely to have a very high bacterial load than young adults. A significantly higher incidence of bacteremia in the fall and winter than in the spring and summer was observed. Bartonella antibodies were detected in 10% (19/193) of kittens and 46.2% (73/158) of young adults, with culture-positive kittens being 9.4 times more likely to be seronegative than young adults.

Zoonotic Bartonella species in cardiac valves of healthy coyotes, California, USA.

We investigated whether Bartonella spp. could cause endocarditis in coyotes or localize to cardiac valves before lesions develop. Bartonella DNA was amplified more often from coyote cardiac valves than spleen. Bartonella infection apparently leads to cardiac valve tropism, which could cause endocarditis, an often lethal complication in mammals, including humans.

Hemin binding protein C is found in outer membrane vesicles and protects Bartonella henselae against toxic concentrations of hemin.

Bartonella species are gram-negative, emerging bacterial pathogens found in two distinct environments. In the gut of the obligately hematophagous arthropod vector, bartonellae are exposed to concentrations of heme that are toxic to other bacteria. In the bloodstream of the mammalian host, access to heme and iron is severely restricted. Bartonellae have unusually high requirements for heme, which is their only utilizable source of iron. Although heme is essential for Bartonella survival, little is known about genes involved in heme acquisition and detoxification. We developed a strategy for high-efficiency transposon mutagenesis to screen for genes in B. henselae heme binding and uptake pathways. We identified a B. henselae transposon mutant that constitutively expresses the hemin binding protein C (hbpC) gene. In the wild-type strain, transcription of B. henselae hbpC was upregulated at arthropod temperature (28°C), compared to mammalian temperature (37°C). In the mutant strain, temperature-dependent regulation was absent. We demonstrated that HbpC binds hemin and localizes to the B. henselae outer membrane and outer membrane vesicles. Overexpression of hbpC in B. henselae increased resistance to heme toxicity, implicating HbpC in protection of B. henselae from the toxic levels of heme present in the gut of the arthropod vector. Experimental inoculation of cats with B. henselae strains demonstrated that both constitutive expression and deletion of hbpC affect the ability of B. henselae to infect the cat host. Modulation of hbpC expression appears to be a strategy employed by B. henselae to survive in the arthropod vector and the mammalian host.

Multiple locus variable number tandem repeat analysis for the characterization of wild feline Bartonella species and subspecies.

Bartonella genus includes an increasing number of species and subspecies, especially among wild felids, the positioning of which, with regards to the zoonotic species Bartonella henselae, is important to determine. The aim of this study was to test the ability of a molecular typing technique to distinguish between various Bartonella isolates obtained from four different species of free-ranging and captive wild felids and to identify key profiles or markers allowing differentiating them from each other and/or from B. henselae or B. koehlerae. A molecular typing technique for B. henselae based on the polymorphism of variable number tandem repeat units (VNTR) called MLVA (Multiple Locus VNTR Analysis) was applied to 24 Bartonella isolates from free-ranging or captive wild felids, 19 of which were obtained from California and five from three countries in Southern Africa, and compared with 49 B. henselae isolates from cats, dog or humans from the United States including the human ATCC (American Type Culture Collection) reference strain, B. henselae Houston 1. MLVA allowed distinguishing Bartonella isolates from wild felids from either B. henselae or B. koehlerae. We confirmed infection of semi-captive cheetahs with an isolate similar to a Californian bobcat isolate. MLVA also confirmed the unique profile of a free-ranging cheetah isolate from Namibia. Specific profiles were observed making MVLA a useful identification/classification tool of these wild felid isolates and suggesting that they are highly adapted to a specific feline reservoir. Finally, circulation of B. henselae isolates between domestic cats, wild felids and humans is likely occurring, based on the close allelic profiles of some isolates.

Bartonella rochalimae in raccoons, coyotes, and red foxes.

To determine additional reservoirs for Bartonella rochalimae, we examined samples from several wildlife species. We isolated B. rochalimae from 1 red fox near Paris, France, and from 11 raccoons and 2 coyotes from California, USA. Co-infection with B. vinsonii subsp. berkhoffii was documented in 1 of the coyotes.

Infective endocarditis in a dog and the phylogenetic relationship of the associated "Bartonella rochalimae" strain with isolates from dogs, gray foxes, and a human.

