Research in Veterinary Science 88 (2010) 379384

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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, ne 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 amplication 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 6072.9% 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. 2009 Elsevier Ltd. All rights reserved.

Article history: Accepted 5 November 2009

Keywords: Bartonella henselae Bartonella clarridgeiae Nested-PCR Oral swabbloodlymph 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 ea, Ctenocephalides felis (Chomel et al., 1996) and human infection occurs through contamination of cat scratches with ea excrement or cat bites, if cat blood or ea 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, Facolt di Medicina e Chirurgia, Policlinico Universitario, Torre Biologica, 2 piano, Universit 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 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 Immunodeciency Virus (FIV) was signicantly 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

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with B. henselae and Bartonella clarridgeiae has been reported (Gureld et al., 2001). Although B. clarridgeiae has also been linked to cases of cat scratch disease (Kordick et al., 1997), the role of this organism in causing human disease in unclear. Veterinarians are increasingly being asked to test pets belonging to owners who have, or are considered most susceptible to Bartonella-related diseases. Currently, the laboratory diagnosis of bartonellosis in cats is based on bacterial culture, serology or polymerase chain reaction (PCR) from blood and tissue specimens. Isolation is the gold standard for proving the infection but the need of specialized media limit to the research eld this technique. Serologic testing usually overestimates active infection and is of limited diagnostic value for bacteraemia because the positive predictive value is only 46.4% and the negative predictive value is 89.7% (Glaus et al., 1997). Nested PCR on DNA extracted from blood has been considered a sensitive method for diagnosis of B. henselae infection (Roy et al., 2001). As no data claiming the molecular evidence of B. henselae and B. clarridgeiae in cats are available in Southern Italy, the aim of this study was to determine the carriage rate and the distribution of these bacteria in different biological samples (oral swabs, blood, ne 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, especially for immunocompromised patients. Evaluation of oral swabs for Bartonella DNA detection could represent an additional, non invasive diagnostic tool useful for enhancing the sensitivity of the test results. 2. Materials and methods 2.1. 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. Data concerning age, breed, gender and neutering status, environmental history (indoor/outdoor, single cat household/multi-cat household) of the cat, history or presence of ea and tick infestation were recorded together with physical examination ndings and laboratory investigations. Both healthy (admitted for vaccination or neutering) and unhealthy cats were enrolled as specied in item 3.1; cats affected by lymphadenomegaly and/or oral pathologies were specically targeted because of a concomitant cytological study on the same cats (data not shown). 2.2. Clinical samples 2.2.1. 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. 2.2.2. Lymph node aspirate A lymph node ne needle aspirate from a submandibular lymph node was taken for molecular analysis and for a cytological evaluation from each cat (not reported here). 2.2.3. Oral swab A dry cotton swab was rolled over the vestibular area (normal cats) or over lesions (cats affected by gingivo-stomatitis). Cytological samples were also carried out from the same mucosal area (data not shown). All clinical samples were stored at 20 C until tested.

