ARTICLE Investigation of Ehrlichia spp., Anaplasma spp. and ...

ARTICLE Investigation of Ehrlichia spp., Anaplasma spp. and Rickettsia spp. in ectoparasites collected from domestic animals, Rio de Janeiro State, Brazil Thayssa Keren S. da Silva1, Carolina M. Blanco1, Maria Ogrzewalska1, Mariana Barbosa de Souza1, Jairo Dias Barreira2, Namir Santos Moreira3, Maria A.gélica Monteiro de Mello Mares-Guia1, Elba Regina S. de Lemos1 Laboratório de Hantaviroses e Rickettsioses, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil 2 Departamento de Microbiologia e Parasitologia do Instituto Biomédico da Universidade do Rio de Janeiro (UNIRIO), Rio de Janeiro, RJ, Brazil 3 Centro Universitário Anhanguera de Niterói, UNIAN, Niterói, RJ, Brazil 1

The aim of this study was to determine the occurrence of emerging arthropod-borne pathogens Anaplasma, Ehrlichia and Rickettsia infection in ticks (Acari: Ixodidae) and fleas (Insecta: Siphonaptera) collected from dogs and horses within municipality of Itaboraí, Rio de Janeiro State, Southern Brazil. Samples from 280 ticks and two fleas were subjected to family or/and genus specific PCR for Anaplasmataceae, Ehrlichia and Rickettsia, followed by DNA sequencing to ensure pathogen identity. In ticks Rhipicephalus sanguineus collected from dogs the DNAof Anaplasma platys and Ehrlichia canis was detected in 6.8% and 2.2% samples respectively. In two R. sanguineus confection with two pathogens was observed. In Dermacentor nitens ticks, collected from horses Francisella-like endosymbiont was found in 42.8% samples. DNA of Rickettsia felis and Wolbachia pipetens was detected in fleas Ctenocephalides canis fleas. No DNA of Rickettsia was found in tested ticks. The findings contribute to our knowledge of tick-borne bacteria, ticks and endosymbionts distribution in Brazil.

Key words: Rhipicephalus, Dermacentor, dogs, Wolbachia, Ctenocephalides, Rickettsia

INTRODUCTION Ticks (Ixodida) and fleas (Siphonaptera) are haematophagous ectoparasites of vertebrates and are responsible for the transmission of a great variety of pathogens to humans, and domestic animals (Sonenshine and Roe 2014). Among tick, the Ixodidae, the most important family for human health, comprehend over 700 species among at least 900 tick species described in many regions of the world (Sonenshine & Roe 2014). The Brazilian tick fauna consists of 70 species (Martins et al. 2014, Labruna et al. 2016) and some of them are recognized as vectors of pathoge-

*Corresponding author: Maria Ogrzewalska

E-mail: [email protected]

nic bacteria with medical and veterinary relevance (Labruna and Machado 2006, Szabó et al. 2013, Vieira et al. 2011). In Brazil the most predominant tick-borne agents belong to the Order Rickettsiales (Anaplasma, Ehrlichia, and Rickettsia), and are detected in both ticks and vertebrate species (Labruna and Machado 2006, Szabó et al. 2013, Vieira et al. 2011). Within Anaplasma genus, Anaplasma phagocytophilum is known to be pathogenic to humans, causing human granulocytotropic anaplasmosis in United States and Europe (Dumler et al. 2005, Dahlgren et al 2011). In Brazil, though A. phagocytophilum was found only infecting dogs and ticks (Santos et al. 2013), but other species such as Anaplasma bovis, Anaplasma marginale and Anaplasma platys have been found infecting domestic animals across the country (Santos and Carvalho 2006, Pohl et al. 2013, Souza et al. 2013). Among Ehrlichia, various strains, geno-

Virus Reviews & Research Vol 22, 2017 types and possibly new species have been recently found in ticks and wild and domestic vertebrates in Brazil (André et al. 2010, Widmer et al. 2011, Braga et al. 2012, Cabezas-Cruz et al. 2012, Almeida et al. 2013, Aguiar et al. 2014). The best known is Ehrlichia canis agent causing canine monocytic ehrlichiosis and till now, no humans cases of ehrlichiosis are known in Brazil (Vieira et al. 2011). Rickettsia rickettsii is undoubtedly the most important bacterium transmitted by ticks to humans causing Brazilian spotted fever (BSF) in Brazil. Though, during the last 15 years the number of other Rickettsia with confirmed and unknown pathogenicity has been detected in various tick species (Labruna, 2009, Labruna et al. 2011, Silva et al. 2011, Blanco et al. 2016). During the recent Q fever surveillance study conducted in the municipality of Itaboraí, State of Rio de Janeiro, ectoparasites were collected from domestic animals (Mares-Guia, 2015). Taking into the consideration that no research about arthropod-borne diseases in the investigated region exists, the aim of this study was to screen the collected ectoparasites for the presence of bacteria from genus Ehrlichia, Anaplasma, and Rickettsia.

