Molecular Evidence for Transstadial Transmission of Ehrlichia canis by Rhipicephalus sanguineus sensu lato Under Field Conditions

Molecular Evidence for Transstadial Transmission of Ehrlichia canis by Rhipicephalus sanguineus... Abstract This study investigated possible transstadial transmission of Ehrlichia canis by Rhipicephalus sanguineus sensu lato collected from shelter dogs and the shelter grounds in Diyarbakır Province of south-eastern Turkey. Totally 225 engorged nymphs were collected from eight infected dogs with E. canis and incubated at 28°C for moulting. Unfed ticks from the shelter grounds comprising 1,800 larvae, 3,100 nymphs, and 85 adults were sorted according to sampling origin, life stage, and sex into 116 pools and screened by 16S rRNA PCR. Nine out of 26 pools of unfed adult ticks were positive for E. canis, with overall infection rate maximum likelihood estimation (MLE) of 4.83 (CI 2.39–8.87). E. canis was detected in three of 12 male pools (MLE 3.22, CI 0.86–8.83) and six of 14 female pools (MLE 6.16, CI 2.59–12.90). No adult pools collected from the shelter grounds were positive. Among 62 unfed nymph pools collected from the shelter, six were infected with E. canis (MLE 0.20, CI 0.08–0.42). No E. canis DNA was detected in any of the larva pools. Our results revealed molecular evidence for transstadial transmission of E. canis by R. sanguineus s.l. both from larva to nymph and from nymph to adult. We found no evidence of transovarial transmission. Ehrlichia canis, Rhipicephalus sanguineus sensu lato, dog, transstadial transmission by R. sanguineus s.l Anaplasmataceae are microorganisms that infect vertebrates and ticks (Dumler 2005) and cause serious disease in hosts worldwide (Nicholson et al. 2010, Dantas-Torres and Otranto 2016). Ehrlichia canis is a tick-borne pathogen that infects dogs in many countries (Sainz et al. 2015), including Turkey (Aktas et al. 2009, 2015). Recently, clinical signs of E. canis infection have been described in humans (Perez et al. 2006, Andrić 2014), suggesting E. canis as a zoonotic agent of canine monocytic ehrlichiosis (CME). Rhipicephalus sanguineus sensu lato is a well-recognized putative vector of pathogenic agents including bacteria, protozoa, nematodes, and viruses affecting dogs and occasionally humans (Dantas-Torres 2010, Dantas-Torres et al. 2012, Dantas-Torres and Otranto 2015, Aktas and Özübek 2017). R. sanguineus s.l. has been recognized as the main tick species for the transmission of E. canis (Groves et al. 1975, Mathew et al. 1996, Bremer et al. 2005, Dantes-Torres et al. 2012, Sainz et al. 2015). Transmission of E. canis by R. sanguineus has been shown under laboratory conditions (Fourie et al. 2013). However, this tick is currently assumed a group (Gray et al. 2013). Molecular phylogenetic analysis revealed the existence of two phylogenetic clades and six different haplotypes (Moraes-Filho et al. 2011). Therefore, there is a consensus that populations of this tick species should be referred to as R. sanguineus s.l. until its taxonomic status is resolved (Dantas-Torres et al. 2017). The findings of an experimental study revealed that one population of R. sanguineus group were competent vectors of E. canis, whereas the other populations were not (Moraes-Filho et al. 2015). CME has been described in Turkey (Aktas 2014, Düzlü et al. 2014, Cetinkaya et al. 2016) but no studies have been reported potential vectors of the disease. This study aimed to examine a possible transstadial route for E. canis transmission by R. sanguineus s.l. under field conditions. Materials and Methods Tick samples used in this study were from a study carried out in Diyarbakır Province (37° 55′ N, 40° 12′ E) of south-eastern Turkey, in a municipal dog shelter in which E. canis infection was reported in dogs (Aktas et al. 2015). A total of 4,985 unfed ticks comprising 85 adults, 3,100 nymphs, and 1,800 larvae were collected from the grounds of the shelter. In addition, eight dogs positive for E. canis by PCR with heavy tick infestation were included in the study. No dog showed clinical signs of disease at the time of inclusion. Throughout May and June 2015, each dog was examined and all ticks were collected and placed in glass vials. Engorged nymphs were incubated for molting to adults, and 225 unfed adults (104 males, 121 females) were obtained (Table 1). The ticks were identified microscopically as R. sanguineus s.l. (Estrada-Peña et al. 2004), and sorted by origin (specific dog or shelter ground), life stage, and sex into 116 pooled samples: 18 larva pools (100 per pool), 62 nymph pools (50 per pool) and 36 adult pools (4–14 per pool). Table 1. Pooled samples of unfed adult Rhipicephalus sanguineus s.l. removed from the Ehrlichia canis-infected dogs as engorged nymphs testing positive for Ehrlichia canis by PCR Animal ID  No. of ticks  No. of pools  Positive pools (%)  MLE (95% CI)  1 AEG1  35  4  0  NAa  2 AEG 7  26  4  1 (25%)  4.06 (0.24–21.11)  3 NO1  21  3  2 (67%)  11.04 (2.31–42.51)  4 NO2  12  1  1 (100%)  NAa  5 NO 3  20  2  2 (100%)  NAa  6 NO 4  17  2  1 (50%)  5.32 (0.39–31.68)  7 NO 5  43  5  0  NAa  8 NO 6  51  5  2 (40%)  4.48 (0.84–15.73)  Total  225  26  9 (35%)  4.83 (2.39–8.87)  Animal ID  No. of ticks  No. of pools  Positive pools (%)  MLE (95% CI)  1 AEG1  35  4  0  NAa  2 AEG 7  26  4  1 (25%)  4.06 (0.24–21.11)  3 NO1  21  3  2 (67%)  11.04 (2.31–42.51)  4 NO2  12  1  1 (100%)  NAa  5 NO 3  20  2  2 (100%)  NAa  6 NO 4  17  2  1 (50%)  5.32 (0.39–31.68)  7 NO 5  43  5  0  NAa  8 NO 6  51  5  2 (40%)  4.48 (0.84–15.73)  Total  225  26  9 (35%)  4.83 (2.39–8.87)  NA, not applicable. aWhen all pools are positive or negative, likelihood methods fail. View Large Genomic DNAs were isolated from 116 tick pools were included in the study. Mitochondrial cytochrome c oxidase 1 (cox1) gene was amplified by PCR (Chitimia et al. 2010). Following cox1 gene PCR, a nested PCR was performed to detect E. canis (Wen et al. 1997). Positive (DNA from E. canis identified by DNA sequencing) and negative (RNase-free water) controls were used. Three products generated from R. sanguineus s.l. pools were sequenced. Also representative amplicons specifically targeting E. canis were sequenced and comparative sequence analyses were performed. Sequence comparison was made by BLASTn. A phylogenetic tree was created using cox1 sequences obtained from tick pools based on a 785 nucleotides in MEGA version 6.0 (Tamura et al. 2013). The evolutionary history was constructed using the maximum likelihood (ML) method based on the Tamura 3-parameter model (Tamura 1992). The percentage of replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates). Maximum likelihood estimation (MLE) method was used to calculate infection rate in tick pools (Biggerstaff 2009). Fischer’s exact test implemented in SPSS 15.00 was used, with P-values < 0.05 considered significant (Daniel and Cross 2013). The study was carried out in compliance with the regulations of animal and welfare issued by the Turkish legislation for the protection of animals (Animal Experiment Ethic Committee: 71156/2009). Results Amplification using species-specific primers for E. canis yielded nested PCR products of the expected size (~390 bp) from positive control samples. Non-template control was PCR-negative. Nine out of 26 pools (35%) representing 225 unfed adult R. sanguineus s.l. from E. canis-infected dogs were found to be infected, with overall infection rate MLE of 4.83 (2.39–8.87). The ticks recovered from two dogs (1, 7) were negative