Caged cat fleas, Ctenocephalides felis (Bouché), were fed on 6 cats; 3 cats were injected with 5 x 10(7) colony forming units of Bartonella henselae intradermally and 3 cats were injected with an equal volume of saline. After the fleas fed for 4 d, 5 groups of 50 B. henselae-exposed fleas were caged and allowed to feed on 5 cats for 6 d. Five cats each were injected intradermally with 1 ml of saline containing 45 mg of feces from B. henselae-exposed fleas. Five cats were fed 50 B. henselae-exposed fleas and 45 mg of fresh feces from B. henselae-exposed fleas. Five cats received all 3 treatments by using fleas and feces collected from cats inoculated with saline (controls). Cats were bled weekly and tested by culture and serology. The cats that were injected with feces from infected fleas were positive by culture for B. henselae at 1 or 2 wk after exposure and were the only cats to become bacteremic or seropositive by week 20.
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.
Capsular polysaccharide (CPS) is a major virulence factor in Vibrio vulnificus, and encapsulated strains have an opaque, smooth (OpS) colony morphology, while nonencapsulated strains have a translucent, smooth (TrS) colony morphology. Previously, we showed that OpS and TrS parental strains can yield a third colony type, rugose (R), and that the resulting strains, with the OpR and TrR phenotypes, respectively, form copious biofilms. Here we show that while OpR and TrR strains both produce three-dimensional biofilm structures that are indicative of rugose extracellular polysaccharide (rEPS) production, OpR strains also retain expression of CPS and are virulent in an iron-supplemented mouse model, while TrR strains lack CPS and are avirulent. Chlorine resistance assays further distinguished OpR and TrR isolates as exposure to 3 g/ml NaOCl eradicated both OpS and OpR strains, while both TrS and TrR strains survived, but at rates which were significantly different from one another. Taken together, these results further emphasize the importance of CPS for virulence of V. vulnificus and establish a correlation between CPS expression and chlorine sensitivity in this organism. Using reverse transcriptase PCR, we also identified a nine-gene cluster associated with both CPS and rEPS expression in V. vulnificus, designated the wcr (capsular and rugose polysaccharide) locus, with expression occurring primarily in R variants. The latter results set the stage for characterization of functional determinants which individually or collectively contribute to expression of multiple EPS forms in this pathogen.Vibrio vulnificus is a gram-negative marine and estuarine bacterium capable of causing severe disease in susceptible individuals. This bacterium is normally found in fish and shellfish, including oysters. Several medical conditions can predispose a person to being susceptible to infection by this organism, including diabetes, liver disease, hemachromatosis, and a compromised immune system. When people with such conditions consume raw oysters or other seafood containing V. vulnificus, they are at risk of developing a rapidly progressing primary septicemia that can be fatal within 24 to 48 h. In an otherwise healthy individual, if a break in the skin is exposed to seawater containing this organism, a severe wound infection may develop that could necessitate amputation if treatment is not begun soon after the onset of symptoms. V. vulnificus does not infect as many people as other members of the genus Vibrio, but it is the leading reported cause of death from the consumption of seafood in the United States (28, 48).V. vulnificus produces several virulence factors, including multiple enzymes, siderophores, RtxA toxin, and a polysaccharide capsule (for a review, see reference 15). While the other factors may assist in virulence, the capsular polysaccharide (CPS) is considered a major virulence factor and has been reported to protect the bacteria from phagocytosis and complement-mediated killing by the host immune system. When grown ...
Bartonella henselae is the causative agent of human cat scratch disease as well as several serious sequelae of infections, including bacillary angiomatosis and bacillary peliosis. Conflicting reports describe the pathogenesis of B. henselae in the cat. In this study, we characterized a strain of B. henselae termed LSU16. This strain was isolated on rabbit blood agar from a naturally infected 10-month-old female cat during a recurrent episode of bacteremia. The bacterial species was confirmed by PCR-restriction fragment length polymorphism analysis. Nine cats were infected intradermally with 5 × 107 CFU of LSU16, and clinical signs, antibody responses, and bacteremia were monitored. All nine cats developed raised, erythematous areas at the site of inoculation within 72 h postinoculation; the swelling peaked at 14 days postinfection and was not palpable by 28 days postinfection. Fever developed in all nine cats between 6 and 16 days postinfection and lasted for 1 to 8 days. Between 6 and 16 days postinfection, all nine cats experienced lethargy which persisted 5 to 18 days. Seven of nine cats were bacteremic by day 7, and all nine cats had become bacteremic by 14 days postinfection. Bacteremia peaked at 14 to 28 days postinfection in all cats. In six of the nine infected cats, bacterial numbers reached nondetectable levels during the 7th week postinfection; however, a single animal maintained bacteremia to 18 weeks postinfection. All nine cats developed strong antibody responses toB. henselae, as determined by Western blot analysis and enzyme-linked immunosorbent assay. Subsequently, three naive cats were injected intradermally with blood from cats infected with LSU16 from a pure culture, and five naive cats were injected with feces from fleas which had been feeding on cats infected with a pure culture of LSU16. These cats developed signs similar to those described in the previous experiment and were euthanized at 5 weeks postinfection. We conclude that B. henselae LSU16 is a virulent strain of B. henselae in cats and propose that the virulence of B. henselae in cats is strain dependent.
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 ؋ 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.Bartonella henselae is a medically important gram-negative, zoonotic pathogen. Cats are the reservoir, and infections are generally asymptomatic. B. henselae causes a diverse and emerging disease spectrum in humans, from self-limiting lymphadenopathy to life-threatening conditions such as bacillary angiomatosis. Cat scratch disease (CSD), a generally benign infection characterized by regional lymphadenopathy and persistent fever, is the most-common manifestation of B. henselae infection in humans. CSD occurs in healthy individuals and affects an estimated 24,000 persons a year in the United States (11). In immunocompromised patients, B. henselae causes more-serious infections, including bacillary angiomatosis and bacillary peliosis, which can be fatal when misdiagnosed and improperly treated. A major contributing factor to misdiagnosis is the inherent difficulty in culturing the bacterium. B. henselae takes an average of 21 days to form colonies during primary isolation on blood agar plates, and no reliable liquid growth medium exists (8). In clinical settings, the protracted growth phase ofte...
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