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

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Abstract

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.

Introduction

Bartonella henselae is a gram-negative, intraerythrocytic, feline-adapted 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 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 with B. henselae and Bartonella clarridgeiae has been reported (Gurfield 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 field 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, 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, 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.

Section snippets

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 flea and tick infestation were recorded together with physical examination findings and laboratory investigations.

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 ⩽1 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

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 classified 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

Acknowledgements

We are grateful to Dr. Massimo Fabbi, Sezione Diagnostica di Pavia, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Ubertini” Pavia, Italy, for providing the strain of B. henselae ATCC 49882. This study was supported in part by research grants (2003 financing) from the University of Messina, Italy.

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