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Occurrence of Bartonella henselae types I and II in Central Italian domestic cats
Research in Veterinary Science 93 (2012) 63–66
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Occurrence of Bartonella henselae types I and II in Central Italian domestic cats Valentina V. Ebani ⇑, Fabrizio Bertelloni, Filippo Fratini Department of Animal Pathology, Prophylaxis and Food Hygiene, Faculty of Veterinary Medicine, University of Pisa, Italy
a r t i c l e
i n f o
Article history: Received 29 April 2011 Accepted 13 July 2011
Keywords: Cat Bartonella henselae Cat scratch disease Bacteraemia Genotype PCR
a b s t r a c t 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. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Cat scratch disease (CSD) is a zoonosis mainly caused by Bartonella henselae. The transmission of this intraerythrocytic proteobacterium among cats is via the flea Ctenocephalides felis (Woestyn et al., 2004). Ticks have been proposed as vectors for transmission of Bartonella species among cats, human beings, dogs, and other mammalian hosts. B. henselae has been detected by PCR in Ixodes ricinus ticks, in which transtadial transmission has been demonstrated (Sanogo et al., 2003; Cotté et al., 2008; Guptill, 2010). The natural infection is usually asymptomatic in cats, or characterized by mild clinical signs that owners do not observe. Lymphoadenopathy, stomatitis, renal, and neurological disease are sometimes observed (Guptill, 2010). Infected cats, even if asymptomatic, may be highly bacteraemic for several months, representing an important reservoir for the bacterium and a risk to humans. Serological tests are not able to detect bacteraemic cats, because seropositive subjects may not be carriers. Humans are infected incidentally, directly by a cat’s bite or scratch or indirectly by cat fleas. In immunocompetent hosts the disease is self-limited to CSD, with an erythematous papule at the scratch or bite site and enlargement of regional lymph nodes. Lymph nodes can develop into abscesses, and symptoms, such as headache, malaise, and fever may be reported. Occasionally the bacterium can spread and cause a more serious symptomatology, ⇑ Corresponding author. Address: Department of Animal Pathology, Prophylaxis and Food Hygiene, Faculty of Veterinary Medicine, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy. Tel.: +39 50 2216968; fax: +39 50 2216941. E-mail address: [email protected] (V.V. Ebani). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.07.013
characterized by osteomyelitis, endocarditis, pulmonitis, meningitis, encephalopathy, neuroretinitis, hepatic, and splenic involvement (Carithers and Margileth, 1991; Raoult et al., 1996; Bass et al., 1997; Arisoy et al., 1999; Robson et al., 1999; Florin et al., 2008). In immunocompromised hosts bartonellae are often involved in angioproliferative disorders such as bacillary angiomatosis and hepatic peliosis (Relman et al., 1991; Tappero et al., 1993). B. henselae contains genetically diverse strains: 16S rRNA types I and II, respectively called Huston I and Marseille II. Cats and humans can be infected with either genotype I or II and occasionally with a co-infection of both types. Types I and II seem to have a different role in human bartonellosis: type I appears more pathogenic than type II (Woestyn et al., 2004; Bouchouicha et al., 2009). Previous studies have demonstrated that the prevalence of B. henselae genotypes varies considerably among cat populations from different geographical areas. In particular, type I is dominant in Asia, while type II is dominant in western United States, western Europe and Australia (Chomel et al., 1999; Maruyama et al., 2000; Bergh et al., 2002; Engvall et al., 2003; Messam et al., 2005). However, within a given country, the prevalence of these genotypes may vary in relationship to the studied geographical areas, as demonstrated in France (Chomel et al., 2006). There is little information on the distribution of B. henselae genotypes among Italian cats (Fabbi et al., 2004). The aim of this research is to verify the distribution of types I and II B. henselae among domestic cats living in Central Italy, using PCR assays. The animals were also tested to verify their bacteraemic status for other Bartonella species with a PCR protocol specific for the Bartonella genus and the presence of antibodies to B. henselae using a indirect immunofluorescence antibody test.