for E. canis DNA, while at least one pool recovered from the remaining six dogs was positive (Table 1). Infection rate MLE varied from 4.06 (0.24–21.11) to 11.04 (2.31–42.51) in the pools. All ticks from dog 5 were positive for E. canis (Table 1). Origin, life stage, and sex of the sampled ticks and frequency of E. canis infection are shown in Table 2. A total of 116 pools, representing 5,210 unfed R. sanguineus s.l. (330 adults, 3,100 nymphs, 1,800 larvae) were screened by the PCR for the presence of bacteria. E. canis was detected in 3 of 12 (25%) male pools (MLE 3.22, CI 0.86–8.83) and 6 of 14 (42.8%) females (MLE 6.16; CI 2.59–12.90) obtained from E. canis-infected dogs. There was no significant difference in infection rates of male and female ticks (P > 0.05). No pool of adult ticks collected from the shelter grounds was positive for E. canis. Of 62 nymph pools collected from the grounds, 6 (9.7%) were infected with E. canis (MLE 0.20, CI 0.08–0.42). The difference in infection rate of adult and nymph stages of ticks was not significant (P > 0.05). E. canis DNA was not detected in the pools consisted from larvae specimens. Table 2. Frequency of Ehrlichia canis in pooled samples of unfed Rhipicephalus sanguineus s.l. collected from dogs (n = 225) and the environment of the dog shelter (n = 4,985) Tick source  No. of ticks  No. of pools  Positive pools (%)  MLE (95% CI)  P-value  Ticks from dogsa   Male  104  12  3 (25)  3.22 (0.86–8.83)  P > 0.05   Female  121  14  6 (42.8)  6.16 (2.59–12.90)  From environment   Male  30  3  0  NAb     Female  55  7  0  NA   Nymph  3,100  62  6 (9.7)  0.20 (0.08–0.42)   Larva  1,800  18  0  NA  Tick source  No. of ticks  No. of pools  Positive pools (%)  MLE (95% CI)  P-value  Ticks from dogsa   Male  104  12  3 (25)  3.22 (0.86–8.83)  P > 0.05   Female  121  14  6 (42.8)  6.16 (2.59–12.90)  From environment   Male  30  3  0  NAb     Female  55  7  0  NA   Nymph  3,100  62  6 (9.7)  0.20 (0.08–0.42)   Larva  1,800  18  0  NA  NA, not applicable. aMolting from the nymph to adult. bWhen all pools are positive or negative, likelihood methods fail. View Large Partial sequence of the 16S rRNA gene determined for E. canis was deposited in the EMBL/GenBank databases (KY847523). BLAST search revealed that the sequence shared 100% identity with E. canis isolate TrKysEcan3 detected in dogs from Turkey (KJ513197) and other corresponding published sequences available in GenBank database. The partial sequences of cox1 gene were also analyzed using BLAST. The cox1 sequences from tick pools shared 99–100% identity with Rhipicephalus sp. I lineage (Hornok et al. 2017). Based on cox1 gene, the phylogenetic tree revealed that our sequences from Turkey clustered with Rhipicephalus sp. I of ‘temperate lineage’ (East Mediterranean clade) (Fig. 1). Fig. 1. View largeDownload slide Phylogenetic tree based on partial (785 bp) cox 1 sequences of R. sanguineus s.l. pools from Turkey and other regions available in GenBank. The evolutionary history was inferred using the maximum likelihood method based on the Tamura 3-parameter model. The percentage of replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates). The three cox 1 sequence of R. sanguineus s.l. obtained in the present study are represented in bold. Fig. 1. View largeDownload slide Phylogenetic tree based on partial (785 bp) cox 1 sequences of R. sanguineus s.l. pools from Turkey and other regions available in GenBank. The evolutionary history was inferred using the maximum likelihood method based on the Tamura 3-parameter model. The percentage of replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates). The three cox 1 sequence of R. sanguineus s.l. obtained in the present study are represented in bold. Discussion E. canis is principally transmitted by R. sanguineus s.l. (Bremer et al. 2005, Dumler 2005, Stich et al. 2008, Moraes-Filho et al. 2015). The bacterium was found infecting R. sanguineus s.l. in Asia, Africa, and South America (Unver et al. 2001, Aguiar et al. 2007, Socolovschi et al. 2012, Ybañez et al. 2012). However, mitochondrial 16S rDNA sequences from R. sanguineus s.l. collected from different geographical regions showed the presence of two phylogenetic lineages (temperate and tropical) (Cicuttin et al. 2015, Dantas-Torres and Otranto 2015). Moreover, operational taxonomic units (Rhipicephalus sp. I-IV) have been reported (Dantas-Torres et al. 2013). A recent study underlined several important aspects concerning the systematic status of the R. sanguineus complex (Hekimoğlu et al. 2016). Our three cox1 sequences from tick pools showed 99–100% similarity with Rhipicephalus sp. I of the ‘temperate lineage’ (Hornok et al. 2017), confirming the presence of Rhipicephalus sp. I in Turkey. Phylogenetic analysis of cox1 sequences from new isolates of R. sanguineus s.l. pools in the present study confirmed the existence of Rhipicephalus sp. I. Presence and distribution of this lineage is of special interest because the lineage of R. sanguineus s.l. ticks in the Mediterranean region is believed to be different from that in tropical areas (Nava et al. 2015). Moraes-Filho et al. (2012) experimentally demonstrated differences in the vector competence to transmit E. canis between the tropical and temperate lineages of R. sanguineus s.l. They found that the tropical lineage is competent vector of E. canis but lineage from temperate areas of South America, which are morphologically and molecularly identical to the R. sanguineus s.l. ticks found in the Mediterranean region are not (Moraes-Filho et al. 2012). Unfed adult R. sanguineus s.l. that had fed as nymphs on E. canis-infected dogs were infected with E. canis, indicating that nymphs can become infected with the pathogen when feeding on an infected dog, and that they may transmit the pathogen to moulted adults, suggesting that transstadial transmission occurs from nymph to adult. This is similar to the previous finding that R. sanguineus s.l. nymphs feeding on clinically infected dogs acquired E. canis infection and transmitted it to the adult stage (Stich et al. 2008, Fourie et al. 2013, Moraes-Filho et al. 2015). In this study, at least one tick pool recovered from each of the six E. canis-infected dogs was positive for E. canis DNA. This is consistent with Ramos et al. (2014) report of A. platys DNA in at least one specimen collected from positive dog. In the present study, infection rate MLE in positive pools ranged from 1.43 (dog 4) to 11.04 (dog 7) (Table 1). The finding is consistent with Aguiar et al. (2007) report of E. canis infection rates ranging from 2.3 to 6.2% in R. sanguineus s.l. under field conditions. An experimental study (Moraes-Filho et al. 2015) on E. canis revealed similar findings, with real-time PCR detecting E. canis DNA in 46% of unfed seven day-post-moulting adult R. sanguineus s.l. that had fed as nymphs on E. canis-infected dogs. Tick pools recovered from two E. canis-infected dogs were negative in this study (Table 1). The dogs were asymptomatic, in agreement with the findings of Lewis et al. (1977). The result is concordant with the reports of low amount of circulating pathogen could affect and hindrance the detection leading to a false negative (Kamani et al. 2013, Cicuttin et al. 2014, Almazán et al. 2016, Carvalho et al. 2017). We speculate that the negative findings in tick pools could be due to insufficient concentrations of circulating pathogens in the clinically healthy dogs to allow detection. Recent molecular characterization of R. sanguineus has