MATERIALS AND METHODS Ectoparasites were collected and identified as part of a previous study conducted in 2015 in the municipality of Itaboraí (Mares-Guia, 2015). In total 280 ticks were individually tested by a battery of PCR for pathogens: Rhipicephalus sanguineus (33 nymphs, 134 females, 98 males) collected from domestic dogs, Dermacentor nitens (seven females) collected from equines and Amblyomma sculptum collected from a dog (one male) and from one equine (five females, two males). A.ditionally to ticks, two fleas Ctenocephalides canis (Siphonaptera: Pulicidae) collected from a dog were also tested for the same pathogens. A.l ectoparasites were screen individually for bacteria from the family Anaplasmataceae targeting the fragment of ~350 bp of the gene 16S rRNA.using primers EHR16SD 5’- GGTA.CYA.A.A. GA.GTCC-3’ and EHER 16SR 5’-TGCA.TCA.CGTTTA.A.-3’ as described by Inokuma et al. (2000). In the case of positive reaction, the samples were submitted to the second reaction targeting a specific portion of the genus Ehrlichia, disulfide bond formation protein-encoding gene (dsb), (Doyle et al. 2005). In this case primers DSB-330 5’-GA.GA.GTTTGA.GA.A.SA.A.A.A.-3’ and DSB-720 5’-CTA.TTTA.TTCTTA.A.TTGA.A.A.C-3’ targeting the fragment of the size of ~409 bp were used in the first reaction and primers DSB380 5’-A.TTTTA.RGA.TTTCCA.TA.TTGG-3’ and DSB-720 targeting the fragment of 349 bp in the second reaction (semi nested PCR reaction) as described by Almeida et al. (2013). For Rickettsia detection, the DNAsamples were tested by PCR using primers CS-78 5’- GCA.GTA.CGGTGA.GA. GTA.T-3’, and CS-323 5’- GCTTCCTTA.A.TTCA.TA.A.CA. GA.-3’ targeting 401-bp fragment of the citrate synthase gene (gltA. of apparently all Rickettsia species (Labruna et al. 2004).

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Negative controls (blanks tubes containing water) and appropriate positive controls samples (Ehrlichia chaffensis, Rickettsia rickettsii and A. bovis) were run together with ticks samples in each PCR essay. A.plicons were visualized on 1,5% agarose gels stained with Gel Red Nucleic A.id Gel StainTM 10000× in DMSO (Biotium, Hayward, CA. USA.. PCR products of the expected size were purified with ExoSA.-IT® (Alfymetrix, Cleveland, OH, USA., and sequenced in both directions in a 96-capillary 3730xl DNAA. alyzer® (A.plied Biosystems, Foster City, CA. USA. according to protocols developed by Otto et al. (2008), using the same primers (forward and reverse) used for the PCR. Partial sequences obtained were analyzed with MEGA.6.0 (Tamura et al. 2013) and submitted to Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

RESULTS A.ter analysis of the partial sequences obtained, DNAof A. platys and E. canis was observed in 6.8% (18/265) and 2.2% (6/265) samples of R. sanguineus, respectively (Table). Coinfection with A. platys and E. canis was detected in two samples. The sequences fragments of 16S rDNAgene of A. platys were identical with each other and 100% (289/289bp) with various sequences of A. platys available in GenBank (ex. KU500914, KP903296, KP903292). Ehrlichia canis 16S rDNAgene fragments were 100% (289/289bp) identical with several sequences of E. canis available in GenBank (KR920044, KP717552, KP182942, EF195134) and with Ehrlichia regneryi (KF843826), Ehrlichia ovina (A.318946) and various Ehrlichia spp. (ex. KP642752, KP717552, KP745632) as well. The dsb gene sequences were likewise identical with each other and 99.6% (314/315bp) identical with E. canis found in the blood donor in Costa Rica (KR732921), and with E. canis found in R. sanguineus from Brazil (KP167596), Cameroon (DQ902687), dog blood from Brazil (GU586135) and complete genome of E. canis (CP000107). In 42.8% (3/7) of D. nitens ticks we unexpectedly obtained sequences 99.6% (288/289 bp) identical with Francisella-like endosymbiont (Thiotrichales: Francisellacae) detected previously in D. nitens (EU332822) from Brazil and Dermacentor albipictus (GU968872) from Canada and 99.3% identical with endosymbiont found in Dermacentor variabilis (A.375404) and D. nitens (A.375400) from the United States and Rhipicephalus microplus (EU332820) from Brazil. DNAof Wolbachia pipientis (Rickettsiales: Rickettsiaceae) was detected in the two C. canis collected on a dog and the partial sequences showed 99.6-100% similarity with some sequences of W. pipientis available in GenBank (LN864488, LN864488, A.632146). A.l the tick tested samples were negative for the presence of bacteria from the genus Rickettsia. However, in one C. canis DNAof Rickettsia felis was detected. The partial sequence was 100% (324/324 bp) similar with the corresponding sequence

