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Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection
Veterinary Microbiology 142 (2010) 346–351
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection A.M.S. Guimaraes a,1, P.E. Branda˜o b, W. Moraes c, S. Kiihl d, L.C. Santos c, C. Filoni e,f,g, Z.S. Cubas c, R.R. Robes h, L.M. Marques a, R.L. Neto a, M. Yamaguti a, R.C. Oliveira a, J.L. Cata˜o-Dias e,i, L.J. Richtzenhain b, J.B. Messick j, A.W. Biondo h, J. Timenetsky a,* a
Departmento de Microbiologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo (USP), Avenida Prof. Lineu Prestes, 1374 Sa˜o Paulo, SP 05508900, Brazil Departamento de Medicina Veterina´ria Preventiva e Sau´de Animal, Faculdade de Medicina Veterina´ria e Zootecnia (FMVZ), USP, Av. Prof. Dr. Orlando Marques de Paiva, 87 Sa˜o Paulo, SP 05508270, Brazil c Departamento de Reservato´rio e A´reas Protegidas, Itaipu Binacional, Rua Terezina, 62 Refu´gio Biolo´gico Bela Vista Vila ‘‘C’’, Nova Foz do Iguac¸u, PR 85866900, Brazil d Johns Hopkins Bloomberg School of Public Health, 615N. Wolfe Street, Baltimore, MD 21205, USA e Departamento de Patologia, FMVZ, USP, Av. Prof. Dr. Orlando Marques de Paiva, 87 Sa˜o Paulo, SP 05508270, Brazil f Instituto Brasileiro para Medicina da Conservac¸a˜o – TRI´ADE, Av. Prof. Elisabete Rolim, 116 Sa˜o Paulo, SP 05514080, Brazil g Faculdade de Medicina Veterina´ria, Universidade Paulista, Rodovia Presidente Dutra Km 157.5 Sa˜o Jose´ dos Campos, SP 12240420, Brazil h Departamento de Medicina Veterina´ria, Universidade Federal do Parana´, Rua dos Funciona´rios, 1540 Curitiba, PR 80035050, Brazil i Fundac¸a˜o Parque Zoolo´gico de Sa˜o Paulo, Av. Miguel Ste´fano, 4241 Sa˜o Paulo, SP 04301-905, Brazil j Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 March 2009 Received in revised form 1 October 2009 Accepted 2 October 2009
Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopardus wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopardus geoffroyi, 17 L. wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDH gene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure] were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher’s exact test. The 635 bp product amplified from 10 samples (10/67 = 14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI = 0.00–0.451, p = 0.0028), however other analyzed variables were not considered risk factors (p > 0.05). Hematological abnormalities were not associated with infection (p > 0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Bartonella Neotropical felids Wild cats Leopardus Panthera Puma
* Corresponding author. Tel.: +55 11 3091 7297; fax: +55 11 3091 7354. E-mail addresses: [email protected], [email protected] (J. Timenetsky). 1 Present address: Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA. 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.10.002
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1. Introduction Bartonella spp. are emerging bacteria that infect mammals. Although domestic cats are considered the main reservoir of Bartonella henselae and Bartonella clarridgeiae (Breitschwerdt, 2008), the epidemiology of bartonellosis in wild felids is poorly understood. Bartonella henselae seropositivity has been described in more than 20 different felid species, including the eight Brazilian neotropical species: Leopardus wiedii, Leopardus colocolo, Leopardus pardalis, Leopardus tigrinus, Leopardus geoffroyi, Puma yagouaroundi, Puma concolor and Panthera onca (Yamamoto et al., 1998; Filoni, 2006). This microorganism also has been isolated and/or detected by PCR in Panthera leo and Acynonyx jubatus (Molia et al., 2004; Pretorius et al., 2004). Non-identified bartonella species were cultured from P. concolor, Lynx rufus, P. leo and A. jubatus (Breitschwerdt and Kordick, 2000; Molia et al., 2004). Just one study has described B. henselae PCR detection in the blood of Brazilian felids (Filoni, 2006). Risk factors for bartonella infection in domestic cats vary according to the studied population and diagnostic test used (Guptill et al., 2004). Bacteremia is common in young cats infested with fleas (Brunt et al., 2006). Experimentally infected domestic cats may develop hematological disorders including anemia, thrombocytopenia, lymphocytosis, neutropenia, and eosinophilia (Breitschwerdt, 2008). Lymphocytosis has been associated with B. henselae seropositivity in pet cats (Breitschwerdt et al., 2005). Risk factors analyses in bartonella infected wild felids are scarce (Yamamoto et al., 1998; Rotstein et al., 2000; Chomel et al., 2004), and hematological abnormalities have never been studied. Therefore, the aim of this study was to detect Bartonella spp. DNA in the blood of captive neotropical felids using PCR and to evaluate risk factors and hematological abnormalities associated with the infection. 2. Material and methods 2.1. Animals and blood collection EDTA blood samples were collected from 57 small neotropical felids; including 1 L. geoffroyi, 17 L. wiedii, 22 L. tigrinus, 14 L. pardalis, and 3 P. yagouaroundi (27 males and 30 females; 34 wild-caught and 23 zoo-born); maintained in captivity at Bela Vista Sanctuary, Itaipu Binacional, Foz do Iguacu, South Brazil on three consecutive mornings of
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November 2006. In July 2007, Bela Vista Sanctuary, Itaipu Binacional, received two large neotropical felids (P. onca) to maintain in captivity: a 6-month-old male captured in Pantanal region and a male adult transferred from another zoo. Blood samples were also collected from these animals. Blood samples from an additional eight large neotropical felids (P. onca) stored in a blood bank (70 8C) at Universidade de Sa˜o Paulo were obtained (3 males and 5 females). These animals were from two different institutions, the Parque Ecolo´gico Municipal de Paulı´nea (Paulı´nea Zoo) and the Fundac¸a˜o Parque Zoolo´gico de Sa˜o Paulo (Sa˜o Paulo Zoo), both located in Sa˜o Paulo State, Southeast Brazil, about 800 miles from the Bela Vista Sanctuary. Blood collection of these animals occurred on different dates prior to 2006. Small neotropical felids from Bela Vista Sanctuary were captured with nets and anesthesized under different protocols with ketamine hydrochloride (10.7–28.10 mg/ kg), xylazine hydrochloride (0.67–1.86 mg/kg) and/or diazepam (0.27–1.10 mg/kg), and atropine sulphate (0.01–0.09 mg/kg). P. onca specimens were initially sedated using darts and then anesthetized using similar protocols. All procedures were performed according to the Guidelines of the Ethical Principles in Animal Research of the School of Veterinary Medicine and Animal Science of Universidade de Sa˜o Paulo. While under anesthesia, blood samples were collected by puncture of jugular or femoral veins and placed in tubes containing EDTA. Blood remaining after performing a complete blood cell count (CBC) was transferred to another tube and frozen at 70 8C pending molecular analysis. 2.2. Hematology Blood samples collected from small neotropical felids in November 2006 at Bela Vista Sanctuary were submitted for a complete blood count (CBC) by manual method on Neubauer’s chamber12 in the Itaipu Clinical Pathology Laboratory. The hematological reference values used in this study were retrieved from Santos (1999) and from the International Species Information System (ISIS, 2002) (Table 1). 2.3. Bacterial strain Lyophilized B. henselae Houston-1 strain was obtained from the Instituto Adolfo Lutz, Sa˜o Paulo, Brazil. The bacteria were hydrated with sterile water and cultured in
Table 1 Hematological reference values for selected small neotropical felid species. Parameter
Puma yagouaroundi Leopardus pardalis Leopardus tigrinus Leopardus wiedii Leopardus geoffroyia
Hematocrit (%)
TPP (g/dL)
Leukocytes (103 cells/mm3)
Males
Females
Males and females
Males and females
38–47 35–44 38–46 40–51 32.9–56.7
41–47 39–46 37–44 32–39
6.14–11.22 6.38–8.08 5.8–7.60 6.13–7.55 6.0–9.0
4.9–12.1 6.2–12.4 4.1–9.6 7.5–11.9 3.0–20.8
Source: Santos (1999). a Reference values for L. geoffroyi were the only species reference values retrieved from the International Species Information System (ISIS), 2002. In this case, both genders were combined for hematocrit analysis.