determined that it consists of a complex of different species that may differ in biological features, including capability to transmit disease agents (Dantas-Torres et al. 2013). All ticks tested in Uruguay were negative (Venzal et al. 2007). A study showed that only tropical species have been found to be infected with E. canis in Argentina (Cicuttin et al. 2015). Recent comprehensive studies on the morphological and genetic diversity of R. sanguineus showed that there are different groups (Rhipicephalus sp. I, and Rhipicephalus sp. IIa, Rhipicephalus sp. IIb) and haplotypes under the ‘temperate lineage’ in Europe, especially Mediterranean countries (Danta-Torres et al. 2013, Hornok et al. 2017, Almeida et al. 2017). Although the sequences obtained in this study were acquired from pools of ticks, we speculate that there may be differences in vector competence for E. canis among populations of R. sanguineus s.l. ticks in Turkey, as exemplified by E. canis and Hepatozoon canis transmission by R. sanguineus s.l. (Venzal et al. 2007, Demoner et al. 2013, Latrofa et al. 2014, Dantas-Torres and Otranto 2015, Moraes-Filho et al. 2015). However, further studies are needed to confirm this assumption. Our observations of unfed nymphs collected from the shelter grounds indicate that E. canis may also be transmitted by this tick from larva to nymph, suggesting that immature stages of the tick are important in the epidemiology of CME, especially in southern Europe, the Mediterranean basin, and the Balkan countries, where larvae and nymphs occur on dogs throughout the year (Lorusso et al. 2010). Ixodid ticks are capable of maintaining tick-borne pathogens in nature through several generations by transovarial passage (Bremer et al. 2005, Dantas-Torres 2007, Fourie et al. 2013). In the present study, 1,800 unfed larvae were screened for E. canis, but no parasite DNA was detected. This may support the long-held assumption that transovarial transmission of E. canis by female R. sanguineus s.l. does not occur. In conclusion, we emphasize that E. canis can be transmitted by nymph and adult stages of R. sanguineus s.l. under field conditions. This suggests that acaricide application should target larvae and nymphs to control CME, as R. sanguineus s.l. is a species of which all parasitic stages feed on dogs. Considering zoonotic relevance of E. canis, it also should reinforce the importance of information to public health authorities. References Cited Aguiar, D. M., Cavalcante G. T., Pinter A., Gennari S. M., Camargo L. M. A., and Labruna M. B.. 2007. Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in dogs and Rhipicephalus sanguineus (Acari: Ixodidae) ticks from Brazil. J. Med. Entomol . 44: 126– 132. Google Scholar CrossRef Search ADS PubMed  Aktas, M. 2014. A survey of ixodid tick species and molecular identification of tick-borne pathogens. Vet. Parasitol . 200: 276– 283. Google Scholar CrossRef Search ADS PubMed  Aktas, M., Altay K., Dumanli N., and Kalkan A.. 2009. Molecular detection and identification of Ehrlichia and Anaplasma species in ixodid ticks. Parasitol. Res . 104: 1243– 1248. Google Scholar CrossRef Search ADS PubMed  Aktas, M., Ozubek S., Altay K., Ipek N. D. S., Balkaya I., Utuk A. E., Kirbas A., Simsek S., and Dumanli N.. 2015. Molecular detection of tick-borne rickettsial and protozoan pathogens in domestic dogs from Turkey. Parasite Vector . 8: 157. Aktas, M., and Özübek S.. 2017. Transstadial transmission of Hepatozoon canis by Rhipicephalus sanguineus (Acari: Ixodidae) in Field Conditions. J. Med. Entomol . 54: 1044– 1048. Google Scholar CrossRef Search ADS PubMed  Almazán C., Gonzalez-Alvarez V. H., de Mera I. G. F., Cabezas-Cruz A., Rodriguez-Martinez R., and de la Fuente J.. 2016. Molecular identification and characterization of Anaplasma platys and Ehrlichia canis in dogs in Mexico. Ticks Tick-Borne Dis . 7: 276– 283. Google Scholar CrossRef Search ADS PubMed  Almeida, C., Simoes R., Coimbra-Dores M. J., Rosa F., and Dias D.. 2017. Mitochondrial DNA analysis of Rhipicephalus sanguineus s.l. from the western Iberian peninsula. Med. Vet. Entomol . 31: 167– 177. Google Scholar CrossRef Search ADS PubMed  Andrić, B. 2014. Diagnostic evaluation of Ehrlichia canis human infections. Open J. Med. Microbiol . 4: 132– 139. Google Scholar CrossRef Search ADS   Biggerstaff, B. J. 2009. PooledInfRate, Version 4.0: a Microsoft® Office Excel© Add-In to compute prevalence estimates from pooled samples . Centers for Disease Control and Prevention, Fort Collins, CO. Bremer, W. G., Schaefer J. J., Wagner E. R., Ewing S. A., Rikihisa Y., Needham G. R., Jittapalapong S., Moore D. L., and Stich R. W.. 2005. Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus. Vet. Parasitol . 131: 95– 105. Carvalho, L., Armua-Fernandez M. T., Sosa N., Felix M. L., and Venzal J. M.. 2017. Anaplasma platys in dogs from Uruguay. Ticks Tick Borne Dis . 8: 241– 245. Google Scholar CrossRef Search ADS PubMed  Cetinkaya, H., Matur E., Akyazi I., Ekiz E. E., Aydin L., and Toparlak M.. 2016. Serological and molecular investigation of Ehrlichia spp. and Anaplasma spp. in ticks and blood of dogs, in the Thrace Region of Turkey. Ticks Tick Borne Dis . 7: 706– 714. Google Scholar CrossRef Search ADS PubMed  Chitimia, L., Lin R. Q., Cosoroaba I., Wu X. Y., Song H. Q., Yuan Z. G., and Zhu X. Q.. 2010. Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox1 and nad5 genes. Exp. Appl. Acarol . 52: 305– 311. Google Scholar CrossRef Search ADS PubMed  Cicuttin, G. L., Brambati D. F., Rodriguez Eugui J. I., Lebrero C. G., De Salvo M. N., Beltran F. J., Gury Dohmen F. E., Jado I., and Anda P.. 2014. Molecular characterization of Rickettsia massiliae and Anaplasma platys infecting Rhipicephalus sanguineus ticks and domestic dogs, Buenos Aires (Argentina). Ticks Tick Borne Dis . 5: 484– 488. Google Scholar CrossRef Search ADS PubMed  Cicuttin, G. L., Tarragona E. L., De Salvo M. N., Mangold A. J., and Nava S.. 2015. Infection with Ehrlichia canis and Anaplasma platys (Rickettsiales: Anaplasmataceae) in two lineages of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) from Argentina. Ticks Tick Borne Dis . 6: 724– 729. Google Scholar CrossRef Search ADS PubMed  Daniel, W. W., and Cross C. L.. 2013. Biostatistics: a foundation for analysis in the health sciences , 10th ed. Wiley, Hoboken, New Jersey, United States of America, 960 pp. Dantas-Torres, F. 2007. Rocky Mountain spotted fever. Lancet. Infect. Dis . 7: 724– 732. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F. 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit. Vectors . 3: 26. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., Chomel B. B., and Otranto D.. 2012. Ticks and tick-borne diseases: a One Health perspective. Trends Parasitol . 28: 437– 446. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., Latrofa M.S., Annoscia G., Giannelli A., Parisi A., and Otranto D.. 2013. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds. Parasit Vectors  6: 213. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., and Otranto D.. 2015. Further thoughts on the taxonomy and vector role of Rhipicephalus sanguineus group ticks. Vet. Parasitol . 208: 9– 13. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., and Otranto D.. 2016. Best practices for preventing vector-borne diseases in dogs and humans. Trends Parasitol . 32: 43– 55. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., Maia C., Latrofa M. S., Annoscia G., Cardoso L., and Otranto D.. 2017. Genetic characterization of Rhipicephalus sanguineus (sensu lato) ticks from dogs in Portugal. Parasit Vectors  10: 133. Google Scholar CrossRef Search ADS PubMed  Demoner Lde, C., Rubini A. S., Paduan Kdos S., Metzger B., de Paula Antunes J.M., Martins T. F., Mathias M. I., and O’Dwyer L. H.. 2013. Investigation of tick vectors of Hepatozoon canis in Brazil. Ticks Tick Borne Dis . 4: 542– 546. Google Scholar CrossRef Search ADS PubMed  Dumler, J. S. 2005. Anaplasma and Ehrlichia infection. Ann. N. Y. Acad. Sci . 1063: 361– 373. Google Scholar CrossRef Search ADS PubMed  Duzlu, O., Inci A., Yildirim A., Onder Z., and Ciloglu A.. 2014. The investigation of some tick-borne protozoon and rickettsial infections in dogs by Real Time PCR and the molecular characterizations of the detected isolates. Ankara Univ. Vet. Fak . 61: 275– 282. Estrada-Peña, A., Bouattour A., Camicas J. L., and Walker A. R.. 2004. Ticks of domestic animals in the Mediterranean Region. A guide of identification of species . University of Zaragoza Press, Zaragoza, Spain. Fourie, J. J., Stanneck D., Luus H. G., Beugnet F., Wijnveld M., and Jongejan F.. 2013. Transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks feeding on dogs and on artificial membranes. Vet. Parasitol . 197: 595– 603. Google Scholar CrossRef Search ADS PubMed  Gray, J., Dantas-Torres F., Estrada-Peña A., and Levin M.. 2013. Systematics and ecology of the brown dog tick, Rhipicephalus sanguineus. Ticks Tick Borne Dis . 4: 171– 180. Google Scholar CrossRef Search ADS PubMed  Groves, M. G., Dennis G. L., Amyx H. L., and Huxsoll D. L.. 1975. Transmission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus). Am. J. Vet. Res . 36: 937– 940. Google Scholar PubMed  Hekimoğlu, O., Sağlam I. K., Özer N., and Estrada-Peña A.. 2016. New molecular data shed light on the global phylogeny and species limits of the Rhipicephalus sanguineus complex. Ticks Tick Borne Dis . 7: 798– 807. Google Scholar CrossRef Search ADS PubMed  Hornok, S., Sandor A. D., Tomanovic S., Beck R., D’Amico G., Kontschan J., Takacs N., Gorfol T., Bendjeddou M. L., Foldvari G., and Farkas R.. 2017. East and west separation of Rhipicephalus sanguineus mitochondrial lineages in the Mediterranean Basin. Parasit Vectors  10: 39. Google Scholar CrossRef Search ADS PubMed  Kamani, J., Baneth G., Mumcuoglu K. Y., Waziri N. E., Eyal O., Guthmann Y., and Harrus S.. 2013. Molecular detection and characterization of tick-borne pathogens in dogs and ticks from Nigeria. PLoS Negl. Trop. Dis . 7: e2108. Google Scholar CrossRef Search ADS PubMed  Latrofa, M. S., Dantas-Torres F., Giannelli A., and Otranto D.. 2014. Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus group ticks. Ticks Tick Borne Dis . 5: 943– 946. Google Scholar CrossRef Search ADS PubMed  Lewis, G. E., Ristic M., Smith R. D., Lincoln T., and Stephenson E. H.. 1977. The brown dog tick Rhipicephalus sanguineus and the dog as experimental hosts of Ehrlichia canis. Am. J. Vet. Res . 38: 953– 1955. Lorusso, V., Dantas-Torres F., Lia R. P., Tarallo V. D., Mencke N., Capelli G., and 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. Google Scholar PubMed  Mathew, J. S., Ewing S. A., Barker R. W., Fox J. C., Dawson J. E., Warner C. K., Murphy G. L., and Kocan K. M.. 1996. Attempted transmission of Ehrlichia canis by Rhipicephalus sanguineus after passage in cell culture. Am. J. Vet. Res . 57: 1594– 1598. Google Scholar PubMed  Moraes-Filho, J., Marcili A., Nieri-Bastos F. A., Richtzenhain L. J., and Labruna M. B.. 2011. Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America. Acta Trop . 117: 51– 55. Google Scholar CrossRef Search ADS PubMed  Moraes-Filho, J., Marcili A., Nieri-Bastos F. A., Richtzenhain L. J., and Labruna M. B.. 2012. Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America. Acta Trop . 117: 51– 55. Google Scholar CrossRef Search ADS   Moraes-Filho, J., Krawczak F. S., Costa F. B., Soares J. F., and Labruna M. B.. 2015. Comparative evaluation of the vector competence of four south american populations of the Rhipicephalus sanguineus group for the bacterium Ehrlichia canis, the agent of Canine Monocytic Ehrlichiosis. Plos One  10: e0139386. Google Scholar CrossRef Search ADS PubMed  Nava, S., Estrada-Pena A., Petney T., Beati L., Labruna M. B., Szabo M. P., Venzal J. M., Mastropaolo M., Mangold A. J., and Guglielmone A. A.. 2015. The taxonomic status of Rhipicephalus sanguineus (Latreille, 1806). Vet. Parasitol . 208: 2– 8. Google Scholar CrossRef Search ADS PubMed  Nicholson, W. L., Allen K. E., McQuiston J. H., Breitschwerdt E. B., and Little S. E.. 2010. The increasing recognition of rickettsial pathogens in dogs and people. Trends Parasitol . 26: 205– 212. Google Scholar CrossRef Search ADS PubMed  Perez, M., Bodor M., Zhang C., Xiong Q., and Rikihisa Y.. 2006. Human infection with Ehrlichia canis accompanied by clinical signs in Venezuela. Ann. N. Y. Acad. Sci . 1078: 110– 117. Google Scholar CrossRef Search ADS PubMed  Ramos, R. A., Latrofa M.S., Giannelli A., Lacasella V., Campbell B. E., Dantas-Torres F., and Otranto D.. 2014. Detection of Anaplasma platys in dogs and Rhipicephalus sanguineus group ticks by a quantitative real-time PCR. Vet. Parasitol . 205: 285– 288. Google Scholar CrossRef Search ADS PubMed  Sainz, A., Roura X., Miro G., Estrada-Pena A., Kohn B., Harrus S., and Solano-Gallego L.. 2015. Guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in Europe. Parasit Vectors  8: 75. Google Scholar CrossRef Search ADS PubMed  Socolovschi, C., Gomez J., Marie J. L., Davoust B., Guigal P. M., Raoult D., and Parola P.. 2012. Ehrlichia canis in Rhipicephalus sanguineus ticks in the Ivory Coast. Ticks Tick Borne Dis . 3: 411– 413. Google Scholar CrossRef Search ADS PubMed  Stich, R. W., Schaefer J. J., Bremer W. G., Needham G. R., and Jittapalapong S.. 2008. Host surveys, ixodid tick biology and transmission scenarios as related to the tick-borne pathogen, Ehrlichia canis. Vet. Parasitol . 158: 256– 273. Google Scholar CrossRef Search ADS PubMed  Tamura, K. 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol. Biol. Evol . 9: 678– 687. Google Scholar PubMed  Tamura, K., Stecher G., Peterson D., Filipski A., and Kumar S.. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Bio. Evol . 30: 2725– 2729. Google Scholar CrossRef Search ADS   Unver, A., Perez M., Orellana N., Huang H., and Rikihisa Y.. 2001. Molecular and antigenic comparison of Ehrlichia canis isolates from dogs, ticks, and a human in Venezuela. J. Clin. Microbiol . 39: 2788– 2793. Google Scholar CrossRef Search ADS PubMed  Venzal, J. M., Estrada-Peña A., Castro O., De Souza C. G., Portillo A., and Oteo J. A.. 2007. Study on seasonal activity in dogs and ehrlichial infectionin Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) from southern Uruguay. Parasitol. Latinoam . 62: 23– 26. Google Scholar CrossRef Search ADS   Wen, B., Rikihisa Y., Mott J. M., Greene R., Kim H. Y., Zhi N., Couto G. C., Unver A., and Bartsch R.. 1997. Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline. J. Clin. Microbiol . 35: 1852– 1855. Google Scholar PubMed  Ybañez, A. P., Perez Z. O., Gabotero S. R., Yandug R. T., Kotaro M., and Inokuma H.. 2012. First molecular detection of Ehrlichia canis and Anaplasma platys in ticks from dogs in Cebu, Philippines. Ticks Tick Borne Dis . 3: 288– 293. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Medical Entomology Oxford University Press

Molecular Evidence for Transstadial Transmission of Ehrlichia canis by Rhipicephalus sanguineus sensu lato Under Field Conditions

Loading next page...