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V.V. Ebani et al. / Research in Veterinary Science 93 (2012) 63–66
2. Material and methods 2.1. Animals Between January 2009 and May 2010, 234 domestic cats were examined to detect B. henselae infected subjects. The animals lived in private houses and/or gardens, in the province of Pisa, Central Italy; 128 were female and 106 male. All pets were asymptomatic. Whole blood and EDTA-blood samples were collected from the cats by clinicians and forwarded to the Clinical Microbiology Laboratory of the Department of Animal Pathology (University of Pisa, Italy). Whole blood samples were centrifuged at 1500g for 15 min to obtain sera, which were stored at 20 °C until tested. Blood samples collected in EDTA were centrifuged at 5000g for 20 min; the supernatant was removed and the lysed erythrocyte concentrate was employed in DNA extraction. 2.2. Serological analysis The indirect immunofluorescence antibody test (IFAT) was performed on B. henselae IFAT slides (MegaScreen B. henselae, MegaCor, Austria) prepared with fixed B. henselae-infected Vero cells. All sera were diluted 1:64 (cut off) in phosphate-buffered saline (PBS, pH 7.2) and incubated on wells of the slides at 37 °C in a humid chamber for 30 min. The slides were rinsed three times in PBST (PBS + 0.4% Tween 80 – Sigma–Aldrich, St. Louis, Mo, USA), once in distilled water and air-dried. Each well of the slides was probed with fluorescein isothiocyanate-conjugated sheep anti-Cat IgG (Sigma–Aldrich) diluted 1:50 in Evans Blue (Sigma–Aldrich) solution and incubated at 37 °C in a humid chamber for 30 min. The slides were washed and dried as described above and examined with a fluorescence microscope. Positive samples were twofold serially diluted to determine the endpoint titer. Scores from 1 to 4 were assigned to the intensity of specific fluorescence and the antibody titer was defined as the major dilution with a P2 score. 2.3. DNA extraction DNA was extracted from centrifuged EDTA blood samples, using the DNeasy Tissue Kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer’s recommendations. The DNA samples were stored at 4 °C until used as template in PCR protocols. 2.4. Polymerase chain reaction DNA samples were employed in a PCR assay in order to identify the Bartonella genus. The primers p24E and p12B, previously described by Relman et al. (1990), were used in this protocol to amplify a 296 bp fragment of the Bartonella 16S rRNA gene. The PCR amplification was performed in 50 ll of reaction mixtures containing 200 lM of deoxynucleoside triphosphates (Qiagen), 0.5 lM of each primer, 1.25 U of Taq polymerase (Qiagen), 5 ll of 10 Qiagen PCR buffer and 2 ll of extracted DNA. PCR amplifications were performed in an automated thermal cycler (Gene-Amp PCR System 2700, Perkin–Elmer, Norwalk, Connecticut, USA) for 40 cycles. Each cycle consisted of denaturation at 95 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 1 min; an initial denaturation of 5 min at 95 °C and a final extension of 5 min at 72 °C were done. PCR controls included a known positive DNA extract and a reagent blank. PCR products were analyzed by electrophoresis on 1.5% agarose (Sigma–Aldrich) gel at 100 V for 45 min; the gel was stained with
ethidium bromide (Sigma–Aldrich) and observed. The DNA molecular weight marker was Gelpilot 100 bp Plus Ladder (Qiagen). All DNA samples were successively tested with a PCR protocol to identify B. henselae and distinguish types I and II. Reverse type-specific primers BH1 or BH2 in combination with the forward broad-host-range primer 16SF were used (Bergmans et al., 1996). The type-specific amplifications of B. henselae DNA, designed type I PCR and type II PCR respectively, were carried out in 50 ll of reaction mixtures containing 200 lM of deoxynucleoside triphosphates (Qiagen), 0.5 lM of BH1 or BH2 primer, 0.5 lM of 16SF, 1.25 U of Taq polymerase (Qiagen), 5 ll of 10 Qiagen PCR buffer and 2 ll of extracted DNA. PCR cycling consisted of 40 cycles under conditions similar to those for the amplification with p12B and p24E primers, but with an annealing at 56 °C for 45 s. The expected size of the product resulting from the two type-specific PCR protocols was 185 bp. A reagent blank was included as negative control. Types I and II PCR reaction mixtures were assembled in two separated laboratories to avoid cross-contaminations. PCR products were analyzed by electrophoresis on 1.5% agarose gel at 100 V for 45 min; gel was stained with ethidium bromide and observed. GelPilot 100 bp Plus Ladder (Qiagen) was used as DNA marker. 3. Results 3.1. Serology The indirect immunofluorescence antibody test detected 78 (33.33%) positive serum samples for antibodies against B. henselae with the following titers: 41 (17.52%) at 1:64, 19 (8.12%) at 1:128, 15 (6.41%) at 1:256, and 3 (1.28%) at 1:512. Among the seropositive samples, 35 (14.96%) were collected from females and 43 (18.37%) from males (Table 1). 3.2. Polymerase chain reaction The first PCR assay disclosed the 296 bp fragment, specific for the Bartonella 16S rRNA gene with the DNA extracted from 26 (11.11%) seropositive cats, 17 (7.26%) female and 9 (3.85%) male. Bartonella DNA was not detected in seronegative subjects. The second PCR method revealed, among the Bartonella 16S rRNA gene positive samples, 16 (6.84%) as belonging to genotype I and 6 (2.56%) to genotype II. Two (0.85%) male cats were positive to both genotypes I and II. Two (0.85%) female cats were 1:64 seropositive and PCR positive to Bartonella sp., but negative to PCR for genotypes I and II. All DNA specimens that were negative for the Bartonella genus with the first PCR assay, were also negative with types I and II PCR. Tables 1 and 2 show the serological and PCR status of the cats.
Table 1 Comparison between antibody titer and PCR results. Antibody titer
Seropositive cats
Bartonella sp. PCR positive cats
1:64
41 (17.52%)
1:128
19 (8.12%)
1:256
15 (6.41%)
1:512
3 (1.28%)
Total
78 (33.33%) F 35 M 43
8 (3.42%) F5M3 11 (4.70%) F7M4 6 (2.56%) F4M2 1 (0.43%) F1M0 26 (11.11%) F 17 M 9
Abbreviations: F, female cats; M, male cats.