The first case of canine endocarditis caused by "Bartonella rochalimae" is reported. By PCR-restriction fragment length polymorphism, sequence, and phylogenetic analyses, Bartonella isolates from a dog with endocarditis, 22 gray foxes, and three dogs, described as B. clarridgeiae like, were confirmed to belong to the new species "B. rochalimae," suggesting canids as the natural reservoir.

Seroprevalence of Bartonella spp. in the endangered island fox (Urocyon littoralis).

Bartonella clarridgeiae-like strains, presently B. rochalimae, were isolated in gray foxes (Urocyon cinereoargenteus) in mainland California. The objective of this study was to investigate the presence of Bartonella infection in the endangered island fox (Urocyon littoralis) found only on the Channel Islands off the Californian coast. Between 2001 and 2004, 263 serum samples were collected. Antibodies against Bartonella vinsonii subsp. berkhoffii (Bvb) and B. clarridgeiae (Bc) were detected using an immuno-fluorescence antibody test. Sixty-eight (25.8%) and 73 (27.7%) foxes were positive for Bvb and Bc, respectively. Seroprevalence was the highest on Santa Cruz Island (n=36, Bvb=80.5%; Bc=86.1%) and Santa Rosa Island (n=38, Bvb=52.6%; Bc=65.8%). On San Miguel and San Clemente Islands, seroprevalence for Bvb was 20% and 17.3% respectively, and 0% and 21.3% for Bc. Prevalence ranged between 0% and 5.1% on San Nicolas and Santa Catalina Islands. Foxes from Santa Rosa and Santa Cruz Islands were 17.5 times and 31.5 times as likely to be seropositive for Bvb and Bc than foxes from the other islands (95% confidence interval [95% CI]=8.5, 36.7; 14.4, 70.2). There were no statistically significant differences for presence of Bartonella antibodies by sex, age, origin (captive vs. wild) or year of blood collection. This is the first report of exposure to Bartonella in the island fox population. Further studies are necessary to isolate these bacteria from foxes and determine factors associated with presence or absence of Bartonella species on specific islands.

P26-based serodiagnosis for Bartonella spp. infection in cats.

Bartonella henselae P26 has been identified as an immunodominant antigen expressed during feline infection. We used antisera from cats experimentally infected with B. henselae (n = 6), B. clarridgeiae (n = 4), or B. koehlerae (n = 2) and from a sample of naturally infected cats (B. henselae, n = 34; B. clarridgeiae, n = 1) to evaluate recombinant P26 (rP26) as a serodiagnostic antigen. Immunoblots using antisera from cats infected with B. henselae and B. clarridgeiae reacted strongly with rP26, whereas B. koehlerae antisera did not. A capture ELISA was designed to evaluate the kinetics of rP26 IgG in sera from experimentally infected cats. For B. henselae and B. clarridgeiae antisera, the kinetic profiles of reactivity were similar for rP26 capture ELISA and Bartonella spp. indirect fluorescence assay. However, for B. koehlerae antisera, reactivity in rP26 capture ELISA was consistently low. The serodiagnostic potential of rP26 capture ELISA was evaluated using sera from cats with known Bartonella sp. exposure histories. All 24 (100%) uninfected cats were seronegative, and 33 of 35 (94.3%) cats bacteremic for Bartonella spp. were seropositive. We propose that rP26-based serology can serve as a useful adjunct tool for the diagnosis of feline infection with B. henselae and B. clarridgeiae, but it may not be useful for feline infection with B. koehlerae.

Evidence of multiple zoonotic agents in a wild rodent community in the eastern Sierra Nevada.

This study aimed to describe the occurrence of Yersinia pestis, Rickettsia rickettsii, Anaplasma phagocytophilum, and ectoparasites in a wild rodent community in the eastern Sierra Nevada. From May to September 2006, rodents were live-trapped, examined for ectoparasites, and blood was collected. All rodents were serologically tested for antibodies to Y. pestis, R. rickettsii, and A. phagocytophilum; in addition, blood samples and ectoparasites were tested by PCR to detect the presence of these zoonotic agents. Overall, 89 rodents, 46 fleas, and four ticks were collected. Antibody prevalence rates observed for rodents were 14% for R. rickettsii or antigenically related spotted-fever group rickettsiae, and 8% for A. phagocytophilum. No samples were positive for antibodies to Y. pestis. Positive PCR results included one yellow-pine chipmunk for Y. pestis (CT=32.8), one golden-mantled ground squirrel for R. rickettsii (CT=33), and one flea found to be co-infected with both R. rickettsii (CT=17) and A. phagocytophilum (CT=36). The results of this study provide evidence of multiple zoonoses overlapping within a single, located rodent community.

