Research in Veterinary Science 88 (2010) 379–384

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Research in Veterinary Science
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Molecular detection of Bartonella henselae and Bartonella clarridgeiae in clinical samples of pet cats from Southern Italy
M.G. Pennisi a, E. La Camera b, L. Giacobbe a, B.M. Orlandella a, V. Lentini c, S. Zummo b, M.T. Fera b,*
a

Department of Veterinary Public Health, University of Messina, 98100 Messina, Italy Department of Pathology and Experimental Microbiology, University of Messina, 98125 Messina, Italy c Department of Animal Biology and Marine Ecology, University of Messina, 98100 Messina, Italy
b

a r t i c l e

i n f o

a b s t r a c t
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. Ó 2009 Elsevier Ltd. All rights reserved.

Article history: Accepted 5 November 2009

Keywords: Bartonella henselae Bartonella clarridgeiae Nested-PCR Oral swab–blood–lymph node samples

1. Introduction Bartonella henselae is a gram-negative, intraerythrocytic, felineadapted fastidious bacterium prevalent among cats throughout most temperate regions of the world. Transmission among cats is mediated by the cat flea, Ctenocephalides felis (Chomel et al., 1996) and human infection occurs through contamination of cat scratches with flea excrement or cat bites, if cat blood or flea excrement contaminates the wound. Substantial evidence has linked B. henselae to various human infectious diseases and it is considered a zoonotic agent (Chomel et al., 2004). Human infections include vasoproliferative illness-bacillary angiomatosis, hepatosplenic granulomatosis, peliosis hepatitis, fever, central nervous disorders and, more commonly, cat scratch disease (Welch et al., 1992; Kordick et al., 1995; Chomel et al., 2006a). Exposure to cats has been proven to be an important acquisition factor for B. henselae

* Corresponding author. Address: Dipartimento di Patologia e Microbiologia Sperimentale, Facoltà di Medicina e Chirurgia, Policlinico Universitario, Torre Biologica, 2° piano, Università di Messina, 98125 Messina, Italy. Tel.: +39 090 2213313; fax: +39 090 2213312. E-mail address: mtfera@unime.it (M.T. Fera). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.11.005

infections. According to the immune status of the host and to the bacterial species and strain, naturally infected cats can develop a chronically recurrent bacteraemia, thus playing a major role as a reservoir for the bacterium (Chomel, 2000; Chomel et al., 2006b). The clinical spectrum of the natural infection in cats is not adequately known because of the high prevalence of B. henselae infection in the cat population. Naturally infected cats seem primarily to be asymptomatic carriers of the bacterium (Chomel et al., 2004; Boulouis et al., 2005; Breitschwerdt and Kordick, 2000). However, sporadic cases of uveitis (Lappin et al., 2000) and two rare cases of valvular endocarditis (Chomel et al., 2003) have been associated with infection caused by B. henselae. According to retrospective studies, seropositive sick cats were more likely to have a variety of kidney and urinary tract diseases and stomatitis. Co-infection of cats with B. henselae and Feline Immunodeficiency Virus (FIV) was significantly associated with gingivitis or lymphoadenomegaly than was either infection alone (Ueno et al., 1996). In experimentally infected cats asymptomatic infection or transient fever, lethargy, anorexia, lymphadenomegaly, mild neurologic signs, myalgia and reproductive disorders have been reported and their severity varied with the strain used for inoculation (Regnery et al., 1996; Guptill et al., 1997; Yamamoto et al., 2002). Co-infection of cats