2.3. DNA extraction DNA was extracted from all materials using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturers instructions. Briey, clinical samples were added to an appropriate volume of phosphate buffered saline (PBS) and homogenized by vortexing. Twenty microlitres of a proteinase K solution (20 mg/ml) and 200 ll of buffer AL provided in the kit were then added, followed by incubation at 56 C for 10 min. Next, 200 ll of ethanol (96%) were added. The mixture was then loaded on the QIAamp spin column and centrifuged at 6000g for 1 min. The QIAamp spin column was placed in a 2 ml collection microtube, and the tube containing the mixture was discarded. The column material was washed (500 ll each) with the rst washing buffer (Buffer AW1) and with the second washing buffer (Buffer AW2) provided in the kit. Finally, the DNA was eluted with 150 ll of a third buffer (Buffer AE) provided in the kit. 2.4. Oligonucleotide primers Two pairs of primers targeting species-specic size differences in the 16S23S rDNA intergenic regions were used in nested-PCR for the detection of B. henselae and B. clarridgeiae. The outer primer pairs P-bhenfa (50 -TCTTCGTTTCTCTTTCTTCA-30 ) and P-benr1 (50 -CAAGCGCGCGCTCTAACC-30 ) gave a 186 bp fragment for B. henselae and 168 bp fragment for B. clarridgeiae. Nested inner primers N-bhenf1a (50 -GATGATCCCAAGCCTTCTGGC-30 ) and N-bhenr (50 - AACCAACTGAGCTACAAGCC-30 ) amplied a 152 bp fragment for B. henselae and a 134 bp for B. clarridgeiae (Rampersad et al., 2005). Additionally, a second primer pair for the pap31 gene, PAPn1 (50 -TTCTAGGAGTTGAAACCGAT-30 ) and PAPn2 (50 -GAAACACCACCAGCAACATA-30 ), as described by Zeaiter et al. (2002), was used to compare all B. henselae positive results of the rst primer sets, obtained from oral swabs samples. The expected size of the product generated with PAPn1- PAPn2 is a 275 bp. All primers were purchased from MWG-Biotech AG. 2.5. PCR protocols Nested PCR protocol for amplication of the B. henselae and B. clarridgeiae DNA was applied to all DNA extracted. The reaction mixture of the rst step, which was made up to 25 ll with sterile water, contained 0.5 pmoles/ml of each primer (P-bhenfa and P-benr1), 0.2 mM of each deoxyribonucleotide triphosphate, 1 PCR buffer (50 mM KCL, 10 mM TrisHCL pH 8.8, 0.1% Triton X-100), 3 mM MgCl2, 0.5 U Taq DNA polymerase and 5 ll extracted DNA. The amplication conditions for the rst step were 94 C for 15 s, 53 C for 30 s, and 72 C for 30 s for 40 cycles, followed by a nal extension step of 72 C for 5 min. The reaction mixture of the second step (25 ll) contained 0.5 pmoles/ml of each primer (N-bhenf1a and N-benr), 0.2 mM of each deoxyribonucleotide triphosphate, 1 PCR buffer (50 mM KCL, 10 mM TrisHCL pH 8.8, 0.1% Triton X-100), 1.5 mM MgCl2, 0.5 U Taq DNA polymerase and 1 ll of the primary reaction mixture. The amplication conditions for the second step were 94 C for 15 s, 57 C for 30 s, and 72 C for 30 s for 40 cycles; the second step was nalized by 72 C for 5 min. In each experiment, puried DNA obtained from a cultured B. henselae Houston-1 (ATCC 49882), B. clarridgeiae (ATCC 51734) and DNAfree water were used as positive and negative controls, respectively. PCR protocol for amplication of the Bartonella pap31 gene was applied to DNA extracted from oral swabs samples. Pap31 PCR amplication 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, annealing

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for 30 s at 58 C, and extension for 45 s at 72 C. The amplication was completed by holding the reaction mixture at 72 C for 7 min to allow complete extension of the PCR products. Detection of PCR products was performed by gel electrophoresis. Samples (15 ll) of nal PCR products were loaded onto 3% agarose gel (MetaPhor Agarose) and subjected to electrophoresis in 1 TAE (0.04 M Tris-acetate, 0.001 M EDTA) buffer for 240 min at 70 V. The gels were stained with ethidium bromide and photographed under UV light trans-illumination. A 25-bp DNA ladder (Promega) was included on each gel as a molecular size standard. 2.6. Sequence analysis Twenty PCR positive products, obtained from the pap31 gene and ten randomly selected nested-PCR positive products obtained from blood, lymph node aspirate and oral swab samples (3, 3 and 4, respectively) were used for nucleotide sequencing. The amplicons were puried by using the Wizard SV Gel and PCR clean-up System (Promega) according to the manufacturers instructions. The sequencing was performed by Genelab c/o ENEA Casaccia Via Anguillarese 301, 00060 S. Maria di Galeria Roma. Blast software (http://www.ncbi.nlm.nih.gov/blast/) was used to conduct homology searches of the GenBank database (Altschul et al., 1997). 2.7. Statistical analysis Statistical analysis was performed using the software GraphPad In-Stat for Windows and P < 0.05 was used to indicate statistical signicance. Comparison of medians of the age of B. henselae positive and B. henselae negative cats was performed with the MannWhitney test. Analysis of B. henselae prevalence related to sex, clinical status, ectoparasite exposure, environmental history, was made with the Fishers exact test. Analysis of prevalence of B. henselae PCR positive results in cats affected by lymphoadenomegaly and in cats with oral pathologies was made with the Fishers exact test. Comparison of results originating from the three different biological samples was made with the chi-square test. To further evaluate the usefulness of oral swabs, comparison of detection of B. henselae expressed as percentages of concordance between the results obtained in oral swabs vs. blood samples and vs. lymph node was performed. 3. Results 3.1. Cat descriptions Of the cats sampled, 72 (84.7%) were domestic short-hair cats and 13 (15.3%) were pedigree (Persian, Balinese, Siamese) or pedigree crosses (Persian, Siamese, Norwegian). Forty-six out of 85 (54%) were females and 39 out of 85 (46%) were males. The age of the cats ranged between 4 months and 15 years (mean 3.87 + 3.65; median 2.5) with 27 cats (31.8%) being 61 year of