Bartonella henselae in small Indian mongooses (Herpestes auropunctatus) from Grenada, West Indies.

Many mammals are established hosts for the vector borne bacterial genus, Bartonella. Small Indian mongooses (Herpestes auropunctatus) have only been reported as a possible host for Bartonella henselae in southern Japan. Confirming Bartonella presence in mongooses from other regions in the world may support their role as potential reservoirs of this human pathogen. Specifically, documenting Bartonella in Caribbean mongooses would identify a potential source of zoonotic risk with mongoose-human contact in the New World. Using serological and molecular techniques, we investigated B. henselae DNA and specific antibody prevalence in 171 mongooses from all six parishes in Grenada, West Indies. Almost a third (32.3%, 54/167) of the tested mongooses were B. henselae seropositive and extracted DNA from 18/51 (35.3%) blood pellets were PCR positive for the citrate synthase (gltA) and/or the ß subunit of RNA polymerase (rpoB) genes. All sequences were identical to B. henselae genotype I, as previously reported from Japan. This study confirms the role of small Indian mongooses as a natural reservoir of B. henselae in the New World.

Detection of Bartonella species, including Candidatus Bartonella ovis sp. nov, in ruminants from Mexico and lack of evidence of Bartonella DNA in saliva of common vampire bats (Desmodus rotundus) predating on them.

Bartonella spp. have been identified in many bat species worldwide, including the zoonotic species, Candidatus Bartonella mayotimonensis. The common vampire bat (Desmodus rotundus) preys preferentially on livestock in Latin America and is frequently infected with Bartonella spp. To determine the potential role of D. rotundus in transmitting Bartonella to livestock, common vampire bats and bat-bitten domestic ruminants from Mexico were tested for Bartonella infection by blood culture or conventional PCR. Furthermore, to explore the possibility of bite transmission during blood feeding, saliva swabs from 35 D. rotundus known to be either Bartonella bacteremic (N = 17) or blood culture negative (N = 18) were tested by PCR to detect the presence of Bartonella DNA. Twenty (17.1%) of 117 sheep and 16 (34.8%) of 46 cattle were Bartonella bacteremic by PCR testing. However, none of them were infected with Bartonella strains previously isolated from vampire bats and none of the 35 D. rotundus saliva swabs tested were PCR positive for Bartonella. All but two animals among those which were Bartonella culture and/or PCR positive, were infected with either B. bovis (cattle) or B. melophagi (sheep). Two sheep were infected by a possible new species, Candidatus Bartonella ovis, being phylogenetically closer to B. bovis than B. melophagi. This study does not support the role of D. rotundus as a reservoir of Bartonella species infecting livestock, which could be transmitted via bite and blood feeding and therefore suggest limited risk of zoonotic transmission of Bartonella from common vampire bats to humans.

Molecular Detection of Bartonella Species in Blood-Feeding Bat Flies from Mexico.

Bartonellae are emerging blood-borne bacteria that have been recovered from a wide range of mammalian species and arthropod vectors around the world. Bats are now recognized as a potential wildlife reservoir for a diverse number of Bartonella species, including the zoonotic Candidatus B. mayotimonensis. These bat-borne Bartonella species have also been detected in the obligate ectoparasites of bats, such as blood-feeding flies, which could transmit these bacteria within bat populations. To better understand this potential for transmission, we investigated the relatedness between Bartonella detected or isolated from bat hosts sampled in Mexico and their ectoparasites. Bartonella spp. were identified in bat flies collected on two bat species, with the highest prevalence in Trichobius parasiticus and Strebla wiedemanni collected from common vampire bats (Desmodus rotundus). When comparing Bartonella sequences from a fragment of the citrate synthase gene (gltA), vector-associated strains were diverse and generally close to, but distinct from, those recovered from their bacteremic bat hosts in Mexico. Complete Bartonella sequence concordance was observed in only one bat-vector pair. The diversity of Bartonella strains in bat flies reflects the frequent host switch by bat flies, as they usually do not live permanently on their bat host. It may also suggest a possible endosymbiotic relationship with these vectors for some of the Bartonella species carried by bat flies, whereas others could have a mammalian host.