The QIAamp spin column was placed in a 2 ml collection microtube. 1997). The reaction mixture of the second step (25 ll) contained 0. followed by incubation at 56 °C for 10 min. In each experiment. single cat household/multi-cat household) of the cat. 2005). obtained from oral swabs samples. 2001). 53 °C for 30 s. Data concerning age. The expected size of the product generated with PAPn1. purified DNA obtained from a cultured B. The outer primer pairs P-bhenfa (50 -TCTTCGTTTCTCTTTCTTCA-30 ) and P-benr1 (50 -CAAGCGCGCGCTCTAACC-30 ) gave a 186 bp fragment for B. 0. Cytological samples were also carried out from the same mucosal area (data not shown). As no data claiming the molecular evidence of B.3. 10 mM Tris–HCL pH 8. 57 °C for 30 s. history or presence of flea and tick infestation were recorded together with physical examination findings and laboratory investigations.2..AACCAACTGAGCTACAAGCC-30 ) amplified a 152 bp fragment for B. serology or polymerase chain reaction (PCR) from blood and tissue specimens. annealing .2. henselae and a 134 bp for B..3.8. Isolation is the gold standard for proving the infection but the need of specialized media limit to the research field this technique. environmental history (indoor/outdoor. Both healthy (admitted for vaccination or neutering) and unhealthy cats were enrolled as specified in item 3.2. and the tube containing the mixture was discarded.1. the laboratory diagnosis of bartonellosis in cats is based on bacterial culture.. which was made up to 25 ll with sterile water.. gender and neutering status. as described by Zeaiter et al.1. PCR protocols Nested PCR protocol for amplification of the B. the aim of this study was to determine the carriage rate and the distribution of these bacteria in different biological samples (oral swabs.4.2 mM of each deoxyribonucleotide triphosphate. The amplification conditions for the first step were 94 °C for 15 s. 0. B. Blood samples Blood samples (1 ml) obtained by aseptic procedure from the jugular veins of cats were placed in serum separator and ethylene-diamine-tetraacetate (EDTA) treated tubes. Finally. Nested PCR on DNA extracted from blood has been considered a sensitive method for diagnosis of B. DNA extraction DNA was extracted from all materials using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s instructions. Currently. henselae and Bartonella clarridgeiae has been reported (Gurfield et al. 1. Twenty microlitres of a proteinase K solution (20 mg/ml) and 200 ll of buffer AL provided in the kit were then added. The amplification conditions for the second step were 94 °C for 15 s.5. 2. cats affected by lymphadenomegaly and/or oral pathologies were specifically targeted because of a concomitant cytological study on the same cats (data not shown). or are considered most susceptible to Bartonella-related diseases. Evaluation of oral swabs for Bartonella DNA detection could represent an additional. 0. henselae and B. henselae positive results of the first primer sets. 1997).2. / Research in Veterinary Science 88 (2010) 379–384 with B. Pennisi et al. Briefly.5 U Taq DNA polymerase and 1 ll of the primary reaction mixture. Additionally. 2001).2 mM of each deoxyribonucleotide triphosphate. fine needle lymph node aspirates) from 85 pet cats recruited in Sicily by using a nested-PCR assay and thus to develop an easy diagnostic strategy for the selection of infection-free cats that are being considered as pets. henselae and B. blood. All clinical samples were stored at À20 °C until tested. clarridgeiae (ATCC 51734) and DNAfree water were used as positive and negative controls.1% Triton X-100). Clinical samples 2. 