Molecular epidemiology of Bartonella species isolated from ground squirrels and other rodents in northern California.

Bartonella spp. are endemic in wild rodents in many parts of the world. A study conducted in two northern California counties (Sonoma and Yolo) sampling California ground squirrels (Otospermophilus beecheyi) and four other rodent species (Peromyscus maniculatus, P. boylii, P. truei and Neotoma fuscipes) led to the isolation of small Gram-negative bacilli which were identified as Bartonella spp. based on colony morphology, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and partial gene sequencing. Overall, Bartonella spp. were isolated from the blood of 71% (32/45) of the ground squirrels and one third (22/66) of the other rodents. PCR-RFLP analysis of the gltA and 16S rRNA genes yielded seven unique profiles, four for the ground squirrels and three for the other rodents. Isolates from each PCR-RFLP profiles were submitted for partial sequencing. Ground squirrel isolates were most closely related to B. washoensis, whereas the other rodent isolates were closest to B. vinsonii subsp. vinsonii and B. vinsonii subsp. arupensis. Two of these three species or subspecies are known zoonotic agents.

Isolation of Bartonella henselae, Bartonella koehlerae subsp. koehlerae, Bartonella koehlerae subsp. bothieri and a new subspecies of B. koehlerae from free-ranging lions (Panthera leo) from South Africa, cheetahs (Acinonyx jubatus) from Namibia and captive cheetahs from California.

Bartonellae are blood- and vector-borne Gram-negative bacteria, recognized as emerging pathogens. Whole-blood samples were collected from 58 free-ranging lions (Panthera leo) in South Africa and 17 cheetahs (Acinonyx jubatus) from Namibia. Blood samples were also collected from 11 cheetahs (more than once for some of them) at the San Diego Wildlife Safari Park. Bacteria were isolated from the blood of three (5%) lions, one (6%) Namibian cheetah and eight (73%) cheetahs from California. The lion Bartonella isolates were identified as B. henselae (two isolates) and B. koehlerae subsp. koehlerae. The Namibian cheetah strain was close but distinct from isolates from North American wild felids and clustered between B. henselae and B. koehlerae. It should be considered as a new subspecies of B. koehlerae. All the Californian semi-captive cheetah isolates were different from B. henselae or B. koehlerae subsp. koehlerae and from the Namibian cheetah isolate. They were also distinct from the strains isolated from Californian mountain lions (Felis concolor) and clustered with strains of B. koehlerae subsp. bothieri isolated from free-ranging bobcats (Lynx rufus) in California. Therefore, it is likely that these captive cheetahs became infected by an indigenous strain for which bobcats are the natural reservoir.

Bartonella clarridgeiae and Bartonella vinsonii subsp. berkhoffii exposure in captive wild canids in Brazil.

SUMMARY Wild canids are potential hosts for numerous species of Bartonella, yet little research has been done to quantify their infection rates in South America. We sought to investigate Bartonella seroprevalence in captive wild canids from 19 zoos in São Paulo and Mato Grosso states, Brazil. Blood samples were collected from 97 wild canids belonging to four different native species and three European wolves (Canis lupus). Indirect immunofluorescent antibody testing was performed to detect the presence of B. henselae, B. vinsonii subsp. berkhoffii, B. clarridgeiae, and B. rochalimae. Overall, Bartonella antibodies were detected in 11 of the canids, including five (12·8%) of 39 crab-eating foxes (Cerdocyon thous), three (11·1%) of 27 bush dogs (Speothos venaticus), two (8·7%) of 23 maned wolves (Chrysocyon brachyurus) and one (12·5%) of eight hoary foxes (Lycalopex vetulus), with titres ranging from 1:64 to 1:512. Knowing that many species of canids make excellent reservoir hosts for Bartonella, and that there is zoonotic potential for all Bartonella spp. tested for, it will be important to conduct further research in non-captive wild canids to gain an accurate understanding of Bartonella infection in free-ranging wild canids in South America.

Bartonella infection in urban and rural dogs from the tropics: Brazil, Colombia, Sri Lanka and Vietnam.

Dogs can be infected by a wide range of Bartonella spp., but limited studies have been conducted in tropical urban and rural dog populations. We aimed to determine Bartonella antibody prevalence in 455 domestic dogs from four tropical countries and detect Bartonella DNA in a subset of these dogs. Bartonella antibodies were detected in 38 (8·3%) dogs, including 26 (10·1%) from Colombia, nine (7·6%) from Brazil, three (5·1%) from Sri Lanka and none from Vietnam. DNA extraction was performed for 26 (63%) of the 41 seropositive and 10 seronegative dogs. Four seropositive dogs were PCR positive, including two Colombian dogs, infected with B. rochalimae and B. vinsonii subsp. berkhoffii, and two Sri Lankan dogs harbouring sequences identical to strain HMD described in dogs from Italy and Greece. This is the first detection of Bartonella infection in dogs from Colombia and Sri Lanka and identification of Bartonella strain HMD from Asia.