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V.V. Ebani et al. / Research in Veterinary Science 93 (2012) 63–66 Table 2 Comparison between serological and PCR results. Animals
Bartonella sp. PCR
Bartonella sp. PCR + B. henselae genotype I
B. henselae genotype II
B. henselae genotypes I and II
B. henselae negative
Total
Seropositive female Seropositive male Seronegative Total
18 34 156 208 (88.89%)
11 5 0 16 (6.84%)
4 2 0 6 (2.56%)
0 2 0 2 (0.85%)
2 0 0 2 (0.85%)
17 9 0 26 (11.11%)
A moderate correlation between IFAT and PCR specific for Bartonella genus was confirmed by the kappa test (k = 0.41). 4. Discussion and conclusion The indirect immunofluorescence antibody test was positive to B. henselae in 33.33% of cases, but only 11.11% of these animals were considered bacteraemic, since they were positive with the PCR assay specific for the Bartonella genus. All cats tested did not show clinical signs of Bartonella infection. These observations supported the hypothesis that cats, even if clinically healthy, often represent a reservoir for Bartonella-associated infection in humans. All seronegative cats were not bacteraemic. On the basis of these results, IFAT seems to have a good specificity. In the case of seropositive animals, the antibody levels detected might be due to a current bacteraemic phase, but more likely a past infection. For these reasons, serological tests can be useful for epidemiological surveys, but not for diagnosis. Detection of B. henselae in blood is the best method to reveal a bacteraemic cat. However, bacteriological examinations have some limits; they require long incubation periods (about 2–4 weeks), and can give false negative results because cultivation on media is very difficult, particularly when a low number of bacteria are present in peripheral blood at the collection time. PCR is more suitable, because it is specific and sensitive and rapidly detects the presence of bartonellae in feline blood samples. Types I and II PCR assays employed during the present survey revealed 6.84% of bacteraemic cats for B. henselae type I and 2.56% for type II. A previous study on stray cats living in northern Italy found a prevalence of 20.6% of type I and 61.1% of type II (Fabbi et al., 2004). Other researchers have found B. henselae type II widespread in western Europe (Bergmans et al., 1997; Heller et al., 1997; Sander et al., 1997; Arvand et al., 2001). The higher incidence of type I found in the present study might be due to the survey being conducted in a different geographic area. In fact, previous surveys have demonstrated that the prevalence of the two genotypes may vary within a country (Chomel et al., 2006). Simultaneous infections of B. henselae types I and II appear to be rare (0.85%) in the feline population studied, even if other surveys have found higher percentages in Europe including Italy (Gurfield et al., 2001; Fabbi et al., 2004). A very low percentage of cats examined resulted PCR positive for Bartonella, but not for B. henselae. It is clear that these animals were infected with another Bartonella agent. In particular, it is possible that they were bacteraemic for Bartonella clarridgeiae, which has been identified as another causative agent of CSD in humans (Clarridge et al., 1995). Cats have been recognized as a reservoir of this bacterium on several occasions and in different parts of the world, including Italy (Chomel et al., 2004; Boulouis et al., 2005; Robinson et al., 2009; Capitta et al., 2010). However, B. clarridgeiae is likely present at very low frequencies in the cat population in Italy (Fabbi et al., 2004). The diagnosis of bartonellosis is very important, because veterinarians are often asked to test ill cats for Bartonella infection, but
also healthy cats belonging to clients with Bartonella-related illness or considered most susceptible to this infection. The typing of B. henselae can be useful, particularly for epidemiological studies, because researches have demonstrated that different types may induce varying pathologic features in infected people. In particular, genotype I seems to be more pathogenic for humans than genotype II (Woestyn et al., 2004; Bouchouicha et al., 2009). The diagnostic method described here is very rapid, because the PCR assays are performed on DNA extracted directly from feline blood samples, and not from Bartonella isolates. This protocol allows the detection of Bartonella-bacteraemic cat in 2 days, and at the same time it is possible to determine if the animal is positive for B. henselae, specifying genotype I or II, or another Bartonella species. References Arisoy, E.S., Correa, A.G., Wagner, M.L., Kaplan, S.L., 1999. Hepatosplenic cat scratch disease in children: selected clinical features and treatment. Clinical Infectious Diseases 28, 778–784. Arvand, M., Klose, A.J., Schwartz-Porsche, D., Hahn, H., Wendt, C., 2001. Genetic variability and prevalence of Bartonella henselae in cats in Berlin, Germany, and analysis of its genetic relatedness to a strain from Berlin that is pathogenic for humans. Journal of Clinical Microbiology 39, 743–746. Bass, J.W., Vincent, J.M., Person, D.A., 1997. The expanding spectrum of Bartonella infections. II. Cat scratch disease. Pediatric Infectious Diseases 16, 163–179. Bergh, K., Bevanger, L., Hanssen, I., Loseth, K., 2002. Low prevalence of Bartonella henselae infections in Norwegian domestic and feral cats. Acta Pathologica, Microbiologica et Immunologica Scandinavica 110, 309–314. Bergmans, A.M.C., Schellekens, J.F.P., Van Embden, J.D.A., Schouls, L.M., 1996. Predominance of two Bartonella henselae variants among cat scratch disease patients in The Netherlands. Journal of Clinical Microbiology 34, 254–260. Bergmans, A.M.C., De Jong, C.M.A., Van Amerongen, G., Schot, C.S., Schouls, L.M., 1997. Prevalence of Bartonella species in domestic cats in The Netherlands. Journal of Clinical Microbiology 35, 2256–2261. Bouchouicha, R., Durand, B., Monteil, M., Chomel, B.B., Berrich, M., Arvand, M., Birtles, R.J., Breitschwerdt, E.B., Koehler, J.E., Maggi, R., Maruyama, S., Kasten, R., Petit, E., Boulouis, H.J., Haddad, N., 2009. Molecular epidemiology of feline and human Bartonella henselae isolates. Emerging Infectious Diseases 15, 813–816. Boulouis, H.J., Chang, C.C., Henn, J.B., Kasten, R.W., Chomel, B.B., 2005. Factors associated with the rapid emergence of zoonotic Bartonella infections. Veterinary Record 36, 383–410. Capitta, P., Zobba, R., Masala, G., Cocco, R., Tola, S., Pinna Parpaglia, M.L., 2010. Isolation and characterization of Bartonella strains in cats. Transboundary and Emerging Diseases 57, 201–204. Carithers, H.A., Margileth, A.M., 1991. Cat scratch disease. Acute encephalopathy and other neurological manifestations. American Journal of Diseases of Children 145, 98–101. Chomel, B.B., Carlos, E.T., Kastenm, R.W., Yamamoto, K., Chang, C.C., Carlos, R.S., Abenes, M.V., Pajares, C.M., 1999. Bartonella henselae and Bartonella clarridgeiae infection in domestic cats from The Philippines. American Journal of Tropical Medicine and Hygiene 60, 593–597. Chomel, B.B., Boulouis, H.J., Breitschwerdt, E.B., 2004. Cat scratch disease and other zoonotic Bartonella infections. Journal of the American Veterinary Medical Association 224, 1270–1279. Chomel, B.B., Kasten, R.W., Henn, J.B., Molia, S., 2006. Bartonella infection in domestic cats and wild felids. Annals of the New York Academy of Sciences 1078, 410–415. Clarridge III, J.E., Raich, T.J., Pirwani, D., Simon, B., Tsai, L., Rodriguez-Barradas, R., Zollo, A., Jones, D.C., Rambo, C., 1995. Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat. Journal of Clinical Microbiology 33, 2107–2113. Cotté, V., Bonnet, S., Le Rhun, D., Le Naour, E., Chauvin, A., Boulouis, H.J., Lecuelle, B., Lilin, T., Vayssier-Taussat, M., 2008. Transmission of Bartonella henselae by Ixodes ricinus. Emerging Infectious Diseases 14, 1074–1080.
