Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil

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Original article

Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil Marcos Rogério André a,∗ , Heitor Miraglia Herrera b , Simone de Jesus Fernandes a , Keyla Cartens Marques de Sousa a , Luiz Ricardo Gonc¸alves a , Iara Helena Domingos c , Gabriel Carvalho de Macedo b , Rosangela Zacarias Machado a a Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias/Universidade Estadual Paulista (FCAV/UNESP), Jaboticabal, SP, Brazil b Universidade Católica Dom Bosco, Campo Grande, MS, Brazil c Centro de Controle de Zoonoses (CCZ), Campo Grande, MS, Brazil

a r t i c l e

i n f o

Article history: Received 23 February 2015 Received in revised form 3 July 2015 Accepted 4 July 2015 Available online xxx Keywords: Brazil Cats Ehrlichia spp. Hepatozoon spp. Piroplasmids

a b s t r a c t Anaplasmataceae agents, piroplasmids and Hepatozoon spp. have emerged as important pathogens among domestic and wild felines. The present work aimed to detect the presence of species belonging to the Anaplasmataceae family, piroplasmas and Hepatozoon spp. DNA in blood samples of domesticated and stray cats in the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Between January and April 2013, whole blood samples were collected from 151 cats (54 males, 95 females and two without gender registration) in the city of Campo Grande, state of Mato Grosso do Sul, Brazil. DNA extracted from cat blood samples was submitted to conventional PCR assays for Theileria/Babesia/Cytauxzoon spp. (18S rRNA, ITS-1), Ehrlichia spp. (16S rRNA, dsb, groESL), Anaplasma spp. (16S rRNA, groESL) and Hepatozoon spp. (18S rRNA) followed by phylogenetic reconstructions. Out of 151 sampled cats, 13 (8.5%) were positive for Ehrlichia spp. closely related to Ehrlichia canis, 1 (0.66%) for Hepatozoon spp. closely related to Hepatozoon americanum and Hepatozoon spp. isolate from a wild felid, 1 (0.66%) for Cytauxzoon sp. closely related do Cytauxzoon felis, and 18 (11.9%) for Babesia/Theileria (one sequence was closely related to Babesia bigemina, eight for Babesia vogeli, five to Theileria spp. from ruminants [Theileria ovis, Theileria lestoquardi] and four to Theileria sp. recently detected in a cat). The present study showed that Ehrlichia spp., piroplasmids (B. vogeli, Theileria spp. and Cytauxzoon spp.) and, more rarely, Hepatozoon spp. circulate among stray and domesticated cats in the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. © 2015 Elsevier GmbH. All rights reserved.

1. Introduction Recently, ticks and tick-borne pathogens are expanding their zoo-geographic range due to climate and environmental changes. Additionally, arthropod vectors have now adapted to a peridomicillary cycle involving cats (Shaw et al., 2001). In this context, species belonging to the Anaplasmataceae family, piroplasmids and Hepatozoon spp. have emerged as important pathogens among wild and domestic carnivores. Piroplasmids (Babesia spp., Theileria spp., and Cytauxzoon spp.) parasitize cats’ blood cells, causing sporadic cases of disease, and

∗ Corresponding author at: Laboratório de Imunoparasitologia, Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias Júlio de Mesquita Filho (UNESP), Campus de Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane, s/n, Zona Rural, CEP 14884-900, Jaboticabal, SP, Brazil. E-mail address: marcos [email protected] (M.R. André).

rarely outbreaks (Carli et al., 2012; Criado-Fornelio, 2012a). These protozoa have a complex life-cycle which includes Ixodid ticks as definitive hosts and felines as intermediate hosts (Criado-Fornelio, 2012a). On the other hand, vectors of feline ehrlichiosis and anaplasmosis, caused by Ehrlichia canis and Anaplasma phagocytophilum, respectively, are still unknown (Almosny et al., 1998; Almosny and Massard, 1999; Stubbs et al., 2000; Bjoersdorff et al., 1999; Tarello, 2005). Exposure to arthropods (ticks and fleas) and ingestion of rodents are suggestive routes of transmission of this disease among cats (Beaufils et al., 1999). Hepatozoonosis, an emergent disease in felines, is transmitted by ingestion of an infected invertebrate definitive host (CriadoFornelio et al., 2003). Even though hepatozoonosis in felines is normally subclinical, pathogenic effects may be exacerbated in stressed, immunocompromised animals or in concomintant infections. The ticks species involved in transmission cycles are still unknown (Criado-Fornelio, 2012b).

http://dx.doi.org/10.1016/j.ttbdis.2015.07.004 1877-959X/© 2015 Elsevier GmbH. All rights reserved.