Bartonellosis, an increasingly recognized zoonosis.

Cat scratch disease is the most common zoonotic infection caused by Bartonella bacteria. Among the many mammals infected with Bartonella spp., cats represent a large reservoir for human infection, as they are the main reservoir for Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. Bartonella spp. are vector-borne bacteria, and transmission of B. henselae by cat fleas occurs mainly through infected flea faeces, although new potential vectors (ticks and biting flies) have been identified. Dogs are also infected with various Bartonella species and share with humans many of the clinical signs induced by these infections. Although the role of dogs as source of human infection is not yet clearly established, they represent epidemiological sentinels for human exposure. Present knowledge on the aetiology, clinical features and epidemiological characteristics of bartonellosis is presented.

Prevalence of Bartonella sp. in United States military working dogs with infectious endocarditis: a retrospective case-control study.

OBJECTIVES: Bartonella infection has been associated with endocarditis in humans, dogs, cats and cattle. In order to evaluate the importance of this pathogen as a possible source of endocarditis in United States military working dogs (MWDs), we performed a retrospective case-control study on 26 dogs with histological diagnosis of culture negative endocarditis (n = 18), endomyocarditis (n = 5) or endocardiosis (n = 3) and 28 control dogs without any histological cardiac lesions. METHODS: DNA was extracted from paraffin embedded cardiac valves and tissues from case and control dogs and submitted to PCR testing with primers targeting the Bartonella gltA gene. PCR-RFLP using four restriction endonucleases and partial sequencing was then performed to determine the Bartonella species involved. RESULTS: Nineteen (73%) cases were PCR positive for Bartonella, including B. henselae (8 dogs), B. vinsonii subsp. berkhoffii (6 dogs), B. washoensis (2 dogs) and B. elizabethae (1 dog). Only one control dog was weakly PCR positive for Bartonella. Based on the type of histological diagnosis, 13 (72.2%) dogs with endocarditis, 3 (60%) dogs with endomyocarditis and all 3 dogs with endocardiosis were Bartonella PCR positive. CONCLUSIONS: Bartonella sp. Infections were correlated with cardiopathies in US military working dogs. Systemic use of insecticides against ectoparasites and regular testing of MWDs for Bartonella infection seem highly appropriate to prevent such life-threatening exposures.

Environmental factors associated with Bartonella vinsonii subsp. berkhoffii seropositivity in free-ranging coyotes from northern California.

Bartonella vinsonii subsp. berkhoffii is a newly recognized pathogen of domestic dogs and humans. Coyotes (Canis latrans) are considered an important reservoir of this bacterium in the western United States, but its vectors are still unknown. Our objective was to identify environmental factors associated with Bartonella antibody prevalence in 239 coyotes from northern California, using an enzyme-linked immunosorbent assay. In addition, associations were evaluated between B. v. berkhoffii and two pathogens with known vectors and habitat requirements, Dirofilaria immitis and Anaplasma phagocytophilum. Overall, B. v. berkhoffii seroprevalence was 28% (95% confidence interval [CI], 22.3%, 33.7%) and Bartonella seropositive coyotes were more likely than seronegative coyotes to be positive for Anaplasma phagocytophilum (Odds ratio = 3.3; 95% CI = 1.8, 5.9) and Dirofilaria immitis (Odds ratio = 2.1; 95% CI = 1.2, 3.8). The most likely geographic clusters of Bartonella and Dirofilaria overlapped. Bartonella seropositivity was associated with higher precipitation (p = 0.003) and proximity to the coast (p = 0.007) in univariate analysis. The association with precipitation varied with season, based on a logistic regression model.

Serosurvey of selected zoonotic agents in polar bears (Ursus maritimus).

Between 1982 and 1999 blood samples were collected from 500 polar bears (Ursus maritimus) captured in the Beaufort and Chukchi seas, to determine the seroprevalence of Brucella species, Toxoplasma gondii, and Trichinella species infections. The bears were classified into four age groups, cubs, yearlings, subadults and adults. Brucella and Toxoplasma antibodies were detected by agglutination (a buffered acidified card antigen and rapid automated presumptive test for brucellosis and a commercial latex agglutination test for toxoplasmosis); an ELISA was used to detect Trichinella antibodies. The overall seroprevalence of Brucella species was 5 per cent, and subadults and yearlings were 2-62 times (95 per cent confidence interval 1.02 to 6.82) more likely to be seropositive for Brucella species than adults and their cubs. The antibody prevalence for Toxoplasma gondii was 6 per cent, and for Trichinella species 55.6 per cent. The prevalence of antibodies to Trichinella species increased with age (P<0.001).

