Abstract Canine tick-borne pathogens are the source of emerging diseases and have important zoonotic relevance. Dogs play a major role in the transmission of several zoonotic tick-borne pathogens, as reservoirs and/or sentinels. To simultaneously detect Anaplasma and Ehrlichia species, a reverse line blot assay was conducted on 219 blood samples collected from autochthonous asymptomatic shelter dogs. One hundred and three (47.0%, CI 40.3–53.9) dogs were positive for one or both rickettsial pathogens. Seventy-one (32.4%, CI 26.3–39.0) dogs were infected with Anaplasma platys and 23 (10.5%, CI 6.8–15.3) with Ehrlichia canis. Concurrent infection with A. platys and E. canis was detected in nine (4.1%, CI 1.9–7.6) dogs. Partial sequences of the 16S ribosomal RNA gene shared 100% identity with the corresponding published sequences for A. platys and E. canis. Infection with Anaplasma phagocytophilum was not detected in the examined dogs. In total, 1018 (range 1–70, mean intensity 13.1, mean abundance 4.6) Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) sensu lato ticks (45.7% nymphs, 54.3% adults) were collected from the dogs. There was no significant association between Anaplasma/Ehrlichia infection and dog sex or age, but a significant correlation was found between rickettsia infection and presence of R. sanguineus. Improved tick control strategies to reduce the risk of these pathogens spreading among dogs and humans are needed in the region. Anaplasma platys, Ehrlichia canis, Rhipicephalus sanguineus sensu lato, dog, Reverse line blot Tick-borne disease is an emerging cosmopolitan problem associated with sub-clinical infections or serious disease in hosts (Nicholson et al. 2010, Dantas-Torres and Otranto 2016). Anaplasmosis and ehrlichiosis are two important tick-borne diseases of dogs caused by a bacterium of the family Anaplasmataceae (Little 2010). Some are particularly significant due to their zoonotic potential because companion animals can be reservoirs for human tick-borne infectious agents (Colwell et al. 2011, Dantas-Torres and Otranto 2015). Anaplasma platys, Anaplasma phagocytophilum, and Ehrlichia canis are the main Anaplasmataceae detected and are associated with acute or non-clinical canine infections in many countries (Little 2010). A. phagocytophilum is the pathogen of human granulocytic anaplasmosis (Bakken and Dumler 2015). A. platys and E. canis are also recognised as emerging human pathogens (Perez et al. 2006, Arraga-Alvarado et al. 2014, Breitschwerdt et al. 2014). A. phagocytophilum is associated with the occurrence of Ixodes ricinus, a common tick species in European countries (Aktas et al. 2010, Estrada-Pena et al. 2013). A. platys and E. canis are transmitted by Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) (Ramos et al. 2014, Moraes-Filho et al. 2015). Apart from a few reports of the occurrence of canine anaplasmosis and ehrlichiosis (Ulutas et al. 2007, Aktas 2014, Aktas et al. 2015, Cetinkaya et al. 2016), information about tick-borne rickettsia in Turkey remains limited. This work aimed to assess the occurrence and frequency of canine rickettsias in dogs from a municipal shelter in the Diyarbakır Province of Turkey and to identify ixodid ticks found on dogs in the region. Materials and Methods In total, 219 dogs from a shelter located in Diyarbakır Province (37° 55′ N, 40° 69 12′ E) of south-eastern Turkey were included in the study. The province has a typical continental climate with extremely hot summers, cold winters, and little precipitation. The mean annual rainfall and temperature are 630 mm and 22.5° C, respectively. Before being taken to the