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Investigation of Ehrlichia spp., Anaplasma spp. and Rickettsia spp. in ectoparasites collected from domestic animals, Rio de Janeiro State, Brazil. • Silva et al.

of the complete genome of R. felis (CP000053). The GenBank nucleotide sequence accession numbers for the partial sequences generated in the present study are:

KY436064 (16S rRNA. A. platys), KY436062 (16S rRNA. E. canis), KY436063 (dsb, E. canis), KY436065 (16S rRNA. Francisella-like endosymbiont), KY436066 (16S rRNA. W. pipientis).

Table. Number of tested and infected ectoparasites collected from domestic animal for the presence of by bacteria Anaplasma, Ehrlichia, Rickettsia and Francisella sp. Itaboraí, RJ, Brazil. Number of infected / Ectoparasites

Number of tested Vertebrate hosts

Order

Parasitiformis

Family

Species

Ixodidae

Rhipicephalus sanguineus

Nymphs

dog

Siphonaptera

Pulicidae

Bacteria detected

ectoparasites A.ults

1/33

17/232

Anaplasma platys

0/33

6/232

Ehrlichia canis

Dermacentor nitens

equine

-

3/7

Francisella sp. endosymbiont

A.blyomma sculptum

dog

-

0/1

-

equine

-

0/7

-

Ctenocephalides canis

dog

-

2/2

Wolbachia pipetens

-

1/2

Rickettsia felis

DISCUSSION In the present study we found 6.8% and 2.2% of R. sanguineus infected with A. platys and E. canis respectively. Tick R. sanguineus was introduced to Brazil with colonization and now is abundant throughout urban and rural areas across the whole country, and is always associated with dogs (Szabó et al. 2013). However, in fact recent analyses revealed that at least two distinct species have been considered under the taxon R. sanguineus in Latin A.erica (Moraes-Filho et al. 2011, Nava et al. 2014). It is also known to be the main vector of E. canis to dogs causing canine monocytic ehrlichiosis (Vieira et al. 2011) and is suspected to be a vector of A. platys agent of canine cyclic thrombocytopenia (Inokuma et al. 2000, Ramos et al. 2014). Molecular detection of A. platys and E. canis in dogs has been conducted in Brazil for many years and has revealed widely variable prevalence, from 8 to 88% and from 0 to 55%, respectively (Dagnone et al. 2003, Bulla et al. 2004, Macieira et al. 2005, Santos et al. 2009, Lasta et al. 2013, Rotondano et al. 2015). On the other hand, few studies concern infection of natural population of R. sanguineus. In general, our results show the high concordance of previous findings revealing usually low prevalence in ticks from 2.3-6.2% in the case of E. canis (Aguiar et al. 2007) and 0-9,4% in the case of A. platys (Inokuma et al. 2000, Sano-

go et al. 2003), however Ramos et al. (2014) found A. platys in 48.3% Rhipicephalus sp. tick. These differences might be related with a different population of R. sanguineus vector competence as showed experimentally by Moraes-Filho et al. (2015). Limited studies indicate that E. canis and A. platys may cause disease in humans. Ehrlichia canis was detected in patients with clinical disease in in Venezuela (Perez et al. 2006) and A. platys in a veterinarian who probably got infected in Grenada (Maggi et al. 2013). A.though both authors suggest R. sanguineus as a potential vector to humans, in Brazil human R. sanguineus tick-biting is a very rare event if one considers the frequent and closed relationship of R. sanguineus and people (Szabó et al. 2013). This fact might be a result of the existence of distinct populations of R. sanguineus across South A.erica as mentioned before (Moraes-Filho et al. 2011, 2015, Nava et al. 2014). Summing up, there are not clear evidences about the role of R. sanguineus as a vector of any rickettsial pathogen transmitted to humans in Brazil (Szabó et al. 2013). In D. nitens ticks we detected bacteria closely related to Francisella-like endosymbiont belonging to the same family as Francisella tularensis, the causative agent of tularemia (Scoles 2004, Sun et al. 2000). Francisella-like endosymbionts have been identified in ticks worldwide (Machado-Ferreira et al. 2009, Carvalho et al. 2011, Wójcik-Fatla et al. 2015) and probably provide