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Columbia agar with 5% of sheep blood at 37 8C in a 5% CO2 atmosphere for 7 days (Breitschwerdt, 2008). 2.4. DNA extraction DNA extraction of small neotropical felids blood samples was performed using a commercially available kit (GE Healthcare Life Sciences, Waukesha, USA) according to the manufacturer’s instructions. DNA extraction of the P. onca blood samples was conducted approximately 1 year later, when these samples became available, and the extraction was done using a different kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany). After culturing B. henselae Houston-1 strain, bacterial DNA was extracted as described by Boom et al. (1990). Using a DU-640 UV/VIS scanning spectrophotometer at a wavelength of 260 nm, DNA in all samples was quantified according to the manufacturer’s instructions (Beckman Coulter, Sa˜o Paulo, Brazil). 2.5. PCR for housekeeping gene DNA extraction from blood was verified by PCR of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using a primer set previously described (Shy and Roy-Burman, 2000). A blood DNA sample from a specific pathogen free (SPF) domestic cat provided by Professor Regina Hofmann-Lehmann, University of Zurich, was used as PCR positive control for the housekeeping gene. Ultrapure water was used as negative control. PCR reactions of 25 mL contained 1 of 10 PCR buffer (200 mM Tris–HCl, 500 mM KCl, pH 8.4), 1.8 mM of 50 mM MgCl2, 0.2 mM of 1 mM dNTPs (dATP, dGTP, dCTP, dTTP), 20 pmol of each primer, 1.25 U of Taq Platinum DNA polymerase (Invitrogen, Sa˜o Paulo, Brazil), water and template DNA. The cycling conditions consisted of 94 8C for 10 min followed by 30 cycles of 94 8C for 1 min, 50.1 8C for 1 min, and 72 8C for 1 min with a final extension of 72 8C for 5 min and a cooling at 4 8C. The predicted products were separated by electrophoresis in a 1% agarose gel containing 5 mg/ml ethidium bromide. Gels were photographed under ultraviolet light with a Vilber Lourmat (Vilber Loumart, Marne la Vallee´, France) imaging system.
from blood of the SPF cat were used as negative controls. Approximately 200 ng of DNA extracted from blood of wild felids were added for testing, as described by Maggi et al. (2006). PCR reactions of 25 mL contained 1 of 10 PCR buffer (200 mM Tris–HCl, 500 mM KCl, pH 8.4), 2.0 mM of 50 mM MgCl2, 0.2 mM of 1 mM dNTPs (dATP, dGTP, dCTP, dTTP), 12.5 pmol of each primer, 0.625 U of Taq Platinum DNA polymerase (Invitrogen, Sa˜o Paulo, Brazil), water and template DNA. The cycling conditions consisted of 94 8C for 5 min followed by 45 cycles of 94 8C for 30 s, 66 8C for 30 s, and 72 8C for 30 s with a final extension of 72 8C for 5 min and a cooling at 4 8C. The predicted products were separated by electrophoresis in a 2% agarose gel and photographed as described above. 2.7. Detection limit and specificity of Bartonella spp. PCR DNA extracted from B. henselae Houston-1 strain was quantified using spectrophotometry and serially diluted from 24 ng to 2.4 fg to determine the limit of detection for the Bartonella spp. PCR. In order to estimate the number of organisms, a genome size of approximately 2 Mb for B. henselae (GenBank accession number: NC 005956) was used for the calculation as described elsewhere (Applied Biosystems, 2003). The specificity of the PCR was also tested against DNA of the following microorganisms: Mycoplasma haemofelis, ‘‘Candidatus Mycoplasma turicensis’’, ‘‘Candidatus Mycoplasma haemominutum’’, Ehrlichia chaffensis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Proteus mirabilis. 2.8. Sequencing of PCR products Bartonella spp. PCR products were purified from the gel using a commercially available kit (Illustra GFX PCR DNA and gel band purification kit, GE Healthcare Life Sciences, Amersham, UK). Purified DNA products were directly sequenced in both directions by a dideoxy terminator method at Laboratory of Molecular Biology at the Departmento de Medicina Veterina´ria Preventiva e Sau´de Animal, Universidade de Sa˜o Paulo. 2.9. Statistical analysis
2.6. PCR for Bartonella spp. The DNA detection of Bartonella spp. in the extracted blood was performed as described by Maggi et al. (2006). The following primer set was used: 50 -CTTCAGATGATGATCCCAAGCCTTYTGGCG-30 and 50 -GAACCGACGACCCCCTGCTTGCAAAGCA-30 . These primers are complementary to the 16S-23S rRNA intergenic spacer region of bartonellas; since this is a hypervariable region, it allows differentiation of Bartonella spp. according to the size of the PCR product. B. henselae and B. clarridgeiae have a product size of 635 bp, whereas Bartonella vinsonii berkhoffii, Bartonella elizabethae, Bartonella quintana and Bartonella bovis have a product size of 725, 673, 520 and 406 bp, respectively. Extracted DNA of B. henselae Houston-1 was used as positive control and ultrapure water and DNA extracted
Statistical analysis of risk factors for infection and hematological abnormalities associated with the infection were performed for all the small neotropical felids (n = 57). The analyses were performed using the software R (R Development Core Team, 2008) and SAS (SAS, 1989). Initially, the population was analyzed for homogeneity related to gender and number of wild-caught/zoo-born animals using the Fisher’s exact test. Risk factors [gender, origin (wild-caught or zoo-born), species and flea exposure (based on medical records and observation at the time of blood collection)] were analyzed using Fisher’s exact test as a univariate analysis and then evaluated in an exact multiple logistic regression. Hematological findings (anemia, hyperproteinemia/polycythemia, leukocytosis and leukopenia) were also evaluated using Fisher’s exact test.