 
/lp/ou_press/molecular-evidence-for-transstadial-transmission-of-ehrlichia-canis-by-iW2mijyRXK
Publisher
Oxford University Press
Copyright
© The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
ISSN
0022-2585
eISSN
1938-2928
D.O.I.
10.1093/jme/tjx217
Publisher site
See Article on Publisher Site

Abstract

Abstract This study investigated possible transstadial transmission of Ehrlichia canis by Rhipicephalus sanguineus sensu lato collected from shelter dogs and the shelter grounds in Diyarbakır Province of south-eastern Turkey. Totally 225 engorged nymphs were collected from eight infected dogs with E. canis and incubated at 28°C for moulting. Unfed ticks from the shelter grounds comprising 1,800 larvae, 3,100 nymphs, and 85 adults were sorted according to sampling origin, life stage, and sex into 116 pools and screened by 16S rRNA PCR. Nine out of 26 pools of unfed adult ticks were positive for E. canis, with overall infection rate maximum likelihood estimation (MLE) of 4.83 (CI 2.39–8.87). E. canis was detected in three of 12 male pools (MLE 3.22, CI 0.86–8.83) and six of 14 female pools (MLE 6.16, CI 2.59–12.90). No adult pools collected from the shelter grounds were positive. Among 62 unfed nymph pools collected from the shelter, six were infected with E. canis (MLE 0.20, CI 0.08–0.42). No E. canis DNA was detected in any of the larva pools. Our results revealed molecular evidence for transstadial transmission of E. canis by R. sanguineus s.l. both from larva to nymph and from nymph to adult. We found no evidence of transovarial transmission. Ehrlichia canis, Rhipicephalus sanguineus sensu lato, dog, transstadial transmission by R. sanguineus s.l Anaplasmataceae are microorganisms that infect vertebrates and ticks (Dumler 2005) and cause serious disease in hosts worldwide (Nicholson et al. 2010, Dantas-Torres and Otranto 2016). Ehrlichia canis is a tick-borne pathogen that infects dogs in many countries (Sainz et al. 2015), including Turkey (Aktas et al. 2009, 2015). Recently, clinical signs of E. canis infection have been described in humans (Perez et al. 2006, Andrić 2014), suggesting E. canis as a zoonotic agent of canine monocytic ehrlichiosis (CME). Rhipicephalus sanguineus sensu lato is a well-recognized putative vector of pathogenic agents including bacteria, protozoa, nematodes, and viruses affecting dogs and occasionally humans (Dantas-Torres 2010, Dantas-Torres et al. 2012, Dantas-Torres and Otranto 2015, Aktas and Özübek 2017). R. sanguineus s.l. has been recognized as the main tick species for the transmission of E. canis (Groves et al. 1975, Mathew et al. 1996, Bremer et al. 2005, Dantes-Torres et al. 2012, Sainz et al. 2015). Transmission of E. canis by R. sanguineus has been shown under laboratory conditions (Fourie et al. 2013). However, this tick is currently assumed a group (Gray et al. 2013). Molecular phylogenetic analysis revealed the existence of two phylogenetic clades and six different haplotypes (Moraes-Filho et al. 2011). Therefore, there is a consensus that populations of this tick species should be referred to as R. sanguineus s.l. until its taxonomic status is resolved (Dantas-Torres et al. 2017). The findings of an experimental study revealed that one population of R. sanguineus group were competent vectors of E. canis, whereas the other populations were not (Moraes-Filho et al. 2015). CME has been described in Turkey (Aktas 2014, Düzlü et al. 2014, Cetinkaya et al. 2016) but no studies have been reported potential vectors of the disease. This study aimed to examine a possible transstadial route for E. canis transmission by R. sanguineus s.l. under field conditions. Materials and Methods Tick samples used in this study were from a study carried out in Diyarbakır Province (37° 55′ N, 40° 12′ E) of south-eastern Turkey, in a municipal dog shelter in which E. canis infection was reported in dogs (Aktas et al. 2015). A total of 4,985 unfed ticks comprising 85 adults, 3,100 nymphs, and 1,800 larvae were collected from the grounds of the shelter. In addition, eight dogs positive for E. canis by PCR with heavy tick infestation were included in the study. No dog showed clinical signs of disease at the time of inclusion. Throughout May and June 2015, each dog was examined and all ticks were collected and placed in glass vials. Engorged nymphs were incubated for molting to adults, and 225 unfed adults (104 males, 121 females) were obtained (Table 1). The ticks were identified microscopically as R. sanguineus s.l. (Estrada-Peña et al. 2004), and sorted by origin (specific dog or shelter ground), life stage, and sex into 116 pooled samples: 18 larva pools (100 per pool), 62 nymph pools (50 per pool) and 36 adult pools (4–14 per pool). Table 1. Pooled samples of unfed adult Rhipicephalus sanguineus s.l. removed from the Ehrlichia canis-infected dogs as engorged nymphs testing positive for Ehrlichia canis by PCR Animal ID  No. of ticks  No. of pools  Positive pools (%)  MLE (95% CI)  1 AEG1  35  4  0  NAa  2 AEG 7  26  4  1 (25%)  4.06 (0.24–21.11)  3 NO1  21  3  2 (67%)  11.04 (2.31–42.51)  4 NO2  12  1  1 (100%)  NAa  5 NO 3  20  2  2 (100%)  NAa  6 NO 4  17  2  1 (50%)  5.32 (0.39–31.68)  7 NO 5  43  5  0  NAa  8 NO 6  51  5  2 (40%)  4.48 (0.84–15.73)  Total  225  26  9 (35%)  4.83 (2.39–8.87)  Animal ID  No. of ticks  No. of pools  Positive pools (%)  MLE (95% CI)  1 AEG1  35  4  0  NAa  2 AEG 7  26  4  1 (25%)  4.06 (0.24–21.11)  3 NO1  21  3  2 (67%)  11.04 (2.31–42.51)  4 NO2  12  1  1 (100%)  NAa  5 NO 3  20  2  2 (100%)  NAa  6 NO 4  17  2  1 (50%)  5.32 (0.39–31.68)  7 NO 5  43  5  0  NAa  8 NO 6  51  5  2 (40%)  4.48 (0.84–15.73)  Total  225  26  9 (35%)  4.83 (2.39–8.87)  NA, not applicable. aWhen all pools are positive or negative, likelihood methods fail. View Large Genomic DNAs were isolated from 116 tick pools were included in the study. Mitochondrial cytochrome c oxidase 1 (cox1) gene was amplified by PCR (Chitimia et al. 2010). Following cox1 gene PCR, a nested PCR was performed to detect E. canis (Wen et al. 1997). Positive (DNA from E. canis identified by DNA sequencing) and negative (RNase-free water) controls were used. Three products generated from R. sanguineus s.l. pools were sequenced. Also representative amplicons specifically targeting E. canis were sequenced and comparative sequence analyses were performed. Sequence comparison was made by BLASTn. A phylogenetic tree was created using cox1 sequences obtained from tick pools based on a 785 nucleotides in MEGA version 6.0 (Tamura et al. 2013). The evolutionary history was constructed using the maximum likelihood (ML) method based on the Tamura 3-parameter model (Tamura 1992). The percentage of replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates). Maximum likelihood estimation (MLE) method was used to calculate infection rate in tick pools (Biggerstaff 2009). Fischer’s exact test implemented in SPSS 15.00 was used, with P-values < 0.05 considered significant (Daniel and Cross 2013). The study was carried out in compliance with the regulations of animal and welfare issued by the Turkish legislation for the protection of animals (Animal Experiment Ethic Committee: 71156/2009). Results Amplification using species-specific primers for E. canis yielded nested PCR products of the expected size (~390 bp) from positive control samples. Non-template control was PCR-negative. Nine out of 26 pools (35%) representing 225 unfed adult R. sanguineus s.l. from E. canis-infected dogs were found to be infected, with overall infection rate