Transmission of Bartonella henselae within Rhipicephalus sanguineus: Data on the Potential Vector Role of the Tick.

Bartonella henselae is a fastidious intraerythrocytic, gram-negative bacteria that causes cat scratch disease in humans. Ixodes ricinus has been confirmed to be a competent vector of B. henselae, and some indirect evidences from clinical cases and epidemiological studies also suggested that some other tick species, including Rhipicephalus sanguineus, may transmit the bacteria. B. henselae has been detected in R. sanguineus but no experimental investigations have been performed to evaluate the vector competency of this tick species regarding B. henselae transmission. To this end, this work aimed to assess the transstadial transmission of B. henselae between larvae and nymphs of R. sanguineus as well as transmission by nymphs infected at the larval stage. Four hundred B. henselae negative larvae were fed with B. henselae-infected blood by using an artificial membrane feeding system. After five days of feeding, B. henselae was detected by PCR in 57.1% (8/14) of engorged larval pools, 66.7% (4/6) of semi-engorged larval pools, and 66.7% (2/3) of larval feces pools. After molting, B. henselae DNA was also detected in 10% (1/10) of nymph pools, but not in tick feces. After a pre-fed step of nymphs infected at the larval stage on non-infected blood meal, B. henselae was detected by PCR in blood sample from the feeder, but no Bartonella colonies could be obtained from culture. These findings showed that B. henselae could be transstadial transmitted from R. sanguineus larvae to nymphs, and also suggest that these nymphs may retransmitted the bacteria through the saliva during their blood meal. This is the first study that validated the artificial membrane feeding system for maintaining R. sanguineus tick colony. It shows the possibility of transstadial transmission of B. henselae from R. sanguineus larvae to nymphs.

A cat's scratch or a wolf's bite?

Bartonella henselae endocarditis mimicking systemic vasculitis has been reported in patients with valvulopathy. Herein, we describe a patient with B. henselae endocarditis involving a prosthetic pulmonic valve with positive anti-dsDNA antibodies misdiagnosed with systemic lupus erythematosus (SLE) based on the revised classification SLE criteria.

The trimeric autotransporter adhesin BadA is required for in vitro biofilm formation by Bartonella henselae.

Bartonella henselae (Bh) is a Gram-negative rod transmitted to humans by a scratch from the common house cat. Infection of humans with Bh can result in a range of clinical diseases including lymphadenopathy observed in cat-scratch disease and more serious disease from persistent bacteremia. It is a common cause of blood-culture negative endocarditis as the bacterium is capable of growing as aggregates, and forming biofilms on infected native and prosthetic heart valves. The aggregative growth requires a trimeric autotransporter adhesin (TAA) called Bartonella adhesin A (BadA). TAAs are found in all Bartonella species and many other Gram-negative bacteria. Using Bh Houston-1, Bh Houston-1 ∆badA and Bh Houston-1 ∆badA/pNS2PTrc badA (a partial complement of badA coding for a truncated protein of 741 amino acid residues), we analyze the role of BadA in adhesion and biofilm formation. We also investigate the role of environmental factors such as temperature on badA expression and biofilm formation. Real-time cell adhesion monitoring and electron microscopy show that Bh Houston-1 adheres and forms biofilm more efficiently than the Bh Houston-1 ∆badA. Deletion of the badA gene significantly decreases adhesion, the first step in biofilm formation in vitro, which is partially restored in Bh Houston-1 ∆badA/pNS2PTrc badA. The biofilm formed by Bh Houston-1 includes polysaccharides, proteins, and DNA components and is susceptible to enzymatic degradation of these components. Furthermore, both pH and temperature influence both badA expression and biofilm formation. We conclude that BadA is required for optimal adhesion, agglutination and biofilm formation.

Prevalence of Bartonella spp. by culture, PCR and serology, in veterinary personnel from Spain.

