Cercopithifilaria bainae in Rhipicephalus sanguineus ...

Ticks and Tick-borne Diseases 8 (2017) 623–625

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Short communication

Cercopithifilaria bainae in Rhipicephalus sanguineus sensu lato ticks from dogs in Brazil

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Marcos Antônio Bezerra Santosa, Islanne Barbosa de Souzaa, Lucia Oliveira de Macedoa, Carlos Alberto do Nascimento Ramosb, Ana Gabriela de Oliveira Regoc, Leucio Câmara Alvesc, ⁎ Rafael Antonio Nascimento Ramosa, , Gílcia Aparecida de Carvalhoa a b c

Unidade Acadêmica de Garanhuns, Universidade Federal Rural de Pernambuco, Garanhuns, Brazil Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Campo Grande, Brazil Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords: Filarial worms Cercopithifilaria bainae Ectoparasites Ticks Vector

Rhipicephalus sanguineus sensu lato (s.l.) ticks act as intermediate host for a range of canine vector-borne pathogens, including nematodes ranked in the genus Cercopithifilaria. Though being the object of several studies in the last years, information on the distribution of these parasites is still lacking. In this study, the occurrence of Cercopithifilaria spp. was investigated in on-host population of R. sanguineus s.l. collected from naturally infested dogs. Ticks (n = 1906, including one larva, 294 nymphs and 1611 adults) were sampled on domestic dogs (n = 155) living in the municipality of Garanhuns (northeastern Brazil). Tick collections (n = 36) were performed every 8 days, from October 2015 to June 2016. Filarioid larvae detected at tick dissection were morphologically and morphometrically identified at species level. At the end of the study, only R. sanguineus s.l. ticks were collected, with the highest number in January 2016 (n = 254) and the lowest in June 2016 (n = 26). Out of 1906 dissected ticks, 2.68% (51/1906) harboured Cercopithifilaria bainae larvae, whose identification was molecularly confirmed, with a nucleotide identity of 99% with C. bainae. Data here reported indicate that, in the study area, R. sanguineus s.l. is the predominant tick infesting domestic dogs. Accordingly, these animals are at a high risk of C. bainae infection.

1. Introduction Ticks are ectoparasites of medical and veterinary concern due to their ability to transmit a range of pathogens to animals and human beings (Dantas-Torres et al., 2012; Ogden and Lindsay, 2016). In Brazil, Rhipicephalus sanguineus sensu lato (s.l.) is the most prevalent species parasitizing dogs (Labruna and Pereira, 2001; Dantas-Torres, 2010), to whom it can transmit several pathogenic microorganisms, including bacteria (Ehrlichia canis) or protozoa (Babesia vogeli and Hepatozoon canis) (Dantas-Torres, 2010; Hubálek and Rudolf, 2012). Nonetheless, the brown dog tick can also harbour and transmit nematodes, such Cercopithifilaria bainae (Ramos et al., 2014), a parasite species that received increasing scientific attention in the last years (Otranto et al., 2012). Cercopithifilaria genus comprises 28 species, three of which (i.e., Cercopithifilaria grassi, C. bainae and Cercopithifilaria sp. 2 sensu Otranto et al., 2013a,b) conclude their life cycles in dogs (Otranto et al., 2011, 2012, 2013a,b). Interestingly, it has been demonstrated that R. sanguineus s.l. act as vector for all these filarial species, which reach

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the infective stage (L3) in approximately 30 days (Brianti et al., 2012). C. bainae is the most prevalent species, and, although being usually apathogenic, it may induce dermatological alterations, histologically appearing as perivascular interstitial dermatitis (Otranto et al., 2012). In addition, a case of chronic polyarthritis has been documented in a dog infected by C. bainae (Gabrielli et al., 2014). The distribution of Cercopithifilaria spp. is poorly known and studies on its epidemiology are limited to a few reports (e.g., Latrofa et al., 2014). Nonetheless, species of Cercopithifilaria were accidentally reported in dogs (Almeida and Vicente, 1984; Ramos et al., 2016) and ticks (Latrofa et al., 2014) from Brazil. However, the seasonal dynamic and exact distribution of these nematodes in Rhipicephalus ticks has not been fully evaluated Hence, the occurrence of Cercopithifilaria spp. was investigated in on-host population of R. sanguineus s.l. ticks collected from dogs naturally infested.

Corresponding author at: Unidade Acadêmica de Garanhuns – Universidade Federal Rural de Pernambuco, Av. Bom Pastor S/N, Boa Vista, Garanhuns CEP: 55292-270, Brazil. E-mail address: [email protected] (R.A.N. Ramos).

http://dx.doi.org/10.1016/j.ttbdis.2017.04.007 Received 3 December 2016; Received in revised form 11 April 2017; Accepted 11 April 2017 Available online 17 April 2017 1877-959X/ © 2017 Elsevier GmbH. All rights reserved.