66
V.V. Ebani et al. / Research in Veterinary Science 93 (2012) 63–66
Engvall, E.O., Brandstrom, B., Fermer, C., Blomqvist, G., Englund, L., 2003. Prevalence of Bartonella henselae in young healthy cats in Sweden. Veterinary Record 152, 366–369. Fabbi, M., De Giuli, L., Tranquillo, M., Dragoni, R., Casiraghi, M., Genchi, C., 2004. Prevalence of Bartonella henselae in italian stray cats: evaluation of serology to assess the risk of transmission of Bartonella to humans. Journal of Clinical Microbiology 42, 264–268. Florin, T.F., Zaoutis, T.E., Zaoutis, L.B., 2008. Beyond cat scratch disease: widening spectrum of Bartonella henselae infection. Pediatrics 121, 1012–1024. Guptill, L., 2010. Bartonellosis. Veterinary Microbiology 140, 347–359. Gurfield, A.N., Boulouis, H.J., Chomel, B.B., Kasten, R.W., Heller, R., Bouillin, C., Gandoin, C., Thibault, D., Chang, C.C., Barrat, F., Piedmont, Y., 2001. Epidemiology of Bartonella infection in domestic cats in France. Veterinary Microbiology 80, 185–198. Heller, R., Artois, M., Xemar, V., De Briel, D., Gehin, H., Jaulhac, B., Monteil, H., Piedmont, Y., 1997. Prevalence of Bartonella henselae and Bartonella clarridgeiae in stray cats. Journal of Clinical Microbiology 35, 1327–1331. Maruyama, S., Nakamura, Y., Kabeya, H., Tanaka, S., Sakai, T., Katsube, Y., 2000. Prevalence of Bartonella henselae, Bartonella clarridgeiae and the 16S rRNA gene types of Bartonella henselae among pet cats in Japan. Journal of Veterinary Medical Science 62, 273–279. Messam, L.L., Kasten, R.W., Ritchie, M.J., Chomel, B.B., 2005. Bartonella henselae and domestic cats, Jamaica. Emerging Infectious Diseases 11, 1146–1147. Raoult, D., Fournier, P.E., Drancourt, M., Marrie, T.J., Etienne, J., Cosserat, J., Cacoub, P., Poinsignon, Y., Leclercq, P., Sefton, M., 1996. Diagnosis of 22 new cases of Bartonella endocarditis. Annals of Internal Medicine 125, 646–652.
Relman, D.A., Loutit, S.J., Schmidt, T.M., Falkow, S., Tompkins, S., 1990. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. New England Journal of Medicine 323, 1573–1580. Relman, D.A., Falkow, S., LeBoit, P.E., Perkocha, L.A., Min, K.W., Welch, D.F., Slater, L.N., 1991. The organism causing bacillary angiomatosis, peliosis hepatis, and fever and bacteremia in immunocompromised patients. New England Journal of Medicine 324, 1514. Robinson, M.T., Hillman, T., Langton, D.A., Shaw, S.E., 2009. Bartonella clarridgeiae in a cat in the UK. Veterinary Record 164, 58–59. Robson, J.M.B., Harte, G.J., Osborne, D.R.S., McCormack, J.G., 1999. Cat scratch disease with paravertebral mass and osteomyelitis. Clinical of Infectious Diseases 28, 274–278. Sander, A., Buhler, C., Pelz, K., Von Cramm, E., Bredt, W., 1997. Detection and identification of two Bartonella henselae variants in domestic cats in Germany. Journal of Clinical Microbiology 35, 584–587. Sanogo, Y.O., Zeaiter, Z., Caruso, G., Merola, F., Shpynov, S., Brouqui, P., Raoult, D., 2003. Bartonella henselae in Ixodes ricinus ticks (Acari: Ixodida) removed from humans, Belluno Province, Italy. Emerging Infectious Diseases 9, 329–332. Tappero, J.W., Mohle-Boetani, J., Koehler, J.E., Swaminathan, B., Barger, T.G., LeBoit, P.E., Smith, L.L., Wenger, J.D., Pinner, R.W., Kemper, C.A., 1993. The epidemiology of bacillary angiomatosis and bacillary peliosis. Journal of the American Medical Association 269, 770–775. Woestyn, S., Olivé, N., Bigaignon, G., Avesani, V., Delmee, M., 2004. Study of genotypes and virB4 secretion gene of Bartonella henselae strains from patients with clinically defined cat scratch disease. Journal of Clinical Microbiology 42, 1420–1427.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Occurrence of Bartonella henselae types I and II in Central Italian domestic cats Valentina V. Ebani ⇑, Fabrizio Bertelloni, Filippo Fratini Department of Animal Pathology, Prophylaxis and Food Hygiene, Faculty of Veterinary Medicine, University of Pisa, Italy
a r t i c l e
i n f o