BACKGROUND: The genus Bartonella includes fastidious, facultative intracellular bacteria mainly transmitted by arthropods and distributed among mammalian reservoirs. Bartonella spp. implicated as etiological agents of zoonoses are increasing. Apart from the classical Bartonella henselae, B. bacilliformis or B. quintana, other species (B. elizabethae, B. rochalimae, B. vinsonii arupensis and B. v. berkhoffii, B. tamiae or B. koehlerae, among others) have also been associated with human and/or animal diseases. Laboratory techniques for diagnosis (culture, PCR assays and serology) usually show lack of sensitivity. Since 2005, a method based on a liquid enrichment Bartonella alphaproteobacteria growth medium (BAPGM) followed by PCRs for the amplification of Bartonella spp. has been developed. We aimed to assess culture, molecular and serological prevalence of Bartonella infections in companion animal veterinary personnel from Spain. METHODS: Each of 89 participants completed a questionnaire. Immunofluorescence assays (IFA) using B. vinsonii berkhoffii (genotypes I, II and III), B. henselae, B. quintana and B. koehlerae as antigens were performed. A cut-off of 1:64 was selected as a seroreactivity titer. Blood samples were inoculated into BAPGM and subcultured onto blood agar plates. Bartonella spp. was detected using conventional and quantitative real-time PCR assays and DNA sequencing. RESULTS: Among antigens corresponding to six Bartonella spp. or genotypes, the lowest seroreactivity was found against B. quintana (11.2%) and the highest, against B. v. berkhoffii genotype III (56%). A total of 27% of 89 individuals were not seroreactive to any test antigen. Bartonella spp. IFA seroreactivity was not associated with any clinical sign or symptom. DNA from Bartonella spp., including B. henselae (n = 2), B. v. berkhoffii genotypes I (n = 1) and III (n = 2), and B. quintana (n = 2) was detected in 7/89 veterinary personnel. PCR and DNA sequencing findings were not associated with clinical signs or symptoms. No co-infections were observed. One of the two B. henselae PCR-positive individuals was IFA seronegative to all tested antigens whereas the other one was not B. henselae seroreactive. The remaining PCR-positive individuals were seroreactive to multiple Bartonella spp. antigens. CONCLUSIONS: High serological and molecular prevalences of exposure to, or infection with, Bartonella spp. were found in companion animal veterinary personnel from Spain. More studies using BAPGM enrichment blood culture and PCR are needed to clarify the finding of Bartonella PCR-positive individuals lacking clinical symptoms.

Bartonella henselae is usually not viable in lymph nodes of patients with cat scratch disease.

Bartonella henselae, the agent of cat scratch disease (CSD), appears to be a common organism responsible for lymphadenitis in both adults and children. There is a very low isolation rate for B. henselae from lymph nodes of patients with CSD. Our objective was to evaluate B. henselae viability in a large series of lymph nodes from patients with CSD. From January to November 2016, we analyzed lymph node biopsy samples from patients diagnosed with CSD. We used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect B. henselae RNA, as well as cultures, histological analyses, and fluorescence in situ hybridization (FISH). We tested 87 lymph nodes positive for B. henselae DNA but only 8 (9%) presented with B. henselae RNA. We did not find a significant difference for the pap threshold cycle (CT) values between RNA-positive and RNA-negative lymph nodes (p = 0.5). Cultures, histological analyses, and FISH were negative for all the tested samples. We provide evidence that B. henselae are not or are rarely viable in most cases in the lymph nodes of patients with CSD.

Infections and cardiovascular disease: is Bartonella henselae contributing to this matter?

Cardiovascular disease is still the major cause of death worldwide despite the remarkable progress in its prevention and treatment. Endothelial progenitor cells (EPCs) have recently emerged as key players of vascular repair and regenerative medicine applied to cardiovascular disease. A large amount of effort has been put into discovering the factors that could aid or impair the number and function of EPCs, and also into characterizing these cells at the molecular level in order to facilitate their therapeutic applications in vascular disease. Interestingly, the major cardiovascular risk factors have been associated with reduced number and function of EPCs. The bacterial contribution to cardiovascular disease represents a long-standing controversy. The discovery that Bartonella henselae can infect and damage EPCs revitalizes the enduring debate about the microbiological contribution to atherosclerosis, thus allowing the hypothesis that this infection could impair the cardiovascular regenerative potential and increase the risk for cardiovascular disease. In this review, we summarize the rationale suggesting that Bartonella henselae could favour atherogenesis by infecting and damaging EPCs, thus reducing their vascular repair potential. These mechanisms suggest a novel link between communicable and non-communicable human diseases, and put forward the possibility that Bartonella henselae could enhance the susceptibility and worsen the prognosis in cardiovascular disease.