M.A.B. Santos et al.

2. Material and methods

Table 1 Tick life stage collected and detection of Cercopithifilaria spp.

2.1. Study area and ethical statement

Tick life stage

The study was conducted in the urban area of the municipality of Garanhuns (Pernambuco, northeastern Brazil, 8°53′25″S and 36°29′34″W). This region is located at 900 m above sea level and presents an average temperature of 22 °C, with maximum in January (24 °C) and minimum in June (19 °C), and average relative humidity of 90%. This study was approved by the Ethics Committee for Animal Experimentation (ECAE) of the Universidade Federal Rural de Pernambuco (number: 99/2016), as this research represented an extension of a previous work on canine visceral leishmaniosis.

Samples

Positivity AF (n)

RF (%)

Engorged larva Engorged nymphs Engorged females Unengorged females Partially engorged females Males

1 294 437 321 204 649

– 11 – 16 – 24

– 3.7 – 5.0 – 3.7

Total

1906

51

–

AF: absolute frequency; RF: relative frequency.

2.2. Tick collection and identification

3. Results

Samplings (n = 36) were performed every ± 8 days from October 2015 to June 2016. Ticks were collected manually on domiciled dogs for a period of 5 min for each animal and placed into plastic vials, being morphologically identified (Walker et al., 2000). In addition, the feeding status of each tick was based on their dimension (Walker et al., 2000).

A total of 1906 ticks (mean ± 12.2 ticks per animal) were collected on 155 dogs during the whole study, with the highest number in January 2016 (n = 254) and the lowest in June 2016 (n = 26). All specimens were identified as R. sanguineus s.l. Data about tick stage collected and positivity is reported in Table 1. All ticks infected by nematode larvae were collected from 16 dogs (i.e., 13.5%). Larvae were morphologically identified as C. bainae and belonged to four different life stages: microfilariae, developing larval stage 1 (DL1), developing larval stage 2 (DL2), and developing larval stage 3 (DL3) (Table 2). After molecular analysis, a DNA sequence of 278 bp was obtained (GenBank accession number: KY083056). An identity of 99% among 12S rRNA sequences of C. bainae from Brazil (Genbank accession number: KX156956) and Europe (KF381408, JF461461 and KP760321) was registered at the BLASTn search. Cladograms clustered C. bainae sequences in two clades according their provenience (Fig. 1). When compared, a single nucleotide difference was observed between the Brazilian and European isolates, with the former showing a thymine vs adenine at the position 163 of DNA sequence.

2.3. Tick dissection and laboratory examination Unengorged specimens were immediately dissected, whereas partially engorged and engorged larvae and nymphs were maintained under controlled conditions (26 ± 2 °C and 70% ± 10% RH) and examined 30 days post-collection, when they moulted to nymphs and adults, respectively. Ticks were dissected on a slide containing a drop of saline solution (NaCl 0.9%) as previously described (Patton et al., 2012). Briefly, the idiosoma was incised with a sterile scalpel, and the body cavity exposed. The tick internal content was mixed with saline and examined under a light microscope at different magnifications (10× and 40×) (Patton et al., 2012). Larvae of Cercopithifilaria spp. were identified at stage level and measured (Brianti et al., 2012). In order to confirm their identity, larvae were further molecularly analyzed. Genomic DNA was extracted using a commercial kit (Qiagen DNeasy Blood & Tissue Kit; Hilden-Germany), following the manufacturer's recommendations. Cercopithifilaria DNA was amplified as previously described (Otranto et al., 2011). The amplicon obtained was purified using CleanSweep PCR Purification (Thermo Fisher Scientific Carlsbad, CA, USA) and sequenced in both directions by the Sanger's method (Sanger et al., 1977) in an automated sequencer ABI-3130 (Applied Biosystems). The identity of the DNA sequences was determined by comparison with sequences available in the GenBank using the BLASTn search tool (Altschul et al., 1990). A phylogenetic tree was constructed using the UPGMA method (Sneath and Sokal, 1973). Sequences of other filarioids available in GenBank database were used for phylogenetic analysis. Bootstrap resampling (1000 replicates) was performed for the statistical support of the reliabilities of the nodes on the trees (Felsenstein, 1985) using the MEGA software version 6.0 (Tamura et al., 2013). Ascaris lumbricoides (KY045803) was used as outgroup.