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Specimens of P. yagouaroundi (n = 3) and L. geoffroyi (n = 1) were combined into a single group due to the low sample size. Confidence intervals of 95% were used and results were reported as statistically significant when p < 0.05. 3. Results All samples successfully amplified the predicted product for GAPDH gene. The Bartonella PCR detection limit was 24 fg of extracted B. henselae DNA, an amount close to that contained in approximately 10 microorganisms. In addition, the reaction did not amplify DNA of any unrelated organisms. Ten samples (14.92%) were positive for Bartonella spp. by PCR, including 9 (15.8%) out of the 57 small neotropical felids [1 L. geoffroyi, 1 P. yagouaroundi, 3 L. tigrinus, 3 L. wiedii, 1 L. pardalis] and 1 out of the 10 P. onca. The bands were approximately 635 bp in size. Positive samples were all identified as B. henselae by DNA sequencing, showing 100% identity to the corresponding fragment of the B. henselae Houston-1 complete genome (embjBX897699). All positive small neotropical felids animals were males, of which 6 (66.6%) were wild-caught, one (11.1%) showed history of flea exposure, none were anemic, two (22.2%) showed polycythemia/hypoproteinemia, four (44.4%) were leukopenic, and none showed leukocytosis. In the test of population homogeneity, there were no statistical differences between the number of males and females and between the number of wild-caught and zooborn animals in the small neotropical felid population maintained at Bela Vista Sanctuary (p > 0.05). The reference intervals for hematologic parameters evaluated in this study are shown in Table 1. Only three felids were anemic (1/3 P. yagouaroundi and 2/14 L. pardalis), however the TPP of these 3 animals were within the reference interval (Santos, 1999). Anemia was statistically associated with the felid species (p = 0.0019), but no association was found with Bartonella spp. detection by PCR (p = 1.0). Twelve (21%) felids were polycythemic and hyperproteinemic (2/22 L. tigrinus, 2/14 L. pardalis, and 8/17 L. wiedii). These changes were not statistically associated with the felid species (p = 0.2140). In addition, polycythemia and hyperproteinemia was not associated with detection of Bartonella spp. infection in these cats (p = 1.0). Eighteen (31.57%) felids presented leukopenia (2/3 P. yagouaroundi, 4/14 L. pardalis, 12/17 L. wiedii); this finding was statistically associated with the felid species (p < 0.0001). On the other hand, nine (15.78%) felids showed leukocytosis (5/22 L. tigrinus, 2/14 L. pardalis, and 2/17 L. wiedii). There was no significant association between leukocytosis and the felid species (p = 0.83). Neither leukopenia nor leukocytosis showed an association with Bartonella spp. DNA detection (p = 0.46 and p = 0.33, respectively). The univariate analysis using Fisher’s exact test showed gender as a risk factor for Bartonella spp. detection in the blood of the 57 small neotropical cats (p = 0.0005, 95% CI = 2.79–1). The results of the exact multiple logistic regression for risk factors are shown in Table 2. Males were
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Table 2 Results of the exact multiple logistic regression of risk factors for Bartonella spp. infection in 57 small neotropical felids maintained at Bela Vista Sanctuary, Itaipu Binacional, Foz do Iguacu, Brazil. Parameter
Odds ratio
95% CI
p-Value
Gender Male vs female Female
14.7 1.0
2.2–1
0.0028
Origin ZB vs WCa WC
1.57 1.0
0.22–12.7
0.8878
Flea exposure Yes vs no No
2.43 1.0
0.02–258
1.0
Species P. yagouaroundi and L. geoffroyi L. tigrinus L. pardalis L. wiedii
1.0 0.195 0.211 0.377
a
0.003–5.169 0.002–7.315 0.0005–9.868
0.5030 0.6490 0.8999
WC = wild-caught felids, ZB = zoo-born felids.
14.7 times more likely to be positive than females and this result was statistically significant (p = 0.003). Based on animal clinical records, 5 (8.8%) out of the 57 small neotropical felids had a history of flea infestation while in captivity or on arrival at the zoo. No fleas were observed on any of the 57 wild cats at the time of blood collection. Origin, species, and flea exposure were not statistically associated with the Bartonella spp. detection by PCR (p > 0.05) (Table 2). 4. Discussion This is the first report of B. henselae DNA detection in the blood of L. pardalis, L. geoffroyi, L. tigrinus, P. yagouaroundi and P. onca. A previous study performed in Brazil documented serological evidence of infection in captive and free-ranging wild felids (Filoni, 2006), but just one captive L. wiedii was positive for B. henselae by real-time PCR (1/109 = 0.92%). Only captive wild felids were tested by real-time PCR in the Filoni’s study. In the present study, 14.92% of the felids showed positive results in a conventional Bartonella spp. PCR. The reason for this discrepancy remains unknown; however, the animals evaluated by Filoni (2006) were maintained in captivity at the Sa˜o Paulo Zoo, while most of the felids studied herein were from the Bela Vista Sanctuary. Therefore, geographic origin (or zoo of origin) as well as felid species differences at these locations may play a role in the frequency of infection. Clinical and hematological abnormalities are infrequent in domestic cats naturally infected with B. henselae (Breitschwerdt, 2008). As with the domestic cat, hematologic abnormalities were not associated with B. henselae infection of small neotropical felids in our study. Polycythemia and hyperproteinemia were used as indicators of dehydration and also were not associated to Bartonella spp. detection; 21% of the wild felids were considered dehydrated. A confirmative approach would be to also consider the urine specific gravity. Stress, environmental temperature and availability of fresh water, should be
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further investigated as reasons for these findings. Leukopenia was statistically associated with the felid species; 70.6% of the L. wiedii were affected. Since these animals showed no signs of illness, the authors believe that the reference interval for white cell counts in the L. wiedii should be re-evaluated. The association of anemia with the felid species was biased by the small sample size of P. yagouaroundi (n = 3). Risk factors associated with positive serology are different from those associated with positive results on culture or PCR (Guptill et al., 2004); comparisons between the results of this study and others using serological tests should be cautiously addressed. Herein, males were 14.7 times more likely to be B. henselae positive by PCR than females. The positive P. onca was also a male. Gender was not associated with infection in free-ranging A. jubatus and P. leo in a molecular study conducted by Molia et al. (2004). Similarly, gender has not been considered a risk factor for bacteremia in domestic cats (Chomel et al., 1995; Gurfield et al., 2001; Guptill et al., 2004). For free-ranging Felis concolor in California however, males were more likely than females to be seropositive for B. henselae (Yamamoto et al., 1998). It remains unclear why males were more predisposed to infection in this study. In contrast to domestic cats (Chomel et al., 1995; Guptill et al., 2004; Gurfield et al., 2001), flea infestation was not associated with Bartonella spp. detection in our study. The studied felids are routinely under a rigorous flea control program. Our ability to find an association was probably confounded by the fact that history of flea exposure was not reported for many of the wild-caught felids and it is likely to have occurred in the wild. The felid’s origin was not associated with Bartonella spp. detection by PCR. Although wild-caught animals had been in captivity for at least 1 year, it is not possible to determine when the infection occurred in the 5 positive wild-caught felids. Since prolonged or relapsing bacteremia and re-infection have been reported in domestic cats (Kordick et al., 1995; Arvand et al., 2008), the felids of this study could have been infected in the wild and still be bacteremic even after a long period in captivity. Interestingly, the positive wild-caught P. onca had been maintained in captivity for only 1 week; the authors thus strongly believe that this animal was infected in the wild. Although the molecular detection of B. henselae DNA suggests bacteremia, it is uncertain the role wild cats might play as a reservoir for infection. The use of culture techniques are needed to further evaluate the presence of viable organisms. Given recent reports of B. henselae infection in species as disparate as porpoise and beef cattle (Cherry et al., 2009; Harms et al., 2008), transmission in nature is likely much more complex than previously suggested. If, similarly to domestic cats, wild cats remain infected for long periods of time, they could be a significant reservoir of the organism in the wild or in captivity.
