Veterinary Microbiology 168 (2014) 225–228

Contents lists available at ScienceDirect

Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Short communication

Bartonella spp. in cats from Buenos Aires, Argentina Gabriel L. Cicuttin a, Diego F. Brambati a, Marı´a F. De Gennaro a, Fernando Carmona a, Marı´a L. Isturiz a, Laura E. Pujol a, Guillermo C. Belerenian a, Horacio Gil b,* a b

Instituto de Zoonosis Luis Pasteur, Buenos Aires, Av. Dı´az Ve´lez 4821, C1405DCD, Ciudad Auto´noma de Buenos Aires, Argentina Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, Ctra/Majadahonda-Pozuelo Km 2,5, Majadahonda 28220, Madrid, Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 August 2013 Received in revised form 3 October 2013 Accepted 13 October 2013

In Argentina, data on the presence of members of the genus Bartonella is scarce. To increase knowledge about these zoonotic pathogens in this country, the presence and variability of Bartonella spp. was investigated in cats and dogs from Buenos Aires. Bartonella spp. was detected in 17.8% of cats, while all dogs tested negative by PCR and Reverse Line Blot. B. henselae was the most frequent species, being detected in 11.9% (14/101), while B. clarridgeiae was found in only 5.9% (6/101) of the cats. Afterwards, B. henselae isolates and positive blood samples were characterized by Multiple Locus Sequence Typing (MLST) and Multiple Locus Variable Number Tandem Repeats Analysis (MLVA). As result, four different MLST sequence types (ST) and eight MLVA profiles were identified. ST 1 was the most frequent variant found in cats, followed by ST 8. Interestingly, some of the MLVA profiles that were detected in this study have been previously associated with human disease, and represents a potential risk of infection. Veterinarians and physicians should consider the presence of these emerging pathogens in their diagnostic routine. ß 2013 Elsevier B.V. All rights reserved.

Keywords: Bartonella henselae Cat Argentina MLVA MLST

1. Introduction More than 30 different species belong to the genus Bartonella. These emerging pathogens present a complex cycle in nature, including different mammal reservoir hosts and hematophagous arthropods which play a vector role. To date, more than half of the species of Bartonella have been associated with human disease (Harms and Dehio, 2012). Among them, B. henselae is the etiological agent of cat scratch disease, whose reservoir host is the feline population and vector is the cat flea (Chomel et al., 1996). Transmission from cats to humans mainly occurs directly by a cat scratch or bite. The main symptom of this zoonotic disease is a

* Corresponding author at: Laboratorio de Espiroquetas y Pato´genos Especiales, Centro Nacional de Microbiologı´a, ISCIII, Ctra MajadahondaPozuelo Km 2.5, 28220 Majadahonda, Madrid, Spain. Tel.: +34 91 822 3752; fax: +34 91 509 7966. E-mail address: [email protected] (H. Gil). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.10.016

regional lymphadenopathy that may be accompanied by other manifestations, such as fever or fatigue, and is usually self-limiting. Also, its dissemination can produce other severe manifestations, such as endocarditis or neurological manifestations, among others (Lamps and Scott, 2004). Although B. henselae is a zoonotic pathogen, recent characterization analyses have shown that some of the B. henselae variants are only found in the feline population, indicating that these variants could represent a group of strains less pathogenic for humans (Arvand et al., 2007; Bouchouicha et al., 2009; Gil et al., 2013; Li et al., 2007). In Argentina, data on the presence of Bartonella is scarce (Correa et al., 2012; Garre et al., 2008). In fact, among animals, only a few dog valvulopathy cases from Buenos Aires caused by Bartonella spp. have been described (Belerenian et al., 2012). For this reason, we have studied the presence of Bartonella spp. in cats and dogs from Buenos Aires, as well as the variability of B. henselae to evaluate its potential risk of infection for humans.

G.L. Cicuttin et al. / Veterinary Microbiology 168 (2014) 225–228

226

Table 1 Prevalence of Bartonella spp. in cats from Buenos Aires. No.

Bartonella spp.

B. henselae

Co-infection B. henselae/B. clarridgeiae

%

n

n

%

n

%

39 62

5 13

12.8 21.0

4 8

10.3 12.9

0 4

0 6.4

1 1

2.6 1.6

Agea Young Adult

51 50

9 9

17.6 18.0

6 6

11.8 12.0

2 2

3.9 4.0

1 1

2.0 2.0

Habitsb In-house Around-the-house Stray

50 44 7

8 8 2

16.0 18.2 28.6

7 4 1

14.0 9.1 14.3

0 3 1

0.0 6.8 14.3

1 1 0

2.0 2.3 0.0

32 27

5 4

15.6 14.8

4 1

12.5 3.7

0 2

0.0 7.4

1 1

3.1 3.7

46 55

11 7

23.9 12.7

8 4

17.4 7.3

2 2

4.3 3.6

1 1

2.2 1.8

101

18

17.8

12

11.9

4

4.0

2

2.0

Sex Male Female

Flea infestationc Yes No Area LSEL Rest Total a b c

n

B. clarridgeiae %

Animals older than one year were considered adults. In-house: pet that does not leave the house/around-the-house: pet goes unchecked/stray: ownerless. Not all owners were consulted.