Please cite this article in press as: André, M.R., et al., Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.07.004

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New Ehrlichia spp. and Anaplasma spp. genotypes have been detected in wild felines maintained in captivity in the state of São Paulo (André et al., 2010a, 2012). Ehrlichia sp. closely related to E. canis has been detected in domestic cats sampled in the cities of Vic¸osa (state of Minas Gerais, southeastern Brazil) (Oliveira et al., 2009), São Luís (state of Maranhão, northeastern Brazil) (Braga et al., 2012) and Cuiabá (state of Mato Grosso, central-midwestern Brazil) (Braga et al., 2014). Recently, Anaplasma sp. closely related to A. phagocytophilum, Babesia vogeli and Theileria sp. closely related to Theileria equi have been detected in stray cats in a zoo in the city of São Paulo, southeastern Brazil (André et al., 2014). While new genotypes of Hepatozoon spp. were reported in wild felids maintained in captivity in zoos in the cities of Jundiaí, Ilha Solteira (state of São Paulo) and Cuiabá (state of Mato Grosso) (André et al., 2010b), Hepatozoon spp. closely related to Hepatozoon canis (Rubini et al., 2006) and Hepatozoon felis (De Bortoli et al., 2011) have been detected in domestic cats in the states of São Paulo and Maranhão (northeastern Brazil), respectively. Although Cytauxzoon sp. has been detected in neotropical wild felids and lions maintained in zoos in cities located in the states of São Paulo (André et al., 2009), Rio de Janeiro (Peixoto et al., 2007) and Distrito Federal (André et al., 2009), there is only a report of this parasite in domestic cats in Brazil, in the state of Rio de Janeiro (Maia et al., 2013). The present work aimed to detect the presence of species belonging to the Anaplasmataceae family, piroplasmas and Hepatozoon spp. DNA in blood samples of domesticated and stray cats in the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil.

2. Material and methods Between January and April 2013, whole blood samples were collected from 151 cats (54 males, 95 females and two without gender registration) in the city of Campo Grande, which is the capital of the state of Mato Grosso do Sul, Brazil. Stray non-domesticated cats (n = 65) were caught by technical staff from the local zoonosis control center (CCZ). Domesticated cats (n = 86) were sampled during pre-surgical procedures for a castration project at the CCZ; these animals were returned to their homes after surgery. Overall, the domesticated cats were in a better physical condition than the nondomesticated animals (Santis et al., 2014). The blood samples were collected in EDTA and stored at −20 ◦ C until DNA extraction. The project was approved by the university’s Ethics Committee under the protocol number 004987/13. DNA was extracted from 200 ␮L of each whole blood sample using the QIAamp DNA blood mini-kit (QIAGEN® , Valencia, California, USA), in accordance with the manufacturer’s instructions. DNA concentration and quality was measured using absorbance ratio between 260/280 nm (Nanodrop, Term Scientific, USA). In order to confirm the presence of amplifiable DNA in samples, an internal control PCR targeting 28S rRNA feline gene was used (Helps et al., 2003) (Table 1). Microtubes containing ultrapure sterile water were intercaled between each series of five cats’ blood samples and submitted to DNA extraction. Each sample of extracted DNA was used as a template in 16S rRNA-based nested PCR assays for Ehrlichia spp. (16S rRNA gene) (Murphy et al., 1998) and Anaplasma spp. (16S rRNA gene) (Massung et al., 1998) (Table 1). E. canis and Anaplasma platys DNA positive controls were obtained from naturally infected dogs from Campo Grande, MS, Brazil (Dagnone et al., 2009). Positive samples were submitted to additional molecular characterization using nested PCR protocols based on omp-1 (Inayoshi et al., 2004), dsb (Doyle et al., 2005) and groESL (Sumner et al., 1997; Lotric-Furlan et al., 1998; Nicholson et al., 1999) (Table 1). Previously described PCR protocols based on ITS-1 region for piroplasmids (Shock et al., 2014) and 18S rRNA gene for