Bartonella henselae and Bartonella clarridgeiae infection in domestic cats from The Philippines.

One hundred seven domestic cats from The Philippines were serologically tested to establish the prevalence of Bartonella infection. A subset of 31 of these cats also had whole blood collected to tentatively isolate Bartonella strains. Bartonella henselae and B. clarridgeiae were isolated from 19 (61%) of these cats. Bartonella henselae type I was isolated from 17 (89%) of the 19 culture-positive cats. Six cats (31%) were infected with B. clarridgeiae, of which four were coinfected with B. henselae. Sixty-eight percent (73 of 107) and 65% (70 of 107) of the cats had antibodies to B. henselae and B. clarridgeiae, respectively, detected by an immunofluorescence antibody (IFA) test at a titer > or = 1:64. When tested by enzyme immunoassay (EIA), 67 cats (62.6%) had antibodies to B. henselae and 71 cats (66.4%) had antibodies to B. clarridgeiae. Compared with the IFA test, the B. henselae EIA had a sensitivity of 90.4% and a specificity of 97%, with positive and negative predictive values of 98.5% and 82.5%, respectively. Similarly, the B. clarridgeiae EIA had a sensitivity of 97% and a specificity of 92% specificity, with positive and negative predictive values of 95.8% and 94.4%, respectively. The presence of antibodies to Bartonella was strongly associated with flea infestation. Domestic cats represent a large reservoir of Bartonella infection in the Philippines.

Prevalence of Bartonella species and 16s rRNA gene types of Bartonella henselae from domestic cats in Thailand.

Prevalence of Bartonella infection among 275 cats in 9 sites from 4 geographical regions (northern area: Chiang Mai; central area: Kanchanaburi, Ratchaburi, and Bangkok; northeastern area: Khon Kaen, Roi Et, Ubon Ratcharthani, and Nakhonratchasima; southern area: Songkhla) of Thailand was investigated. Overall, Bartonella species were isolated from 27.6% (76 of 275) of the cats. The isolation rate varied from 12.8% (5 of 39) in Songkhla (southern area) to 50.0% (26 of 52) in Khon Kaen (northeastern area). Bartonella henselae and B. clarridgeiae were isolated from 82.9% (63 of 76) and 11.8% (9 of 76) of the Bartonella-positive cats, respectively. Coinfection with both species was found in 5.3% (4 of 76) of the bacteremic cats. Of the 67 bacteremic cats from which B. henselae was isolated, 48 (71.6%) and 13 (19.4%) were infected with only Type I and Type II, respectively. Coinfection with both types was observed in 9.0% (6 of 67) of the B. henselae-positive cats. To our knowledge, this is the first report on the presence of Bartonella infection in domestic cats from Thailand, which constitute a large reservoir of Bartonella infection in this country.

Genomic diversity of Bartonella henselae isolates from domestic cats from Japan, the USA and France by pulsed-field gel electrophoresis.

The genomic DNA diversity of 27 Bartonella henselae and three B. clarridgeiae isolates from 18 domestic cats from Japan, the USA and France was investigated by pulsed-field gel electrophoresis (PFGE) with NotI, AscI and SmaI restriction enzymes. A great diversity of genomic patterns was found for all B. henselae, but none for B. clarridgeiae isolates. The DNA size of B. henselae and B. clarridgeiae isolates were 1.7-2.9 and 1.7Mbp, respectively. All 13 Japanese cat isolates were identified as B. henselae type I. Furthermore, three of the four Japanese cats harbored genetically different B. henselae type I isolates, suggesting for the first time co-infection with various type I isolates. One French cat and one American cat were co-infected with B. henselae and B. clarridgeiae. B. henselae type I and type II were mainly grouped in two different clusters by PFGE using SmaI endonuclease in the dendrogram.

Genotypic and phenotypic analysis of Salmonella strains associated with an outbreak of equine neonatal salmonellosis.

