Molecular Identification and Prevalence of Ehrlichia canis and Rhipicephalus sanguineus (Acari: Ixodidae) Infecting Pet Dogs in Wenzhou, China

1College of Animal Science, Wenzhou Vocational College of Science and Technology, Wenzhou 325006, People’s Republic of China 2Wuhan Business University, Wuhan 430056, People’s Republic of China 3Jiangxi Animal Disease Prevention and Control Center, Nanchang 330096, People’s Republic of China 4Qilu Animal Health Products Co., LTD., Jinan 250100, People’s Republic of China 5Agriculturral Service Center of Street Office of Datang affiliated to The People’s Government of Zhuji Municipality, Zhuji 311801, People’s Republic of China 6College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China.


INTRODUCTION
T he Ixodidae ticks are obligate hematophagous ectoparasites, which are distributed globally . These ticks are considered the most important vectors of animal or wild lives lives' pathogens, and the second most important vector (next to mosquitoes) of human pathogens for the transmission of various kinds of tickborne diseases (TBDs) caused by these pathogens (bacteria, viruses and parasites) Papa et al., 2017; O n l i n e

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symptoms of CME in dogs are high fever, depression, lethargy, myalgia, anorexia, lymphadenomegaly, anemia, splenomegaly, and thrombocytopenia (Mircean et al., 2012;Escribano et al., 2017). Human E. canis infection was first isolated from an asymptomatic human in Venezuela in 1996 and detected as clinical ehrlichiosis in Venezuela in 2006 (Perez et al., 1996;Pére et al., 2006). Since then, E. canis has been identified as a causative agent of human ehrlichiosis in Brazil, USA and Costa Rica (Diniz et al., 2007;Nicholson et al., 2010;Bouza-Mora et al., 2017). Previously, 36 tick species from more than 119 tick species were documented and demonstrated to transmit one or more diseases in China (Yu et al., 2015;Zhang et al., 2017;Zhao et al., 2020). The prevalence of E. canis infection in dogs was found to be 2.04%-40% in some regions in China . However, until now, there is very little information about the tick species and E. canis infection in pet dogs in Wenzhou (northern latitude: 27°3´-28°36´; east longitude: 119°37´-121°18´), which is situated to the southeast of the Zhejiang province in China. The present study was undertaken to determine the prevalence and molecular identification of Ehrlichia canis and the vector hipicephalus sanguineus in pet dogs in Wenzhou, China.

Ethics statement
Samples were obtained after due permission from the relevant institutions. All procedures were performed under the instructions and approval of the Laboratory Animals Research Centre of Zhejiang province in China and the Ethics Committee of the Wenzhou Vocational College of Science and Technology.

Serum samples and ticks collection
A total of 309 blood samples were collected from pet dogs (male 173, female 136) from the animal hospitals in Wenzhou, China during 2017-2018. The gender and age information were recorded in detail. Each blood sample was centrifuged at 1000×g for 15 min, and serum was separated and stored at -70°C for further analysis. Ticks were obtained from pet dogs without obvious symptoms visiting the University Veterinary Hospital, and ticks were washed by double distilled water. Then these ticks were identified morphologically by using a light microscope (Lu et al., 2017). Ticks from the same dogs were pooled together. All the ticks were stored at -70°C until further testing.

Determination of antibodies against E. canis
Serums from pet dogs were examined for antibodies against E. canis by commercial rapid in-clinic ELISA (enzyme-linked immunosorbent assay) kits (SNAP® 4Dx®, IDEXX Laboratories, Westbrook, Maine, USA), according to the manufacturer's instructions. Each serum sample (150 µL) was mixed with 200 µL of the testing reagent and loaded into the device detection well.

DNA isolation, amplification and sequencing
The pooled adult ticks were washed thrice using distilled water and then cut into pieces using sterilized scissors, as reported previously (Licari et al., 2017). The genomic DNA (gDNA) of ticks was prepared by using a DNA extraction reagent kit (TIANamp Genomic DNA Kit, Tiangen Biotech CO., LTD, Wuhan, China) according to the manufacturer's instructions. The eluted DNA was kept at -20°C prior to PCR analysis.

Sequence alignment and phylogenetic analysis
Multiple alignments were performed for the COI and dsb sequences relative to reference sequences available at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) by DNAMAN (5.2.9 Demo version). The phylogenetic analysis was conducted by the NJ (neighbor-joining) algorithm to determine the ticks and rickettsial species, respectively, using the MEGA (version 6.0) software, and the distances were computed using the Tajima-Nei means. The stability of branches was assessed after bootstrapping with 1000 replicates.

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Statistical analysis Statistical analysis was performed by employing the chi-square test with the IBM SPSS (Statistical Analysis System, Version 20.0.) software. The differences were considered significant for P < 0.05.