Babesia/Theileria spp. (Jefferies et al., 2007), Cytauxzoon felis (Birkenheuer et al., 2006) and Hepatozoon spp. (Ujvari et al., 2004) were used for DNA amplification (Table 1). Babesia sp. (André et al., 2012), Cytauxzoon sp. (André et al., 2009) and Hepatozoon sp. (André et al., 2010b) DNA samples obtained from naturally infected wild felids were also used as positive controls. Ultra-pure sterile water was used as negative control in all PCR assays described above. In each set of reactions, five tubes containing ultra-pure water were used as controls. In order to prevent PCR contamination, DNA extraction, reaction setup, PCR amplification and electrophoresis were performed in separated rooms. The reaction products (fragments of 358 bp for 16S rRNA Ehrlichia spp., 409 bp for dsb Ehrlichia spp., 800 bp for 18S rRNA Babesia spp./Theileria spp., 400 bp for ITS Cytauxzoon spp., and 600 bp for Hepatozoon spp.) were purified using Silica Bead DNA Gel Extraction Kit (Fermentas, São Paulo, SP, Brazil). Purified amplified DNA fragments from positive samples were submitted to sequence confirmation in an automatic sequencer (ABI Prism 310 Genetic Analyser–Applied Byosystem/Perkin Elmer). Consensus sequences were obtained through the analysis of the sense and antisense sequences using the CAP3 program (http://mobyle.pasteur. fr/cgi-bin/MobylePortal/portal.py). Comparisons with sequences deposited in GenBank were done using the basic local alignment search tool (BLAST) (Altschul et al., 1990). The sequences were aligned with sequences published in GenBank using Clustal/W (Thompson et al., 1994) in Bioedit v. 7.0.5.3 (Hall, 1999). Phylogenetic inference was based on Bayesian (BI) and maximum likelihood (ML) inference. The Bayesian inference (BI) analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) on the CIPRES Science Gateway (Miller et al., 2010) using the best models elected by the program MEGA-4 version 4.0 (Kumar et al., 2004) under the Corrected Akaike Information Criterion (AICc). Markov chain Monte Carlo (MCMC) simulations were run for 109 generations with a sampling frequency of every 100 generations and a burn-in of 25%. The Maximum-likelihood (ML) phylogenies were inferred with RAxML-HPC BlackBox 7.6.3 (Stamatakis et al., 2008) (which includes an estimation of bootstrap node support) through the CIPRES Science Gateway (Miller et al., 2010), using a GTRGAMMA model of evolution and 1000 bootstrapping replicates. The trees were examined in Treegraph 2.0.56-381 beta (Stover and Muller, 2010). Logistic regression models were employed to assess the effect of the putative predictor variables (i.e., gender, area of activity and the interaction between them) on the logit of the probability relative to the positive PCR assays for Ehrlichia sp. (16S rRNA), Ehrlichia sp. (dsb gene), Babesia sp. (18S rRNA), Babesia sp., (ITS-1 region), Theileria sp. (18S rRNA), Cytauxzoon sp. (ITS region) and Hepatozoon sp. (18S rRNA). All analyses were carried out using R 3.0.2 software (R Core Team, 2013).

3. Results All DNA samples amplified the predicted product for feline 28S rDNA, which indicates a successful DNA extraction. Out of 151 sampled cats, 13 (8.5%) were positive for Ehrlichia spp. 16SrRNA (eight [5.29%] females (three [1.98%] domesticated and five [3.31%] stray) and four (2.64%) males (three [1.98%] domesticated and one [0.66%] stray) and one cat (0.66%) without gender and area of activity records. The analysis on 13 sequenced products based on the 16S rRNA region (GenBank accession numbers KP65973, KP659738, KP659739, KP659740, KP659741, KP659742, KP659743, KP659744, KP659745, KP659746, KP659747, KP659748, and KP659749) showed 100% sequence identity with E. canis (GenBank accession number KJ995844). The fragments of Ehrlichia spp. 16S rRNA gene found in sampled cats were in the same clade as other

Please cite this article in press as: André, M.R., et al., Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.07.004