2. Materials and methods From September 2011 to January 2012, blood samples were collected from 101 cats and 94 dogs from two different areas of Buenos Aires (348360 1400 S and 588220 5400 W), Argentina. The first area corresponded to two neighborhoods with a low socioeconomic level (LSEL), under the areas of influence of primary care health centers N8 18 (348400 2900 S, 588270 5500 W) and 24 (348390 3400 S, 588270 1600 W). The second area corresponded to the rest of the city, compose of districts with different socioeconomic levels. Specimens were obtained by jugular venipuncture, collected in tubes with EDTA and kept at -70 8C until they were processed. Data from sex, age, flea infestation (yes/ no) and animal habits (animals in-house, around-thehouse or stray) were recorded (Table 1). DNA was obtained from the blood samples using the QIAcube automatic extractor (QIAGEN, Hilde, Germany) following the manufacturer’s instructions. Then, between 100 and 300 ng of DNA was tested by a PCR combined with a Reverse Line Blot (RLB) for the specific detection of Bartonella (Garcı´a-Esteban et al., 2008; Gil et al., 2010). Positive controls (1000, 100 and 10 genomic equivalents of B. schoenbuchensis) and negative controls (water) were included in each experiment. For culturing, a volume of 100 ml of each PCR positive sample was plated in triplicate in Columbia agar plates (Oxoid, Basingstoke, UK), one supplemented with 5% of defibrinated sheep blood (Oxoid), one with 5% of defibrinated horse blood (Oxoid) and one with 5% of defibrinated horse blood plus hemin. The three were incubated at 35 8C in a moist atmosphere with 5% CO2 for 4 weeks. B. henselae isolates and PCR-positive samples were characterized by Multiple Locus Sequence Typing (MLST), sequencing the eight housekeeping genes described

previously (Arvand et al., 2007; Gil et al., 2013). The identified sequence types (ST) were assigned according with the B. henselae MLST online database (http:// bhenselae.mlst.net). The same specimens were also studied by Multiple Locus Variable Number Tandem Repeat Analysis (MLVA), determining the number of copies in five loci (BHV-A to E) (Bouchouicha et al., 2009; Gil et al., 2013). The identified profiles were assigned and deposited in the B. henselae MLVA online database (http://mlva.u-psud.fr/). Data were analyzed by x2 and the Fisher’ exact test using the statistical package SPSS (SPSS Ibe´rica, Madrid, Spain). 3. Results Regarding the molecular detection, 17.8% (18/101) of the felines were positive to Bartonella spp., B. henselae being detected in 11.9% (14/101) and B. clarridgeiae in 5.9% (6/101) (Table 1). Co-infection was detected in 2.0% (2/ 101) of the cats. By contrast, all the canine specimens were negative to Bartonella spp., although a positive hybridization signal with the specific probe for Ochrobactrum grignonense (Gil et al., 2010) was observed in one of the dog samples. No statistical significance was observed between the Bartonella PCR results and the sex, age, flea infestation or habits of the tested animals (p > 0.05). Interestingly, the prevalence of B. henselae in cats was higher in the LSEL (17%) than in other neighborhoods (7.4%) but it was not statistically significant (Fisher exact’s test: p = 0.1555). Overall, seven B. henselae and one B. clarridgeiae isolates were obtained from 18 Bartonella positive cats, representing a 50% efficiency of isolation for B. henselae (7/14) and 16.7% for B. clarridgeiae (1/6). A total of seven B. henselae isolates and five blood samples from 12 positive cats were characterized by MLST.

G.L. Cicuttin et al. / Veterinary Microbiology 168 (2014) 225–228

227

Table 2 MLST and MLVA characterization results. MLST ST

a

1 1 1 1 1 8 8 5 6 a b c d

MLVA BHV-Ab

BHV-B

BHV-C

BHV-D

BHV-E

Profile no.c

No. cats

14 14 14 13 13 14 14 14 10

38 35 20 20 20 31 26 32 NDd

10 10 10 10 10 6 6 8 ND

7 7 6 6 3 5 5 3 ND

2 2 5 2 2 5 5 4 ND

205 206 207 208 209 199 210 211 –

1 2 2 1 1 2 1 1 1

ST was assigned according to the data available on the website (http://bhenselae.mlst.net). BHV: B. henselae variable number tandem repeats. Number of copies identified in each locus. Profile numbers were assigned according to the data available on the website (http://mlva.u-psud.fr). ND: target not amplified.