10 mM Tris–HCL pH 8. 2.380 M.7% (Glaus et al. a second primer pair for the pap31 gene. Pap31 PCR amplification gene was carried out under the following conditions: an initial 3 min of denaturation at 94 °C was followed by 44 cycles of denaturation for 30 s at 94 °C. Lymph node aspirate A lymph node fine needle aspirate from a submandibular lymph node was taken for molecular analysis and for a cytological evaluation from each cat (not reported here). clarridgeiae DNA was applied to all DNA extracted. henselae and 168 bp fragment for B. 1Â PCR buffer (50 mM KCL. The reaction mixture of the first step. Oligonucleotide primers Two pairs of primers targeting species-specific size differences in the 16S–23S rDNA intergenic regions were used in nested-PCR for the detection of B. henselae and B. 2..2. Oral swab A dry cotton swab was rolled over the vestibular area (normal cats) or over lesions (cats affected by gingivo-stomatitis).8. Nested inner primers N-bhenf1a (50 -GATGATCCCAAGCCTTCTGGC-30 ) and N-bhenr (50 . was used to compare all B.PAPn2 is a 275 bp. clarridgeiae in cats are available in Southern Italy. clarridgeiae.1% Triton X-100). Serologic testing usually overestimates active infection and is of limited diagnostic value for bacteraemia because the positive predictive value is only 46. Next. Although B. Veterinarians are increasingly being asked to test pets belonging to owners who have. clinical samples were added to an appropriate volume of phosphate buffered saline (PBS) and homogenized by vortexing. Materials and methods 2.5 U Taq DNA polymerase and 5 ll extracted DNA. All primers were purchased from MWG-Biotech AG.5 pmoles/ml of each primer (N-bhenf1a and N-benr). clarridgeiae has also been linked to cases of cat scratch disease (Kordick et al. clarridgeiae (Rampersad et al. and 72 °C for 30 s for 40 cycles. 2. 1Â PCR buffer (50 mM KCL. 2. and 72 °C for 30 s for 40 cycles.G.1. contained 0. especially for immunocompromised patients.5 pmoles/ml of each primer (P-bhenfa and P-benr1).5 mM MgCl2. 2. PAPn1 (50 -TTCTAGGAGTTGAAACCGAT-30 ) and PAPn2 (50 -GAAACACCACCAGCAACATA-30 ). respectively. breed. Cats A total of 85 pet cats (39 males and 46 females) were recruited in this study between November 2003 and June 2006 at the Small Animal Clinic of the Faculty of Veterinary Medicine of the University of Messina. the DNA was eluted with 150 ll of a third buffer (Buffer AE) provided in the kit.4% and the negative predictive value is 89. 3 mM MgCl2. 2. henselae Houston-1 (ATCC 49882). The mixture was then loaded on the QIAamp spin column and centrifuged at 6000g for 1 min. (2002). 200 ll of ethanol (96%) were added. The column material was washed (500 ll each) with the first washing buffer (Buffer AW1) and with the second washing buffer (Buffer AW2) provided in the kit. clarridgeiae. 0. the second step was finalized by 72 °C for 5 min. non invasive diagnostic tool useful for enhancing the sensitivity of the test results. followed by a final extension step of 72 °C for 5 min. henselae infection (Roy et al. PCR protocol for amplification of the Bartonella pap31 gene was applied to DNA extracted from oral swabs samples. the role of this organism in causing human disease in unclear. 0. 0.