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Table 1 Oligonucleotides sequences used in PCR assays for tick-borne agents in domestic cats from Campo Grande, state of Mato Grosso do Sul, Brazil. Primers and agent Internal control PCR 28S rDNAFw 28S rDNARv Ehrlichia canis dsb-330 dsb-728 Ehrlichia spp. ECC ECB Nested ECAN-5 HE3 Ehrlichia spp. conP28-F1 conP28-R1 Nested conP28-F2 conP28-R2 Anaplasma spp. gE3a gE10R Nested gE2 gE9f Ehrlichia spp. and Anaplasma spp. HS1a EHR-CS778R Nested HS43 HSVR Piroplasmids ITS-15C ITS-13B Nested ITS-15D ITS-13C Babesia/Theileria spp. BTF1 BTR1 Nested BTF2 BTR2 Cytauxzoon felis CY-R CY-F Hepatozoon spp. HepF300 HepR900

Gene

Oligonucleotide sequence

Ref.

28S rRNA

5 -AGCAGGAGGTGTTGGAAGAG-3 5 -AGG GAGAGCCTAAATCAAAGG-3

Helps et al. (2003)

dsb

5 -GATGATGTCTGAAGATATGAAACAAAT-3 5 -CTGCTCGTCTATTTTACTTCTTAAAGT-3

Doyle et al. (2005)

5 -GAACGAACGCTGGCGGCAAGC-3 5 -CGTATTACCGCGGCTGCTGGCA-3 16S rRNA

Murphy et al. (1998) 5 -CAA TTATTTATAGCCTCTGGCTATAGGA-3 5 -TATAGGTACCGTCATTATCTTCCCTAT-3 5 -AT(C/T)AGT(G/C)AAA(A/G)TA(T/C)(A/G)T(G/A)CCAA-3 5 -TTA(G/A)AA(A/G)G(C/T)AAA(C/T)CT(T/G)CCTCC-3

omp-1

Inayoshi et al. (2004) 5 CAATGG(A/G)(T/A)GG(T/C)CC(A/C)AGA(AG)TAG-3 5 -TTCC(T/C)TG(A/G)TA(A/G)G(A/C)AA(T/G)TTTAGG-3 5 -CACATGCAAGTCGAACGGATTATTC-3 5 -TTCCGTTAAGAAGGATCTAATCTCC´ı-3

16S rRNA

Massung et al. (1998) 5 -GGCAGTATTAAAAGCAGCTCCAGG-3 5 -AACGGATTATTCTTTATAGCTTGCT-3 5 -AITGGGCTGGTAITGAAAT-3 5 -CCICCIGGIACIAIACCTTC-3

Sumner et al. (1997) Nicholson et al. (1999)

5 -AT(A/T)GC(A/T)AA(G/A)GAAGCATAGTC-3 5 -CTCAACAGCAGCTCTAGTAGC-3

Lotric-Furlan et al. (1998)

groESL

5 -CGATCGAGTGATCCGGTGAATTA-3 5 -GCTGCGTCCTTCATCGTTGTG-3 Shock et al. (2014)

ITS-1 5 -AAGGAAGGAGAAGTCGTAACAAGG-3 5 -TTGTGTGAGCCAAGACATCCA-3 5 -GGCTCATTACAACAGTTATAG-3 5 -CCCAAAGACTTTGATTTCTCTC-3 18S rRNA

Jefferies et al. (2007) 5 -CCGTGCTAATTGTAGGGC TAATAC-3 5 -GGACTACGACGGTATCTGATCG-3

18S rRNA

5 -GCGAATCGCATTGCTTTATGCT-3 5 -CCAAATGATACTCCGGAAAGAG-3

Birkenheuer et al. (2006)

18S rRNA

5 -GTTTCTGACCTATCAGCTTTCGACG-3 5 -CAAATCTAAGAATTTCACCTCTGAC-3

Ujvari et al. (2004)

isolates of E. canis, supported by bootstrap values in Maximum Likehood and Posterior Probability (PP) in the Bayesian analysis, using A. phagocytophilum, Rickettsia spp. and Neoehrlichia mikurensis as outgroups (Fig. 1). Only one cat (0.66%) (without gender and area of activity records) was positive for Ehrlichia spp. (dsb gene). Unfortunately, it was not possible to deposit the dsb sequence in GenBank database, since the trimmed sequence size was lower than 200 bp. The analysis on one sequenced product based on the dsb gene showed 100% sequence identity with E. canis (GenBank accession number GU586135). The fragment of Ehrlichia spp. dsb gene found in one sampled cat was in the same clade as other isolates of E. canis, supported by bootstrap values in Maximum Likelihood and Posterior Probability (PP) in the Bayesian analysis, using Ehrlichia ruminantium as outgroup (Fig. 2). All 13 cats positive in 16S rRNA Ehrlichia spp. PCR showed negative results in omp-1 and groESL nested PCR assays. None of sampled cats were positive for Anaplasma spp. in PCR.