Isolates of Salmonella choleraesuis serotype ohio (S. ohio) recovered during an outbreak of equine neonatal salmonellosis on a Thoroughbred farm were compared with isolates of the same serotype from various animal, feed and environmental sources. Biochemical profiles, antimicrobial susceptibility patterns, phage susceptibility, plasmid profiles, restriction endonuclease analysis and ribotyping were used to compare relatedness of the strains. A total of 46 outbreak and non-outbreak associated isolates of S. ohio were studied. Differences in antimicrobial susceptibility patterns, phage susceptibility and plasmid profiles were useful for differentiating outbreak isolates from other equine isolates as well as bovine, porcine and some poultry isolates. Feed and other poultry isolates, most in geographic proximity to the outbreak, were indistinguishable from outbreak isolates by any of the methods employed. Investigative studies on the farm along with results of genotypic and phenotypic analysis of isolates suggested that contaminated feed was the most likely source of Salmonella in this outbreak.

Epidemiology of Bartonella infection in domestic cats in France.

Blood samples were collected between February and June 1996 from a convenience sample of 436 domestic French cats living in Paris and its environs and were tested for Bartonella bacteremia and seropositivity. Seventy-two cats (16.5%) were Bartonella bacteremic, of which 36 cats (50%) were infected with Bartonella henselae type II (B.h. II) only, 15 cats (21%) were infected with Bartonella clarridgeiae (B.c.) only, and 11 cats (15%) were infected with B. henselae type I (B.h. I) only. Eight cats (11%) were co-infected with B. henselae and B. clarridgeiae (B.h. II/B.c.: five cats; B.h. I/B.c.: three cats). Two cats (2.8%) were concurrently bacteremic with B. henselae types I and II. Risk factors associated with bacteremia included ownership for <6months (prevalence ratio (PR)=1.80; 95% confidence interval (CI)=1.13-2.85), adoption from the pound or found as a stray (PR=1.67, 95% CI=1.05-2.65), and cohabitation with one or more cats (PR=1.60, 95% CI=1.01-2.53). Bartonella antibodies to either B. henselae or B. clarridgeiae were detected in 179 cats (41.1%). Risk factors associated with seroposivity paralleled those for bacteremia, except for lack of association with time of ownership. Prevalence ratios of bacteremic or seropositive cats increased with the number of cats per household (p=0.02). The lack of antibodies to B. henselae or B. clarridgeiae was highly predictive of the absence of bacteremia (predictive value of a negative test=97.3%). Multiple logistic regression analysis indicated that bacteremia, after adjustment for age and flea infestation, and positive serology, after adjustment for age, were associated with origin of adoption and number of cats in the household. Flea infestation was associated with positive serology.

Prevalence of Bartonella infection in wild African lions (Panthera leo) and cheetahs (Acinonyx jubatus).

Bartonella species are emerging pathogens that have been isolated worldwide from humans and other mammals. Our objective was to estimate the prevalence of Bartonella infection in free-ranging African lions (Panthera leo) and cheetahs (Acinonyx jubatus). Blood and/or serum samples were collected from a convenience sample of 113 lions and 74 cheetahs captured in Africa between 1982 and 2002. Whole blood samples available from 58 of the lions and 17 of the cheetahs were cultured for evidence of Bartonella spp., and whole blood from 54 of the 58 lions and 73 of the 74 cheetahs tested for the presence of Bartonella DNA by TaqMan PCR. Serum samples from the 113 lions and 74 cheetahs were tested for the presence of antibodies against Bartonella henselae using an immunofluorescence assay. Three (5.2%) of the 58 lions and one (5.9%) of the 17 cheetahs were bacteremic. Two lions were infected with B. henselae, based on PCR/RFLP of the citrate synthase gene. The third lion and the cheetah were infected with previously unidentified Bartonella strains. Twenty-three percent of the 73 cheetahs and 3.7% of the 54 lions tested by TaqMan PCR were positive for Bartonella spp. B. henselae antibody prevalence was 17% (19/113) for the lions and 31% (23/74) for the cheetahs. The prevalence of seropositivity, bacteremia, and positive TaqMan PCR was not significantly different between sexes and age categories (juvenile versus adult) for both lions and cheetahs. Domestic cats are thus no longer the only known carriers of Bartonella spp. in Africa. Translocation of B. henselae seronegative and TaqMan PCR negative wild felids might be effective in limiting the spread of Bartonella infection.

Experimental and natural infection with Bartonella henselae in domestic cats.