1) (73. Detection of PCR products was performed by gel electrophoresis. Seventeen cats (20%) were clinically healthy (admitted for vaccination or neutering) and 68 (80%) cats had clinical signs at the time of sampling.4%).65. 3. ectoparasite exposure. lymph node was performed.9) Oral swab (%) 51 11 40 28 23 27 24 (60.2%) of the 85 cats lived or had lived outdoors and 27 (31.ncbi.3) (84. Blast software (http://www.6) (75. henselae negative cats was performed with the Mann–Whitney test.. with 27 cats (39. of cats Positive results obtained from at least one sample (%) 71 16 55 36 35 39 32 (83. clarridgeiae.nlm. Norwegian).1) (80. respiratory signs (10. clinical status. / Research in Veterinary Science 88 (2010) 379–384 381 for 30 s at 58 °C.7%) were indoor cats.8%).5) with 27 cats (31.5) Total Asymptomatic Symptomatic Oral pathology No oral pathology Lymphadenomegaly No lymphadenomegaly 85 17 68 44 41 46 39 . Sequence analysis Twenty PCR positive products. The sequencing was performed by Genelab c/o ENEA Casaccia Via Anguillarese 301.7%).5%) of the 85 cats were reported to be more or less frequently infested with fleas and 18 out of these 54 cats (33.04 M Tris-acetate. Female cats showed a 82. comparison of detection of B.M.0) (58.8%) being 61 year of age. blood samples and vs.6% prevalence (33 positive samples out of 39). oral pathologies (64. Siamese.6. respectively) were used for nucleotide sequencing. AF312495. The sequences deduced by the pap31-based PCR identified B.0) (68. The amplicons were purified by using the Wizard SV Gel and PCR clean-up System (Promega) according to the manufacturer’s instructions. 39 out of 71 (54. n. as an amplification band of 152 bp size marker relative to B.87 + 3. Clinical signs No. B.3%) were pedigree (Persian. Siamese) or pedigree crosses (Persian. otitis (10.9%).7%) were domestic short-hair cats and 13 (15.3%). lymph node aspirate and oral swab samples (3. was made with the Fisher’s exact test. 60 blood samples. henselae expressed as percentages of concordance between the results obtained in oral swabs vs. ocular signs (7. The gels were stained with ethidium bromide and photographed under UV light trans-illumination. The pap31-based PCR assay has confirmed with success the occurrence of B.7%) showing both lymphadenomegaly and oral pathologies. Fifty-eight (68. 72. henselae positive and B. no PCR product was obtained for B.G. with no significant difference (Table 1).7) (61. Positive and negative controls showed expected results in the primary and in the nested reactions. henselae PCR positive products from each diagnostic sample clinical (%) Blood (%) 60 12 48 30 30 34 26 (70. median 2.6% prevalence (38 positive samples out of 46) and male cats a 84. Balinese. BX897699). Results 3.6%) and higher than those from oral swabs (51/ 85. 70.9%) similar to those obtained from blood samples (60/85.nih.7) (58.05 was used to indicate statistical significance. 34 cats were from a single cat household (40%) and 51 cats out of 85 (60%) were from a multicat household. of B.7) (73.3%). Fifty-four (63.9) (87. henselae (GenBank accession number DQ529247.gov/blast/) was used to conduct homology searches of the GenBank database (Altschul et al. 3 and 4.9) (66. The amplification was completed by holding the reaction mixture at 72 °C for 7 min to allow complete extension of the PCR products.1.2. 3. Maria di Galeria Roma. henselae PCR positive results in cats affected by lymphoadenomegaly and in cats with oral pathologies was made with the Fisher’s exact test. henselae positive cats showed positive PCR result in all of the three clinical samples. AJ457178. Comparison of results originating from the three different biological samples was made with the chi-square test. skin lesions (5. 72 (84. Statistical analysis Statistical analysis was performed using the software GraphPad In-Stat for Windows and P < 0. Analyzing the detection result. environmental history. To further evaluate the usefulness of oral swabs.0) (69.001 M EDTA) buffer for 240 min at 70 V. 1997). B.7.6) (72. 51 oral swabs were positive in the nested reaction. obtained from the pap31 gene and ten randomly selected nested-PCR positive products obtained from blood. henselae was observed.3%) were also infected with ticks. 0.5) (69. 2.1) (73.5) (94. Analysis of B. A 25-bp DNA ladder (Promega) was included on each gel as a molecular size standard. but n.7) (82. Results of PCR reactions were confirmed by subsequent sequence analysis. and extension for 45 s at 72 °C. Samples (15 ll) of final PCR products were loaded onto 3% agarose gel (MetaPhor Agarose) and subjected to electrophoresis in 1Â TAE (0. Alignment of 16S–23S gene sequences of Bartonella spp. henselae DNA in oral swab samples examined (GenBank accession number DQ351240 and AF308167).66 years and the neg- Table 1 Analysis of Bartonella henselae detection results in both asymptomatic and symptomatic cats. The age of the cats ranged between 4 months and 15 years (mean 3. and other miscellaneous conditions (36. henselae positive cats had a median age of 1. henselae-specific DNA was found in 83. However. Pennisi et al. with no significant difference related to sex.6) (56. Cat descriptions Of the cats sampled. PCR amplification of B. 00060 S.6) Lymph node aspirate (%) 62 14 48 32 30 32 30 (72. 60%) of cats. Analysis of prevalence of B. Detection result was negative in all of the clinical samples from 14 cats. henselae prevalence related to sex.6%). AF312496. In most cases each single cat had a combination of different clinical signs. Forty-six out of 85 (54%) were females and 39 out of 85 (46%) were males.9%) B. The 68 cats with clinical signs were affected by lymphadenomegaly (67.9) (81. 62 lymph node aspirates and n. gastro-intestinal signs (7.0) (63.5% (71/85) of all cats (Table 1).5) (76. henselae in oral swab samples already detected as positive by the other two pairs of primers targeting species-specific size differences in the 16S–23S rDNA intergenic regions. When PCR positive results with any of the three different samples tested were considered as a whole. available in GenBank showed that there was a complete homology of nested amplified products with the species B.0) No. Comparison of medians of the age of B.8) (85.1) (69. henselae DNA yielded positive products from lymph node aspirate specimens (62/85.6) (68. PCR amplifications and sequencing None of the samples subjected to PCR were positive in the primary reaction. 2.4%).