Out of 151 sampled cats, 18 (11.9%) were positive in Babesia/Theileria spp. 18S rRNA nested PCR. After sequencing, nine (5.96%) were positive for Babesia spp: three (1.98%) females (one [0.66%] domesticated and two [1.32%] stray) and six (3.97%) males (four [2.64%] domesticated and two [1.32%] stray). The analysis on nine sequenced products based on the 18S rRNA region showed 99% sequence identity with B. vogeli (GenBank accession number JX112785) in eight cats (GenBank accession numbers KP410274, KP410275, KP410276, KP410278, KP410279, KP410280, KP410281, and KP410282), and 100% sequence identity with Babesia bigemina (GenBank accession number JX495402) in one cat (GenBank accession number: KP410277). Nine (5.96%) were positive for Theileria spp.: three (1.98%) males (one [0.66%] stray and two [1.32%] domesticated) and six (3.97%) females (five [3.31%] domesticated and one [0.66%] stray). The analysis on nine sequenced products based on the 18S rRNA region showed 99% sequence identity with Theileria sp. recently detected in a domestic cat in São Paulo

Please cite this article in press as: André, M.R., et al., Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.07.004

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Fig. 1. Phylogenetic trees of Ehrlichia spp. (16SrRNA) based on Bayesian inference (A) and maximum likelihood (B). Numbers correspond to BI posterior probabilities and ML bootstrap values over 50, respectively.

Fig. 2. Phylogenetic trees of Ehrlichia spp. (dsb gene) based on Bayesian inference (A) and maximum likelihood (B). Numbers correspond to BI posterior probabilities and ML bootstrap values over 50, respectively.

state, Brazil, (GenBank accession number KF970930) in seven cats (GenBank accession numbers KP410267, KP410268, KP410269, KP410270, KP410271, KP410272, KP410273), and 99% sequence identity with Theileria cervi (GenBank accession number AY735134) in two cats (GenBank accession numbers: KP402163, KP402164). The phylogenetic analyses using both Maximum Likelihood and Bayesian methods grouped one sequence with B. bigemina and eight sequences with B. vogeli, using Adelina sp., Eimeria sp., Isospora sp., and Sarcocystis sp. as outgroup (Fig. 3). Although BLAST analysis showed a high percent of similarity (99%) and closely positioning of some sequences with T. equi, some sequences amplified from cats’ blood samples were separated from this equid piroplasmid by bootstraps in Maximum Likelihood and Posterior Probability in Bayesian analyses dendograms. Furthermore, although BLAST analysis showed a high percent of similarity (99%) of some sequences with T. cervi, Theileria 18S rRNA sequences from sampled cats

grouped with Theileria ovis and Theileria lestoquardi but separated from them by high bootstrap values (99–100), using Adelina sp., Eimeria sp., Isospora sp., and Sarcocystis sp. as outgroup (Fig. 3). Attempts to amplify another gene (ˇ-tubulin) from these piroplasmid species did not generate amplicons for further phylogenetic reconstruction (data not-shown). Two cats (1.3%) were positive to ITS-1 piroplasmid nested PCR (one stray female and one domesticated male), whose amplicons after sequencing showed 99% sequence identity (GenBank accession number: KP683155) with B. vogeli (GenBank accession number JX861394) and 99% sequence identity (GenBank accession number: KP683154) with Cytauxzoon sp. (FJ876452), respectively. The fragment of Babesia sp. ITS-1 region found in one cat was in the same clade as other isolates of B. vogeli and separated from B. canis by bootstrap values in Maximum Likelihood and Posterior Probability in Bayesian analyses, using Cyclospora cayetanensis as outgroup (Fig. 4). The fragment of

Please cite this article in press as: André, M.R., et al., Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.07.004

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Fig. 3. Phylogenetic trees of Babesia spp. and Theileria spp. (18SrRNA) based on Bayesian inference (A) and maximum likelihood (B). Numbers correspond to BI posterior probabilities and ML bootstrap values over 50, respectively.

