First molecular evidence of Hepatozoon canis infection in Rhipicephalus sanguineus (sensu lato) ticks in Iran

Hepatozoon canis is a protozoan that is transmitted by the ixodid ticks. Ingesting the tick or a section of the tick organ which is infected by the mature oocysts containing infectious sporozoite is the main source of infection in dogs. Canine hepatozoonosis infects dogs in Iran, but the vector for Hepatozoon protozoa from Iran has never been demonstrated. The present study aims to detect H. canis in dogs and vector ticks in Iran.


Background
Canine hepatozoonosis is a tick-borne disease of domestic and wild canids that is caused by Hepatozoon canis and Hepatozoon americanum [1]. The rst report of hepatozoonsis in canids caused by H. canis was in India in 1905 and then it was found in other countries [2]. But H. americanum was rst noticed predominantly in the southern United States as distinct specie [1]. H. canis causes subclinical infection whereas the Canine hepatozoonosis caused by H. americanum can be fatal in some cases [3][4][5]. Canine hepatozoonosis has been seen in different countries of Asia, Europe, Latin America, and Africa [6].
Dogs are mainly infected by ingesting infected ticks. In the vector, infective oocysts of parasite which has numerous sporocysts with sporozoites was produced in the hemocoel [7]. Among various vectors, Rhipicephalus sanguineus is regarded as the main biological vector for H. canis, but other species can also be considered as vectors. Several de nitive hosts have been approved for H. canis among which one can name Rhipicephalus microplus, Haemaphysalis ava, Amblyomma ovale, and Haemaphysalis longicornis ticks ( The diagnosis of H. canis is conducted by microscopic analysis of blood smears stained with Giemsa and it leads to the recognition of H. canis gamonts in the neutrophil's cytoplasm and in some rare cases, it is used to identify in monocytes [12]. Various epidemiological examinations of hepatozoonosis in canids have used different serological tests like IFAT, western blot and ELISA [1]. Recent studies have shown that PCR and sequence analysis are providing accurate information regarding the prevalence of protozoan by detecting the DNA of H. canis in both the peripheral blood of infected dogs and in infected ticks [1,13].
The recent parasitological, and molecular surveys have revealed H. canis infection in stray dogs from the northeastern and central of Iran [14,15], but the tick vectors were not determined. In the current study, we used 3 different diagnostic methods, microscopic, serologic, and molecular techniques to investigate the occurrence of H. canis in dogs, and from the Ixodid ticks removed from the dogs of Urmia, Northwest of Iran.

Study Area
The study was conducted from 2018 to 2019 in Urmia, West Azerbaijan province located in northwestern Iran (37º 32´ N and 45º 04´ E). The study area was approximately 8000 km 2 (Fig. 1). The study area has witnessed varying temperatures in different seasons in the range of -3.8 to +23.4° C. The area is located on the borders of Turkey and Iraq [16].
Sampling 240 dogs (103 stray, 99 shelter, and 44 pets) were randomly selected. The data regarding the sex, age, tick burden and dog's lifestyle were recorded. Blood samples were collected from the cephalic vein of dogs and placed into EDTA and serum tubes. Furthermore, blood smears were prepared from each blood sample. The whole skin of the dogs was carefully examined for ticks and the collected ticks were kept in 70% ethanol. The obtained samples were maintained under cool conditions prior to being sent to the laboratory of parasitology, Urmia university. Sera were separated by centrifuging the serum tubes. The tubes of sera and blood EDTA were stored at -20° C prior to molecular and serological examinations.

Microscopical Examination
Stained thin blood smears with 10% Giemsa solution were analyzed to nd Hepatozoon spp. gamonts. Parasitemia was calculated by counting the number of infected neutrophils in at least 50 examined microscopic elds of vision [14].
Identi cation of Ticks 141 ticks (84 fed males, 26 fully engorged females, and 31 partially engorged females) were obtained from 99 dogs sampled. Then, the ticks were kept in vials with mesh cover which had plaster bottoms.
Morphological features of the ticks were used for the identi cation of their specimen which was formerly explained by Walker et al. and Estrada-Pena et al. [17,18]. Feeding status and the sex (fed males, semiengorged females, fully engorged females, unfed males) were the criteria used for classi cation of the ticks. Table 3 presents detailed information about the ticks used in the current study.

Serology for H. canis
The indirect uorescent antibody test (IFAT) was used to determine the serology of H. canis [19]. Blood samples were obtained from dogs and allowed to clot. subsequently, they were centrifuged for the separation of the serum. The obtained serum samples were stored at -20 °C until use. Hepatozoon spp. gametocyte antigen for IFAT was prepared by collecting 40 ml of heparinized peripheral blood from an infected dog with 18.5% parasitemia. The method described by Baneth et al. was used to separate white blood cells on Ficoll-Hypaque density gradients [20]. The nal pellet was resuspended in PBS containing 1.5% bovine serum albumin (BSA), and thin blood smears were prepared on glass slides by the method of Baneth et al. (1998). Sera were diluted at 1:32 as the cut off titer for IgG seropositivity determined earlier [21,22]. Rabbit-antidog IgG uorescein conjugate (Sigma-Aldrich, St Louis, MO, USA) was used at 1:100 dilution [19].