As a result, four different STs were identified (Table 2), ST 1 being the most frequent (58.3%, 7/12), followed by ST 8 (25%, 3/12). Afterwards, the same isolates and the blood samples save one were also characterized by MLVA). Eight different profiles were identified (Table 2), none of which had been described previously, except for profile 199 (Table 2) that had been detected in four Spanish patients diagnosed with cat scratch disease (Gil et al., 2013). The 38 copies found in the locus BHV-B of one of the profiles corresponded to a new allele (Table 2). In addition, nine of 11 B. henselae variants showed 14 copies in the locus BHVA that has been associated with infection in humans (Bouchouicha et al., 2009). 4. Conclusions To date, this is the first description of Bartonella spp. in cats and the first study of B. henselae characterization in Argentina. The prevalence found in cats is within the range found in South America and other regions of the world (Boulouis et al., 2005; Crissiuma et al., 2011; Ferre´s et al., 2005; Staggemeier et al., 2010). The differences in the prevalence are associated with the type of the feline population studied. In fact, stray cats present higher prevalence than pet cats (Boulouis et al., 2005). In our study, cats from LSEL neighborhoods presented a higher percentage of infection although it was not statistically significant. Surprisingly, although different Bartonella species have been described in canines, none of the dogs analyzed in our study tested positive. This indicates that the canine population seems to play a less important role as reservoir host of Bartonella compared to the feline population in Buenos Aires. These data are in contrast with a previous survey performed in dogs with valvulopathies from Buenos Aires (Belerenian et al., 2012). Valve tissues of these animals, which presented proliferative lesions, were analyzed by PCR (molecular detection performed in CDC, Fort Collins, CO, USA), detecting six animals positive to B. henselae and one co-infected with B. henselae and B. vinsonii subsp. berkhoffii. No blood or serum samples were analyzed from these animals. Although the animals in both studies came from the same districts, the different

studied dog population studied (sick and old dogs) and type of analyzed samples (valve tissues) can explain this discrepancy. Information about the B. henselae variants in South America is being provided here for the first time. It is noteworthy that the worldwide distribution of B. henselae variants is not homogeneous. ST 1 is the predominant variant found in Asia and is similar to the distribution found in the feline population we have studied (Arvand et al., 2007), and it is in contrast with the distribution in Europe, where ST 5 and ST 7 are predominant (Arvand et al., 2007; Chaloner et al., 2011; Gil et al., 2013). Interestingly, some of the variants found in our study have been previously associated with human disease. ST 1 and ST 8, the main variants found here, as well as the MLVA profiles with 14 copies in the locus BHV-A, and the MLVA profile 199, have been detected in cat scratch disease patients (Arvand et al., 2007; Bouchouicha et al., 2009; Chaloner et al., 2011; Gil et al., 2013). Therefore, the circulation of these B. henselae variants strengthens the importance of the feline population as a source of zoonotic agents and represents a potential risk of infection. In fact, veterinarian and physicians should consider the presence of these zoonotic pathogens in their diagnostic routine. Acknowledgments This study was supported by a grant from the Fondo de Investigacio´n Sanitaria of ISCIII (PI10/00051) and a research ˜ ola de Cooperacio´n fellowship from the Agencia Espan Internacional (MAEC-AECID, program II.B 2011–2012). References Arvand, M., Feil, E.J., Giladi, M., Boulouis, H.J., Viezens, J., 2007. Multi-locus sequence typing of Bartonella henselae isolates from three continents reveals hypervirulent and feline-associated clones. PLoS ONE 2, e1346. Belerenian, G., Pucheta, C., Medina-Bouquet, O., Fermepin, M., Venu´tolo, M.L., Iachini, R., Otros, O., 2012. First report of coinfection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii in canine aortic valve endocarditis. In: Matamoro, L.A. (Ed.), National Congress of the Association of Specialized Veterinary in Pets Animals. p. 232. 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,

