Parasitology Research

, Volume 112, Issue 8, pp 3013–3017

Tick vectors of Cercopithifilaria bainae in dogs: Rhipicephalus sanguineus sensu lato versus Ixodes ricinus

Authors

  • Rafael Antonio Nascimento Ramos
    • Dipartimento di Medicina VeterinariaUniversità degli Studi di Bari
  • Alessio Giannelli
    • Dipartimento di Medicina VeterinariaUniversità degli Studi di Bari
  • Emanuele Brianti
    • Dipartimento di Sanità Pubblica Veterinaria, Facoltà di Medicina VeterinariaUniversità degli Studi di Messina
  • Giada Annoscia
    • Dipartimento di Medicina VeterinariaUniversità degli Studi di Bari
  • Cinzia Cantacessi
    • Centre for Biodiscovery and Molecular Development of TherapeuticsJames Cook University
  • Filipe Dantas-Torres
    • Dipartimento di Medicina VeterinariaUniversità degli Studi di Bari
    • Centro de Pesquisas Aggeu Magalhães (Fiocruz-PE)
    • Dipartimento di Medicina VeterinariaUniversità degli Studi di Bari
Original Paper

DOI: 10.1007/s00436-013-3474-4

Cite this article as:
Ramos, R.A.N., Giannelli, A., Brianti, E. et al. Parasitol Res (2013) 112: 3013. doi:10.1007/s00436-013-3474-4

Abstract

Recently, dermal microfilariae of a Cercopithifilaria species (Spirurida, Onchocercidae), namely Cercopithifilaria bainae , were detected in dogs from several geographical areas of the Mediterranean basin. Evidence from both laboratory and field studies support the role of the brown dog tick, Rhipicephalus sanguineus sensu lato, as an intermediate host of this nematode. In the present study, we investigated the competence of Ixodes ricinus nymphs as vectors of C. bainae. On November 2012, fully engorged nymphs of I. ricinus (n = 174) and R. sanguineus s.l. (n = 10) were collected from a dog infected by C. bainae. The presence of C. bainae in I. ricinus was assessed by both microscopic dissection of specimens and detection of nematode DNA (PCR), at days 3, 10, 20 and 30 (T1–T4) post-collection; due to the small number of specimens available, R. sanguineus s.l. were examined using the same methods at T4 only. No developing larva of C. bainae was detected in I. ricinus specimens at different time points (T1–T4), even if four of these specimens were PCR-positive at T1. Seven out of ten R. sanguineus s.l. were positive for C. bainae third-stage larvae (L3) at both microscopical and molecular analysis at T4. This study indicates that C. bainae does not develop in I. ricinus nymphs, which preclude the role of this tick as an intermediate host of this parasite. Data presented herein provide new insights into the biology of this filarioid species and will lead to a better understanding of the role of different tick species as vectors of nematodes.

Introduction

Among filarioids infecting dogs, those transmitted by culicid vectors (e.g. Dirofilaria immitis and Dirofilaria repens) have been extensively studied (reviewed by Genchi et al. 2009). Recently, much attention has been paid to species of the genus Cercopithifilaria, whose microfilariae can be detected beneath the skin of dogs (Otranto et al. 2013a). A recent case report described the presence of short microfilariae infecting a dog from Sicily (i.e. Cercopithifilaria sp. sensu; Otranto et al. 2011), the recently redescribed C. bainae (Otranto et al. 2013b). Experimental evidence suggests that the ‘brown dog tick’ (R. sanguineus s.l.) acts as a vector and intermediate host for this nematode; in the tick vector, the microfilariae develop to infective larval stages (L3) within approximately 30 days (Brianti et al. 2012).

In southern Europe, the wide distribution of R. sanguineus s.l. overlaps that of C. bainae, with prevalence of infection in dogs reaching 13.9 % in Italy (Otranto et al. 2012a). A molecular analysis of a portion of the cytochrome oxidase subunit 1 (cox1) gene revealed that populations of C. bainae are characterised by a large number of haplotypes (n = 14), which suggests a close affiliation between this nematode, R. sanguineus s.l. and the vertebrate definitive host (Otranto et al. 2012b).

