Molecular detection of Bartonella henselae and Bartonella clarridgeiae in clinical samples of pet cats from Southern Italy

Bartonella henselae is considered an emerging pathogen of veterinary and medical interest that can be occasionally transmitted to humans. Cats are considered to be the only reservoir host for B. henselae. In this study, we used a nested-PCR assay to investigate the prevalence of B. henselae and Bartonella clarridgeiae DNA in peripheral blood samples, fine needle lymph node aspirate specimens and oral swabs from 85 cats in order to develop an easy diagnostic strategy for the selection of infection-free cats that are being considered as pets, especially for immunocompromised patients. Overall, molecular analysis showed that 71 cats (83.5%) tested PCR positive for the presence of B. henselae DNA. PCR amplification of DNA B. henselae produced positive products from lymph node aspirate specimens (62/85; 72.9%) similar to those obtained from blood samples (60/85; 70.6%) and higher than those from oral swabs (51/85; 60%) of cats. No PCR product was obtained for B. clarridgeiae. The simultaneous analysis of three different clinical samples in our study increased the diagnostic possibilities for B. henselae infection in the examined cats from 60–72.9% to 83.5%. Lymph node aspirates were found to be the most effective clinical samples for the detection of B. henselae and blood samples were the next best. Oral swab samples were used in this study with good results when considered in combination with blood and/or lymph node aspiration. The use of nested-PCR assay on these three clinical samples may enhance the diagnostic sensitivity for bartonellosis in cats irrespective of the clinical status of animals.     

Molecular evidence of vector-borne pathogens in dogs and cats and their ectoparasites in Algiers,Algeria

In Algeria, only limited information is currently available on the prevalence of emergent canine and feline vector-borne diseases. The aim of the present work was to detect by qPCR vector-associated bacteria in stray dogs and cats and their ectoparasites from Algiers.18/117 (15.38%) dogs and 2/107 (1.87%) cats were positive for at least one vector-borne agent. Coxiella burnetii and Bartonella henselae were identified in 1/117 (0.85%) dog individually. Ehrlichia canis DNA was detected in 17/117 (14.52%) dogs. 1/107 (0.93%) cat was positive to C. burnetii and another 1/107 (0.93%) to B. henselae.DNA of Rickettsia massiliae, Rickettsia conorii and E. canis was detected in Rhipicephalus sanguineus. Cat fleas were infected with Rickettsia felis, B. henselae and Bartonella clarridgeiae. B. vinsonii subsp. berkhoffii was identified in Xenopsylla cheopis collected from dogs.The findings of this study indicate that dogs and cats from Algeria are exposed to multiple tick and flea-borne pathogens.     

Prevalence of Bartonella species,hemoplasmas, and Rickettsia felis DNA in blood and fleas of cats in Bangkok,Thailand

Flea infestations are common in Thailand, but little is known about the flea-borne infections. Fifty flea pools and 153 blood samples were collected from client-owned cats between June and August 2009 from veterinary hospitals in Bangkok, Thailand. Total DNA was extracted from all samples, and then assessed by conventional PCR assays. The prevalence rates of Bartonella spp. in blood and flea samples were 17% and 32%, respectively, with DNA of Bartonella henselae and Bartonella clarridgeiae being amplified most commonly. Bartonella koehlerae DNA was amplified for the first time in Thailand. Hemoplasma DNA was amplified from 23% and 34% of blood samples and flea pools, respectively, with ‘Candidatus Mycoplasma haemominutum’ and Mycoplasma haemofelis being detected most frequently. All samples were negative for Rickettsia felis. Prevalence rate of B. henselae DNA was increased 6.9 times in cats with flea infestation. Cats administered flea control products were 4.2 times less likely to be Bartonella-infected.     

The prevalence of Bartonella, hemoplasma, and Rickettsia felis infections in domestic cats and in cat fleas in Ontario

The prevalence of persistent bacteremic Bartonella spp. and hemoplasma infections was determined in healthy pet cats in Ontario. Blood samples from healthy cats sent to a diagnostic laboratory for routine health assessment over the course of 1 y were tested for Bartonella spp. using both polymerase chain reaction (PCR) and blood culture, and for the presence of hemoplasma by PCR. The overall prevalence of Bartonella spp. by PCR and by culture combined was 4.3% (28/646) [3.7% (24/646) Bartonella henselae, 0.6% (4/646) Bartonella clarridgeiae]. The novel B. henselae PCR developed for this study demonstrated nearly twice the sensitivity of bacterial isolation. The overall prevalence of hemoplasma was 4% (30/742) [3.3% (25/742) Candidatus Mycoplasma haemominutum, 0.7% (5/742) Mycoplasma haemofelis]. There was no significant difference between the prevalence of infection by season or by age (≤ 2 y, > 2 y). Candidatus Mycoplasma turicensis was identified, for the first time in Canada, in 1 cat. The prevalence of Bartonella (58%) and hemoplasma (47% M. haemofelis, 13% M. haemominutum) in blood from a small sampling (n = 45) of stray cats was considerably higher than that found in healthy pet cats.     