Assessment of persistence of Bartonella henselae in Ctenocephalides felis.

Bartonella henselae (Rhizobiales: Bartonellaceae) is a Gram-negative fastidious bacterium of veterinary and zoonotic importance. The cat flea Ctenocephalides felis (Siphonaptera: Pulicidae) is the main recognized vector of B. henselae, and transmission among cats and humans occurs mainly through infected flea feces. The present study documents the use of a quantitative molecular approach to follow the daily kinetics of B. henselae within the cat flea and its excreted feces after exposure to infected blood for 48 h in an artificial membrane system. B. henselae DNA was detected in both fleas and feces for the entire life span of the fleas (i.e., 12 days) starting from 24 h after initiation of the blood meal.

A first Japanese case of Bartonella henselae-induced endocarditis diagnosed by prolonged culture of a specimen from the excised valve.

Bartonella henselae, the causative agent of cat scratch disease, is increasingly recognized as a cause of culture-negative endocarditis. This report describes the first Japanese case, which was diagnosed after a prolonged culture of the excised aortic valve. High IgG and IgM titers to B. henselae pointed to a subacute course of the disease.

Bartonella henselae: subversion of vascular endothelial cell functions by translocated bacterial effector proteins.

Bartonella henselae (Bh) is a worldwide distributed zoonotic pathogen. Depending on the immune status of the infected individual this bacterium can cause a wide spectrum of clinical manifestations, ranging from cat scratch disease (CSD) to bacillary angiomatosis (BA) and bacillary peliosis (BP). BA and BP are characterized by tumor-like lesions at the skin or in the inner organs, respectively. These structures display pathological sprouting of capillaries with enlarged and hyperproliferated vascular endothelial cells (ECs) that are frequently found in close association with bacteria. Here we review the cellular changes observed upon Bh infection of ECs in vitro and outline the role of the VirB type IV secretion system (T4SS) and its translocated effector proteins in the modulation of EC signalling cascades. The current model how this virulence system could contribute to the vasoproliferative activity of Bh is described.

From cat scratch disease to endocarditis, the possible natural history of Bartonella henselae infection.

BACKGROUND: Most patients with infectious endocarditis (IE) due to Bartonella henselae have a history of exposure to cats and pre-existing heart valve lesions. To date, none of the reported patients have had a history of typical cat scratch disease (CSD) which is also a manifestation of infection with B. henselae. CASE PRESENTATION: Here we report the case of a patient who had CSD and six months later developed IE of the mitral valve caused by B. henselae. CONCLUSION: Based on this unique case, we speculate that CSD represents the primary-infection of B. henselae and that IE follows in patients with heart valve lesions.

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.

Emergence of distinct genetic variants in the population of primary Bartonella henselae isolates.

Bartonella henselae isolates from different hosts display a marked genetic heterogeneity, as determined by pulsed-field gel electrophoresis (PFGE). The aim of the present study was to determine whether different genetic variants may coexist within the population of distinct B. henselae isolates and could be detected by PFGE. Three primary B. henselae isolates and the B. henselae reference strains ATCC 49793 and 49882 were subjected as single colony derived cultures in quadruplicate to PFGE analysis upon restriction with SmaI or NotI. Up to 4 fragment differences were found among the cultures obtained from each primary isolate, indicating the coexistence of genetic variants in the population of primary B. henselae isolates. The clonal relatedness of the genetic variants was confirmed by arbitrarily primed PCR and multi-locus sequence typing. In contrast to the primary isolates, no variants were detected among the single colony derived cultures of the high-passage ATCC strains. We hypothesized that the coexistence of different genetic variants may represent a feature that is restricted to primary or low-passage B. henselae isolates. The primary isolates were serially passed in vitro and then subjected as single colony derived cultures to PFGE analysis, which now revealed identical patterns among the quadruplicate cultures of each high-passage isolate. These results suggest that the population of a primary B. henselae isolate is composed of distinct genetic variants, which may disappear upon repeated passages on artificial culture media. Generation of genetic variants by B. henselae may represent an escape mechanism to circumvent the host specific immune responses.