Domestic cats were experimentally infected with culture propagated Bartonella henselae by intradermal (i.d.) and intravenous (i.v.) routes. Cats were more efficiently infected by the i.d. (8/8 cats) than by the i.v. (2/16) route. Bacteremia was detected 1-3 weeks following inoculation and lasted for most cats for 1-8 months. However, one naturally infected cat was observed for 24 months and was found to be cyclically bacteremic, with bacterial levels varying one hundred fold or more from one period to another. No clinical or hematologic abnormalities were observed in any of the infected cats, even at the peak of bacteremia. Two cats that had become abacteremic were resistant to reinfection when inoculated with B. henselae a second time. Horizontal transmission through intimate contact between bacteremic and susceptible cats did not occur, and antibody positive bacteremic queens did not transmit the infection to their kittens in utero, peri-partum or post-partum. Only four of the 18 kittens acquired detectable levels of maternal antibody following nursing, which disappeared by 6 weeks of age. These studies indicate that B. henselae exists in an almost perfect host-parasite relationship with its feline host, but that most cats can ultimately rid themselves of the infection. The susceptibility of cats to intradermal infection and the lack of direct cat-cat transmission are compatible with possible arthropod vectors.

Homologous protection but lack of heterologous-protection by various species and types of Bartonella in specific pathogen-free cats.

Cat-scratch disease (CSD) is caused by Bartonella henselae, and possibly by B. clarridgeiae. In immuno-compromised persons, B. henselae is one of the agents causing bacillary angiomatosis. Domestic cats are the main reservoir of these bacteria, which are transmitted primarily from cat to cat by fleas. Possible strategies to prevent the spread of infection among cats are to eliminate flea infestation or to prophylactically immunize cats. In order to develop an appropriate vaccine, it is important to determine if cats become resistant to re-infection by the same strain or various types or species of Bartonella. In a series of experiments, 21 SPF cats were experimentally infected by the intradermal route with 10(5)-10(10) colony-forming units/ml of either B. henselae type II (17 cats), or a new strain 'Humboldt' isolated from a mountain lion (4 cats). The cats were bled weekly to every other week for determination of bacteremia and specific antibody production. After they cleared their infection, they were challenged by a homologous or heterologous strain of Bartonella: 10 cats were challenged with B. henselae type II, three cats with B. henselae type I, four cats with B. clarridgeiae and four cats with the 'Humboldt' strain. Seven of these cats received a third inoculum dose resulting in three cats sequentially infected with sequence B. henselae type II/B. henselae type II/'Humboldt', two cats with sequence B. henselae type II/'Humboldt'/B. clarridgeiae, and two cats with the sequence 'Humboldt'/B. henselae type II/'Humboldt'. All cats challenged with a homologous strain remained abacteremic after challenge and had an increased IgG antibody titer. All cats challenged with either a different Bartonella species or type became bacteremic. The few cats receiving a third inoculum with a strain homologous to the initial strain remained abacteremicafter that challenge. All cats infected with B. clarridgeiae suffered relapsing bacteremia compared to only 36% of the B. henselae infected cats and 22% of the 'Humboldt'-infected cats (p=0.008). The duration of bacteremia was significantly longer in B. henselae primary-infected cats (mean: 34 weeks) than B. henselae heterologously challenged cats (mean: 9 weeks) (p=0.014). These data clearly indicate the lack of cross-protection between B. henselae and B. clarridgeiae and furthermore, indicate the lack of protection between B. henselae types I and II, and a wildlife isolate. A vaccine strategy for CSD prevention in domestic cats will require a multivalent vaccine approach.

Validation for use with coyotes (Canis latrans) of a commercially available enzyme-linked immunosorbent assay for Dirofilaria immitis.

Serological tests offer a potentially powerful tool for monitoring parasites in wildlife populations. However, such tests must be validated before using them with target wildlife populations. We evaluated in coyotes (Canis latrans) the performance of a commercially available serological test used to detect canine heartworm (Dirofilaria immitis) in domestic dogs. We obtained 265 coyote carcasses and serological specimens from 54 additional coyotes from several regions of California, USA. We necropsied coyotes to determine the adult heartworm infection status. Blood was collected at necropsy on filter paper strips and allowed to dry; it was later eluted in a buffer solution, and the supernatant was tested for heartworm. Receiver operating characteristic (ROC) analysis was used to assess discriminatory power of the test and indicated a 93% probability that a randomly selected infected coyote would exhibit a higher enzyme-linked immunosorbent assay (ELISA) value than a randomly selected uninfected coyote. We estimated specificity at 96% (95% CI: 92-98%) for 165 uninfected coyotes and sensitivity at 85% (77-91%) for 100 infected coyotes, results similar to published values for the commercial serological test used with dog serum or plasma. Test performance was similar for filter paper specimens and supernatant of frozen whole blood collected in EDTA tubes (i.e. hemolyzed plasma). We found no difference in test performance among geographic or demographic coyote groups. Our findings support application of the test to filter paper or standard serological specimens for detection of heartworm in coyote populations.