shelter, the animals had freely roamed urban and rural areas and apparently did not receive any tick or flea control treatment. On the basis of behaviour and appearance, the dogs were healthy. Sampling was performed from May to June 2015. The sampled dogs were divided into categories based on sex, age, and presence or absence of tick infestation. Age of the dogs was estimated from body size and teeth (6 mo–1 yr and 1–7 yr). Blood samples (3 ml) were collected in EDTA-tubes and stored at −20°C until processing. Each dog was examined for ticks, with attention paid to the ears, neck, inguinal region, and under the tail. In total, 1018 ticks were removed, and identified following Estrade-Peña et al. (2004). Prevalence and intensity of the tick infestations were estimated (Bush et al. 1997). Genomic DNA was extracted with a QIAamp DNA Blood Minikit (Qiagen, Hilden, Germany) from the collected 219 blood samples. A nested polymerase chain reaction (PCR) was utilized using universal primers with described protocols indicated in Table 1. Nested PCR products were hybridized on the reverse line blot (RLB) membrane. Details of primer pairs and probes are provided in Table 1. PCR was performed as previously described (Aktas et al. 2015). PCR products were electrophoresed in 1.5% agarose gel. DNA from E. canis, A. phagocytophilum, and A. platys, previously detected by RLB and DNA sequencing (GenBank accession nos. KP745632, KP745629, KF038320, respectively), were used as positive controls. RNase-free water and canine genomic DNA from blood of an uninfected dog were used as negative controls. Five microliter of PCR product was electrophorezed, and the remaining products were stored at +4°C until used in the RLB. Table 1. Primers and probes used in this study Primer and probe specificity Nucleotid sequence (5′–3′) Target gene Primer name Product size Reference Primer Anaplasma spp., Ehrlichia spp. First reaction TGATCCTGGCTCAGAACGAACG TACCTTGTTACGACTT 16S rRNA EC12A EC9 1462 Chen et al. (1994) Second reaction GGAATTCAGAGTTGGATCMTGGYTCAG Biotin-CGGGATCCCGAGTTTGCCGGGACTTYTTCT 16S rRNA 16S8FE BGA1B-new 492–498 Bekker et al. (2002) Probe Ehrlichia/Anaplasma catch-all AmMC6-GGGGGAAAGATTTATCGCTA Bekker et al.(2002) Anaplasma/Ehrlichia AmMC6-TTATCGCTATTAGATGAGCC Schouls et al. (1999) E. canis AmMC6-TCTGGCTATAGGAAATTGTTA Schouls et al. (1999) A. platys AmMC6-GATTTTTGTCGTAGCTTGCTATG Anda et al. (2006) A. phagocytophilum 1 AmMC6-TTGCTATAAAGAATAATTAGTGG Schouls et al.(1999) A. phagocytophilum 3 AmMC6-TTGCTATGAAGAATAATTAGTGG Schouls et al.(1999) A. phagocytophilum 5 AmMC6-TTGCTATAAAGAATAGTTAGTGG Schouls et al.(1999) A. phagocytophilum 7 AmMC6-TTGCTATAGAGAATAGTTAGTGG Schouls et al.(1999) A. phagocytophilum A-HGE AmMC6-GCTATAAAGAATAGTTAGTGG Schouls et al.(1999) A. phagocytophilum A-D- HGE AmMC6-GCTATGAAGAATAGTTAGTG Schouls et al.(1999) Primer and probe specificity Nucleotid sequence (5′–3′) Target gene Primer name Product size Reference Primer Anaplasma spp., Ehrlichia spp. First reaction TGATCCTGGCTCAGAACGAACG TACCTTGTTACGACTT 16S rRNA EC12A EC9 1462 Chen et al. (1994) Second reaction GGAATTCAGAGTTGGATCMTGGYTCAG Biotin-CGGGATCCCGAGTTTGCCGGGACTTYTTCT 16S rRNA 16S8FE BGA1B-new 492–498 Bekker et al. (2002) Probe Ehrlichia/Anaplasma catch-all AmMC6-GGGGGAAAGATTTATCGCTA Bekker et al.(2002) Anaplasma/Ehrlichia AmMC6-TTATCGCTATTAGATGAGCC Schouls et al. (1999) E. canis AmMC6-TCTGGCTATAGGAAATTGTTA Schouls et al. (1999) A. platys AmMC6-GATTTTTGTCGTAGCTTGCTATG Anda et al. (2006) A. phagocytophilum 1 AmMC6-TTGCTATAAAGAATAATTAGTGG Schouls et al.(1999) A. phagocytophilum 3 AmMC6-TTGCTATGAAGAATAATTAGTGG Schouls et al.