Article history: Received 29 April 2011 Accepted 13 July 2011
Keywords: Cat Bartonella henselae Cat scratch disease Bacteraemia Genotype PCR
a b s t r a c t 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. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Cat scratch disease (CSD) is a zoonosis mainly caused by Bartonella henselae. The transmission of this intraerythrocytic proteobacterium among cats is via the flea Ctenocephalides felis (Woestyn et al., 2004). Ticks have been proposed as vectors for transmission of Bartonella species among cats, human beings, dogs, and other mammalian hosts. B. henselae has been detected by PCR in Ixodes ricinus ticks, in which transtadial transmission has been demonstrated (Sanogo et al., 2003; Cotté et al., 2008; Guptill, 2010). The natural infection is usually asymptomatic in cats, or characterized by mild clinical signs that owners do not observe. Lymphoadenopathy, stomatitis, renal, and neurological disease are sometimes observed (Guptill, 2010). Infected cats, even if asymptomatic, may be highly bacteraemic for several months, representing an important reservoir for the bacterium and a risk to humans. Serological tests are not able to detect bacteraemic cats, because seropositive subjects may not be carriers. Humans are infected incidentally, directly by a cat’s bite or scratch or indirectly by cat fleas. In immunocompetent hosts the disease is self-limited to CSD, with an erythematous papule at the scratch or bite site and enlargement of regional lymph nodes. Lymph nodes can develop into abscesses, and symptoms, such as headache, malaise, and fever may be reported. Occasionally the bacterium can spread and cause a more serious symptomatology, ⇑ Corresponding author. Address: Department of Animal Pathology, Prophylaxis and Food Hygiene, Faculty of Veterinary Medicine, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy. Tel.: +39 50 2216968; fax: +39 50 2216941. E-mail address: [email protected] (V.V. Ebani). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.07.013
characterized by osteomyelitis, endocarditis, pulmonitis, meningitis, encephalopathy, neuroretinitis, hepatic, and splenic involvement (Carithers and Margileth, 1991; Raoult et al., 1996; Bass et al., 1997; Arisoy et al., 1999; Robson et al., 1999; Florin et al., 2008). In immunocompromised hosts bartonellae are often involved in angioproliferative disorders such as bacillary angiomatosis and hepatic peliosis (Relman et al., 1991; Tappero et al., 1993). B. henselae contains genetically diverse strains: 16S rRNA types I and II, respectively called Huston I and Marseille II. Cats and humans can be infected with either genotype I or II and occasionally with a co-infection of both types. Types I and II seem to have a different role in human bartonellosis: type I appears more pathogenic than type II (Woestyn et al., 2004; Bouchouicha et al., 2009). Previous studies have demonstrated that the prevalence of B. henselae genotypes varies considerably among cat populations from different geographical areas. In particular, type I is dominant in Asia, while type II is dominant in western United States, western Europe and Australia (Chomel et al., 1999; Maruyama et al., 2000; Bergh et al., 2002; Engvall et al., 2003; Messam et al., 2005). However, within a given country, the prevalence of these genotypes may vary in relationship to the studied geographical areas, as demonstrated in France (Chomel et al., 2006). There is little information on the distribution of B. henselae genotypes among Italian cats (Fabbi et al., 2004). The aim of this research is to verify the distribution of types I and II B. henselae among domestic cats living in Central Italy, using PCR assays. The animals were also tested to verify their bacteraemic status for other Bartonella species with a PCR protocol specific for the Bartonella genus and the presence of antibodies to B. henselae using a indirect immunofluorescence antibody test.