Virus Reviews & Research Vol 22, 2017 nutrients absent in blood nevertheless essential for normal ticks development (Scoles 2004, Gerhart et al. 2016). Recent analysis of sequenced genome of the Francisella-like endosymbionts found in A.blyomma species showed that it undergone minimal genome reduction, but the virulence genes contain inactivating mutations. It indicates that Francisella-like endosymbionts possibly evolved recently from a pathogenic strain of Francisella, indicating that tick endosymbionts can evolve from mammalian pathogens (Gerhart et al. 2016). Few occurrences of Francisella spp. have been described in the Southern Hemisphere (Venzal et al. 2008). To our knowledge this is the second molecular detection of Francisella-like endosymbionts in Brazilian ticks after Machado-Ferreira et al. (2009) who detected these endosymbionts in A.blyomma dubitatum, D. nitens, and Rhipicephalus microplus collected from domestic animals in the states of Minas Gerais and Mato Grosso. The amplification of the genus Francisella in the present study was not truly surprising result. A.though broad-spectrum primers (EHR16SD and EHER 16SR) for Ehrlichia-like bacteria were designed only for members of the family Anaplasmataceae (Inokuma et al. 2000), previous studies showed that their specificity is not absolute (Parola et al. 2003) and might occur while targeting conserved genes such as the 16S rDNA Likewise, Venzal et al. (2008) detected Francisella sp. in A.blyomma triste in Uruguay using the same par of primers. In dog fleas C. canis DNAof W. pipientis and R. felis was detected. Fleas C. canis are cosmopolitan species that are found in South A.erica on pet animals. Besides the dermatological implications, these ectoparasites can transmit several disease agents to animals and humans. In Brazil, C. canis are found frequently infected with R. felis (Horta et al. 2006) what is in concordance of or results. Rickettsia felis has been detected infecting Ctenocephalides spp. world widely and it is presently recognized as a causative agent of flea-borne spotted fever (Brown and Macaluso 2016), however, the global scarcity of R. felis clinical cases deserves further attention of its real pathogenicity to humans. Wolbachia pipientis group bacteria are obligate intracellular endosymbionts with an extremely broad host range mainly arthropods (Jeyaprakash and Hoy 2000). The bacterium can induce various alterations in hosts, such as reproductive, cytoplasmic incompatibility, parthenogenesis, feminization and male-killing (Stouthamer et al. 1999, Werren et al. 2008). It has received much recent attention due to its medical relevance as can provide host protection against some viruses and other pathogens (reviewed in Hedges et al. 2008) being for example tested as means to reduce transmission of arbovirus from mosquitoes to humans (Moreira et al. 2009, Dutra et al. 2016). Wolbachia sp. was detected in cat fleas Ctenocephalides felis (Jeyaprakash and Hoy 2000, Rolain et al. 2003, Oteo et al. 2014). To our knowledge this is the first record of W. pipientis in C. canis in Brazil. The role of Wolbachia

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in the biology of C. canis is not known and requires further investigations. No DNAof Rickettsia was found in sampled ticks. Rhipicephalus sanguineus is known as the vector for Rickettsia conorii, the cause of Mediterranean spotted fever in people in southern Europe and northern A.rica and may be involved in transmission of R. rickettsia in USA.resulting in outbreaks and fatal cases of Rocky Mountain spotted fever (Demma et al, 2005, Folkema et al. 2012). Although R. rickettsii have been found in R. sanguineus in Brazil, the involvement of this tick specie in the transmission of rickettsia in Brazilian territory is by now speculative but demands awareness for its potential (Cunha et al, 2009, Moraes-Filho et al, 2009, Pacheco et al 2011, Ogrzewalska et al 2012, Szabó et al. 2013). CONCLUSIONS We detected circulation of the following organisms: A. platys and E. canis in natural population of R. sanguineus ticks and DNAfrom bacteria genetically close with Francisella-like endosymbiont in D. nitens ticks, and R. felis and W. pipetens in C. canis fleas. These findings are a valuable input to better understand the occurrence and distribution of arthropod borne pathogens and endosymbionts in Brazil.

ACKNOWLEDGEMENTS We are grateful to the Genomic Platform – DNASequencing – RPT01A.(Rede de Plataformas Tecnológicas FIOCRUZ). We also thank Rubens Pinto de Mello and Catarina Macedo for their assistance in the arthropod capture expeditions.

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Funding Information The authors thank to FAPERJ (E-26/010.001567/2014) and CNPQ (407664/2012-2 APQ) for the financial support.