age. Fifty-eight (68.2%) of the 85 cats lived or had lived outdoors and 27 (31.7%) were indoor cats; 34 cats were from a single cat household (40%) and 51 cats out of 85 (60%) were from a multicat household. Fifty-four (63.5%) of the 85 cats were reported to be more or less frequently infested with eas and 18 out of these 54 cats (33.3%) were also infected with ticks. Seventeen cats (20%) were clinically healthy (admitted for vaccination or neutering) and 68 (80%) cats had clinical signs at the time of sampling. The 68 cats with clinical signs were affected by lymphadenomegaly (67.6%), oral pathologies (64.7%), respiratory signs (10.3%), otitis (10.3%), gastro-intestinal signs (7.4%), ocular signs (7.4%), skin lesions (5.9%), and other miscellaneous conditions (36.8%). In most cases each single cat had a combination of different clinical signs, with 27 cats (39.7%) showing both lymphadenomegaly and oral pathologies. 3.2. PCR amplications and sequencing None of the samples subjected to PCR were positive in the primary reaction, but n. 60 blood samples, n. 62 lymph node aspirates and n. 51 oral swabs were positive in the nested reaction, as an amplication band of 152 bp size marker relative to B. henselae was observed. However, no PCR product was obtained for B. clarridgeiae. Positive and negative controls showed expected results in the primary and in the nested reactions. The pap31-based PCR assay has conrmed with success the occurrence of B. henselae in oral swab samples already detected as positive by the other two pairs of primers targeting species-specic size differences in the 16S23S rDNA intergenic regions. Results of PCR reactions were conrmed by subsequent sequence analysis. Alignment of 16S23S gene sequences of Bartonella spp. available in GenBank showed that there was a complete homology of nested amplied products with the species B. henselae (GenBank accession number DQ529247, AF312496, AF312495, AJ457178, BX897699). The sequences deduced by the pap31-based PCR identied B. henselae DNA in oral swab samples examined (GenBank accession number DQ351240 and AF308167). When PCR positive results with any of the three different samples tested were considered as a whole, B. henselae-specic DNA was found in 83.5% (71/85) of all cats (Table 1). PCR amplication of B. henselae DNA yielded 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, with no signicant difference (Table 1). Analyzing the detection result, 39 out of 71 (54.9%) B. henselae positive cats showed positive PCR result in all of the three clinical samples. Detection result was negative in all of the clinical samples from 14 cats. Female cats showed a 82.6% prevalence (38 positive samples out of 46) and male cats a 84.6% prevalence (33 positive samples out of 39), with no signicant difference related to sex. B. henselae positive cats had a median age of 1.66 years and the neg-

Table 1 Analysis of Bartonella henselae detection results in both asymptomatic and symptomatic cats. Clinical signs No. of cats Positive results obtained from at least one sample (%) 71 16 55 36 35 39 32 (83.5) (94.1) (80.9) (81.8) (85.3) (84.7) (82.0) No. of B. henselae PCR positive products from each diagnostic sample clinical (%) Blood (%) 60 12 48 30 30 34 26 (70.6) (75.0) (69.6) (68.1) (73.1) (73.9) (66.6) Lymph node aspirate (%) 62 14 48 32 30 32 30 (72.9) (87.5) (69.6) (72.7) (73.1) (69.5) (76.9) Oral swab (%) 51 11 40 28 23 27 24 (60.0) (68.7) (58.0) (63.6) (56.0) (58.7) (61.5)

Total Asymptomatic Symptomatic Oral pathology No oral pathology Lymphadenomegaly No lymphadenomegaly