Experimental infection of domestic cats with passaged genotype I Bartonella henselae.

The influence of in vitro passage on Bartonella henselae pathogenesis in cats has not been thoroughly evaluated. Our objective was to examine the bacterial kinetics and humoral immune responses in cats experimentally infected with three different in vitro passages of B. henselae F1, a genotype I strain of feline origin. The F1 strain was in vitro passaged 20 and 40 times, and each was inoculated into a group of 5 cats. The kinetics of bacteremia and the feline humoral immune response to bacterial antigens were compared to a previous study involving a group of six cats inoculated with the original F1 strain. Among the three groups of cats, the kinetics of bacteremia profiles and the humoral immune responses to B. henselae lysates were similar. The influence of passage on bacterial membrane proteins was examined. In vitro passage altered the expression of 4/17 (23.5%) bacterial membrane proteins and 6/15 (40%) bacterial membrane antigens. An association between poor seroreactivity to three lysate antigens (15-, 18- and 45kDa), prolonged bacteremia and decreased serum bactericidal activity was noted. Our data show that in vitro passage of B. henselae did not alter the kinetics of bacteremia, including the occurrence of relapsing bacteremia, in experimentally infected cats. This suggests that highly passaged strains may not be suitable for future vaccination studies. Furthermore, in vitro passage results in phenotypic and antigenic changes in the bacterial membrane protein profile, which warrants caution in the interpretation of studies involving passaged B. henselae strains.

Bartonella henselae infection in splenectomized domestic cats previously infected with hemotropic Mycoplasma species.

Cat scratch disease is caused by Bartonella henselae and the domestic cat represents its main reservoir. In immunocompromised patients, infection with B. henselae is characterized by more severe clinical forms than in non-immunocompromised individuals. The objective of the present study was to investigate the characteristics of B. henselae (Houston-I strain) infection in four splenectomized and three non-splenectomized cats, five of which were chronically infected with 'Candidatus Mycoplasma haemominutum'. No major clinical signs were observed in either group of cats. Cats in both splenectomized and non-splenectomized groups became bacteremic within a week post-inoculation. Although bacteremia was on average 10 days longer in the splenectomized cats, that difference was not statistically significant (P=0.72). In both groups, the level of bacteremia peaked within the same time frame; however, the level of bacteremia was about 10-fold higher in the splenectomized cats (P=0.007). Such a difference could be associated with a reduced immune response to the infection, especially a reduced ability to phagocytize Bartonella organisms in the splenectomized cats. Concurrent infection with 'Candidatus M. haemominutum' did not appear to alter the course of infection.

Prevalence and Potential Risk Factors for Bartonella Infection in Tunisian Stray Dogs.

Bartonellae are blood-borne and vector-transmitted pathogens, some are zoonotic, which have been reported in several Mediterranean countries. Transmission from dogs to humans is suspected, but has not been clearly demonstrated. Our objectives were to determine the seroprevalence of Bartonella henselae, Bartonella vinsonii subsp. berkhoffii, Bartonella clarridgeiae, and Bartonella bovis (as a proxy for Candidatus Bartonella merieuxii) in stray dogs from Tunisia, identify the Bartonella species infecting the dogs and evaluate potential risk factors for canine infection. Blood samples were collected between January and November 2013 from 149 dogs in 10 Tunisian governorates covering several climatic zones. Dog-specific and geographic variables were analyzed as potential risk factors for Bartonella spp. seropositivity and PCR-positivity. DNA was extracted from the blood of all dogs and tested by PCR for Bartonella, targeting the ftsZ and rpoB genes. Partial sequencing was performed on PCR-positive dogs. Twenty-nine dogs (19.5%, 95% confidence interval: 14-27.4) were seropositive for one or more Bartonella species, including 17 (11.4%) for B. vinsonii subsp. berkhoffii, 14 (9.4%) for B. henselae, 13 (8.4%) for B. clarridgeiae, and 7 (4.7%) for B. bovis. Statistical analysis revealed a few potential risk factors, mainly dog's age and breed, latitude and average winter temperature. Twenty-two (14.8%) dogs, including 8 of the 29 seropositive dogs, were PCR-positive for Bartonella based on the ftsZ gene, with 18 (81.8%) of these 22 dogs also positive for the rpoB gene. Partial sequencing showed that all PCR-positive dogs were infected with Candidatus B. merieuxii. Dogs from arid regions and regions with cold average winter temperatures were less likely to be PCR-positive than dogs from other climatic zones. The widespread presence of Bartonella spp. infection in Tunisian dogs suggests a role for stray dogs as potential reservoirs of Bartonella species in Tunisia.