henselae antibodies in 46. Chomel et al. there is no linear association between the antibody titre and the level of bacteraemia (Fabbi et al. in some cats.. compared to 32 out of the 39 (82%) cats without lymphadenomegaly (not a significant difference). This allowed us to determine the diagnostic value of the PCR assay in three different clinical samples of cats classified according to their clinical status. indoor/outdoor.7% (lymphadenomegaly) and 77. and diagnosis of B. As B. 1999.5%). henselae seroprevalence in Europe. PCR amplification of B. Conversely.3) 33 (71. Of the 46 cats with lymphadenomegaly. henselae infection in the cat study population in Sicily was clearly higher than those previously obtained in other Italian regions. (2002). There is a substantial difference in prevalence rates for B.3%) cats. but a comparison is irrelevant because of the different techniques used.6) 31 (67. no reports have been published on molecular evidence of B. No significant association could be made between our DNA B.5%) with lymphadenomegaly and of 30 of the 39 cats (76.4) 32 (72. Bacteraemia can last for months and. Other authors have reported different percentage values for B. Percentages of concordance among cats with the two more represented clinical signs ranged between 67. henselae DNA was amplified from lymph nodes of 32 of the 46 cats (69. henselae positive as were 40 cats out of 51 (78. henselae infection can only be confirmed after increasing titres have been noted in a second serum sample.. 2002.3%) cats without oral pathologies. 39 (84. in the Southern Italy was assessed by using a nested-PCR assay.. henselae seroprevalence in cats were reported in Switzerland (8. henselae and B. 2001).. henselae infection reported by serological and molecular assays. Haimerl et al. It is well known that serological studies only document exposure to infection and negative results do not exclude the presence of acute infection in cats. henselae and B.4% (lymphadenomegaly) and 72. Exposure to B. henselae is intraerythrocytic. clarridgeiae in pet cats from the Messina area in Sicily. (2006) also reported the highest seroprevalence (71. Climatic differences among these countries are one possible explanation for difference in prevalence rates reported by serological and molecular assays for B. compared to 35 out of 41 (85. Solano-Gallego et al. Our results support previous research which asserts that the infection with Bartonella . henselae seroprevalence (Jameson et al. henselae DNA in 71 cats (83. henselae DNA in the blood of a cat population from Sicily is higher than that (15%) reported by Solano-Gallego et al. 2009) saliva tested positive more often than blood in both feral (44. 36 (81. henselae DNA in blood and in oral swab samples were obtained from 60 of the 85 (70. henselae. The B. Pennisi et al.8%) and pet cats (33.3%) from Korea.. The prevalence of B. of cats 85 46 44 Clinical signs Asymptomatic and symptomatic Lymphadenomegaly Oral pathologies Oral swab– blood (%) 60 (70. Jacomo et al. Of the 44 cats with oral pathologies. Sero-epidemiological and bacteriological studies have demonstrated the worldwide distribution of B.7%) were positive for B. even many years. single cat/multi-cat status of cats and clinical signs.4%) living in a multicat household (not a significant difference).8%) were positive for B. Previous epidemiological studies have reported young cats as being the highest risk group for B. the cats may become adapted to the chronic bacteraemia (Rolain et al. No. Pinna Parpaglia et al. (2004) concerning the very low frequencies of B. the B. In the very early stages the antibody titre for both IgG and IgM might still be low. (2002) detected B. Moreover. henselae positive results and gender. opposite to what observed in our study where 60% of oral swabs were positive. The high prevalence rate of B. The B. 2003. respectively. 2001). Concordant positive results concerning the detection of B. probably because of the overall high prevalence of the infection. 