85 17 68 44 41 46 39

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Table 2 Comparison of detection of Bartonella henselae in three clinical materials of 85 cats expressed as percentages of total and partial concordances. No. of cats 85 46 44 Clinical signs Asymptomatic and symptomatic Lymphadenomegaly Oral pathologies Oral swab blood (%) 60 (70.6) 31 (67.4) 32 (72.7) Oral swablymph node aspirate (%) 64 (75.3) 33 (71.7) 34 (77.2)

ative cats a median age of 4.00 years, with a difference borderline signicant (P = 0.0572). Forty-nine out of the 58 (84.5%) outdoor cats were B. henselae positive as were 22 out of 27 (81.5%) indoor cats (not a signicant difference). Thirty-one out of 34 (91.2%) cats living in single cat households were B. henselae positive as were 40 cats out of 51 (78.4%) living in a multicat household (not a significant difference). The prevalence of B. henselae DNA in cats not reported with eas (26/31; 83.8%) was similar to that detected in those with eas (45/54; 83.3%) (not a signicant difference). The analysis of B. henselae detection results in both symptomatic and asymptomatic cats is shown in Table 1. B. henselae DNA was amplied from 16 out of 17 asymptomatic cats (94.1%) and from 55 out of 68 cats (80.9%) with clinical signs. The B. henselae detection results analyzed according to clinical signs showed no signicant difference. Of the 44 cats with oral pathologies, 36 (81.8%) were positive for B. henselae, compared to 35 out of 41 (85.3%) cats without oral pathologies. PCR amplication of B. henselae DNA from oral swabs occurred in 28 of the 44 cats (66.3%) with oral pathologies and in 23 of the 41 cats (56%) without oral pathologies (not a signicant difference). Of the 46 cats with lymphadenomegaly, 39 (84.7%) were positive for B. henselae, compared to 32 out of the 39 (82%) cats without lymphadenomegaly (not a signicant difference). B. henselae DNA was amplied from lymph nodes of 32 of the 46 cats (69.5%) with lymphadenomegaly and of 30 of the 39 cats (76.9%) with no lymphadenomegaly (not a signicant difference) (Table 1). Concordant positive results concerning the detection of B. henselae DNA in blood and in oral swab samples were obtained from 60 of the 85 (70.6%) cats examined (Table 2). Lymph node aspirates results were concordant with those from swab samples in 64 out of 85 (75.3%) cats. Percentages of concordance among cats with the two more represented clinical signs ranged between 67.4% (lymphadenomegaly) and 72.7% (oral pathologies) for blood and oral swab samples and between 71.7% (lymphadenomegaly) and 77.2% (oral pathologies) for lymph node aspirate and oral swab samples (Table 2). 4. Discussion In this study the prevalence of B. henselae and B. clarridgeiae in pet cats from the Messina area in Sicily, in the Southern Italy was assessed by using a nested-PCR assay. The majority of these cats were clinically ill cats. This allowed us to determine the diagnostic value of the PCR assay in three different clinical samples of cats classied according to their clinical status. Overall, molecular analysis showed that the PCR scored positive with B. henselae DNA in 71 cats (83.5%). A lack of B. clarridgeiae PCR products is consistent with the results reported by Fabbi et al. (2004) concerning the very low frequencies of B. clarridgeiae in cat populations in Northern Italy. To our knowledge, no reports have been published on molecular evidence of B. henselae and B. clarridgeiae in cats in Sicily. Exposure to B. 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., 2002; Fabbi et al., 2004; Pinna Parpaglia et al., 2007). Seroreactivity reported by Fabbi et al. (2004) in a population