(1999) A. phagocytophilum 5 AmMC6-TTGCTATAAAGAATAGTTAGTGG Schouls et al.(1999) A. phagocytophilum 7 AmMC6-TTGCTATAGAGAATAGTTAGTGG Schouls et al.(1999) A. phagocytophilum A-HGE AmMC6-GCTATAAAGAATAGTTAGTGG Schouls et al.(1999) A. phagocytophilum A-D- HGE AmMC6-GCTATGAAGAATAGTTAGTG Schouls et al.(1999) AmMC6, C6 amino linker linked to the 5′. View Large RLB assay was performed on the nested PCR amplicons as previously reported (Aktas and Ozubek 2015a,b; Aktas et al. 2015). To confirm PCR results, two amplicons comprising A. platys and E. canis were sequenced and comparative sequence analyses were performed. Shortly after, PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen GmbH) by following the manufacturer’s instructions and sequenced by the commercial company. They were manually edited by Chromas-Lite (www.technelysium.com.au). The prevalence and 95% binomial exact CIs were calculated using Sourceforge net (http://sampsize.sourceforge.net/iface). The data obtained from dogs were compared by Pearson chi-square tests using SPSS 15.00 software for correlated proportions. Association of rickettsial pathogens with sex, age, and presence of tick infestation was assessed. P-values ≤0.05 were considered significant. This study was carried out according to the regulations on animal welfare issued by the Turkish legislation for the protection of animals (Animal Experiment Ethics Committee, 71156/2009). Results Amplification using primers 16S8FE/BGA1B-new yielded PCR products of the expected size (~500 bp) from positive control samples. The PCR products obtained from both positive controls and field samples were hybridized and observed for signal to catch all, genus- and species-specific oligonucleotide probes in RLB (Table 1). All positive products (positive control and field samples) produced a signal. Frequencies of single and combined A. paltys and E. canis in dogs relative to sex, age, and presence of tick infestation are shown in Table 2. About 103 (47.0%, CI 40.3–53.9) out of 219 blood samples were positive by the 16S rRNA PCR for Anaplasmataceae. Seventy-one (32.4%, CI 26.3–39.0) and 23 (10.5%, CI 6.8–15.3) dogs were also found positive for A. platys and E. canis, respectively in RLB assay. Co-existence of A. platys and E. canis was detected in nine (4.1%, CI 1.9–7.6) dogs. No dog was positive for A. phagocytophilum. Table 2. Frequency (%), 95% CI (lower, upper intervals) and association of canine tick-borne rickettsial pathogens detected by RLB relative to sex, age, and presence or absence of tick infestation (Rhipicephalus sanguineus sensu lato) Variables Sample Anaplasma/Ehrlichia (%, 95% CI) P-value Identified pathogen Co-infection (%, 95% CI) Single infection (%, 95% CI) A. platys E. canis A. platys + E. canis 219 103 (47.0, 40.3–53.9) 71 (32.4, 26.3–39.0) 23 (10.5, 6.8–15.3) 9 (4.1, 1.9–7.6) Sex Female 109 55 (50.4, 40.7–60.2) > 0.05 39 (35.8, 26.8–45.5) 11 (10.1, 5.1–17.3) 5 (4.6, 1.5–10.4) Male 110 48 (43.6, 34.2–53.4) 32 (29.1, 20.8–38.5) 12 (10.9, 5.8–18.3) 4 (3.6, 1.0–9.0) Age Juvenile (6–12 mo) 69 27 (39.1, 27.6–51.6) > 0.05 16 (23.2, 13.9–34.9) 8 (11.6, 5.1–21.6) 3 (4.3, 0.9–12.2) Adult (1–7 yr) 150 76 (50.7, 42.4–58.9) 55 (36.7, 28.9–44.9) 15 (10, 5.7–15.9) 6 (4, 1.5–8.5) Presence of ticks Absent 141 48 (34.0, 26.3–42.5) < 0.05 33 (23.4, 16.7–31.3) 12 (8.5, 4.5–14.4) 3 (2.1, 0.4–6.1) Present 78 55 (70.5, 59.1–80.3) 38 (48.7, 37.2–60.3) 11 (14.1, 7.2–23.8) 6 (7.7, 2.9–16.0) Variables Sample Anaplasma/Ehrlichia (%, 95% CI) P-value Identified pathogen Co-infection (%, 95% CI) Single infection (%, 95% CI) A. platys E. canis A. platys + E. canis 219 103 (47.0, 40.3–53.9) 71 (32.4, 26.3–39.0) 23 (10.5, 6.8–15.3) 9 (4.1, 1.9–7.6) Sex Female 109 55 (50.4, 40.7–60.2) > 0.05 39 (35.8, 26.8–45.5) 11 (10.1, 5.1–17.3) 5 (4.6, 1.5–10.4) Male 110 48 (43.6, 34.2–53.4) 32 (29.1, 20.8–38.5) 12 (10.9, 5.8–18.3) 4 (3.6, 1.0–9.0) Age Juvenile (6–12 mo) 69 27 (39.1, 27.6–51.6) > 0.05 16 (23.2, 13.9–34.9) 8 (11.6, 5.1–21.6) 3 (4.3, 0.9–12.2) Adult (1–7 yr) 150 76 (50.7, 42.4–58.9) 55 (36.7, 28.9–44.9) 15 (10, 5.7–15.9) 6 (4, 1.5–8.5) Presence of ticks Absent 141 48 (34.0, 26.3–42.5) < 0.05 33 (23.4, 16.7–31.3) 12 (8.5, 4.5–14.4) 3 (2.1, 0.4–6.1) Present 78 55 (70.5, 59.1–80.3) 38 (48.7, 37.2–60.3) 11 (14.1, 7.2–23.8) 6 (7.7, 2.9–16.0) View Large No significant differences were observed in tick-borne pathogen positivity of male and female dogs (P > 0.05). The difference in infection rate of young and adult dogs was also not significant (P > 0.05). Among the 103 PCR-positive dogs, 55 (70.5%, CI 59.1–80.3) were infested with R. sanguineus s.l. (45.7% nymphs, 54.3% adults). Presence of this tick was significantly associated with canine rickettsia infection (P < 0.05). Seventy-eight out of 219 (35.6%) examined dogs carried at least one tick. Abundance (mean number of ticks per dog) and intensity (mean number of ticks per infested dog) were 4.64 and 13.05, respectively. The number of ticks per animal ranged from 1 to 70. All ticks were identified as R. sanguineus s.l. The partial sequences of the 16S rRNA genes determined for A. platys (1389 bp) and E. canis (1380 bp) were deposited in the EMBL/GenBank databases under accession numbers KY594914 and KY594915, respectively. A BLAST search showed the obtained sequences to be 100% identical to the corresponding published sequences for A. platys strain Okinawa 1 from Japan (AF536828) and E. canis isolate TrKysEcan3 from Turkey (KJ513197). Discussion We described here a molecular survey detecting canine tick-borne rickettsial agents and found shelter dogs in Diyarbakır Province, Turkey to be exposed to A. platys and E. canis. Both pathogens from dogs and ticks were previously reported in Turkey (Ulutas et al. 2007, Sen et al. 2011, Aktas 2014). However, a high infection frequency (47.0%, CI 40.3–53.9) was detected compared with previous reports in Turkey of 14.5% (Duzlu et al. 2014), 5.4% (Aktas et al. 2015), and 6% (Çetinkaya et al. 2016), 6.4% in Nigeria (Dahmani et al. 2015), 1.9% in Portugal (Maia et al. 2015), 3.7% in Iran (Maazi et al. 2014), 14.5% in Algeria (Bessas et al. 2016), and 6.5% in India (Abd Rani et al. 2011). The differences in frequency may be related to geographic region, environmental conditions, sample size, sampling periods, or diagnostic methods, as well as to the primary characteristics of the targeted dog population such as outdoor, indoor, shelter, stray, or pet (Inokuma et al. 2006, Volgina et al. 2013, Dahmani et al. 2015, Cetinkaya et al. 2016). High abundance and intensity of R. sanguineus s.l., a vector of A. platys and E. canis (Dantas-Torres 2008, 2010, Ramos et al. 2014, Dantas-Torres and Otranto 2015), might explain the higher positive rate of these pathogens in this study. Probes specific for A. phagocytophilum were included in our study, but no positive signals were obtained by PCR. This result is not surprising, as A. phagocytophilum is transmitted by Ixodes (Silaghi et al. 2012), which has never been reported on domestic animals, including dogs, in the studied area (Aktas et al. 2006; 2013). Similar findings have been reported in a study conducted in Maio Island of Cape Verde, where the only hard tick reported is R. sanguineus s.l. (Lauzi et al. 2016). R. sanguineus is common in the Mediterranean countries. This tick species