Cercopithifilaria nematodes infect a wide range of carnivores and ungulates, with up to 28 species described (Bain et al. 2002), and only six known arthropod vector species (Table 1). For instance, Cercopithifilaria rugosicauda, a parasite of the European deer (Capreolus capreolus), is vectored by Ixodes ricinus (Winkhardt 1980). I. ricinus is a widespread tick species and one the most important vectors of pathogens to animals and humans (Dantas-Torres et al. 2012; Hartelt et al. 2008), which is likely a consequence of its low degree of host specificity. In southern Italy, I. ricinus lives in wooded environments with high humidity and medium to low temperatures (Dantas-Torres and Otranto 2013). Interestingly, cases of C. bainae infection in dogs have been found in areas where I. ricinus is present (Otranto et al. 2012a), but the role of this tick as an intermediate host of C. bainae is currently unknown. In the present study, we investigate the development of C. bainae in I. ricinus and R. sanguineus s.l. nymphs collected from a naturally infested dog.
Table 1

Cercopithifilaria species and their vectors

Species

Definitive host(s)

Vector

Reference

Cercopithifilaria rugosicauda

Roe deer

Ixodes ricinus

Winkhardt (1980)

Cercopithifilaria grassii

Dog

Rhipicephalus sanguineus

Bain et al. (1982)

Cercopithifilaria roussilhoni

Rodent

Rhipicephalus sanguineus

Bain et al. (1986)

Cercopithifilaria dermicola

Bovine

Hyalomma truncatum

Bain and Denke (1986)

Cercopithifilaria johnstoni

Rodent, marsupial

Ixodes thichosuri

Spratt and Haicock (1988)

Cercopithifilaria bainae

Dog

Rhipicephalus sanguineus

Brianti et al. (2012)

Material and methods

Dog and tick samples

On November 2012, ticks were collected from a 2-year-old, neutered female dog from the municipality of Putignano (province of Bari) that had visited the Gallipoli Cognato Forest with her owner (Matera province, Basilicata region, southern Italy). The dog was naturally infected by C. bainae, as confirmed by microscopic examination of sediments obtained from skin samples. Briefly, a piece of skin (0.5 × 0.5 × 0.6 cm) was collected from the interscapular region, using a disposable scalpel, and subsequently immersed in saline solution for 10 min at 37 °C. Then, 20 μl of the sediment were observed under a light microscope, where dermal microfilariae were detected, counted (i.e. 19 microfilariae/20 μl) and identified morphologically and molecularly as C. bainae (Otranto et al. 2011, 2012a, 2013b).

Fully engorged nymphs were manually collected from the dog and placed in individual vials, sealed with a cotton plug. In the laboratory, ticks were identified as I. ricinus (n = 174) and R. sanguineus s.l. (n = 10) according to established morphological keys (Manilla 1998; Walker et al. 2000). I. ricinus ticks were mostly collected from interscapular and rump regions, whereas R. sanguineus s.l. specimens from the ears. Pools of ten ticks each were transferred into glass vials and maintained under controlled conditions of temperature and humidity (i.e. I. ricinus 20 ± 1 °C, relative humidity (RH) > 80 %; R. sanguineus s.l. 26 ± 1 °C, RH > 70 %) until dissection. Ticks were monitored daily, and their moulting period and mortality rate were recorded.

Tick dissection and microscopic examination

In order to detect larvae of C. bainae, I. ricinus nymphs (n = 160) were dissected at pre-determined time points (i.e. at day 3, 10, 20 and 30 (T1–T4) post-collection). Only ten R. sanguineus s.l. specimens were retrieved on animal and dissected 30 days (T4) post-collection. Briefly, ticks were placed onto glass slides containing a drop of 0.9 % physiological saline solution, dissected with a sterile scalpel and examined immediately under a light microscope, at different magnifications. All nematode larvae detected were photographed and subsequently morphometrically identified (Otranto et al. 2011; Brianti et al. 2012), using the Leica LAS AF version 4.1 software. I. ricinus ticks that died prior to dissection (n = 14) were placed in individual vials containing 200 μl of phosphate buffered saline and stored at −20 °C until molecular analysis (see below).

Molecular examination

Genomic DNA was extracted from individual specimens using the guanidine isothiocyanate-phenol technique (Sangioni et al. 2005). A portion of the cox1 (~304 bp) gene was amplified using specific primers (i.e. CbCox1F/NTR) using reaction procedures and an amplification protocol previously described (Otranto et al. 2011). Amplicons were purified using Ultrafree-DA columns (Amicon, Millipore; Bedford, USA) and sequenced directly using the Taq DyeDeoxyTerminator Cycle Sequencing Kit (v.2, Applied Biosystems) in an automated sequencer (ABI-PRISM 377). Sequences were aligned using ClustalW programme (Larkin et al. 2007) and compared with those available in GenBankTM database by BLASTn analysis.