Acknowledgements The authors are in debt to Aricelma P. Franc¸a for valuable laboratory assistance and to all veterinarians and
staff of Itaipu Binacional, Universidade Federal do Parana´, Fundac¸a˜o Parque Zoolo´gico de Sa˜o Paulo and Parque Ecolo´gico Municipal de Paulı´nea, especially to Carlos F. da Silva, Marcos J. de Oliveira, Rosana P. de Almeida, Sandra H. R. Correˆa, Mara C. Marques, Jose´ D.L. Fedullo, Rodrigo H.F. Teixeira, and Marcelo Q. Telles. The authors also thank Marco A. Gioso and Joa˜o L. Rossi Jr from the Laborato´rio de Odontologia Comparada, Faculdade de Medicina Veterina´ria, Universidade de Sa˜o Paulo for obtaining blood samples from the jaguars from the Paulı´nea Zoo; Regina Hofmann-Lehmann from Clinical Laboratory, Vetsuisse Faculty, University of Zurich for the SPF cat DNA blood sample, and Pedro P. Diniz from College of Veterinary Medicine, North Carolina State University for assistance. This study was conducted by Ana M.S. Guimaraes as partial fulfillment of the requirements for her master degree at Universidade de Sa˜o Paulo and was funded by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES). References Applied Biosystems, 2003. Creating standard curves with genomic DNA or plasmid DNA templates for use in quantitative PCR. Available from: http://www.appliedbiosystems.com/support/tutorials/pdf/quant_ pcr.pdf (accessed November 2007). Arvand, M., Viezens, J., Berghoff, J., 2008. Prolonged Bartonella henselae bacteremia caused by reinfection in cats. Emerg. Infect. Dis. 14, 152– 154. Breitschwerdt, E.B., 2008. Feline bartonellosis and cat scratch disease. Vet. Immunol. Immunopathol. 123, 167–171. Breitschwerdt, E.B., Kordick, D.L., 2000. Bartonella infection in animals: carriership, reservoir potential, pathogenicity, and zoonotic potential for human infection. Clin. Microbiol. Rev. 13, 428–438. Breitschwerdt, E.B., Levine, J.F., Radulovic, S., Hanby, S.B., Kordick, D.L., LaPerle, K.M.D., 2005. Bartonella henselae and Rickettsia seroreactivity in a sick cat population from North Carolina. Int. J. Appl. Res. Vet. Med. 3, 287–302. Boom, R., Sol, C.J.A., Salmans, M.M.M., Jansen, C.L., Werthiem Van Dillen, P.M.E., Van Der Noordaa, J., 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495–503. Brunt, J., Guptill, L., Kordick, D.L., Kudrak, S., Lappin, M.R., 2006. American Association of Feline Practitioners 2006 panel report on diagnosis, treatment, and prevention of Bartonella spp. infections. J. Fel. Med. Surg. 8, 213–226. Cherry, N.A., Maggi, R.G., Cannedy, A.L., Breitschwerdt, E.B., 2009. PCR detection of Bartonella bovis and Bartonella henselae in the blood of beef cattle. Vet. Microbiol. 135, 308–312. Chomel, B.B., Abbott, R.C., Kasten, R.W., Floyd-Howkins, K.A., Kass, P.H., Glaser, C.A., Pedersen, N.C., Koehler, J.E., 1995. Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers. J. Clin. Microbiol. 9, 2445– 2450. Chomel, B.B., Kikuchi, Y., Marterson, J.S., Roelke-Parker, M.E., Chang, C.C., Kasten, R.W., Foley, J.E., Laudre, J., Murphy, K., Swift, P.K., Kramer, V.L., O’Brien, S.J., 2004. Seroprevalence of Bartonella infection in American free-ranging and captive pumas (Felis concolor) and bobcats (Lynx rufus). Vet. Res. 35, 233–241. Filoni, C., 2006. Exposic¸a˜o de felı´deos selvagens a agentes infecciosos selecionados. Ph.D. Thesis. Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil. Guptill, L., Wu, C.C., HogenEsch, H., Slater, L.N., Geickman, N., Dunham, A., Syme, H., Glickman, L., 2004. Prevalence, risk factors, and genetic diversity of Bartonella henselae infection in pet cats in four regions of the United States. J. Clin. Microbiol. 42, 652–659. 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., Piemont, Y., 2001. Epidemiology of Bartonella infection in domestic cats in France. Vet. Microbiol. 80, 185–198. Harms, C.A., Magi, R.G., Breitschwerdt, E.B., Clemon-Chevis, C.L., Solangi, M., Rotstein, D.S., Fair, P.A., Hansen, L.J., Honhn, A.A., Lowewell, G.N., McLellan, W.A., Pabst, D.A., Rowles, T.K., Schwacke, L.H., Townsend, F.I., Wells, R.S., 2008. Bartonella species detection in captive, stranded and free-ranging cetaceans. Vet. Res. 39, 59–66.