is associated with shelters such as kennels, gardens, or cracks in walls of human habitations (Gray et al. 2013). The abundance of infestation by R. sanguineus in dogs can vary geographically and seasonally, as well as depending upon the application of acaricides (Dantas-Torres 2010, Lorusso et al. 2010, Gray et al. 2013). In this study, the occurrence of R. sanguineus s.l. confirms a previous report identifying the majority of ticks found on dogs as this species (Aktas et al. 2013). Tick-transmitted infections generally involve multiple pathogens (De Tommasi et al. 2013, Sainz et al. 2015). Reverse line blotting is a powerful and practical tool allowing simultaneous detection of tick-borne pathogens (Schouls et al. 1999, Bekker et al. 2002). Our survey revealed nine dogs (4.1%, CI 1.9–7.6) presenting co-infections with A. platys and E. canis (Table 2). Similar findings have been reported in a previous study in Turkey (Pasa et al. 2009). Co-infection was also reported by Yisaschar-Mekuzas et al. (2013), Aktas et al. (2015), and Aktas and Ozubek (2015b). Our survey found no significant differences in the prevalence of bacteria in adult and young dogs (P > 0.05), or in dogs of different genders (P > 0.05). These results agree with findings that the frequency of infection is not related to sex and age (Santos et al. 2013, Maazi et al. 2014, Dahmani et al. 2015, Lauzi et al. 2016, Maia et al. 2015), suggesting that tick intensity and geographic distribution affect the prevalence of pathogens in all dogs, regardless of sex or age. A correlation between the occurrence of A. platys and E. canis and the presence of R. sanguineus s.l. was observed (P < 0.05), consistent with a survey conducted on dogs in which 155/286 samples showed antibodies reactive to E. canis. Among the seropositive dogs, 58% had ticks at the time of sampling (M’ghirbi et al. 2009). In Sudan, the frequency of E. canis and A. platys was found to be 80.8 and 24.4%, respectively, and R. sanguineus was found on all dogs (Inokuma et al. 2006). On the contrary, in Algeria, frequency of E. canis and A. platys was found to be 6.3% (7/110) and 5.4% (4/110), respectively. The low prevalence was attributed to the fact that the survey was carried out in late winter and early spring, periods of low tick activity (Dahmani et al. 2015). These results suggest that infestation by ticks is a main determinant of tick-borne rickettsial infection rates. E. canis is reported to be transmitted by R. sanguineus s.l. (Moraes-Filho et al. 2015), which is also presumed to be the main vector of A. platys (Sanogo et al. 2003). However, the only study attempting to confirm R. sanguineus as vector of A. platys was unsuccessful (Simpson et al. 1991), possibly due to the low sensitivity of the detection method or the tick strain or species used (Ramos et al. 2014). Further studies of the role of R. sanguineus s.l. as a vector of A. platys and E. canis in field conditions are needed to confirm our results. A. platys and E. canis were detected in apparently healthy dogs from a municipal dog shelter where dogs were in close contact and therefore at an increased risk of exposure to ticks, which, in turn, increases the risk of human infection. The prevalence of the pathogens in dogs in the region is high. Our results reinforce the importance of introducing effective control strategies throughout the country. 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Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: email@example.com.
Journal of Medical Entomology – Oxford University Press
Published: Mar 1, 2018
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