MLE of 4.83 (2.39–8.87). The ticks recovered from two dogs (1, 7) were negative for E. canis DNA, while at least one pool recovered from the remaining six dogs was positive (Table 1). Infection rate MLE varied from 4.06 (0.24–21.11) to 11.04 (2.31–42.51) in the pools. All ticks from dog 5 were positive for E. canis (Table 1). Origin, life stage, and sex of the sampled ticks and frequency of E. canis infection are shown in Table 2. A total of 116 pools, representing 5,210 unfed R. sanguineus s.l. (330 adults, 3,100 nymphs, 1,800 larvae) were screened by the PCR for the presence of bacteria. E. canis was detected in 3 of 12 (25%) male pools (MLE 3.22, CI 0.86–8.83) and 6 of 14 (42.8%) females (MLE 6.16; CI 2.59–12.90) obtained from E. canis-infected dogs. There was no significant difference in infection rates of male and female ticks (P > 0.05). No pool of adult ticks collected from the shelter grounds was positive for E. canis. Of 62 nymph pools collected from the grounds, 6 (9.7%) were infected with E. canis (MLE 0.20, CI 0.08–0.42). The difference in infection rate of adult and nymph stages of ticks was not significant (P > 0.05). E. canis DNA was not detected in the pools consisted from larvae specimens. Table 2. Frequency of Ehrlichia canis in pooled samples of unfed Rhipicephalus sanguineus s.l. collected from dogs (n = 225) and the environment of the dog shelter (n = 4,985) Tick source  No. of ticks  No. of pools  Positive pools (%)  MLE (95% CI)  P-value  Ticks from dogsa   Male  104  12  3 (25)  3.22 (0.86–8.83)  P > 0.05   Female  121  14  6 (42.8)  6.16 (2.59–12.90)  From environment   Male  30  3  0  NAb     Female  55  7  0  NA   Nymph  3,100  62  6 (9.7)  0.20 (0.08–0.42)   Larva  1,800  18  0  NA  Tick source  No. of ticks  No. of pools  Positive pools (%)  MLE (95% CI)  P-value  Ticks from dogsa   Male  104  12  3 (25)  3.22 (0.86–8.83)  P > 0.05   Female  121  14  6 (42.8)  6.16 (2.59–12.90)  From environment   Male  30  3  0  NAb     Female  55  7  0  NA   Nymph  3,100  62  6 (9.7)  0.20 (0.08–0.42)   Larva  1,800  18  0  NA  NA, not applicable. aMolting from the nymph to adult. bWhen all pools are positive or negative, likelihood methods fail. View Large Partial sequence of the 16S rRNA gene determined for E. canis was deposited in the EMBL/GenBank databases (KY847523). BLAST search revealed that the sequence shared 100% identity with E. canis isolate TrKysEcan3 detected in dogs from Turkey (KJ513197) and other corresponding published sequences available in GenBank database. The partial sequences of cox1 gene were also analyzed using BLAST. The cox1 sequences from tick pools shared 99–100% identity with Rhipicephalus sp. I lineage (Hornok et al. 2017). Based on cox1 gene, the phylogenetic tree revealed that our sequences from Turkey clustered with Rhipicephalus sp. I of ‘temperate lineage’ (East Mediterranean clade) (Fig. 1). Fig. 1. View largeDownload slide Phylogenetic tree based on partial (785 bp) cox 1 sequences of R. sanguineus s.l. pools from Turkey and other regions available in GenBank. The evolutionary history was inferred using the maximum likelihood method based on the Tamura 3-parameter model. The percentage of replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates). The three cox 1 sequence of R. sanguineus s.l. obtained in the present study are represented in bold. Fig. 1. View largeDownload slide Phylogenetic tree based on partial (785 bp) cox 1 sequences of R. sanguineus s.l. pools from Turkey and other regions available in GenBank. The evolutionary history was inferred using the maximum likelihood method based on the Tamura 3-parameter model. The percentage of replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates). The three cox 1 sequence of R. sanguineus s.l. obtained in the present study are represented in bold. Discussion E. canis is principally transmitted by R. sanguineus s.l. (Bremer et al. 2005, Dumler 2005, Stich et al. 2008, Moraes-Filho et al. 2015). The bacterium was found infecting R. sanguineus s.l. in Asia, Africa, and South America (Unver et al. 2001, Aguiar et al. 2007, Socolovschi et al. 2012, Ybañez et al. 2012). However, mitochondrial 16S rDNA sequences from R. sanguineus s.l. collected from different geographical regions showed the presence of two phylogenetic lineages (temperate and tropical) (Cicuttin et al. 2015, Dantas-Torres and Otranto 2015). Moreover, operational taxonomic units (Rhipicephalus sp. I-IV) have been reported (Dantas-Torres et al. 2013). A recent study underlined several important aspects concerning the systematic status of the R. sanguineus complex (Hekimoğlu et al. 2016). Our three cox1 sequences from tick pools showed 99–100% similarity with Rhipicephalus sp. I of the ‘temperate lineage’ (Hornok et al. 2017), confirming the presence of Rhipicephalus sp. I in Turkey. Phylogenetic analysis of cox1 sequences from new isolates of R. sanguineus s.l. pools in the present study confirmed the existence of Rhipicephalus sp. I. Presence and distribution of this lineage is of special interest because the lineage of R. sanguineus s.l. ticks in the Mediterranean region is believed to be different from that in tropical areas (Nava et al. 2015). Moraes-Filho et al. (2012) experimentally demonstrated differences in the vector competence to transmit E. canis between the tropical and temperate lineages of R. sanguineus s.l. They found that the tropical lineage is competent vector of E. canis but lineage from temperate areas of South America, which are morphologically and molecularly identical to the R. sanguineus s.l. ticks found in the Mediterranean region are not (Moraes-Filho et al. 2012). Unfed adult R. sanguineus s.l. that had fed as nymphs on E. canis-infected dogs were infected with E. canis, indicating that nymphs can become infected with the pathogen when feeding on an infected dog, and that they may transmit the pathogen to moulted adults, suggesting that transstadial transmission occurs from nymph to adult. This is similar to the previous finding that R. sanguineus s.l. nymphs feeding on clinically infected dogs acquired E. canis infection and transmitted it to the adult stage (Stich et al. 2008, Fourie et al. 2013, Moraes-Filho et al. 2015). In this study, at least one tick pool recovered from each of the six E. canis-infected dogs was positive for E. canis DNA. This is consistent with Ramos et al. (2014) report of A. platys DNA in at least one specimen collected from positive dog. In the present study, infection rate MLE in positive pools ranged from 1.43 (dog 4) to 11.04 (dog 7) (Table 1). The finding is consistent with Aguiar et al. (2007) report of E. canis infection rates ranging from 2.3 to 6.2% in R. sanguineus s.l. under field conditions. An experimental study (Moraes-Filho et al. 2015) on E. canis revealed similar findings, with real-time PCR detecting E. canis DNA in 46% of unfed seven day-post-moulting adult R. sanguineus s.l. that had fed as nymphs on E. canis-infected dogs. Tick pools recovered from two E. canis-infected dogs were negative in this study (Table 1). The dogs were asymptomatic, in agreement with the findings of Lewis et al. (1977). The result is concordant with the reports of low amount of circulating pathogen could affect and hindrance the detection leading to a false negative (Kamani et al. 2013, Cicuttin et al. 2014, Almazán et al. 2016, Carvalho et al. 2017). We speculate that the negative findings in tick pools could be due to insufficient concentrations of circulating pathogens in the clinically healthy dogs to