Novel chemically modified liquid medium that will support the growth of seven bartonella species.

Bacteria of the genus Bartonella, a member of the Alphaproteobacteria, are fastidious, gram-negative, aerobic bacilli that comprise numerous species, subspecies, and subtypes. In human and veterinary medicine, species isolation remains a vital component of the diagnostic and therapeutic management of Bartonella infection. We describe a novel, chemically modified, insect-based liquid culture medium that supports the growth of at least seven Bartonella species. This medium will also support cocultures consisting of different Bartonella species, and it facilitated the primary isolation of Bartonella henselae from blood and aqueous fluid of naturally infected cats. This liquid growth medium may provide an advantage over conventional direct blood agar plating for the diagnostic confirmation of bartonellosis.

Infection of human CD34+ progenitor cells with Bartonella henselae results in intraerythrocytic presence of B. henselae.

Although there is evidence that endothelial cells are important targets for human pathogenic Bartonella species, the primary niche of infection is unknown. Here we elucidated whether human CD34+ hematopoietic progenitor cells (HPCs) internalize B. henselae and may serve as a potential niche of the pathogen. We showed that B. henselae does not adhere to or invade human erythrocytes. In contrast, B. henselae invades and persists in HPCs as shown by gentamicin protection assays, confocal laser scanning microscopy (CLSM), and electron microscopy (EM). Fluorescence-activated cell sorting (FACS) analysis of glycophorin A expression revealed that erythroid differentiation of HPCs was unaffected following infection with B. henselae. The number of intracellular B. henselae continuously increased over a 13-day period. When HPCs were infected with B. henselae immediately after isolation, intracellular bacteria were subsequently detectable in differentiated erythroid cells on day 9 and day 13 after infection, as shown by CLSM, EM, and FACS analysis. Our data provide, for the first time, evidence that a bacterial pathogen is able to infect and persist in differentiating HPCs, and suggest that HPCs might serve as a potential primary niche in Bartonella infections.

[Evaluation of isolation media for the detection of Bartonella henselae--isolation of Bartonella henselae from domestic cats].

Bartonella henselae is a causative agent of cat scratch disease. We preliminarily tested four media for the bacterial growth, including agar plates with sheep, horse or rabbit blood, and chocolate agar. Of these media, rabbit blood and chocolate agar plate were found to be more excellent for the growth than the medium with sheep or horse blood. Blood samples from 60 domestic cats in Yamaguchi Prefecture were then cultured using 7% rabbit blood agar plates and BACTEC9050 (BD), automated blood culture microbial detection system. B. henselae was isolated from six of the 60 (10%) blood samples. Tiny colonies of B. henselae were visible on the agar medium after one week of culture at 35 degrees C in the 5% CO2 atmosphere. BACTEC 9050 detected B. henselae in one of the 10 blood samples and it took two weeks to detect the bacteria automatically, though gram stain failed to show organisms in the blood culture bottle. In conclusion, rabbit blood or chocolate agar and incubation of agar media more than one week and of BACTEC more than two weeks are recommended for the detection of B. henselae.

The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells.

Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.

Growth characteristics of Bartonella henselae in a novel liquid medium: primary isolation, growth-phase-dependent phage induction, and metabolic studies.

Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 x 10(8) CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.

Phase variation in Bartonella henselae.