Fig. 4. Phylogenetic trees of Babesia spp. (ITS) based on Bayesian inference (A) and maximum likelihood (B). Numbers correspond to BI posterior probabilities and ML bootstrap values over 50, respectively.

Please cite this article in press as: André, M.R., et al., Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.07.004

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Fig. 5. Phylogenetic trees of Cytauxzoon spp. (ITS) based on Bayesian inference (A) and maximum likelihood (B). Numbers correspond to BI posterior probabilities and ML bootstrap values over 50, respectively.

Cytauxzoon sp. ITS region found in one cat was in the same clade as other isolates of C. felis in Maximum Likelihood and Posterior Probability in Bayesian analyses, using C. cayetanensis, Sarcocystis anasi and Eimeria anseris as outgroups (Fig. 5). One female domesticated cat (0.66%) was positive for Hepatozoon spp. The analysis on sequenced product based (GenBank accession number: KP410283) on 18S rRNA showed 99% sequence identity with H. felis (GenBank accession number AB771560). The fragment of Hepatozoon spp. 18S rRNA gene found in one cat was grouped with H. americanum (JX415176) and Hepatozoon sp. detected in a little spotted cat in Brazil (FJ876444) in Maximum Likelihood and Posterior Probability in Bayesian analyses, using Adelina sp., Eimeria sp., Isospora sp., and Sarcocystis sp. as outgroup (Fig. 6). Logistic regression results revealed no significant interaction between gender and area of activity on the probability of positive PCR assays for Ehrlichia sp. (16S rRNA), Ehrlichia sp. (dsb gene), Babesia sp. (18S rRNA), Babesia sp. (ITS-1 region), Theileria sp. (18S rRNA), Cytauxzoon sp. (ITS region) and Hepatozoon sp. (18S rRNA). 4. Discussion The present study showed that Ehrlichia spp., piroplasmids (B. vogeli, Theileria spp. and Cytauxzoon spp.) and, more rarely, Hepatozoon spp. circulate among stray and domesticated cats in midwestern Brazil. Ehrlichia spp. DNA closely related to E. canis was found in blood samples of 13 (8.5%) cats in the present study. In the last years, Ehrlichia spp. has been detected in domestic and wild felines in Brazil, by molecular and serological techniques. In the state of Minas Gerais, southeastern Brazil, 16S rRNA-Ehrlichia spp. DNA, showing identity with E. canis by BLAST analysis, was detected in three out of 15 cats attended at Teaching Veterinary Hospital in the city of Vic¸osa (Oliveira et al., 2009). Braga et al. (2012) detected the presence of 16S rRNA-Ehrlichia spp. DNA and antibodies to Ehrlichia spp. in 1% and 5.5%, respectively, out of 200 stray and domesticated cats sampled in northeastern region of Brazil (Braga et al., 2012). Recently, Ehrlichia sp. dsb DNA showing identity to E. canis and antibodies to Ehrlichia spp. have been detected in 9.4% and 41.5%, respectively, out of 212 cats sampled in the state of Mato Grosso, midwestern Brazil (Braga et al., 2014). New genotypes of Ehrlichia spp., detected through PCR assays based on 16S rRNA and omp-1 genes and followed by phylogenetic reconstructions, have been reported in wild felids in captivity in zoos in Brazil (André et al., 2010a, 2012). On the other hand, Ehrlichia spp. has been infrequently detected in cats in others regions of the world, such as in the USA (Breitschwerdt et al., 2002), Taiwan (Yin-Chiachun et al., 2003) and Spain (Tabar et al., 2008). Feline ehrlichiosis has been associated with fever, apathy, anorexia, weight loss, palor, lymphadenopathy, splenomegaly, normocytic normochromic