allow detection. Recent molecular characterization of R. sanguineus has determined that it consists of a complex of different species that may differ in biological features, including capability to transmit disease agents (Dantas-Torres et al. 2013). All ticks tested in Uruguay were negative (Venzal et al. 2007). A study showed that only tropical species have been found to be infected with E. canis in Argentina (Cicuttin et al. 2015). Recent comprehensive studies on the morphological and genetic diversity of R. sanguineus showed that there are different groups (Rhipicephalus sp. I, and Rhipicephalus sp. IIa, Rhipicephalus sp. IIb) and haplotypes under the ‘temperate lineage’ in Europe, especially Mediterranean countries (Danta-Torres et al. 2013, Hornok et al. 2017, Almeida et al. 2017). Although the sequences obtained in this study were acquired from pools of ticks, we speculate that there may be differences in vector competence for E. canis among populations of R. sanguineus s.l. ticks in Turkey, as exemplified by E. canis and Hepatozoon canis transmission by R. sanguineus s.l. (Venzal et al. 2007, Demoner et al. 2013, Latrofa et al. 2014, Dantas-Torres and Otranto 2015, Moraes-Filho et al. 2015). However, further studies are needed to confirm this assumption. Our observations of unfed nymphs collected from the shelter grounds indicate that E. canis may also be transmitted by this tick from larva to nymph, suggesting that immature stages of the tick are important in the epidemiology of CME, especially in southern Europe, the Mediterranean basin, and the Balkan countries, where larvae and nymphs occur on dogs throughout the year (Lorusso et al. 2010). Ixodid ticks are capable of maintaining tick-borne pathogens in nature through several generations by transovarial passage (Bremer et al. 2005, Dantas-Torres 2007, Fourie et al. 2013). In the present study, 1,800 unfed larvae were screened for E. canis, but no parasite DNA was detected. This may support the long-held assumption that transovarial transmission of E. canis by female R. sanguineus s.l. does not occur. In conclusion, we emphasize that E. canis can be transmitted by nymph and adult stages of R. sanguineus s.l. under field conditions. This suggests that acaricide application should target larvae and nymphs to control CME, as R. sanguineus s.l. is a species of which all parasitic stages feed on dogs. Considering zoonotic relevance of E. canis, it also should reinforce the importance of information to public health authorities. References Cited Aguiar, D. M., Cavalcante G. T., Pinter A., Gennari S. M., Camargo L. M. A., and Labruna M. B.. 2007. Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in dogs and Rhipicephalus sanguineus (Acari: Ixodidae) ticks from Brazil. J. Med. Entomol . 44: 126– 132. Google Scholar CrossRef Search ADS PubMed  Aktas, M. 2014. A survey of ixodid tick species and molecular identification of tick-borne pathogens. Vet. Parasitol . 200: 276– 283. Google Scholar CrossRef Search ADS PubMed  Aktas, M., Altay K., Dumanli N., and Kalkan A.. 2009. Molecular detection and identification of Ehrlichia and Anaplasma species in ixodid ticks. Parasitol. Res . 104: 1243– 1248. Google Scholar CrossRef Search ADS PubMed  Aktas, M., Ozubek S., Altay K., Ipek N. D. S., Balkaya I., Utuk A. E., Kirbas A., Simsek S., and Dumanli N.. 2015. Molecular detection of tick-borne rickettsial and protozoan pathogens in domestic dogs from Turkey. Parasite Vector . 8: 157. Aktas, M., and Özübek S.. 2017. Transstadial transmission of Hepatozoon canis by Rhipicephalus sanguineus (Acari: Ixodidae) in Field Conditions. J. Med. Entomol . 54: 1044– 1048. Google Scholar CrossRef Search ADS PubMed  Almazán C., Gonzalez-Alvarez V. H., de Mera I. G. F., Cabezas-Cruz A., Rodriguez-Martinez R., and de la Fuente J.. 2016. Molecular identification and characterization of Anaplasma platys and Ehrlichia canis in dogs in Mexico. Ticks Tick-Borne Dis . 7: 276– 283. Google Scholar CrossRef Search ADS PubMed  Almeida, C., Simoes R., Coimbra-Dores M. J., Rosa F., and Dias D.. 2017. Mitochondrial DNA analysis of Rhipicephalus sanguineus s.l. from the western Iberian peninsula. Med. Vet. Entomol . 31: 167– 177. Google Scholar CrossRef Search ADS PubMed  Andrić, B. 2014. Diagnostic evaluation of Ehrlichia canis human infections. Open J. Med. Microbiol . 4: 132– 139. Google Scholar CrossRef Search ADS   Biggerstaff, B. J. 2009. PooledInfRate, Version 4.0: a Microsoft® Office Excel© Add-In to compute prevalence estimates from pooled samples . Centers for Disease Control and Prevention, Fort Collins, CO. Bremer, W. G., Schaefer J. J., Wagner E. R., Ewing S. A., Rikihisa Y., Needham G. R., Jittapalapong S., Moore D. L., and Stich R. W.. 2005. Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus. Vet. Parasitol . 131: 95– 105. Carvalho, L., Armua-Fernandez M. T., Sosa N., Felix M. L., and Venzal J. M.. 2017. Anaplasma platys in dogs from Uruguay. Ticks Tick Borne Dis . 8: 241– 245. Google Scholar CrossRef Search ADS PubMed  Cetinkaya, H., Matur E., Akyazi I., Ekiz E. E., Aydin L., and Toparlak M.. 2016. Serological and molecular investigation of Ehrlichia spp. and Anaplasma spp. in ticks and blood of dogs, in the Thrace Region of Turkey. Ticks Tick Borne Dis . 7: 706– 714. Google Scholar CrossRef Search ADS PubMed  Chitimia, L., Lin R. Q., Cosoroaba I., Wu X. Y., Song H. Q., Yuan Z. G., and Zhu X. Q.. 2010. Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox1 and nad5 genes. Exp. Appl. Acarol . 52: 305– 311. Google Scholar CrossRef Search ADS PubMed  Cicuttin, G. L., Brambati D. F., Rodriguez Eugui J. I., Lebrero C. G., De Salvo M. N., Beltran F. J., Gury Dohmen F. E., Jado I., and Anda P.. 2014. Molecular characterization of Rickettsia massiliae and Anaplasma platys infecting Rhipicephalus sanguineus ticks and domestic dogs, Buenos Aires (Argentina). Ticks Tick Borne Dis . 5: 484– 488. Google Scholar CrossRef Search ADS PubMed  Cicuttin, G. L., Tarragona E. L., De Salvo M. N., Mangold A. J., and Nava S.. 2015. Infection with Ehrlichia canis and Anaplasma platys (Rickettsiales: Anaplasmataceae) in two lineages of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) from Argentina. Ticks Tick Borne Dis . 6: 724– 729. Google Scholar CrossRef Search ADS PubMed  Daniel, W. W., and Cross C. L.. 2013. Biostatistics: a foundation for analysis in the health sciences , 10th ed. Wiley, Hoboken, New Jersey, United States of America, 960 pp. Dantas-Torres, F. 2007. Rocky Mountain spotted fever. Lancet. Infect. Dis . 7: 724– 732. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F. 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit. Vectors . 3: 26. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., Chomel B. B., and Otranto D.. 2012. Ticks and tick-borne diseases: a One Health perspective. Trends Parasitol . 28: 437– 446. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., Latrofa M.S., Annoscia G., Giannelli A., Parisi A., and Otranto D.. 2013. Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds. Parasit Vectors  6: 213. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., and Otranto D.. 2015. Further thoughts on the taxonomy and vector role of Rhipicephalus sanguineus group ticks. Vet. Parasitol . 208: 9– 13. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., and Otranto D.. 2016. Best practices for preventing vector-borne diseases in dogs and humans. Trends Parasitol . 