Prevalence of Bartonella infection in domestic cats in Denmark

Whole blood and serum from 93 cats (44 pets and 49 shelter/stray cats) from Denmark were tested for the presence of feline Bartonella species by culture and for the presence of Bartonella antibodies by serology. Bartonella henselae was isolated from 21 (22.6%) cats. Bacteremia prevalence was not statistically different between shelter/stray cats (13/49, 26.5%) and pet cats (8/44, 18.2%), but varied widely by geographical origin of the cats, even after stratification for cat origin or age (p < 0.001). All isolates but one were B. henselae type II. The only cat bacteremic with B. henselae type I was not co-infected with B. henselae type II. None of the cats was harboring either B. clarridgeiae or B. koehlerae. Almost half (42/92, 45.6%) of the cats were seropositive for B. henselae and antibody prevalence was similar in shelter/stray cats (23/49, 46.9%) and pet cats (19/43, 44.2%). This is the first report of isolation of B. henselae from domestic cats in Denmark. This study also indicates that domestic cats, including pet cats, constitute a large Bartonella reservoir in Denmark.     

Bartonella spp. in cats from Buenos Aires,Argentina

In Argentina, data on the presence of members of the genus Bartonella is scarce. To increase knowledge about these zoonotic pathogens in this country, the presence and variability of Bartonella spp. was investigated in cats and dogs from Buenos Aires. Bartonella spp. was detected in 17.8% of cats, while all dogs tested negative by PCR and Reverse Line Blot. B. henselae was the most frequent species, being detected in 11.9% (14/101), while B. clarridgeiae was found in only 5.9% (6/101) of the cats. Afterwards, B. henselae isolates and positive blood samples were characterized by Multiple Locus Sequence Typing (MLST) and Multiple Locus Variable Number Tandem Repeats Analysis (MLVA). As result, four different MLST sequence types (ST) and eight MLVA profiles were identified. ST 1 was the most frequent variant found in cats, followed by ST 8. Interestingly, some of the MLVA profiles that were detected in this study have been previously associated with human disease, and represents a potential risk of infection. Veterinarians and physicians should consider the presence of these emerging pathogens in their diagnostic routine.     

Flea-borne pathogens in the cat flea Ctenocephalides felis and their association with mtDNA diversity of the flea host

Flea-borne pathogens were screened from 100 individual cat fleas using a PCR approach, of which 38 % were infected with at least one bacterium. Overall, 28 % of the flea samples were positive for Bartonella as inferred from ITS DNA region. Of these, 25 % (7/28) were identified as Bartonella clarridgeiae, 42.9 % (12/28) as Bartonella henselae consisted of two different strains, and 32.1 % (9/28) as Bartonella koehlerae, which was detected for the first time in Malaysia. Sequencing of gltA amplicons detected Rickettsia DNA in 14 % of cat flea samples, all of them identified as Rickettsia asembonensis (100 %). None of the flea samples were positive for Mycoplasma DNA in 16S rRNA gene detection. Four fleas were co-infected with Bartonella and Rickettsia DNAs. Statistical analyses reveal no significant association between bacterial infection and mtDNA diversity of the cat flea. Nevertheless, in all types of pathogen infections, infected populations demonstrated lower nucleotide and haplotype diversities compared to uninfected populations. Moreover, lower haplotype numbers were observed in infected populations.     