4. Discussion This study assessed the occurrence of C. bainae in on-host R. sanguineus s.l. population of dogs from Brazil, which were exposed to tick infestation during the whole study period. The highest and lowest number of ticks was recorded on January and June, which represent the most hot/dry and cold/wet months in this area, respectively. Our finding therefore suggests that the peak in tick parasitism occurs during the warmer season, as already observed in other R. sanguineus s.l. endemic areas (Lorusso et al., 2010). This finding suggest that the infestation dynamic of the brown dog tick in tropical areas is subjected to a seasonal variation, with periods when animals are more at risk for the infestation and transmission of tick-borne pathogens. However, it cannot excluded that the abundance of adult ticks, when compared to immature stages, was related to the easiness of detecting female and male ixodids, which are bigger than larvae or nymphs. The percentage of ticks positive for C. bainae at the dissection (i.e., Table 2 Cercopithifilaria bainae larvae retrieved at different developmental stages at the dissection of Rhipicephalus sanguineus s.l. Microfilariae, developing first stage larvae (DL1), second stage larvae (L2), and third stage larvae (L3).

2.4. Data analysis Descriptive statistical analysis was performed to analyze the results obtained. The Lilliefors test was used to verify the normality of the data and the Chi-square test (χ2) with Yates correction (5% significance level) used to compare the positivity between the tick stages infected with Cercopithifilaria spp. The BioEstat software (version 5.3) was used for these analyses (Ayres et al., 2007). 624

Larval stage

Number of larvae

Mean length μm ( ± SD)

Mean width μm ( ± SD)

Microfilariae DL1 L2 L3

23 99 14 27

185.22 ( ± 1.78) 232.91 ( ± 9.84) 759.71 ( ± 16.5) 1638.22 ( ± 170)

5.6 ( ± 1.2) 11.6 ( ± 3.35) 26.85 ( ± 3.92) 26.83 ( ± 4.1)


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Brucato, G., Cauquil, L., Giannetto, S., Bain, O., 2012. Rhipicephalus sanguineus (Ixodida, Ixodidae) as intermediate host of a canine neglected filarial species with dermal microfilariae. Vet. Parasitol. 183, 330–337. Dantas-Torres, F., Figueiredo, L.A., Faustino, M.A.G., 2004. Ectoparasitos de cães provenientes de alguns municípios da região metropolitana do Recife, Pernambuco, Brasil. Braz. J. Vet. Parasitol. 13, 151–154. Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit. Vectors 3, 26. Dantas-Torres, F., Chomel, B.B., Otranto, D., 2012. Ticks and tick-borne diseases: a one health perspective. Trends Parasitol. 28, 437–446. Dantas-Torres, F., Latrofa, M.S., Annoscia, G., Giannelli, A., Parisi, A., Otranto, D., 2013. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds. Parasit. Vectors 6, 213. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Gabrielli, S., Giannelli, A., Brianti, E., Dantas-Torres, F., Bufalini, M., Fraulo, M., Torre, F.L., Ramos, R.A.N., Cantacessi, C., Latrofa, M.S., Cancrini, G., Otranto, D., 2014. Chronic polyarthritis associated to Cercopithifilaria bainae infection in a dog. Vet. Parasitol. 205, 401–404. Hubálek, Z., Rudolf, I., 2012. Tick-borne viruses in Europe. Parasitol. Res. 111, 9–36. Labruna, M.B., Pereira, M.C., 2001. Carrapato em cães no Brasil. Clin. Vet. 30, 24–32. Latrofa, M.S., Dantas-Torres, F., Giannelli, A., Otranto, D., 2014. Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus group ticks. Ticks Tick Borne Dis. 5, 943–946. Lorusso, V., Dantas-Torres, F., Lia, R.P., Tarallo, V.D., Mencke, N., Capelli, G., Otranto, D., 2010. Seasonal dynamics of the brown dog tick, Rhipicephalus sanguineus, on a confined dog population in Italy. Med. Vet. Entomol. 24, 309–315. Ogden, N.H., Lindsay, L.R., 2016. Effects of climate and climate change on vectors and vector-borne diseases: ticks are different. Trends Parasitol. 32, 646–656. Otranto, D., Brianti, E., Dantas-Torres, F., Weigl, S., Latrofa, M.S., Gaglio, G., Cauquil, L., Giannetto, S., Bain, O., 2011. Morphological and molecular data on the dermal microfilariae of a species of Cercopithifilaria from a dog in Sicily. Vet. Parasitol. 182, 221–229. Otranto, D., Brianti, E., Latrofa, M.S., Annoscia, G., Weigl, S., Lia, R.P., Gaglio, G., Napoli, E., Giannetto, S., Papadopoulos, E., Mirò, G., Dantas-Torres, F., Bain, O., 2012. On a Cercopithifilaria sp. transmitted by Rhipicephalus sanguineus: a neglected, but widespread filarioid of dogs. Parasit. Vectors 5, 1. Otranto, D., Brianti, E., Dantas-Torres, F., Miró, G., Latrofa, M.S., Mutafchiev, Y., Bain, O., 2013a. Species diversity of dermal microfilariae of the genus Cercopithifilaria infesting dogs in the Mediterranean region. Parasitology 140, 99–108. Otranto, D., Varcasia, A., Solinas, C., Scala, A., Brianti, E., Dantas-Torres, F., Annoscia, G., Martin, C., Mutafchiev, Y., Bain, O., 2013b. Redescription of Cercopithifilaria bainae Almeida & Vicente, 1984 (Spirurida, Onchocercidae) from a dog in Sardinia, Italy. Parasit. Vectors 6, 132. Patton, T.G., Dietrich, G., Brandt, K., Dolan, M.C., Piesman, J., Gilmore, R.D., 2012. Saliva, salivary gland, and hemolymph collection from Ixodes scapularis ticks. J. Vis. Exp. 60, 1–6. Ramos, R.A.N., Giannelli, A., Carbone, D., Baneth, G., Dantas-Torres, F., Otranto, D., 2014. Occurrence of Hepatozoon canis and Cercopithifilaria bainae in an off-host population of Rhipicephalus sanguineus sensu lato ticks. Ticks Tick Borne Dis. 5, 311–314. Ramos, R.A., Oliveira Rêgo, A.G., Farias Firmino, E.D., Ramos, C.A., Carvalo, G.A., Dantas-Torres, F., Otranto, D., Alves, L.C., 2016. Filarioids infecting dogs in northeastern Brazil. Vet. Parasitol. 226, 26–29. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U. S. A. 74, 5463–5467. Sneath, P.H.A., Sokal, R.R., 1973. Numerical Taxonomy. Freeman, San Francisco, pp. 573. Szabó, M.P., Mangold, A.J., João, C.F., Bechara, G.H., Guglielmone, A.A., 2005. Biological and DNA evidence of two dissimilar populations of the Rhipicephalus sanguineus tick group (Acari: Ixodidae) in South America. Vet. Parasitol. 130, 131–140. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Walker, J.B., Keirans, J.E., Horak, I.G., 2000. The Genus Rhipicephalus, (Acari, Ixodidae): A Guide to the Brown Ticks of the World. Cambridge University Press, Cambridge, United Kingdom, pp. 382–391.