RESULTS
In the current study, 1.29% (4/309) serum samples were found to be positive for E. canis (Table I). The seroprevalence was 1.73% and 0.74% in male and female pet dogs, respectively, with no obvious difference in the two genders (p = 0.441, i.e. > 0.05) (Fig. 1). The prevalence was 2.80% and 1.41% in ≤ 1 and 1-3-year-old animals, respectively, with no clear difference among the dogs of the different ages (p = 0.539, i.e. > 0.05) ( Table I). A total of 17 out of 309 (5.5%) dogs were found to be infected by ticks (Table I). The prevalence was 5.20% and 5.88% in male and female dogs, respectively. The prevalence was 9.35% and 6.86% in dogs ≤ 1 and ≥ 5 years of age, respectively. The prevalence of tick infection was significantly higher in dogs with an age ≤ 1 year (p < 0.01) and < 5 years (p < 0.05) than in dogs with an age > 1 year and ≤ 3 years, respectively (Table I). There were noteworthy differences between the prevalence of R. sanguineus and E. canis infection in female dogs (p < 0.05). Difference between the prevalence of R. sanguineus and E. canis infection in dogs with an age ≤ 1 year (p < 0.05) and > 5 years (p < 0.01) was found to be significant (Table I).
COI genes of 17 pooled tick samples and dsb genes from only one sample were amplified. For testing dogs whose tick pool samples were not analyzed for dsb genes, we utilized the commercial Canine Distemper Virus/ Canine parvovirus Antigen Test Kits (Korea); it was found that these dogs were infected with either CDV or CPV. The COI gene and dsb gene sequences were submitted to the GenBank of NCBI with accession numbers of MG969504-MG969508.
The sequence alignment phylogenetic analysis showed that the tick infecting pet dogs in the Wenzhou region was R. sanguineus (Fig. 1). The Wenzhou isolates had a high homology, i.e. a 99.7%-99.8% homology to one other, while the homology of the present isolates to previously reported isolates was 99.1%-100% (Fig. 1). The rickettsia derived from R. sanguineus in the current study were identified as E. canis via phylogenetic analysis (Fig. 2). The Wenzhou E. canis isolate presented a high similarity of 98.7%-99.7% to the previously published isolates (Fig. 2).
The available reference sequences of COI are shown in Figure 1. The reference sequences of dsb are shown in Figure 2. Fig. 1. Phylogenetic tree for R. sanguineus based on partial COI gene sequences. Phylogenetic tree based on partial COI gene sequences of R. sanguineus was constructed using the neighbor-joining method with Kimura two-parameter analysis and bootstrap analysis of 1000 replicates. The numbers on the branches indicate the percentage of replicates that reproduced the topology for each clad. The green rhombi indicate the sequences acquired from the current study (Ascaris suum was used as an outgroup).

DISCUSSION
The importance of the potential distribution of the ticks and their species lays a foundation for possible threat prediction and implementation of suitable epidemiologic strategies using DNAMAN (5.2.9 Demo version) (Campos-Calderón et al., 2016). The diversity of ticks is mainly affected by biotic (vegetation, host) and abiotic (climate, temperature) factors (Campos-Calderón et al., 2016). Therefore, it is necessary to monitor tick infections in pet dogs from the Wenzhou region with an average temperature of 17.3-19.4°C and rainfall of 1113-2494 mm. In the current research, it was seen that the prevalence of tick infection in dogs was 5.50%. The molecular characterization of parasites is crucial for understanding the epidemiology of their infections and their control . Previously, genomic regions such as COI, COII, NADH dehydrogenase subunit 1, and the internal transcribed spacers of nuclear ribosomes have been used for phylogenetic studies of different parasitic species (Li et al., , 2017. In the present study, R. sanguineus, with a high homology to previously reported isolates with regards to the COI gene was detected (Fig. 1). This is consistent with results from a previous study, where R. sanguineus was reported in an imported dog (Wright et al., 2018).
E. canis causes CME, which severely affects domestic dogs (Montenegro et al., 2017). In the present study, the serum prevalence of E. canis infection in dogs was 1.29%, which is in line with its prevalence in dogs (1.3%) in south-eastern China . However, the current prevalence of infection was notably lower than the prevalence of E. canis infection in dogs from Italy (7.6%), Thailand (9.88%), and Costa Rica (38.2%) (Montenegro et al., 2017;Piantedosi et al., 2017;Kaewmongkol et al., 2017). This difference may be because of the difference in climate, temperature, and host abundance (Campbell-Lendrum et al., 2015). To confirm E. canis infection, we amplified the dsb genes of E. canis from the pooled tick samples; we obtained one E. canis sequence, which had a high similarity of 98.7%-99.7% to previously reported isolates, as revealed by the analysis of pair-wise distance of the COI gene from R. sanguineus (Fig. 2).
A previous study indicated that dogs in subtropical climates may be at higher risk for infection by R. sanguineus (Aktas et al., 2017). The current study found that R. sanguineus infects pet dogs in Wenzhou, which has a subtropical climate, contributing to the spread of ticks. From the present results, we can demonstrate that these ticks may pose a threat to public health, as they carry zoonotic TBD pathogens such as E. canis. Therefore, efficient precautions should be taken against the potential threat posed by the ticks, especially to pet owners.

O n l i n e F i r s t A r t i c l e
Prevalence of Rhipicephalus Sanguineus in Pet Drogs