Molecular and serological diagnosis of Bartonella infection in 61 dogs from the United States

Background: Molecular diagnosis of canine bartonellosis can be extremely challenging and often requires the use of an enrichment culture approach followed by PCR amplification of bacterial DNA. Hypotheses: (1) The use of enrichment culture with PCR will increase molecular detection of bacteremia and will expand the diversity of Bartonella species detected. (2) Serological testing for Bartonella henselae and Bartonella vinsonii subsp. berkhoffii does not correlate with documentation of bacteremia. Animals: Between 2003 and 2009, 924 samples from 663 dogs were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Diseases Diagnostic Laboratory for diagnostic testing with the Bartonellaα‐Proteobacteria growth medium (BAPGM) platform. Test results and medical records of those dogs were retrospectively reviewed. Methods: PCR amplification of Bartonella sp. DNA after extraction from patient samples was compared with PCR after BAPGM enrichment culture. Indirect immunofluorescent antibody assays, used to detect B. henselae and B. vinsonii subsp. berkhoffii antibodies, were compared with PCR. Results: Sixty‐one of 663 dogs were culture positive or had Bartonella DNA detected by PCR, including B. henselae (30/61), B. vinsonii subsp. berkhoffii (17/61), Bartonella koehlerae (7/61), Bartonella volans‐like (2/61), and Bartonella bovis (2/61). Coinfection with more than 1 Bartonella sp. was documented in 9/61 dogs. BAPGM culture was required for PCR detection in 32/61 cases. Only 7/19 and 4/10 infected dogs tested by IFA were B. henselae and B. vinsonii subsp. berkhoffii seroreactive, respectively. Conclusions and Clinical Importance: Dogs were most often infected with B. henselae or B. vinsonii subsp. berkhoffii based on PCR and enrichment culture, coinfection was documented, and various Bartonella species were identified. Most infected dogs did not have detectable Bartonella antibodies.     

A review of bacterial pathogens in Ctenocephalides felisin New Zealand

The cat flea, Ctenocephalides felis, is the recognised vector of Bartonella henselae, B. clarridgeiae and Rickettsia felis. Although these Gram-negative bacteria were only described in the last decade, they are already known to cause a variety of diseases in people, particularly children and the immunosuppressed. Such diseases include cat-scratch disease, bacillary angiomato- sis, endocarditis, bacteraemia, encephalopathy, neuroretinitis, osteomyelitis and peliosis hepatis. Although most infections in cats and dogs appear to be subclinical, recent studies have provided growing evidence that the bartonellas can also cause serious problems in pets, including hepatitis, endocarditis, central nervous system (CNS) signs, lymphadenopathy, uveitis, cataracts and reproductive failure. In 2004, DNA of B. henselae, B. clarridgeiae and R. felis was demonstrated in cat fleas from New Zealand and pets and their owners in the country are thus at risk of infection. While flea control programmes have traditionally been advocated by veterinarians to prevent pruritis and tapeworms in pets, they should now also be recommended to prevent infections with the new flea-borne bacterial pathogens. To raise awareness of the organisms amongst veterinarians and animal health workers, this review describes: the biology of the organisms; clinical and laboratory features of infections in cats, dogs and people; diagnosis; and possible treatments and control of infections with these organisms.     

Evidence of Bartonella henselae infection in cats and dogs in the United Kingdom

Sera from cats and dogs in the UK were tested by ELISA for antibodies to Bartonella henselae. Seropositivity was confirmed in 28 of 69 pet cats (40.6 per cent), 33 of 79 feral cats (41.8 per cent) and three of 100 pet dogs. Reactivity to specific B. henselae antigens was confirmed by Western blotting and demonstrated that consistent antigenic bands were bound by sera from the cats and dogs.     

Prevalence of Bartonella species, haemoplasmas and Toxoplasma gondii in cats in Scotland

The objective of this study was to determine the prevalence rates for select infectious agents of cats presented to the Royal (Dick) School of Veterinary Studies at the University of Edinburgh, Scotland. Whole blood, serum, and oral mucosal and nail bed swabs were collected. While Ehrlichia species, Anaplasma species or Rickettsia felis DNA were not amplified from any cat, 44.2% of the cats had evidence of infection or exposure to either a Bartonella species (15.3% were seropositive and 5.8% polymerase chain reaction (PCR) positive), a haemoplasma (28.6% PCR positive), and/or Toxoplasma gondii (19.2% seropositive). No Bartonella species DNA was amplified from the nail or oral mucosal swabs despite a 5.8% amplification rate from the blood samples. This finding likely reflects the absence of Ctenocephalides felis infection from our study population, as this organism is a key component for Bartonella species translocation in cats. The results from this study support the use of flea control products to lessen exposure of cats (and people) to Bartonella species and support discouraging the feeding of raw meat to cats and preventing them from hunting to lessen T gondii infection.     