Bartonella henselae is a fastidious, Gram-negative bacterial pathogen of cats and humans. Previous workers have shown that serial passage in vitro leads to attenuation of virulence-associated attributes such as expression of pili, invasion of human epithelial cell lines and the stimulation of endothelial cell proliferation. In contrast to the published data, it was found that pilin expression is frequently preserved in organisms which have undergone phase variation in vitro. Transition from a slow-growing, dry agar-pitting (DAP) to a faster-growing, smooth non-agar-pitting (SNP) form appears to occur predictably and may reflect competition between two populations growing at different rates. Better survival of the slower-growing (DAP) form may explain its relatively easy retrieval from piliated SNP populations allowed to age on solid media. Pilin expression is associated with auto-agglutination in liquid suspension or broth cultures, and appears to be necessary but not sufficient for expression of the agar-pitting phenotype and for the formation of biofilms. Outer-membrane protein variation is seen in association with phase variation, but lipopolysaccharide expression is preserved in piliated as well as extensively passaged non-piliated isolates. The EagI/HhaI infrequent restriction site-PCR fingerprint, which has been previously used to discriminate between serotypes Marseille and Houston, is shown to alter with phase variation in vitro, and there is evidence that genetic change accompanies these events. The extent of genetic and phenotypic variability of phase-variant B. henselae has previously been underestimated. It may lead to new insights into the pathogenicity of this organism, and must be considered when interpreting data arising from such studies.

Infection and re-infection of domestic cats with various Bartonella species or types: B. henselae type I is protective against heterologous challenge with B. henselae type II.

Four Bartonella species have been isolated from domestic cats, of which two serotypes/genotypes of Bartonella henselae and possibly B. clarridgeiae are human pathogens, causing cat scratch disease (CSD).Our objectives were to evaluate infection and potential cross-protection during re-infection in domestic cats with various Bartonella species or types.Thirty-six cats were primarily inoculated with B. henselae type I (n=16), B. henselae type II (n=10), B. clarridgeiae (n=6) or B. koehlerae (n=4). They were challenged with B. henselae type I (n=15), B. henselae type II (n=13) or B. clarridgeiae (n=8). All 36 cats became bacteremic (1.25x10(2)-1.44x10(6)CFU/ml) and bacteremia lasted from 37 to 582 days. Duration of bacteremia for cats inoculated with B. henselae type I was shorter than for cats inoculated with either B. henselae type II (P=0.025) or B. clarridgeiae (P=0.011). After challenge, 26 cats became bacteremic. Among the nine cats primarily inoculated with B. henselae type I and challenged with B. henselae type II, six cats stayed abacteremic. The three bacteremic cats had a transient low-level bacteremia. No bacteremia was observed in three cats primarily inoculated with B. henselae type I and challenged with another strain of B. henselae type I. Bacteremia levels in the 26 cats were significantly lower than for primary inoculation (P=0.022) and its duration was shorter (P=0.012). Among the eight cats challenged with B. clarridgeiae, duration of bacteremia in the four cats primarily inoculated with B. henselae type I was shorter than in the four cats primarily inoculated with B. henselae type II (P=0.01). Bartonella clarridgeiae inoculated cats were more likely to have relapses for both primary and secondary infections. This is the first demonstration of cross-protection, evidenced by absence of bacteremia, in cats primarily infected with B. henselae type I and challenged with B. henselae type II, whereas no cross-protection was previously shown for cats primarily infected with B. henselae type II and challenged with B. henselae type I. Such results are of major importance for future feline Bartonella vaccine development.

Studies on the growth of Bartonella henselae in the cat flea (Siphonaptera: Pulicidae).

Two out of three pools of cat fleas, Ctenocephalides felis (Bouche), that were fed Bartonella henselae-positive cat blood for 3 d and then bovine blood for 3 d, were polymerase chain reaction (PCR) positive for B. henselae. In a second experiment, three cats were inoculated with a streptomycin-resistant strain of B. henselae. After the cats were inoculated, caged cat fleas were fed on the cats during three different periods, and then pooled and transferred to noninfected recipient cats. In the first trial, the bacteria in the flea feces were below level of detection when the fleas were transferred from the infected cats to the recipient cat. After the fleas had fed on the recipient cat for 6 d, a bacteria level of 4.00 x 10(3) CFU/ mg was detected in the flea feces. Subsequently, the bacteria level increased for 4 d and then declined. In another experiment, the bacteria level in the flea feces was 1.80 x 10(3) CFU/mg at 2 h after collection and 3.33 x 10(2) CFU/mg at 72 h after collection. These data indicated that this strain of B. henselae can persist in flea feces in the environment for at least 3 d, and that B. henselae can multiply in the cat flea.