A.M.S. Guimaraes et al. / Veterinary Microbiology 142 (2010) 346–351 ISIS, 2002. Reference for Physiological Values in Captive Wildlife, CD-ROM. International Species Inventory System, Apple Valley, Minnesota. Kordick, D.L., Wilson, K.H., Sexton, D.J., Hadfield, T.L., Berkhoff, H.A., Breitschwerdt, E.B., 1995. Prolonged Bartonella bacteremia in cats associated with cat-scratch disease patients. J. Clin. Microbiol. 33, 3245–3251. Maggi, R.G., Diniz, P.P., Cadenas, M.B., Breitschwerdt, E.E., 2006. The use of molecular diagnostic techniques to detect Anaplasma, Bartonella and Erlichia species in arthropods or patients. In: The International Canine Vector-Borne Disease Symposium, Billesley, UK, pp. 8–13. Molia, S., Chomel, B.B., Kasten, R.W., Leutenegger, C.M., Steele, B.R., Marker, L., Martenson, J.S., Keet, D.F., Bengis, R.G., Peterson, R.P., Munson, L., O’Brien, S.J., 2004. Prevalence of Bartonella infection in wild African lions (Panthera leo) and cheetahs (Acinonyx jubatus). Vet. Microbiol. 100, 31–41. Pretorius, A.M., Kuyl, J., Isherwood, D.R., Birtles, R.J., 2004. Bartonella henselae in African lion, South Africa. Emerg. Infect. Dis. 10, 2257–2258.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection A.M.S. Guimaraes a,1, P.E. Branda˜o b, W. Moraes c, S. Kiihl d, L.C. Santos c, C. Filoni e,f,g, Z.S. Cubas c, R.R. Robes h, L.M. Marques a, R.L. Neto a, M. Yamaguti a, R.C. Oliveira a, J.L. Cata˜o-Dias e,i, L.J. Richtzenhain b, J.B. Messick j, A.W. Biondo h, J. Timenetsky a,* a
Departmento de Microbiologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo (USP), Avenida Prof. Lineu Prestes, 1374 Sa˜o Paulo, SP 05508900, Brazil Departamento de Medicina Veterina´ria Preventiva e Sau´de Animal, Faculdade de Medicina Veterina´ria e Zootecnia (FMVZ), USP, Av. Prof. Dr. Orlando Marques de Paiva, 87 Sa˜o Paulo, SP 05508270, Brazil c Departamento de Reservato´rio e A´reas Protegidas, Itaipu Binacional, Rua Terezina, 62 Refu´gio Biolo´gico Bela Vista Vila ‘‘C’’, Nova Foz do Iguac¸u, PR 85866900, Brazil d Johns Hopkins Bloomberg School of Public Health, 615N. Wolfe Street, Baltimore, MD 21205, USA e Departamento de Patologia, FMVZ, USP, Av. Prof. Dr. Orlando Marques de Paiva, 87 Sa˜o Paulo, SP 05508270, Brazil f Instituto Brasileiro para Medicina da Conservac¸a˜o – TRI´ADE, Av. Prof. Elisabete Rolim, 116 Sa˜o Paulo, SP 05514080, Brazil g Faculdade de Medicina Veterina´ria, Universidade Paulista, Rodovia Presidente Dutra Km 157.5 Sa˜o Jose´ dos Campos, SP 12240420, Brazil h Departamento de Medicina Veterina´ria, Universidade Federal do Parana´, Rua dos Funciona´rios, 1540 Curitiba, PR 80035050, Brazil i Fundac¸a˜o Parque Zoolo´gico de Sa˜o Paulo, Av. Miguel Ste´fano, 4241 Sa˜o Paulo, SP 04301-905, Brazil j Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 March 2009 Received in revised form 1 October 2009 Accepted 2 October 2009
Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopardus wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopardus geoffroyi, 17 L. wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDH gene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure] were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher’s exact test. The 635 bp product amplified from 10 samples (10/67 = 14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI = 0.00–0.451, p = 0.0028), however other analyzed variables were not considered risk factors (p > 0.05). Hematological abnormalities were not associated with infection (p > 0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Bartonella Neotropical felids Wild cats Leopardus Panthera Puma
* Corresponding author. Tel.: +55 11 3091 7297; fax: +55 11 3091 7354. E-mail addresses: [email protected], [email protected] (J. Timenetsky). 1 Present address: Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA. 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.10.002
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1. Introduction Bartonella spp. are emerging bacteria that infect mammals. Although domestic cats are considered the main reservoir of Bartonella henselae and Bartonella clarridgeiae (Breitschwerdt, 2008), the epidemiology of bartonellosis in wild felids is poorly understood. Bartonella henselae seropositivity has been described in more than 20 different felid species, including the eight Brazilian neotropical species: Leopardus wiedii, Leopardus colocolo, Leopardus pardalis, Leopardus tigrinus, Leopardus geoffroyi, Puma yagouaroundi, Puma concolor and Panthera onca (Yamamoto et al., 1998; Filoni, 2006). This microorganism also has been isolated and/or detected by PCR in Panthera leo and Acynonyx jubatus (Molia et al., 2004; Pretorius et al., 2004). Non-identified bartonella species were cultured from P. concolor, Lynx rufus, P. leo and A. jubatus (Breitschwerdt and Kordick, 2000; Molia et al., 2004). Just one study has described B. henselae PCR detection in the blood of Brazilian felids (Filoni, 2006). Risk factors for bartonella infection in domestic cats vary according to the studied population and diagnostic test used (Guptill et al., 2004). Bacteremia is common in young cats infested with fleas (Brunt et al., 2006). Experimentally infected domestic cats may develop hematological disorders including anemia, thrombocytopenia, lymphocytosis, neutropenia, and eosinophilia (Breitschwerdt, 2008). Lymphocytosis has been associated with B. henselae seropositivity in pet cats (Breitschwerdt et al., 2005). Risk factors analyses in bartonella infected wild felids are scarce (Yamamoto et al., 1998; Rotstein et al., 2000; Chomel et al., 2004), and hematological abnormalities have never been studied. Therefore, the aim of this study was to detect Bartonella spp. DNA in the blood of captive neotropical felids using PCR and to evaluate risk factors and hematological abnormalities associated with the infection. 2. Material and methods 2.1. Animals and blood collection EDTA blood samples were collected from 57 small neotropical felids; including 1 L. geoffroyi, 17 L. wiedii, 22 L. tigrinus, 14 L. pardalis, and 3 P. yagouaroundi (27 males and 30 females; 34 wild-caught and 23 zoo-born); maintained in captivity at Bela Vista Sanctuary, Itaipu Binacional, Foz do Iguacu, South Brazil on three consecutive mornings of
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November 2006. In July 2007, Bela Vista Sanctuary, Itaipu Binacional, received two large neotropical felids (P. onca) to maintain in captivity: a 6-month-old male captured in Pantanal region and a male adult transferred from another zoo. Blood samples were also collected from these animals. Blood samples from an additional eight large neotropical felids (P. onca) stored in a blood bank (70 8C) at Universidade de Sa˜o Paulo were obtained (3 males and 5 females). These animals were from two different institutions, the Parque Ecolo´gico Municipal de Paulı´nea (Paulı´nea Zoo) and the Fundac¸a˜o Parque Zoolo´gico de Sa˜o Paulo (Sa˜o Paulo Zoo), both located in Sa˜o Paulo State, Southeast Brazil, about 800 miles from the Bela Vista Sanctuary. Blood collection of these animals occurred on different dates prior to 2006. Small neotropical felids from Bela Vista Sanctuary were captured with nets and anesthesized under different protocols with ketamine hydrochloride (10.7–28.10 mg/ kg), xylazine hydrochloride (0.67–1.86 mg/kg) and/or diazepam (0.27–1.10 mg/kg), and atropine sulphate (0.01–0.09 mg/kg). P. onca specimens were initially sedated using darts and then anesthetized using similar protocols. All procedures were performed according to the Guidelines of the Ethical Principles in Animal Research of the School of Veterinary Medicine and Animal Science of Universidade de Sa˜o Paulo. While under anesthesia, blood samples were collected by puncture of jugular or femoral veins and placed in tubes containing EDTA. Blood remaining after performing a complete blood cell count (CBC) was transferred to another tube and frozen at 70 8C pending molecular analysis. 2.2. Hematology Blood samples collected from small neotropical felids in November 2006 at Bela Vista Sanctuary were submitted for a complete blood count (CBC) by manual method on Neubauer’s chamber12 in the Itaipu Clinical Pathology Laboratory. The hematological reference values used in this study were retrieved from Santos (1999) and from the International Species Information System (ISIS, 2002) (Table 1). 2.3. Bacterial strain Lyophilized B. henselae Houston-1 strain was obtained from the Instituto Adolfo Lutz, Sa˜o Paulo, Brazil. The bacteria were hydrated with sterile water and cultured in