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2. Material and methods 2.1. Animals Between January 2009 and May 2010, 234 domestic cats were examined to detect B. henselae infected subjects. The animals lived in private houses and/or gardens, in the province of Pisa, Central Italy; 128 were female and 106 male. All pets were asymptomatic. Whole blood and EDTA-blood samples were collected from the cats by clinicians and forwarded to the Clinical Microbiology Laboratory of the Department of Animal Pathology (University of Pisa, Italy). Whole blood samples were centrifuged at 1500g for 15 min to obtain sera, which were stored at 20 °C until tested. Blood samples collected in EDTA were centrifuged at 5000g for 20 min; the supernatant was removed and the lysed erythrocyte concentrate was employed in DNA extraction. 2.2. Serological analysis The indirect immunofluorescence antibody test (IFAT) was performed on B. henselae IFAT slides (MegaScreen B. henselae, MegaCor, Austria) prepared with fixed B. henselae-infected Vero cells. All sera were diluted 1:64 (cut off) in phosphate-buffered saline (PBS, pH 7.2) and incubated on wells of the slides at 37 °C in a humid chamber for 30 min. The slides were rinsed three times in PBST (PBS + 0.4% Tween 80 – Sigma–Aldrich, St. Louis, Mo, USA), once in distilled water and air-dried. Each well of the slides was probed with fluorescein isothiocyanate-conjugated sheep anti-Cat IgG (Sigma–Aldrich) diluted 1:50 in Evans Blue (Sigma–Aldrich) solution and incubated at 37 °C in a humid chamber for 30 min. The slides were washed and dried as described above and examined with a fluorescence microscope. Positive samples were twofold serially diluted to determine the endpoint titer. Scores from 1 to 4 were assigned to the intensity of specific fluorescence and the antibody titer was defined as the major dilution with a P2 score. 2.3. DNA extraction DNA was extracted from centrifuged EDTA blood samples, using the DNeasy Tissue Kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer’s recommendations. The DNA samples were stored at 4 °C until used as template in PCR protocols. 2.4. Polymerase chain reaction DNA samples were employed in a PCR assay in order to identify the Bartonella genus. The primers p24E and p12B, previously described by Relman et al. (1990), were used in this protocol to amplify a 296 bp fragment of the Bartonella 16S rRNA gene. The PCR amplification was performed in 50 ll of reaction mixtures containing 200 lM of deoxynucleoside triphosphates (Qiagen), 0.5 lM of each primer, 1.25 U of Taq polymerase (Qiagen), 5 ll of 10 Qiagen PCR buffer and 2 ll of extracted DNA. PCR amplifications were performed in an automated thermal cycler (Gene-Amp PCR System 2700, Perkin–Elmer, Norwalk, Connecticut, USA) for 40 cycles. Each cycle consisted of denaturation at 95 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 1 min; an initial denaturation of 5 min at 95 °C and a final extension of 5 min at 72 °C were done. PCR controls included a known positive DNA extract and a reagent blank. PCR products were analyzed by electrophoresis on 1.5% agarose (Sigma–Aldrich) gel at 100 V for 45 min; the gel was stained with
ethidium bromide (Sigma–Aldrich) and observed. The DNA molecular weight marker was Gelpilot 100 bp Plus Ladder (Qiagen). All DNA samples were successively tested with a PCR protocol to identify B. henselae and distinguish types I and II. Reverse type-specific primers BH1 or BH2 in combination with the forward broad-host-range primer 16SF were used (Bergmans et al., 1996). The type-specific amplifications of B. henselae DNA, designed type I PCR and type II PCR respectively, were carried out in 50 ll of reaction mixtures containing 200 lM of deoxynucleoside triphosphates (Qiagen), 0.5 lM of BH1 or BH2 primer, 0.5 lM of 16SF, 1.25 U of Taq polymerase (Qiagen), 5 ll of 10 Qiagen PCR buffer and 2 ll of extracted DNA. PCR cycling consisted of 40 cycles under conditions similar to those for the amplification with p12B and p24E primers, but with an annealing at 56 °C for 45 s. The expected size of the product resulting from the two type-specific PCR protocols was 185 bp. A reagent blank was included as negative control. Types I and II PCR reaction mixtures were assembled in two separated laboratories to avoid cross-contaminations. PCR products were analyzed by electrophoresis on 1.5% agarose gel at 100 V for 45 min; gel was stained with ethidium bromide and observed. GelPilot 100 bp Plus Ladder (Qiagen) was used as DNA marker. 3. Results 3.1. Serology The indirect immunofluorescence antibody test detected 78 (33.33%) positive serum samples for antibodies against B. henselae with the following titers: 41 (17.52%) at 1:64, 19 (8.12%) at 1:128, 15 (6.41%) at 1:256, and 3 (1.28%) at 1:512. Among the seropositive samples, 35 (14.96%) were collected from females and 43 (18.37%) from males (Table 1). 3.2. Polymerase chain reaction The first PCR assay disclosed the 296 bp fragment, specific for the Bartonella 16S rRNA gene with the DNA extracted from 26 (11.11%) seropositive cats, 17 (7.26%) female and 9 (3.85%) male. Bartonella DNA was not detected in seronegative subjects. The second PCR method revealed, among the Bartonella 16S rRNA gene positive samples, 16 (6.84%) as belonging to genotype I and 6 (2.56%) to genotype II. Two (0.85%) male cats were positive to both genotypes I and II. Two (0.85%) female cats were 1:64 seropositive and PCR positive to Bartonella sp., but negative to PCR for genotypes I and II. All DNA specimens that were negative for the Bartonella genus with the first PCR assay, were also negative with types I and II PCR. Tables 1 and 2 show the serological and PCR status of the cats.
Table 1 Comparison between antibody titer and PCR results. Antibody titer
Seropositive cats
Bartonella sp. PCR positive cats
1:64
41 (17.52%)
1:128
19 (8.12%)
1:256
15 (6.41%)
1:512
3 (1.28%)
Total
78 (33.33%) F 35 M 43
8 (3.42%) F5M3 11 (4.70%) F7M4 6 (2.56%) F4M2 1 (0.43%) F1M0 26 (11.11%) F 17 M 9
Abbreviations: F, female cats; M, male cats.