Occurrence of Bartonella henselae types I and II in Central Italian domestic cats

Serological and molecular surveys were conducted to determine the occurrence of Bartonella henselae in domestic cats in Central Italy. Samples from 234 pet cats were tested for B. henselae antibodies by indirect immunofluorescence with 78 (33.3%) positive. A PCR assay specific for the Bartonella 16S rRNA gene was carried out on DNA samples extracted from blood of the 234 cats; 26 (11.1%) of the seropositive cats were positive. Two PCR protocols, which discriminate genotypes I and II of B. henselae, were performed on all DNA samples. Sixteen (6.8%) cats were infected by genotype I, 6 (2.5%) by genotype II, and two males (0.8%) by both genotypes. Two female (0.8%) cats which were Bartonella sp. PCR positive, gave negative results with the types I and II PCR. This protocol facilitates the direct and rapid detection of Bartonella DNA in feline blood samples, and differentiates B. henselae genotypes.     

Prevalence of hemoplasmas and Bartonella species in client-owned cats in Beijing and Shanghai,China

A year-round molecular epidemiological survey (2017 to 2018) was conducted on three hemoplasmas and two Bartonella species with zoonotic potential in client-owned cats in Beijing and Shanghai. Among 668 specimens, the overall hemoplasma-positive rate was 4.9% (3.4% for Candidatus Mycoplasma haemominutum, 0.9% for Mycoplasma haemofelis and 1.2% for Candidatus Mycoplasma turicensis). The overall Bartonella-positive rate was 8.5% (4.8% for B. henselae and 4.3% for B. clarridgeiae). Age, breed, ectoparasiticide use and stray history, but not city, season and gender, were significantly associated with the positive rates of one or more pathogens. This is also the first report on the prevalence of Candidatus Mycoplasma turicensis in cats in China.     

Seroprevalence of and risk factors for Bartonella henselae and Bartonella quintana infections among pet cats in Jordan

To determine the seroprevalences of Bartonella henselae and Bartonella quintana antibodies in Jordanian pet cats, serum samples from 153 cats from three geographical regions were analyzed. Seroprevalences were determined by indirect immunofluorescence. The true seroprevalences to B. henselae and B. quintana were 32 and 1.5%, respectively. The seroprevalence of B. henselae-specific antibodies in cats from northern Jordan was significantly higher than in cats from the central or southern parts of Jordan. The seroprevalence to B. henselae increased to age 2 years. Odds of seropositivity were higher in cats living outdoors, showing hunting behavior, having a flea infestation and of a mixed breed. No association was detected with sex.     

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.     

Bartonella henselae seroprevalence in cattle breeders and veterinarians in the rural areas of Aydin and Denizli, Turkey

Bartonella henselae infections are usually detected among people who have close contact with animals. Veterinarians and cattle breeders, in particular, are considered as the risk groups for B. henselae infections. In this study, the seroprevalence of antibodies to B. henselae was investigated in these two groups of subjects in the two cities of Aydin and Denizli, which are located in the same region in the southwest of Turkey. Total antibodies to B. henselae were evaluated by indirect immunofluorescence assay in serum samples taken from 63 cattle breeders and 27 veterinarians. Twenty samples (22.2%) were found to react on 1/64 titre with B. henselae antigens. Bartonella henselae seroprevalence was found to be significantly related to age (P = 0.033) and higher in those living in Aydin (P = 0.047). Age was the only independent factor in multivariate analysis (P = 0.008). Seroprevalence was found to be 2‐fold higher in those people who had had tick contact (P = 0.093). In conclusion, the physicians in the region should consider B. henselae infection among veterinarians and breeders in their differential diagnosis list of fever of unknown origin.     

Molecular evidence for Bartonella spp. in cat and dog fleas from Germany and France

Nine hundred and fifty-two fleas were collected from 148 cats and 133 dogs at 18 widely distributed geographic locations in Germany and France and examined for the presence of six different Bartonella spp. (Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella elizabethae, Bartonella henselae, Bartonella quintana, Bartonella vinsonii subsp. berkhoffii) by PCR. Thirty-five specimens (3.7%) tested positive for either B. henselae (14 positive fleas) or B. clarridgeiae (21 positive fleas). DNA of other Bartonella spp. were not detected. Bartonella clarridgeiae was the dominating species in samples from France (19 out of 22 positive fleas), whereas B. henselae was more frequent in Germany (11 out of 13 positive fleas). With 3.5% (22 out of 632 fleas) in France and 4.1% (13 out of 320 fleas) in Germany, the overall prevalences of pathogen did not vary significantly between the flea populations of both countries. 5.4% of cats in France versus 16.1% of cats from Germany were infested by fleas carrying Bartonella, whereas 9.5% of dogs in France but none of the examined dogs from Germany were infested by Bartonella positive fleas. The molecular evidence of Bartonella infections reveals that agents of zoonotic potential are established in flea populations in Germany and France and that the spectrum of species can vary significantly from country to country.     