of stray and domestic cats from nine areas of Northern Italy was 39% and 43.5%, respectively. A serologic study of cats in Sardinia identied an overall seroprevalence of 21.5% (Pinna Parpaglia et al., 2007) similar to that referred in Tuscany by Ebani et al. (2002). The prevalence of B. henselae infection in the cat study population in Sicily was clearly higher than those previously obtained in other Italian regions, but a comparison is irrelevant because of the different techniques used. Sero-epidemiological and bacteriological studies have demonstrated the worldwide distribution of B. henselae infection in both stray and pet cats. Solano-Gallego et al. (2006) also reported the highest seroprevalence (71.4%) of B. henselae in cats in Europe. Other authors have reported different percentage values for B. henselae seroprevalence in Europe. In Denmark, Chomel et al. (2002) detected B. henselae antibodies in 46.7% of the cats studied. The B. henselae seroprevalence reported in cats was similar in Denmark and other European countries (Barnes et al., 2000; Gureld et al., 2001). Lower percentages of B. henselae seroprevalence in cats were reported in Switzerland (8.3%) and in Germany (15%) (Glaus et al., 1997; Haimerl et al., 1999). A serologic study in pet cats from USA and some areas of Canada reported a 27.9% B. henselae seroprevalence (Jameson et al., 1995). The overall seroprevalence of cats in Taiwan and in the Philippines was 23.7% and 68%, respectively (Chomel et al., 1999; Chang et al., 2006). There is a substantial difference in prevalence rates for B. henselae infection reported by serological and molecular assays. 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. In the very early stages the antibody titre for both IgG and IgM might still be low, and diagnosis of B. henselae infection can only be conrmed after increasing titres have been noted in a second serum sample. Moreover, there is no linear association between the antibody titre and the level of bacteraemia (Fabbi et al., 2004). Bacteraemia can last for months and, in some cats, even many years. As B. henselae is intraerythrocytic, the cats may become adapted to the chronic bacteraemia (Rolain et al., 2001; Jacomo et al., 2002). It is understandable that a serological study does not strictly correlate to PCR analysis. 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., 2001). Our molecular prevalence (70.6%) for B. henselae DNA in the blood of a cat population from Sicily is higher than that (15%) reported by Solano-Gallego et al. (2006) in a cat population from Mallorca and than that reported by Kim et al. (2009) in feral (41.8%) and pet cats (33.3%) from Korea. In the latter study (Kim et al., 2009) saliva tested positive more often than blood in both feral (44.1%) and pet cats (43.5%), opposite to what observed in our study where 60% of oral swabs were positive. Climatic differences among these countries are one possible explanation for difference in prevalence rates reported by serological and molecular assays for B. henselae infection. The high prevalence rate of B. henselae infection reported in this study correlates with warm temperatures and high humidity in the area of the investigation. Conversely, the B. henselae positivity rate in ea-infested cats was not higher than that in the ea-free ones examined in our study. These data do not compare favourably with previous reports from other countries (Maruyama et al., 2003; Fabbi et al., 2004). Previous epidemiological studies have reported young cats as being the highest risk group for B. henselae infection (Guptill et al., 2004). No signicant association could be made between our DNA B. henselae positive results and gender, sex, indoor/outdoor, single cat/multi-cat status of cats and clinical signs, probably because of the overall high prevalence of the infection. Our results support previous research which asserts that the infection with Bartonella

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spp. was not overrepresented in a population of cats with gingivostomatitis (Quimby et al., 2008). Conversely, a study in Switzerland and Germany reported an increased frequency of stomatitis or urological diseases in Bartonella seroreactive sick cats (Glaus et al., 1997). The simultaneous analysis of three different clinical samples (blood, lymph node and oral swab) in our study increased the diagnostic possibilities of B. henselae detection in the cats examined from 60% to 83.5%. Lymph node aspirates were found to be the most effective clinical sample and blood was the next best for the detection of B. henselae. Seven cats with a B. henselae DNA negative result from oral swabs had at least one B. henselae DNA positive result from blood sample or lymph node sample and 13 more cats with negative results from oral swabs had positive ones from both blood and lymph node samples. In any case, we consider oral swab results favourably, particularly if compared with blood. In 8 out of the 85 tested cats (9.4%) PCR B. henselae positive products were obtained successfully in oral swabs even though PCR assay from blood samples failed. In 1 of the 85 cats examined, the oral swab was the only B. henselae DNA positive clinical sample. To exclude the possibility that the band obtained from this single cat was a false positive, DNA sequencing was performed and the identity of the fragment was conrmed to be from B. henselae (GenBank accession number DQ529247). Moreover the oral swab is an easier procedure than taking blood and, in most cases, than lymph node ne needle aspiration in cats not having lymphadenomegaly. In this latter case, testing both blood and oral samples may easily enhance the sensitivity of PCR testing. These results suggest that the use of nested-PCR assay by oral swabs together with blood sample and/or lymph node ne needle aspiration may improve sensitivity in the diagnosis of bartonellosis in cats, irrespective of the clinical status of animals. Acknowledgements We are grateful to Dr. Massimo Fabbi, Sezione Diagnostica di Pavia, Istituto Zooprolattico Sperimentale della Lombardia e dellEmilia Romagna Bruno Ubertini Pavia, Italy, for providing the strain of B. henselae ATCC 49882. This study was supported in part by research grants (2003 nancing) from the University of Messina, Italy. References
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