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V.V. Ebani et al. / Research in Veterinary Science 93 (2012) 63–66 Table 2 Comparison between serological and PCR results. Animals
Bartonella sp. PCR
Bartonella sp. PCR + B. henselae genotype I
B. henselae genotype II
B. henselae genotypes I and II
B. henselae negative
Total
Seropositive female Seropositive male Seronegative Total
18 34 156 208 (88.89%)
11 5 0 16 (6.84%)
4 2 0 6 (2.56%)
0 2 0 2 (0.85%)
2 0 0 2 (0.85%)
17 9 0 26 (11.11%)
A moderate correlation between IFAT and PCR specific for Bartonella genus was confirmed by the kappa test (k = 0.41). 4. Discussion and conclusion The indirect immunofluorescence antibody test was positive to B. henselae in 33.33% of cases, but only 11.11% of these animals were considered bacteraemic, since they were positive with the PCR assay specific for the Bartonella genus. All cats tested did not show clinical signs of Bartonella infection. These observations supported the hypothesis that cats, even if clinically healthy, often represent a reservoir for Bartonella-associated infection in humans. All seronegative cats were not bacteraemic. On the basis of these results, IFAT seems to have a good specificity. In the case of seropositive animals, the antibody levels detected might be due to a current bacteraemic phase, but more likely a past infection. For these reasons, serological tests can be useful for epidemiological surveys, but not for diagnosis. Detection of B. henselae in blood is the best method to reveal a bacteraemic cat. However, bacteriological examinations have some limits; they require long incubation periods (about 2–4 weeks), and can give false negative results because cultivation on media is very difficult, particularly when a low number of bacteria are present in peripheral blood at the collection time. PCR is more suitable, because it is specific and sensitive and rapidly detects the presence of bartonellae in feline blood samples. Types I and II PCR assays employed during the present survey revealed 6.84% of bacteraemic cats for B. henselae type I and 2.56% for type II. A previous study on stray cats living in northern Italy found a prevalence of 20.6% of type I and 61.1% of type II (Fabbi et al., 2004). Other researchers have found B. henselae type II widespread in western Europe (Bergmans et al., 1997; Heller et al., 1997; Sander et al., 1997; Arvand et al., 2001). The higher incidence of type I found in the present study might be due to the survey being conducted in a different geographic area. In fact, previous surveys have demonstrated that the prevalence of the two genotypes may vary within a country (Chomel et al., 2006). Simultaneous infections of B. henselae types I and II appear to be rare (0.85%) in the feline population studied, even if other surveys have found higher percentages in Europe including Italy (Gurfield et al., 2001; Fabbi et al., 2004). A very low percentage of cats examined resulted PCR positive for Bartonella, but not for B. henselae. It is clear that these animals were infected with another Bartonella agent. In particular, it is possible that they were bacteraemic for Bartonella clarridgeiae, which has been identified as another causative agent of CSD in humans (Clarridge et al., 1995). Cats have been recognized as a reservoir of this bacterium on several occasions and in different parts of the world, including Italy (Chomel et al., 2004; Boulouis et al., 2005; Robinson et al., 2009; Capitta et al., 2010). However, B. clarridgeiae is likely present at very low frequencies in the cat population in Italy (Fabbi et al., 2004). The diagnosis of bartonellosis is very important, because veterinarians are often asked to test ill cats for Bartonella infection, but
also healthy cats belonging to clients with Bartonella-related illness or considered most susceptible to this infection. The typing of B. henselae can be useful, particularly for epidemiological studies, because researches have demonstrated that different types may induce varying pathologic features in infected people. In particular, genotype I seems to be more pathogenic for humans than genotype II (Woestyn et al., 2004; Bouchouicha et al., 2009). The diagnostic method described here is very rapid, because the PCR assays are performed on DNA extracted directly from feline blood samples, and not from Bartonella isolates. This protocol allows the detection of Bartonella-bacteraemic cat in 2 days, and at the same time it is possible to determine if the animal is positive for B. henselae, specifying genotype I or II, or another Bartonella species. References Arisoy, E.S., Correa, A.G., Wagner, M.L., Kaplan, S.L., 1999. Hepatosplenic cat scratch disease in children: selected clinical features and treatment. Clinical Infectious Diseases 28, 778–784. Arvand, M., Klose, A.J., Schwartz-Porsche, D., Hahn, H., Wendt, C., 2001. Genetic variability and prevalence of Bartonella henselae in cats in Berlin, Germany, and analysis of its genetic relatedness to a strain from Berlin that is pathogenic for humans. Journal of Clinical Microbiology 39, 743–746. Bass, J.W., Vincent, J.M., Person, D.A., 1997. The expanding spectrum of Bartonella infections. II. Cat scratch disease. Pediatric Infectious Diseases 16, 163–179. Bergh, K., Bevanger, L., Hanssen, I., Loseth, K., 2002. Low prevalence of Bartonella henselae infections in Norwegian domestic and feral cats. Acta Pathologica, Microbiologica et Immunologica Scandinavica 110, 309–314. Bergmans, A.M.C., Schellekens, J.F.P., Van Embden, J.D.A., Schouls, L.M., 1996. Predominance of two Bartonella henselae variants among cat scratch disease patients in The Netherlands. Journal of Clinical Microbiology 34, 254–260. Bergmans, A.M.C., De Jong, C.M.A., Van Amerongen, G., Schot, C.S., Schouls, L.M., 1997. Prevalence of Bartonella species in domestic cats in The Netherlands. Journal of Clinical Microbiology 35, 2256–2261. Bouchouicha, R., Durand, B., Monteil, M., Chomel, B.B., Berrich, M., Arvand, M., Birtles, R.J., Breitschwerdt, E.B., Koehler, J.E., Maggi, R., Maruyama, S., Kasten, R., Petit, E., Boulouis, H.J., Haddad, N., 2009. Molecular epidemiology of feline and human Bartonella henselae isolates. Emerging Infectious Diseases 15, 813–816. Boulouis, H.J., Chang, C.C., Henn, J.B., Kasten, R.W., Chomel, B.B., 2005. Factors associated with the rapid emergence of zoonotic Bartonella infections. Veterinary Record 36, 383–410. Capitta, P., Zobba, R., Masala, G., Cocco, R., Tola, S., Pinna Parpaglia, M.L., 2010. Isolation and characterization of Bartonella strains in cats. Transboundary and Emerging Diseases 57, 201–204. Carithers, H.A., Margileth, A.M., 1991. Cat scratch disease. Acute encephalopathy and other neurological manifestations. American Journal of Diseases of Children 145, 98–101. Chomel, B.B., Carlos, E.T., Kastenm, R.W., Yamamoto, K., Chang, C.C., Carlos, R.S., Abenes, M.V., Pajares, C.M., 1999. Bartonella henselae and Bartonella clarridgeiae infection in domestic cats from The Philippines. American Journal of Tropical Medicine and Hygiene 60, 593–597. Chomel, B.B., Boulouis, H.J., Breitschwerdt, E.B., 2004. Cat scratch disease and other zoonotic Bartonella infections. Journal of the American Veterinary Medical Association 224, 1270–1279. Chomel, B.B., Kasten, R.W., Henn, J.B., Molia, S., 2006. Bartonella infection in domestic cats and wild felids. Annals of the New York Academy of Sciences 1078, 410–415. Clarridge III, J.E., Raich, T.J., Pirwani, D., Simon, B., Tsai, L., Rodriguez-Barradas, R., Zollo, A., Jones, D.C., Rambo, C., 1995. Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat. Journal of Clinical Microbiology 33, 2107–2113. Cotté, V., Bonnet, S., Le Rhun, D., Le Naour, E., Chauvin, A., Boulouis, H.J., Lecuelle, B., Lilin, T., Vayssier-Taussat, M., 2008. Transmission of Bartonella henselae by Ixodes ricinus. Emerging Infectious Diseases 14, 1074–1080.