anemia, thrombocytopenia, leukopenia, increased serum activity of transaminases and alkaline phosphatase (Almosny et al., 1998; Almosny and Massard, 1999; Stubbs et al., 2000). Vectors involved in the transmission cycles of Ehrlichia spp. in Brazil are still unknown. Although Anaplasma spp. was not detected in the present study, Anaplasma sp. closely related to A. phagocytophilum has been recently detected among stray cats in a zoo in southeastern Brazil (André et al., 2014). Besides, Anaplasma sp. closely related to A. platys was detected in a cat showing inclusions in platelets in the state of Pernambuco, northeastern Brazil (Lima et al., 2010). In the state of Rio de Janeiro, 12 out of 91 cats were positive in a specific nested PCR assay for A. platys; four of these positive cats showed inclusions in platelets (Correa et al., 2011). Although negative results in PCR assays targeting omp-1 and groESL genes precluded additional phylogenetic inferences about the real identity of Ehrlichia sp. detected in sampled cats in the present study, our findings strongly suggest that E. canis, or at least, a very closely related agent, circulate in cats in Brazil based on 16S rRNA and dsb phylogenetic inferences. Studies aiming to isolate and investigate the pathogenic potential of this agent are much needed. Regarding the molecular detection of piroplasmids, sequencing showed the presence of Babesia spp. DNA closely related to B. bigemina and B. vogeli in blood samples from one and eight cats, respectively. The positioning of Babesia sp. found in sampled cats closely to B. vogeli was confirmed through two different molecular markers (18S rRNA and ITS) using two different phylogenetic methods (Maximum Likelihood and Bayesian analysis). Recently, B. vogeli was detected in blood samples from stray domestic cats in a Brazilian zoo (André et al., 2014). Also, B. vogeli DNA has already been detected in cat’s blood samples from Trinidad (Georges et al., 2008), Thailand (Simking et al., 2010) and Portugal (Vilhena et al., 2013). André et al. (2011) reported the detection of Babesia spp. DNA closely related to B. leo in a pampas cat, a neotropical wild felid species, maintained in captivity in a Brazilian zoo in state of São Paulo. Besides, Theileria spp. DNA was detected in nine cats in the present study. Interestingly, four sequences obtained in the presenty study were positioned together with Theileria sp. recently detected in a stray cat sampled in a zoo in southeastern Brazil (André et al., 2014). Babesia vulpes sp. Nov. (Baneth et al., 2015) (formerly Theileria annae) was detected in a cat sampled at southern Europe (Criado-Fornelio et al., 2003). The positioning of one Babesia sequence closely to B. bigemina and five Theileria sequences to Theileria spp. from ruminants (T. ovis, T. lestoquardi) in the present study highlights the occurrence of novel genotypes of piroplasmids circulating in cats phylogenetically related to piroplasmids found in wild and domestic ruminants. Feline piroplasmosis is associated with anorexia, weight loss, gastrointestinal alterations, anemia, thrombocytopenia, leukocytosis or leukopenia, and jaundice (Criado-Fornelio, 2012a; Vilhena et al., 2013). Our findings also

Please cite this article in press as: André, M.R., et al., Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.07.004

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Fig. 6. Phylogenetic trees of Hepatozoon spp. (18SrRNA) based on Bayesian inference (A) and maximum likelihood (B). Numbers correspond to BI posterior probabilities and ML bootstrap values over 50, respectively.

emphasize the occurrence of B. vogeli, a canid piroplasmid, in cats in Brazil, corroborating with previous studies conducted in Brazil (André et al., 2014) others regions of the world (Georges et al., 2008; Simking et al., 2010; Vilhena et al., 2013). Additionally, the present work highlights the diversity of piroplasmids circulating in cats in Brazil and the need for molecular confirmation of piroplasmas found in erythrocytes of cats’ blood smears. Herein, one cat was positive for Cytauxzoon sp. based on ITS PCR. The pathogenicity of Cytauxzoon spp. genotypes that circulate in domestic cats and wild felids in Brazil is still unknown. Indeed, little is known about the epidemiology of cytauxzoonosis in Brazil. Even though a fatal case of cytauxzoonosis have been reported in a captive reared lionesses (Panthera leo) in a zoo in the state of Rio de Janeiro (Peixoto et al., 2007), André et al. (2009) detected the parasite using molecular techniques in asymptomatic wild felids in captivity in zoos from Brasilia and cities located in the state of São Paulo. Recently, Cytauxzoon sp. DNA was detected in a cat co-infected with ‘Candidatus Mycoplasma haemominutum’ in the state of Rio de Janeiro (Maia et al., 2013). Our findings corroborate with previous studies carried on in Brazil, which strongly suggest that C. felis, or at least, an agent closely related to C. felis, circulate more often on wild felids compared to cats in our country. However, it seems that the Cytauxzoon spp. isolates in Brazil are different enough from those found in the USA, since fatal cases of cytauxzoonosis in domestic cats Brazil have not been reported. While Dermacentor variabilis (Blouin et al., 1984) and Amblyomma americanum (Reichard et al., 2010) are the tick species incriminated as vectors of C. felis in the USA, vectors involved in transmission cycles of Cytauxzoon spp. in Brazil are still unknown. Hepatozoon spp. was detected in one female domesticated cat in the present study using 18S rRNA PCR. Previously in Brazil, Hepatozoon spp. closely related to H. canis (Rubini et al., 2006) and H. felis (De Bortoli et al., 2011) have been detected in cats in the states of São Paulo and Maranhão, respectively. Herein, the detected Hepatozoon spp. sequence grouped with H. americanum and Hepatozoon sp. recently found in a wild felid in Brazil (André et al., 2010a,b) in both ML and Bayesian analysis, which highlights the possibility of occurrence of a diversity of genotypes of this parasite among carnivores in South America, corroborating with previous findings reported by Criado-Fornelio et al. (2006) and André et al. (2010a,b). Indeed, phylogenetic reconstructions using other genes are much needed for the assessment of the diversity of this group of parasites among domestic and wild animals. Although hepatozoonosis in cats