2004.. The analysis of B.. To our knowledge. henselae infection (Guptill et al. Chang et al.2%) cats living in single cat households were B.2) ative cats a median age of 4.2% (oral pathologies) for lymph node aspirate and oral swab samples (Table 2).3%) with oral pathologies and in 23 of the 41 cats (56%) without oral pathologies (not a significant difference).. 2006). Our molecular prevalence (70. clarridgeiae in cat populations in Northern Italy. The prevalence of B. Fabbi et al. henselae in cats from different geographic regions of Italy has already been evaluated in previous reports but by serological and bacteriological testing (Ebani et al. 2004).5% (Pinna Parpaglia et al. respectively (Chomel et al.9%) with clinical signs. 1995).6%) cats examined (Table 2). B. 1999).5%) indoor cats (not a significant difference). molecular analysis showed that the PCR scored positive with B. 1997.1%) and pet cats (43. In the latter study (Kim et al. Seroreactivity reported by Fabbi et al.. henselae seroprevalence reported in cats was similar in Denmark and other European countries (Barnes et al.382 M.3%) and in Germany (15%) (Glaus et al. A serologic study in pet cats from USA and some areas of Canada reported a 27.00 years. Forty-nine out of the 58 (84.4%) of B. clarridgeiae PCR products is consistent with the results reported by Fabbi et al. henselae. Lymph node aspirates results were concordant with those from swab samples in 64 out of 85 (75. henselae positivity rate in flea-infested cats was not higher than that in the flea-free ones examined in our study. 83. henselae infection. henselae positive as were 22 out of 27 (81. 2004). Gurfield et al. Thirty-one out of 34 (91.0572).G... henselae detection results analyzed according to clinical signs showed no significant difference. The overall seroprevalence of cats in Taiwan and in the Philippines was 23.5%. 2007). PCR testing has been successful as a method of diagnosis for Bartonella infection in humans and proven more sensitive than bacterial culture in experimentally infected cats (Roy et al. henselae DNA was amplified from 16 out of 17 asymptomatic cats (94.. henselae DNA in cats not reported with fleas (26/31. In Denmark. 4. Overall.. henselae infection reported in this study correlates with warm temperatures and high humidity in the area of the investigation.7) 34 (77.3%) (not a significant difference)..1%) and from 55 out of 68 cats (80.9%) with no lymphadenomegaly (not a significant difference) (Table 1).9% B... It is understandable that a serological study does not strictly correlate to PCR analysis. A serologic study of cats in Sardinia identified an overall seroprevalence of 21. 2002). henselae in cats in Europe. These data do not compare favourably with previous reports from other countries (Maruyama et al. Fabbi et al. 2000. 2001.. henselae DNA from oral swabs occurred in 28 of the 44 cats (66.5%) outdoor cats were B. 2007) similar to that referred in Tuscany by Ebani et al. (2009) in feral (41. Discussion In this study the prevalence of B.8%) was similar to that detected in those with fleas (45/54.6%) for B.7) Oral swab–lymph node aspirate (%) 64 (75. sex.7% of the cats studied. Lower percentages of B.. 2004). clarridgeiae in cats in Sicily. (2004) in a population of stray and domestic cats from nine areas of Northern Italy was 39% and 43.5%).7% and 68%. henselae infection in both stray and pet cats. henselae detection results in both symptomatic and asymptomatic cats is shown in Table 1. 83.7% (oral pathologies) for blood and oral swab samples and between 71.. The majority of these cats were clinically ill cats. B. / Research in Veterinary Science 88 (2010) 379–384 Table 2 Comparison of detection of Bartonella henselae in three clinical materials of 85 cats expressed as percentages of total and partial concordances. (2006) in a cat population from Mallorca and than that reported by Kim et al. with a difference borderline significant (P = 0. A lack of B.

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