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V.V. Ebani et al. / Research in Veterinary Science 93 (2012) 63–66
Engvall, E.O., Brandstrom, B., Fermer, C., Blomqvist, G., Englund, L., 2003. Prevalence of Bartonella henselae in young healthy cats in Sweden. Veterinary Record 152, 366–369. Fabbi, M., De Giuli, L., Tranquillo, M., Dragoni, R., Casiraghi, M., Genchi, C., 2004. Prevalence of Bartonella henselae in italian stray cats: evaluation of serology to assess the risk of transmission of Bartonella to humans. Journal of Clinical Microbiology 42, 264–268. Florin, T.F., Zaoutis, T.E., Zaoutis, L.B., 2008. Beyond cat scratch disease: widening spectrum of Bartonella henselae infection. Pediatrics 121, 1012–1024. Guptill, L., 2010. Bartonellosis. Veterinary Microbiology 140, 347–359. Gurfield, A.N., Boulouis, H.J., Chomel, B.B., Kasten, R.W., Heller, R., Bouillin, C., Gandoin, C., Thibault, D., Chang, C.C., Barrat, F., Piedmont, Y., 2001. Epidemiology of Bartonella infection in domestic cats in France. Veterinary Microbiology 80, 185–198. Heller, R., Artois, M., Xemar, V., De Briel, D., Gehin, H., Jaulhac, B., Monteil, H., Piedmont, Y., 1997. Prevalence of Bartonella henselae and Bartonella clarridgeiae in stray cats. Journal of Clinical Microbiology 35, 1327–1331. Maruyama, S., Nakamura, Y., Kabeya, H., Tanaka, S., Sakai, T., Katsube, Y., 2000. Prevalence of Bartonella henselae, Bartonella clarridgeiae and the 16S rRNA gene types of Bartonella henselae among pet cats in Japan. Journal of Veterinary Medical Science 62, 273–279. Messam, L.L., Kasten, R.W., Ritchie, M.J., Chomel, B.B., 2005. Bartonella henselae and domestic cats, Jamaica. Emerging Infectious Diseases 11, 1146–1147. Raoult, D., Fournier, P.E., Drancourt, M., Marrie, T.J., Etienne, J., Cosserat, J., Cacoub, P., Poinsignon, Y., Leclercq, P., Sefton, M., 1996. Diagnosis of 22 new cases of Bartonella endocarditis. Annals of Internal Medicine 125, 646–652.
Relman, D.A., Loutit, S.J., Schmidt, T.M., Falkow, S., Tompkins, S., 1990. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. New England Journal of Medicine 323, 1573–1580. Relman, D.A., Falkow, S., LeBoit, P.E., Perkocha, L.A., Min, K.W., Welch, D.F., Slater, L.N., 1991. The organism causing bacillary angiomatosis, peliosis hepatis, and fever and bacteremia in immunocompromised patients. New England Journal of Medicine 324, 1514. Robinson, M.T., Hillman, T., Langton, D.A., Shaw, S.E., 2009. Bartonella clarridgeiae in a cat in the UK. Veterinary Record 164, 58–59. Robson, J.M.B., Harte, G.J., Osborne, D.R.S., McCormack, J.G., 1999. Cat scratch disease with paravertebral mass and osteomyelitis. Clinical of Infectious Diseases 28, 274–278. Sander, A., Buhler, C., Pelz, K., Von Cramm, E., Bredt, W., 1997. Detection and identification of two Bartonella henselae variants in domestic cats in Germany. Journal of Clinical Microbiology 35, 584–587. Sanogo, Y.O., Zeaiter, Z., Caruso, G., Merola, F., Shpynov, S., Brouqui, P., Raoult, D., 2003. Bartonella henselae in Ixodes ricinus ticks (Acari: Ixodida) removed from humans, Belluno Province, Italy. Emerging Infectious Diseases 9, 329–332. Tappero, J.W., Mohle-Boetani, J., Koehler, J.E., Swaminathan, B., Barger, T.G., LeBoit, P.E., Smith, L.L., Wenger, J.D., Pinner, R.W., Kemper, C.A., 1993. The epidemiology of bacillary angiomatosis and bacillary peliosis. Journal of the American Medical Association 269, 770–775. Woestyn, S., Olivé, N., Bigaignon, G., Avesani, V., Delmee, M., 2004. Study of genotypes and virB4 secretion gene of Bartonella henselae strains from patients with clinically defined cat scratch disease. Journal of Clinical Microbiology 42, 1420–1427.