Fig. 1. Phylogenetic tree based on Cercopithifilaria bainae and other filarioids 12S rRNA gene sequence. Sequences were compared using the UPGMA method. The percentage of replicates trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Scale bar represents the number of mutations per sequence position.

2.68%) is lower than that recorded in Italy, where a positivity rate of 5.05% was registered (Otranto et al., 2012). However, in the latter case, this information was assessed only using molecular tools (Otranto et al., 2012). Interestingly, the majority of tick stages positive for the pathogens were adults, either unengorged (78.43%) or moulted from engorged nymphs (21.57%). This suggests the nymphal stage has a key role in the transstadial transmission of the parasite (Ramos et al., 2014). The molecular analysis of C. bainae detected in ticks from dogs living in northeastern Brazil confirmed its genetic divergence from the European isolates, as a likely consequence of the nematode genetic segregation prompted by the geographic isolation of R. sanguineus s.l. ticks (Szabó et al., 2005; Dantas-Torres et al., 2013). The characterization of other gene sequences may better clarify the differences observed in the present study. Data herein reported indicate that R. sanguineus s.l. is the predominant tick parasitizing dogs in urban areas of Brazil (Dantas-Torres et al., 2004) and that animals living in these areas are at a high risk of tick infestation and C. bainae infection. In addition, this study provides evidence that the population of R. sanguineus found in northeastern Brazil may act as intermediate host of C. bainae. Acknowledgments Authors would like to thank Professor Domenico Otranto and Alessio Giannelli (University of Bari, Italy) for their suggestions on the manuscript. References Almeida, G.L.G., Vicente, J.J., 1984. Cercopithifilaria bainae parasita de Canis familiaris (L.) (Nematoda Filarioidea). Atas Soc. Biol. Rio Jan. 24, 18. 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. Ayres, M., Ayres-Júnior, D.L., Ayres, A.A.S., 2007. BioEstat−Aplicações estatísticas nas áreas de ciências biomédicas Ong Mamiraua, Belém, PA (2007) . Brianti, E., Otranto, D., Dantas-Torres, F., Weigl, S., Latrofa, M.S., Gaglio, G., Napoli, E.,

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