228

G.L. Cicuttin et al. / Veterinary Microbiology 168 (2014) 225–228

S., Kasten, R., Petit, E., Boulouis, H.J., Haddad, N., 2009. Molecular epidemiology of feline and human Bartonella henselae isolates. Emerg. Infect. Dis. 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. Vet. Res. 36, 383–410. Chaloner, G.L., Harrison, T.G., Coyne, K.P., Aanensen, D.M., Birtles, R.J., 2011. Multilocus sequence typing of Bartonella henselae in the United Kingdom indicates that only a few, uncommon sequence types are associated with zoonotic disease. J. Clin. Microbiol. 49, 2132–2137. Chomel, B.B., Kasten, R.W., Floyd-Hawkins, K., Chi, B., Yamamoto, K., Roberts-Wilson, J., Gurfield, A.N., Abbott, R.C., Pedersen, N.C., Koehler, J.E., 1996. Experimental transmission of Bartonella henselae by the cat flea. J. Clin. Microbiol. 34, 1952–1956. Correa, F.G., Pontes, C.L., Verzola, R.M., Mateos, J.C., Velho, P.E., Schijman, A.G., Selistre-de-Araujo, H.S., 2012. Association of Bartonella spp bacteremia with Chagas cardiomyopathy, endocarditis and arrythmias in patients from South America. Braz. J. Med. Biol. Res. 45, 644– 651. Crissiuma, A., Favacho, A., Gershony, L., Mendes-de-Almeida, F., Gomes, R., Mares-Guia, A., Rozental, T., Barreira, J., Lemos, E., Labarthe, N., 2011. Prevalence of Bartonella species DNA and antibodies in cats (Felis catus) submitted to a spay/neuter program in Rio de Janeiro. Brazil. J. Feline. Med. Surg. 13, 149–151. Ferre´s, M., Abarca, K., Godoy, P., Garcı´a, P., Palavecino, E., Mendez, G., Valde´s, A., Ernst, S., Thibaut, J., Koberg, J., Chanqueo, L., Vial, P.A., 2005. Presence of Bartonella henselae in cats: natural reservoir quantification and human exposition risk of this zoonoses in Chile. Rev. Med. Chil. 133, 1465–1471.

Garcı´a-Esteban, C., Gil, H., Rodrı´guez-Vargas, M., Gerrikagoitia, X., Barandika, J., Escudero, R., Jado, I., Garcı´a-Amil, C., Barral, M., Garcı´aPe´rez, A.L., Bhide, M., Anda, P., 2008. Molecular method for Bartonella species identification in clinical and environmental samples. J. Clin. Microbiol. 46, 776–779. Garre, L., Guaraglia, W., Cuatz, D., Kaufman, S., Gil, H., De Rosa, A.F., 2008. Infective endocarditis due to Bartonella quintana. Medicina (B Aires) 68, 144–146. Gil, H., Escudero, R., Pons, I., Rodrı´guez-Vargas, M., Garcı´a-Esteban, C., Rodrı´guez-Moreno, I., Garcı´a-Amil, C., Lobo, B., Valca´rcel, F., Pe´rez, A., Jı´menez, S., Jado, I., Juste, R.A., Segura, F., Anda, P., 2013. Distribuction of Bartonella henselae variants in patients, reservoir hosts and vectors in Spain. PLoS ONE 8, e68248. Gil, H., Garcı´a-Esteban, C., Barandika, J.F., Peig, J., Toledo, A., Escudero, R., Jado, I., Rodrı´guez-Vargas, M., Garcı´a-Amil, C., Lobo, B., Roales, P., Rodrı´guez-Moreno, I., Olmeda, A.S., Garcı´a-Pe´rez, A.L., Anda, P., 2010. Variability of Bartonella genotypes among small mammals in Spain. Appl. Environ. Microbiol. 76, 8062–8070. Harms, A., Dehio, C., 2012. Intruders below the radar: molecular pathogenesis of Bartonella spp. Clin. Microbiol. Rev. 25, 42–78. Lamps, L.W., Scott, M.A., 2004. Cat-scratch disease: historic, clinical, and pathologic perspectives. Am. J. Clin. Pathol. 121 (Suppl.) S71–S80. Li, W., Raoult, D., Fournier, P.E., 2007. Genetic diversity of Bartonella henselae in human infection detected with multispacer typing. Emerg. Infect. Dis. 13, 1178–1183. Staggemeier, R., Venker, C.A., Klein, D.H., Petry, M., Spilki, F.R., Cantarelli, V.V., 2010. Prevalence of Bartonella henselae and Bartonella clarridgeiae in cats in the south of Brazil: a molecular study. Mem. Inst. Oswaldo Cruz. 105, 873–878.

Bartonella spp. in cats from Buenos Aires, Argentina.

In Argentina, data on the presence of members of the genus Bartonella is scarce. To increase knowledge about these zoonotic pathogens in this country,...
249KB Sizes 0 Downloads 0 Views