Table 1 Hematological reference values for selected small neotropical felid species. Parameter
Puma yagouaroundi Leopardus pardalis Leopardus tigrinus Leopardus wiedii Leopardus geoffroyia
Hematocrit (%)
TPP (g/dL)
Leukocytes (103 cells/mm3)
Males
Females
Males and females
Males and females
38–47 35–44 38–46 40–51 32.9–56.7
41–47 39–46 37–44 32–39
6.14–11.22 6.38–8.08 5.8–7.60 6.13–7.55 6.0–9.0
4.9–12.1 6.2–12.4 4.1–9.6 7.5–11.9 3.0–20.8
Source: Santos (1999). a Reference values for L. geoffroyi were the only species reference values retrieved from the International Species Information System (ISIS), 2002. In this case, both genders were combined for hematocrit analysis.
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Columbia agar with 5% of sheep blood at 37 8C in a 5% CO2 atmosphere for 7 days (Breitschwerdt, 2008). 2.4. DNA extraction DNA extraction of small neotropical felids blood samples was performed using a commercially available kit (GE Healthcare Life Sciences, Waukesha, USA) according to the manufacturer’s instructions. DNA extraction of the P. onca blood samples was conducted approximately 1 year later, when these samples became available, and the extraction was done using a different kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany). After culturing B. henselae Houston-1 strain, bacterial DNA was extracted as described by Boom et al. (1990). Using a DU-640 UV/VIS scanning spectrophotometer at a wavelength of 260 nm, DNA in all samples was quantified according to the manufacturer’s instructions (Beckman Coulter, Sa˜o Paulo, Brazil). 2.5. PCR for housekeeping gene DNA extraction from blood was verified by PCR of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using a primer set previously described (Shy and Roy-Burman, 2000). A blood DNA sample from a specific pathogen free (SPF) domestic cat provided by Professor Regina Hofmann-Lehmann, University of Zurich, was used as PCR positive control for the housekeeping gene. Ultrapure water was used as negative control. PCR reactions of 25 mL contained 1 of 10 PCR buffer (200 mM Tris–HCl, 500 mM KCl, pH 8.4), 1.8 mM of 50 mM MgCl2, 0.2 mM of 1 mM dNTPs (dATP, dGTP, dCTP, dTTP), 20 pmol of each primer, 1.25 U of Taq Platinum DNA polymerase (Invitrogen, Sa˜o Paulo, Brazil), water and template DNA. The cycling conditions consisted of 94 8C for 10 min followed by 30 cycles of 94 8C for 1 min, 50.1 8C for 1 min, and 72 8C for 1 min with a final extension of 72 8C for 5 min and a cooling at 4 8C. The predicted products were separated by electrophoresis in a 1% agarose gel containing 5 mg/ml ethidium bromide. Gels were photographed under ultraviolet light with a Vilber Lourmat (Vilber Loumart, Marne la Vallee´, France) imaging system.
from blood of the SPF cat were used as negative controls. Approximately 200 ng of DNA extracted from blood of wild felids were added for testing, as described by Maggi et al. (2006). PCR reactions of 25 mL contained 1 of 10 PCR buffer (200 mM Tris–HCl, 500 mM KCl, pH 8.4), 2.0 mM of 50 mM MgCl2, 0.2 mM of 1 mM dNTPs (dATP, dGTP, dCTP, dTTP), 12.5 pmol of each primer, 0.625 U of Taq Platinum DNA polymerase (Invitrogen, Sa˜o Paulo, Brazil), water and template DNA. The cycling conditions consisted of 94 8C for 5 min followed by 45 cycles of 94 8C for 30 s, 66 8C for 30 s, and 72 8C for 30 s with a final extension of 72 8C for 5 min and a cooling at 4 8C. The predicted products were separated by electrophoresis in a 2% agarose gel and photographed as described above. 2.7. Detection limit and specificity of Bartonella spp. PCR DNA extracted from B. henselae Houston-1 strain was quantified using spectrophotometry and serially diluted from 24 ng to 2.4 fg to determine the limit of detection for the Bartonella spp. PCR. In order to estimate the number of organisms, a genome size of approximately 2 Mb for B. henselae (GenBank accession number: NC 005956) was used for the calculation as described elsewhere (Applied Biosystems, 2003). The specificity of the PCR was also tested against DNA of the following microorganisms: Mycoplasma haemofelis, ‘‘Candidatus Mycoplasma turicensis’’, ‘‘Candidatus Mycoplasma haemominutum’’, Ehrlichia chaffensis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Proteus mirabilis. 2.8. Sequencing of PCR products Bartonella spp. PCR products were purified from the gel using a commercially available kit (Illustra GFX PCR DNA and gel band purification kit, GE Healthcare Life Sciences, Amersham, UK). Purified DNA products were directly sequenced in both directions by a dideoxy terminator method at Laboratory of Molecular Biology at the Departmento de Medicina Veterina´ria Preventiva e Sau´de Animal, Universidade de Sa˜o Paulo. 2.9. Statistical analysis
2.6. PCR for Bartonella spp. The DNA detection of Bartonella spp. in the extracted blood was performed as described by Maggi et al. (2006). The following primer set was used: 50 -CTTCAGATGATGATCCCAAGCCTTYTGGCG-30 and 50 -GAACCGACGACCCCCTGCTTGCAAAGCA-30 . These primers are complementary to the 16S-23S rRNA intergenic spacer region of bartonellas; since this is a hypervariable region, it allows differentiation of Bartonella spp. according to the size of the PCR product. B. henselae and B. clarridgeiae have a product size of 635 bp, whereas Bartonella vinsonii berkhoffii, Bartonella elizabethae, Bartonella quintana and Bartonella bovis have a product size of 725, 673, 520 and 406 bp, respectively. Extracted DNA of B. henselae Houston-1 was used as positive control and ultrapure water and DNA extracted
Statistical analysis of risk factors for infection and hematological abnormalities associated with the infection were performed for all the small neotropical felids (n = 57). The analyses were performed using the software R (R Development Core Team, 2008) and SAS (SAS, 1989). Initially, the population was analyzed for homogeneity related to gender and number of wild-caught/zoo-born animals using the Fisher’s exact test. Risk factors [gender, origin (wild-caught or zoo-born), species and flea exposure (based on medical records and observation at the time of blood collection)] were analyzed using Fisher’s exact test as a univariate analysis and then evaluated in an exact multiple logistic regression. Hematological findings (anemia, hyperproteinemia/polycythemia, leukocytosis and leukopenia) were also evaluated using Fisher’s exact test.