Results

Out of 174 I. ricinus collected, 160 (91.9 %) nymphs survived the observation period, albeit none moulted into adult ticks. None of the R. sanguineus s.l. specimens died, and all of them moulted into adults (moulting period, 16 ± 2 days). No developing larva of C. bainae was detected in I. ricinus, at any time point. However, four I. ricinus specimens were PCR-positive for C. bainae at T1. Dead nymphs tested were PCR-negative. Conversely, microscopic examination of seven out of ten R. sanguineus s.l. (i.e. four males and three females) at T4 revealed the presence of L3 of C. bainae, with infection intensity ranging from one to 24 larvae per tick. Larvae were 1,742.5 (± 70.3) μm long, 25.3 (± 1.5) μm wide in average and characterised by a shallow oral cavity lacking buccal capsule. The oesophagus measured 317.5 (± 28.1) μm in length, and it was characterised by an anterior muscular and a glandular posterior part. The tail was slightly bent ventrally, with rounded extremity ornate with three (i.e. two short lateral and one elongated central) conical lappets. Based on the above measurements and morphological features, the identification of the larvae as L3s of C. bainae was confirmed (Fig. 1). Sequences derived from the amplicons displayed an overall homology of 100 % with a C. bainae sequence deposited in GenBank (JF461457).
https://static-content.springer.com/image/art%3A10.1007%2Fs00436-013-3474-4/MediaObjects/436_2013_3474_Fig1_HTML.gif
Fig. 1

Third-stage larvae (L3) of Cercopithifilaria bainae detected in an adult of Rhipicephalus sanguineus sensu lato. (a) (bar = 200 μm). (b) cephalic region (bar = 50 μm); (c) caudal region (bar = 50 μm), note the presence of three conical lappets, the shorter laterals and the elongated central

Discussion

The present study provides evidence that I. ricinus nymphs, unlike R. sanguineus s.l., do not allow development of microfilariae of C. bainae. Despite the small number of R. sanguineus s.l. collected, all specimens moulted to adult ticks and survived until dissection, in accordance with previous observations (Dantas-Torres et al. 2010). The low rate of mortality in I. ricinus (8.1 %) was unlikely to be linked to the ingestion of C. bainae larvae, since none of the dead nymphs tested was positive for the presence of nematode.

The unsuitability of I. ricinus as an intermediate host of C. bainae may be associated with the fact that this species requires 120 days, including the phase of winter diapause, to moult from nymphs to adults (Dusbábek 1996), which differs from R. sanguineus s.l. (i.e. about 16 ± 2 days). Accordingly, the development of C. rugosicauda from L1 to L3 in I. ricinus nymphs requires 56–67 days (Winkhardt 1980), whereas C. bainae develops in R. sanguineus s.l. within ~30 days (Brianti et al. 2012), therefore resulting in rather different development times of C. bainae larvae in ticks and of tick nymphs into adults, with ratios of almost 1:2 and 2:1 for C. bainae and C. rugosicauda, in their respective tick vectors.

Based on this observation, it could be speculated that the synchrony between the development of arthropod-borne filarioids and that of the different tick species ultimately favours the maintenance and transmission of these nematodes in an environment where suitable definitive hosts are present. In addition, data above also indicate a high degree of co-evolution between these filarioids and their vectors, as suggested by the fact that three cytochrome c oxidase 1 (cox1) haplotypes of C. bainae were found exclusively in ticks in two southern Italian regions, even if several other haplotypes were found in dogs and ticks in the same regions (Otranto et al. 2012b). The fact that a large number of L3 (up to 24) was detected in a single R. sanguineus s.l. confirms that infection by C. bainae is well tolerated by this tick species and it does not affect the survival rate and the moult to the adult stage (Brianti et al. 2012). The high infection rate with C. bainae detected in R. sanguineus s.l. nymphs was likely a consequence of the large number of microfilariae present in the dog’s skin (19 microfilariae/20 μl) at the time ticks were collected.

Data from the present study, together with knowledge of the occurrence of I. ricinus in temperate areas of Europe (Beugnet and Mariè 2009), suggest that the presence of C. bainae in centre and north Europe is unlikely. This hypothesis is supported by the fact that only sporadic cases of infestation by R. sanguineus s.l. have been reported in central and northern Europe to date (Gray et al. 2009). Nonetheless, it has been speculated that an increase in the mean temperature during summer could prompt the establishment of R. sanguineus s.l. populations in temperate regions of northern Europe (Gray et al. 2009). Therefore, the spread of C. bainae and other pathogens that may be transmitted by R. sanguineus s.l. (Dantas-Torres et al. 2012) in these areas cannot be ruled out. Nonetheless, the possible role of other ticks (e.g., Dermacentor spp.) in the transmission of C. bainae should be investigated. These new insights into the biology of this neglected filarioid will lead to a better understanding of the role of different ticks as vectors of nematodes.

Acknowledgments

Authors thank Lénaïg Halos and Federic Beugnet (Merial, France) for partially supporting this research and Luciana Aguiar Figueredo for her thorough suggestions on the manuscript.

Copyright information

© Springer-Verlag Berlin Heidelberg 2013