Bartonella infection in domestic cats and wild felids.

Bartonella are vector-borne, fastidious Gram-negative bacteria causing persistent bacteremia in their reservoir hosts. Felids represent a major reservoir for several Bartonella species. Domestic cats are the main reservoir of B. henselae, the agent of cat-scratch disease. Prevalence of infection is highest in warm and humid climates that are optimal for the survival of cat fleas, as fleas are essential for the transmission of the infection. Flea feces are the likely infectious substrate. Prevalence of B. henselae genotypes among cat populations varies worldwide. Genotype Houston I is more prevalent in the Far East and genotype Marseille is dominant in western Europe, Australia, and the western United States. Cats are usually asymptomatic, but uveitis, endocarditis, neurological signs, fever, necrotic lesions at the inoculation site, lymphadenopathy, and reproductive disorders have been reported in naturally or experimentally infected cats. Domestic cats are also the reservoir of B. clarridgeiae and co-infection has been demonstrated. B. koehlerae has been isolated from domestic cats, and was identified in cat fleas and associated with a human endocarditis case. B. bovis was isolated from a few cats in the United States and B. quintana DNA was recently identified in a cat tooth. Bartonella spp. have also been isolated from free-ranging and captive wild felids from North America and Africa. Whereas, B. henselae was identified in African lions and a cheetah, some strains specific to these wild cats have also been identified, leading to the concept of a B. henselae group including various subspecies, as previously described for B. vinsonii.

Antibodies to Bartonella vinsonii subsp. Berkhoffii in Moroccan dogs.

Bartonella vinsonii subsp. berkhoffii is a fastidious microorganism that has been recognized as an emerging human and canine pathogen. We report for the first time on the prevalence of antibodies to B. vinsonii subsp. berkhoffii in domestic dogs from Morocco. The overall seroprevalence was 38% (56 of 147 dogs tested). Most of the seropositive dogs were stray dogs from Rabat (36%, 8 of 22) and Khenifra (47%, 47 of 101). Antibodies against B. vinsonii subsp. berkhoffii were found infrequently among pet dogs from Rabat (4%, 1 of 24).

Co-detection of Bartonella henselae, Borrelia burgdorferi, and Anaplasma phagocytophilum in Ixodes pacificus ticks from California, USA.

Presence of Bartonella DNA was explored in 168 questing adult Ixodes pacificus ticks from Santa Cruz County, California. Bartonella henselae type I DNA was amplified from 11 ticks (6.55%); previously, two (1.19%) were found to be infected with Borrelia burgdorferi and five (2.98%) with Anaplasma phagocytophilum. Detection of B. henselae was not dependent on co-infection. The present study offers additional evidence that Ixodes spp. ticks may act as hosts and possibly vectors for B. henselae.

First Pediatric Case of Tularemia after a Coyote Bite.

Bite-transmitted tularemia is a rare event in humans and most of the cases have been associated with cat bites. We report the first pediatric case of tularemia caused by a coyote (Canis latrans) bite. Coyotes can be healthy carriers of Francisella tularensis and transmit this infectious agent through a bite. Pediatricians should be aware of this risk after a carnivore bite and implement appropriate antibiotic therapy, as amoxicillin/clavulanate potassium (Augmentin) may have prolonged the typical two to three days' incubation period commonly observed for tularemia after an animal bite and was not effective in preventing clinical signs in this child. Finally, it emphasizes again the importance of early and late serum samples for appropriate serodiagnostic.