Prevalence of DNA of Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum,' Anaplasma phagocytophilum, and species of Bartonella, Neorickettsia, and Ehrlichia in cats used as blood donors in the United States

OBJECTIVE: To identify the prevalence of DNA of Mycoplasma haemofelis; 'Candidatus Mycoplasma haemominutum'; Anaplasma phagocytophilum; and species of Bartonella, Neorickettsia, and Ehrlichia in blood of cats used as blood donors in the United States. DESIGN: Prospective study. ANIMALS: 146 cats that were active blood donors. PROCEDURES: Environmental history was requested for each blood-donor cat from which a blood sample (mixed with EDTA) was available. Polymerase chain reaction assays capable of amplifying the DNA of the microorganisms of interest following DNA extraction from blood were performed. RESULTS: Overall, DNA of one or more of the infectious agents was detected in blood samples from 16 of 146 (11%) feline blood donors. Twenty-eight laboratory-reared cats housed in a teaching hospital had negative results for DNA of all organisms investigated. The DNA of at least 1 infectious agent was amplified from blood samples collected from 16 of 118 (13.6%) community-source cats; assay results were positive for 'Candidatus M haemominutum,' M haemofelis, or Bartonella henselae alone or in various combinations. Of the community-source cats allowed outdoors (n = 61) or with known flea exposure (44), DNA for a hemoplasma or B henselae was detected in 21.3% and 22.7%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: When community-source cats, cats allowed outdoors, or cats exposed to fleas are to be used as blood donors, they should be regularly assessed for infection with M haemofelis, 'Candidatus M haemominutum,' and Bartonella spp, and flea-control treatment should be regularly provided.     

Detection and characterization of feline Bartonella henselae in the Czech Republic

The aims of the study were to characterize isolates of Bartonella henselae and to determine the prevalence of bacteremic domestic cats in urban and suburban parts of Prague, Czech Republic. Five (18%) gram-negative fastidious bacterial single-cat isolates were recovered from 27 hemocultures incubated without previous freezing. Four of these isolates originated from flea infested stray cats (n=6) and one from a shelter cat without any ectoparasites (n=21). None of the 34 previously frozen specimens from flea free pet cats yielded any bacteria. All five isolates were catalase and oxidase negative. Their enzymatic activity, RFLP profile of citrate synthetase gene (gltA) and DNA-DNA hybridization results were typical of B. henselae. According to their PvuII and BglI ribotypes the isolates could be allocated to two homogeneous groups. Ribotype HindIII and RFLP of 16S-23S rRNA spacer region analysis gave unique profiles different from those of Bartonella quintana, Bartonella elizabethae and Bartonella clarridgeiae. The 16S rRNA type-specific amplification revealed an identical profile typical of B. henselae genotype II for all the cat isolates studied. Pulsed-field gel electrophoresis (PFGE) assigned a different profile to each of the isolates studied. Determination of the enzymatic activity, RFLP of gltA gene, RFLP of 16S-23S rRNA spacer region, and HindIII ribotype could be efficient tools for identification of B. henselae isolates. Ribotyping (PvuII, BglI), 16S rRNA typing and PFGE may be useful methods to prospect ecology and epidemiology of the agent.     

A serological investigation of Bartonella henselae infection in cats in Turkey

Bartonella henselae is the causative agent of cat scratch disease (CSD) in humans. Cats are the main reservoir of this bacterium and may infect humans through scratches and bites. The purpose of this study was to determine the B. henselae seroprevalence in cats in Turkey. A total of 298 cats blood samples were collected from six different provinces of Turkey. Sera were tested for the presence of anti-B. henselae IgG antibodies by indirect fluorescent antibody test (IFA). The seroprevalence of B. henselae was 27.9% (83/298) for the cats examined in this study. The seroprevalence of cats by province was significantly higher in Bursa (41.3%), Adana (33.9%), Aydin (27.5%) and Burdur (32.3%) than in Kayseri (17.9%) and Istanbul (12.5%). Statistically significant differences were not observed between cat sexes and living conditions of cats. The results revealed that B. henselae is an important zoonotic pathogen in Turkey.