is characterized by subclinical infection, pathogenic effects may be exacerbated in stressed, immunocompromised animals, concomintant and retroviral infections (Baneth et al., 1998; Criado-Fornelio et al., 2003). 5. Conclusions The present study showed that Ehrlichia spp., piroplasmids (B. vogeli, Theileria spp. and Cytauxzoon spp.) and, more rarely, Hepatozoon spp. circulate among stray and domesticated cats in Brazil. The pathogenic effects of these isolates in cats are still unknown. Infections caused by tick-borne agents (piroplasmids, Anaplasma spp., Ehrlichia spp. and Hepatozoon spp.) among other vector-borne pathogens (hemoplasmas and Bartonella spp.) should be included in the differential diagnosis of cats showing unspecific clinical signs aiming to establish proper therapy and control. Acknowledgments The authors would like to thank Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for providing financial support (2013/09889-0) and Centro de Controle de Zoonoses de Campo Grande (CCZ) for technical assistance. References Almosny, N.R.P., Massard, C.L., 1999. Erliquiose felina – review. Clín. Vet. 4 (23), 30–32. Almosny, N.R.P., Almeida, L.E., Moreira, N.M., Massard, C.L., 1998. Erliquiose clinica em gato (Felis catus). Rev. Bras. Cienc. Vet. 5 (2), 82–83. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. André, M.R., Denardi, N.C.B., Sousa, K.C.M., Gonc¸alves, L.R., Henrique, P.C., Ontivero, C.R.G.R., Gonzalez, I.H.L., Nery, C.V.C., Chagas, C.R.F., Monticelli, C., Santis, A.C.A., Machado, R.Z., 2014. Arthropod-borne pathogens circulating in free-roaming domestic cats in a zoo environment in Brazil. Ticks Tick Borne Dis. 5, 545–551. André, M.R., Dumler, J.S., Scorpio, D.G., Teixeira, R.H.F., Allegretti, S.M., Machado, R.Z., 2012. Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores. Ticks Tick Borne Dis. 3 (4), 247–253. André, M.R., Adania, C.H., Teixeira, R.H.F., Allegretti, S.M., Machado, R.Z., 2011. Molecular and serological detection of Babesia spp. in neotropical and exotic carnivores in Brazilian zoos. J. Zoo Wildl. Med. 42 (1), 139–143. André, M.R., Adania, C.H., Machado, R.Z., Allegretti, S.M., Felippe, P.A.N., Silva, K.F., Nakaghi, A.C.H., 2010a. Molecular and serologic detection of Ehrlichia spp. in endangered Brazilian wild captive felids. J. Wildl. Dis. 46 (3), 1017–1023. André, M.R., Adania, C.H., Teixeira, R.H.F., Vargas, G.H., Falcade, M., Sousa, L., Salles, A.R., Allegretti, S.M., Felippe, P.A.N., Machado, R.Z., 2010b. Molecular detection

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Please cite this article in press as: André, M.R., et al., Tick-borne agents in domesticated and stray cats from the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks Tick-borne Dis. (2015), http://dx.doi.org/10.1016/j.ttbdis.2015.07.004