32: 43– 55. Google Scholar CrossRef Search ADS PubMed  Dantas-Torres, F., Maia C., Latrofa M. S., Annoscia G., Cardoso L., and Otranto D.. 2017. Genetic characterization of Rhipicephalus sanguineus (sensu lato) ticks from dogs in Portugal. Parasit Vectors  10: 133. Google Scholar CrossRef Search ADS PubMed  Demoner Lde, C., Rubini A. S., Paduan Kdos S., Metzger B., de Paula Antunes J.M., Martins T. F., Mathias M. I., and O’Dwyer L. H.. 2013. Investigation of tick vectors of Hepatozoon canis in Brazil. Ticks Tick Borne Dis . 4: 542– 546. Google Scholar CrossRef Search ADS PubMed  Dumler, J. S. 2005. Anaplasma and Ehrlichia infection. Ann. N. Y. Acad. Sci . 1063: 361– 373. Google Scholar CrossRef Search ADS PubMed  Duzlu, O., Inci A., Yildirim A., Onder Z., and Ciloglu A.. 2014. The investigation of some tick-borne protozoon and rickettsial infections in dogs by Real Time PCR and the molecular characterizations of the detected isolates. Ankara Univ. Vet. Fak . 61: 275– 282. Estrada-Peña, A., Bouattour A., Camicas J. L., and Walker A. R.. 2004. Ticks of domestic animals in the Mediterranean Region. A guide of identification of species . University of Zaragoza Press, Zaragoza, Spain. Fourie, J. J., Stanneck D., Luus H. G., Beugnet F., Wijnveld M., and Jongejan F.. 2013. Transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks feeding on dogs and on artificial membranes. Vet. Parasitol . 197: 595– 603. Google Scholar CrossRef Search ADS PubMed  Gray, J., Dantas-Torres F., Estrada-Peña A., and Levin M.. 2013. Systematics and ecology of the brown dog tick, Rhipicephalus sanguineus. Ticks Tick Borne Dis . 4: 171– 180. Google Scholar CrossRef Search ADS PubMed  Groves, M. G., Dennis G. L., Amyx H. L., and Huxsoll D. L.. 1975. Transmission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus). Am. J. Vet. Res . 36: 937– 940. Google Scholar PubMed  Hekimoğlu, O., Sağlam I. K., Özer N., and Estrada-Peña A.. 2016. New molecular data shed light on the global phylogeny and species limits of the Rhipicephalus sanguineus complex. Ticks Tick Borne Dis . 7: 798– 807. Google Scholar CrossRef Search ADS PubMed  Hornok, S., Sandor A. D., Tomanovic S., Beck R., D’Amico G., Kontschan J., Takacs N., Gorfol T., Bendjeddou M. L., Foldvari G., and Farkas R.. 2017. East and west separation of Rhipicephalus sanguineus mitochondrial lineages in the Mediterranean Basin. Parasit Vectors  10: 39. Google Scholar CrossRef Search ADS PubMed  Kamani, J., Baneth G., Mumcuoglu K. Y., Waziri N. E., Eyal O., Guthmann Y., and Harrus S.. 2013. Molecular detection and characterization of tick-borne pathogens in dogs and ticks from Nigeria. PLoS Negl. Trop. Dis . 7: e2108. Google Scholar CrossRef Search ADS PubMed  Latrofa, M. S., Dantas-Torres F., Giannelli A., and Otranto D.. 2014. Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus group ticks. Ticks Tick Borne Dis . 5: 943– 946. Google Scholar CrossRef Search ADS PubMed  Lewis, G. E., Ristic M., Smith R. D., Lincoln T., and Stephenson E. H.. 1977. The brown dog tick Rhipicephalus sanguineus and the dog as experimental hosts of Ehrlichia canis. Am. J. Vet. Res . 38: 953– 1955. Lorusso, V., Dantas-Torres F., Lia R. P., Tarallo V. D., Mencke N., Capelli G., and 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. Google Scholar PubMed  Mathew, J. S., Ewing S. A., Barker R. W., Fox J. C., Dawson J. E., Warner C. K., Murphy G. L., and Kocan K. M.. 1996. Attempted transmission of Ehrlichia canis by Rhipicephalus sanguineus after passage in cell culture. Am. J. Vet. Res . 57: 1594– 1598. Google Scholar PubMed  Moraes-Filho, J., Marcili A., Nieri-Bastos F. A., Richtzenhain L. J., and Labruna M. B.. 2011. Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America. Acta Trop . 117: 51– 55. Google Scholar CrossRef Search ADS PubMed  Moraes-Filho, J., Marcili A., Nieri-Bastos F. A., Richtzenhain L. J., and Labruna M. B.. 2012. Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America. Acta Trop . 117: 51– 55. Google Scholar CrossRef Search ADS   Moraes-Filho, J., Krawczak F. S., Costa F. B., Soares J. F., and Labruna M. B.. 2015. Comparative evaluation of the vector competence of four south american populations of the Rhipicephalus sanguineus group for the bacterium Ehrlichia canis, the agent of Canine Monocytic Ehrlichiosis. Plos One  10: e0139386. Google Scholar CrossRef Search ADS PubMed  Nava, S., Estrada-Pena A., Petney T., Beati L., Labruna M. B., Szabo M. P., Venzal J. M., Mastropaolo M., Mangold A. J., and Guglielmone A. A.. 2015. The taxonomic status of Rhipicephalus sanguineus (Latreille, 1806). Vet. Parasitol . 208: 2– 8. Google Scholar CrossRef Search ADS PubMed  Nicholson, W. L., Allen K. E., McQuiston J. H., Breitschwerdt E. B., and Little S. E.. 2010. The increasing recognition of rickettsial pathogens in dogs and people. Trends Parasitol . 26: 205– 212. Google Scholar CrossRef Search ADS PubMed  Perez, M., Bodor M., Zhang C., Xiong Q., and Rikihisa Y.. 2006. Human infection with Ehrlichia canis accompanied by clinical signs in Venezuela. Ann. N. Y. Acad. Sci . 1078: 110– 117. Google Scholar CrossRef Search ADS PubMed  Ramos, R. A., Latrofa M.S., Giannelli A., Lacasella V., Campbell B. E., Dantas-Torres F., and Otranto D.. 2014. Detection of Anaplasma platys in dogs and Rhipicephalus sanguineus group ticks by a quantitative real-time PCR. Vet. Parasitol . 205: 285– 288. Google Scholar CrossRef Search ADS PubMed  Sainz, A., Roura X., Miro G., Estrada-Pena A., Kohn B., Harrus S., and Solano-Gallego L.. 2015. Guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in Europe. Parasit Vectors  8: 75. Google Scholar CrossRef Search ADS PubMed  Socolovschi, C., Gomez J., Marie J. L., Davoust B., Guigal P. M., Raoult D., and Parola P.. 2012. Ehrlichia canis in Rhipicephalus sanguineus ticks in the Ivory Coast. Ticks Tick Borne Dis . 3: 411– 413. Google Scholar CrossRef Search ADS PubMed  Stich, R. W., Schaefer J. J., Bremer W. G., Needham G. R., and Jittapalapong S.. 2008. Host surveys, ixodid tick biology and transmission scenarios as related to the tick-borne pathogen, Ehrlichia canis. Vet. Parasitol . 158: 256– 273. Google Scholar CrossRef Search ADS PubMed  Tamura, K. 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol. Biol. Evol . 9: 678– 687. Google Scholar PubMed  Tamura, K., Stecher G., Peterson D., Filipski A., and Kumar S.. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Bio. Evol . 30: 2725– 2729. Google Scholar CrossRef Search ADS   Unver, A., Perez M., Orellana N., Huang H., and Rikihisa Y.. 2001. Molecular and antigenic comparison of Ehrlichia canis isolates from dogs, ticks, and a human in Venezuela. J. Clin. Microbiol . 39: 2788– 2793. Google Scholar CrossRef Search ADS PubMed  Venzal, J. M., Estrada-Peña A., Castro O., De Souza C. G., Portillo A., and Oteo J. A.. 2007. Study on seasonal activity in dogs and ehrlichial infectionin Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) from southern Uruguay. Parasitol. Latinoam . 62: 23– 26. Google Scholar CrossRef Search ADS   Wen, B., Rikihisa Y., Mott J. M., Greene R., Kim H. Y., Zhi N., Couto G. C., Unver A., and Bartsch R.. 1997. Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline. J. Clin. Microbiol . 35: 1852– 1855. Google Scholar PubMed  Ybañez, A. P., Perez Z. O., Gabotero S. R., Yandug R. T., Kotaro M., and Inokuma H.. 2012. First molecular detection of Ehrlichia canis and Anaplasma platys in ticks from dogs in Cebu, Philippines. Ticks Tick Borne Dis . 3: 288– 293. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Journal

Journal of Medical EntomologyOxford University Press

Published: Mar 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off