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Specimens of P. yagouaroundi (n = 3) and L. geoffroyi (n = 1) were combined into a single group due to the low sample size. Confidence intervals of 95% were used and results were reported as statistically significant when p < 0.05. 3. Results All samples successfully amplified the predicted product for GAPDH gene. The Bartonella PCR detection limit was 24 fg of extracted B. henselae DNA, an amount close to that contained in approximately 10 microorganisms. In addition, the reaction did not amplify DNA of any unrelated organisms. Ten samples (14.92%) were positive for Bartonella spp. by PCR, including 9 (15.8%) out of the 57 small neotropical felids [1 L. geoffroyi, 1 P. yagouaroundi, 3 L. tigrinus, 3 L. wiedii, 1 L. pardalis] and 1 out of the 10 P. onca. The bands were approximately 635 bp in size. Positive samples were all identified as B. henselae by DNA sequencing, showing 100% identity to the corresponding fragment of the B. henselae Houston-1 complete genome (embjBX897699). All positive small neotropical felids animals were males, of which 6 (66.6%) were wild-caught, one (11.1%) showed history of flea exposure, none were anemic, two (22.2%) showed polycythemia/hypoproteinemia, four (44.4%) were leukopenic, and none showed leukocytosis. In the test of population homogeneity, there were no statistical differences between the number of males and females and between the number of wild-caught and zooborn animals in the small neotropical felid population maintained at Bela Vista Sanctuary (p > 0.05). The reference intervals for hematologic parameters evaluated in this study are shown in Table 1. Only three felids were anemic (1/3 P. yagouaroundi and 2/14 L. pardalis), however the TPP of these 3 animals were within the reference interval (Santos, 1999). Anemia was statistically associated with the felid species (p = 0.0019), but no association was found with Bartonella spp. detection by PCR (p = 1.0). Twelve (21%) felids were polycythemic and hyperproteinemic (2/22 L. tigrinus, 2/14 L. pardalis, and 8/17 L. wiedii). These changes were not statistically associated with the felid species (p = 0.2140). In addition, polycythemia and hyperproteinemia was not associated with detection of Bartonella spp. infection in these cats (p = 1.0). Eighteen (31.57%) felids presented leukopenia (2/3 P. yagouaroundi, 4/14 L. pardalis, 12/17 L. wiedii); this finding was statistically associated with the felid species (p < 0.0001). On the other hand, nine (15.78%) felids showed leukocytosis (5/22 L. tigrinus, 2/14 L. pardalis, and 2/17 L. wiedii). There was no significant association between leukocytosis and the felid species (p = 0.83). Neither leukopenia nor leukocytosis showed an association with Bartonella spp. DNA detection (p = 0.46 and p = 0.33, respectively). The univariate analysis using Fisher’s exact test showed gender as a risk factor for Bartonella spp. detection in the blood of the 57 small neotropical cats (p = 0.0005, 95% CI = 2.79–1). The results of the exact multiple logistic regression for risk factors are shown in Table 2. Males were
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Table 2 Results of the exact multiple logistic regression of risk factors for Bartonella spp. infection in 57 small neotropical felids maintained at Bela Vista Sanctuary, Itaipu Binacional, Foz do Iguacu, Brazil. Parameter
Odds ratio
95% CI
p-Value
Gender Male vs female Female
14.7 1.0
2.2–1
0.0028
Origin ZB vs WCa WC
1.57 1.0
0.22–12.7
0.8878
Flea exposure Yes vs no No
2.43 1.0
0.02–258
1.0
Species P. yagouaroundi and L. geoffroyi L. tigrinus L. pardalis L. wiedii
1.0 0.195 0.211 0.377
a
0.003–5.169 0.002–7.315 0.0005–9.868
0.5030 0.6490 0.8999
WC = wild-caught felids, ZB = zoo-born felids.
14.7 times more likely to be positive than females and this result was statistically significant (p = 0.003). Based on animal clinical records, 5 (8.8%) out of the 57 small neotropical felids had a history of flea infestation while in captivity or on arrival at the zoo. No fleas were observed on any of the 57 wild cats at the time of blood collection. Origin, species, and flea exposure were not statistically associated with the Bartonella spp. detection by PCR (p > 0.05) (Table 2). 4. Discussion This is the first report of B. henselae DNA detection in the blood of L. pardalis, L. geoffroyi, L. tigrinus, P. yagouaroundi and P. onca. A previous study performed in Brazil documented serological evidence of infection in captive and free-ranging wild felids (Filoni, 2006), but just one captive L. wiedii was positive for B. henselae by real-time PCR (1/109 = 0.92%). Only captive wild felids were tested by real-time PCR in the Filoni’s study. In the present study, 14.92% of the felids showed positive results in a conventional Bartonella spp. PCR. The reason for this discrepancy remains unknown; however, the animals evaluated by Filoni (2006) were maintained in captivity at the Sa˜o Paulo Zoo, while most of the felids studied herein were from the Bela Vista Sanctuary. Therefore, geographic origin (or zoo of origin) as well as felid species differences at these locations may play a role in the frequency of infection. Clinical and hematological abnormalities are infrequent in domestic cats naturally infected with B. henselae (Breitschwerdt, 2008). As with the domestic cat, hematologic abnormalities were not associated with B. henselae infection of small neotropical felids in our study. Polycythemia and hyperproteinemia were used as indicators of dehydration and also were not associated to Bartonella spp. detection; 21% of the wild felids were considered dehydrated. A confirmative approach would be to also consider the urine specific gravity. Stress, environmental temperature and availability of fresh water, should be
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further investigated as reasons for these findings. Leukopenia was statistically associated with the felid species; 70.6% of the L. wiedii were affected. Since these animals showed no signs of illness, the authors believe that the reference interval for white cell counts in the L. wiedii should be re-evaluated. The association of anemia with the felid species was biased by the small sample size of P. yagouaroundi (n = 3). Risk factors associated with positive serology are different from those associated with positive results on culture or PCR (Guptill et al., 2004); comparisons between the results of this study and others using serological tests should be cautiously addressed. Herein, males were 14.7 times more likely to be B. henselae positive by PCR than females. The positive P. onca was also a male. Gender was not associated with infection in free-ranging A. jubatus and P. leo in a molecular study conducted by Molia et al. (2004). Similarly, gender has not been considered a risk factor for bacteremia in domestic cats (Chomel et al., 1995; Gurfield et al., 2001; Guptill et al., 2004). For free-ranging Felis concolor in California however, males were more likely than females to be seropositive for B. henselae (Yamamoto et al., 1998). It remains unclear why males were more predisposed to infection in this study. In contrast to domestic cats (Chomel et al., 1995; Guptill et al., 2004; Gurfield et al., 2001), flea infestation was not associated with Bartonella spp. detection in our study. The studied felids are routinely under a rigorous flea control program. Our ability to find an association was probably confounded by the fact that history of flea exposure was not reported for many of the wild-caught felids and it is likely to have occurred in the wild. The felid’s origin was not associated with Bartonella spp. detection by PCR. Although wild-caught animals had been in captivity for at least 1 year, it is not possible to determine when the infection occurred in the 5 positive wild-caught felids. Since prolonged or relapsing bacteremia and re-infection have been reported in domestic cats (Kordick et al., 1995; Arvand et al., 2008), the felids of this study could have been infected in the wild and still be bacteremic even after a long period in captivity. Interestingly, the positive wild-caught P. onca had been maintained in captivity for only 1 week; the authors thus strongly believe that this animal was infected in the wild. Although the molecular detection of B. henselae DNA suggests bacteremia, it is uncertain the role wild cats might play as a reservoir for infection. The use of culture techniques are needed to further evaluate the presence of viable organisms. Given recent reports of B. henselae infection in species as disparate as porpoise and beef cattle (Cherry et al., 2009; Harms et al., 2008), transmission in nature is likely much more complex than previously suggested. If, similarly to domestic cats, wild cats remain infected for long periods of time, they could be a significant reservoir of the organism in the wild or in captivity.
Acknowledgements The authors are in debt to Aricelma P. Franc¸a for valuable laboratory assistance and to all veterinarians and
staff of Itaipu Binacional, Universidade Federal do Parana´, Fundac¸a˜o Parque Zoolo´gico de Sa˜o Paulo and Parque Ecolo´gico Municipal de Paulı´nea, especially to Carlos F. da Silva, Marcos J. de Oliveira, Rosana P. de Almeida, Sandra H. R. Correˆa, Mara C. Marques, Jose´ D.L. Fedullo, Rodrigo H.F. Teixeira, and Marcelo Q. Telles. The authors also thank Marco A. Gioso and Joa˜o L. Rossi Jr from the Laborato´rio de Odontologia Comparada, Faculdade de Medicina Veterina´ria, Universidade de Sa˜o Paulo for obtaining blood samples from the jaguars from the Paulı´nea Zoo; Regina Hofmann-Lehmann from Clinical Laboratory, Vetsuisse Faculty, University of Zurich for the SPF cat DNA blood sample, and Pedro P. Diniz from College of Veterinary Medicine, North Carolina State University for assistance. This study was conducted by Ana M.S. Guimaraes as partial fulfillment of the requirements for her master degree at Universidade de Sa˜o Paulo and was funded by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES). References Applied Biosystems, 2003. Creating standard curves with genomic DNA or plasmid DNA templates for use in quantitative PCR. Available from: http://www.appliedbiosystems.com/support/tutorials/pdf/quant_ pcr.pdf (accessed November 2007). Arvand, M., Viezens, J., Berghoff, J., 2008. Prolonged Bartonella henselae bacteremia caused by reinfection in cats. Emerg. Infect. Dis. 14, 152– 154. Breitschwerdt, E.B., 2008. Feline bartonellosis and cat scratch disease. Vet. Immunol. Immunopathol. 123, 167–171. Breitschwerdt, E.B., Kordick, D.L., 2000. Bartonella infection in animals: carriership, reservoir potential, pathogenicity, and zoonotic potential for human infection. Clin. Microbiol. Rev. 13, 428–438. Breitschwerdt, E.B., Levine, J.F., Radulovic, S., Hanby, S.B., Kordick, D.L., LaPerle, K.M.D., 2005. Bartonella henselae and Rickettsia seroreactivity in a sick cat population from North Carolina. Int. J. Appl. Res. Vet. Med. 3, 287–302. Boom, R., Sol, C.J.A., Salmans, M.M.M., Jansen, C.L., Werthiem Van Dillen, P.M.E., Van Der Noordaa, J., 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495–503. Brunt, J., Guptill, L., Kordick, D.L., Kudrak, S., Lappin, M.R., 2006. American Association of Feline Practitioners 2006 panel report on diagnosis, treatment, and prevention of Bartonella spp. infections. J. Fel. Med. Surg. 8, 213–226. Cherry, N.A., Maggi, R.G., Cannedy, A.L., Breitschwerdt, E.B., 2009. PCR detection of Bartonella bovis and Bartonella henselae in the blood of beef cattle. Vet. Microbiol. 135, 308–312. Chomel, B.B., Abbott, R.C., Kasten, R.W., Floyd-Howkins, K.A., Kass, P.H., Glaser, C.A., Pedersen, N.C., Koehler, J.E., 1995. Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers. J. Clin. Microbiol. 9, 2445– 2450. Chomel, B.B., Kikuchi, Y., Marterson, J.S., Roelke-Parker, M.E., Chang, C.C., Kasten, R.W., Foley, J.E., Laudre, J., Murphy, K., Swift, P.K., Kramer, V.L., O’Brien, S.J., 2004. Seroprevalence of Bartonella infection in American free-ranging and captive pumas (Felis concolor) and bobcats (Lynx rufus). Vet. Res. 35, 233–241. Filoni, C., 2006. Exposic¸a˜o de felı´deos selvagens a agentes infecciosos selecionados. Ph.D. Thesis. Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil. Guptill, L., Wu, C.C., HogenEsch, H., Slater, L.N., Geickman, N., Dunham, A., Syme, H., Glickman, L., 2004. Prevalence, risk factors, and genetic diversity of Bartonella henselae infection in pet cats in four regions of the United States. J. Clin. Microbiol. 42, 652–659. 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., Piemont, Y., 2001. Epidemiology of Bartonella infection in domestic cats in France. Vet. Microbiol. 80, 185–198. Harms, C.A., Magi, R.G., Breitschwerdt, E.B., Clemon-Chevis, C.L., Solangi, M., Rotstein, D.S., Fair, P.A., Hansen, L.J., Honhn, A.A., Lowewell, G.N., McLellan, W.A., Pabst, D.A., Rowles, T.K., Schwacke, L.H., Townsend, F.I., Wells, R.S., 2008. Bartonella species detection in captive, stranded and free-ranging cetaceans. Vet. Res. 39, 59–66.
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