DNA extraction, PCR and Sequencing
DNA from the dog's blood and ticks were extracted using a commercial kit [Molecular Biological System Transfer (MBST), Tehran, Iran], following the manufacturer's instructions and kept at −20 • C until use. A pair of primers, HepF (5´-ATACATGAGCAAAATCTCAAC-3´) and HepR (5´-CTTATTCCATGCTGCAG-3´), were used to amplify a 660 bp fragment of the 18S rRNA gene of Hepatozoon species in dog blood samples and tick's specimen. The primer's speci city and sensitivity and the PCR condition have been described previously by Inokuma et al. (2002). PCR was carried out in 20 μl total reaction volume containing 1 μl (10 pmol) of each primer and 1 μl of extracted DNA, and 16 μl of distilled water were added to a Reddy® to use PCR master mix (Sinaclon Co., Tehran, Iran, Cat. No.: MM2061) with a nal concentration of 250 μM of each dNTP in 10 mMTris-HCl, pH 9.0, 30 mM KCl, 1.5 mM MgCl2, and 1 U Taq DNA polymerase. Cycling condition for Hepatozoon was 95°C for 5 min, followed by 34 cycles at 95°C for 30 sec, 72°C for 5 min and 72°C for 5 min with a nal extension step of 72°C for 10 min. The PCR products were separated by electrophoresis on 1% agarose gel in TAE (Tris-acetate-EDTA) buffer and visualized using ethidium bromide and UV illumination [14]. The positive control for H. canis was provided from faculty of Veterinary Medicine, University of Tehran, Iran. Distilled water used as negative control in PCR ampli cation.
To con rm the results and identify species, positive PCR products detected in dogs and ticks were puri ed and subjected to sequencing with both forward and reverse primers by Sinaclon Co., Tehran, Iran. The sequencing results were analysed using MEGA 6 software and compared with each other and the reference sequence in the GenBank database by Basic Local Alignment Search Tool (BLAST) analysis (www.ncbi.nlm.nih.gov). The phylogenetic tree was constructed using MEGA 6 software based on the maximum likelihood method based on the Tamura-Nei model [23], with bootstrap 1,000 (1,000 replicates). The nucleotide sequences of Babesia canis were used as an outgroup (Fig. 2).

Statistical Analysis
The results were analyzed by SPSS software (ver. 21.0). The Chi-square test was used to analyze the relationship between several risk factors including sex, age, and tick burden with the rate of infection in dogs and the values less than 0.05 was considered signi cant (P<0.05). Cohen's Kappa test was used to evaluate the agreement of three diagnostic methods and the values obtained for Kappa were categorized as low (0.2 < ĸ < 0.4), moderate (0.4 < ĸ < 0.6), good (0.6 < ĸ < 0.8), or excellent (ĸ > 0.8) [24]. The method proposed by Kahl et al. was used to analyze the prevalence of infestation, the mean density, and the intensity of infestation of adult ticks on dogs [25].

Results
The results of the study showed that the gamonts of Hepatozoon spp. were found by microscopic methods in 5 out of 246 (2.03%) blood smears of dogs and the percentage of neutrophils infected with gamonts was between 0.001 and 0.003 (Fig. 2). IFAT revealed the seropositive reaction in 31 dogs (12.6%). The results of PCR also showed the infection of 9.34% (23/246) dogs with H. canis (Fig. 3).
A comparison of results obtained by PCR and IFAT indicated that 21 (67.74%) IFAT-positive sample were also positive by PCR (Table 1). While 10 (32.25%) samples were positive only by IFAT and 2 (9.30%) samples were positive only by PCR. The rate of Hepatozoon positive dogs determined by microscopy, PCR and IFAT differed signi cantly between the three tests (P < 0.05). A poor agreement (ĸ values,0.252 and 0.335) with a highly signi cant difference was observed between microscopic examination with IFAT and PCR methods (P = 0.000), whereas a good agreement (ĸ values, 0.751) with a highly signi cant difference was observed between IFAT and PCR (P = 0.000) ( Table 1).    Table 2, the prevalence of H. canis is the highest in stray dogs and the statistical analysis revealed signi cant difference between them (P < 0.05).  (Fig .3).
The partial sequences of the 18S rRNA gene for H. canis which was isolated in this study were submitted to GenBank database and they are recorded with MT810118 (dog), and MT810115 (tick) accession numbers. A BLAST search performed with the 18S rRNA gene sequences of Hepatozoon species isolated from dogs and ticks showed 99-100% similarity with the corresponding H. canis isolates deposited in the GenBank database. Phylogenetic analyses revealed that the obtained H. canis sequences clustered in the group with H. canis sequences and sequences were clearly distinct from Hepatozoon americanum, and Hepatozoon felis (Fig. 4).

Discussion
H. canis has long been recognized to cause disease in dogs in all over the world [26,27]. H. Canis infection was rst reported by Khoshnegah et al. (2009) in an eleven-year-old male dog in Iran. They observed H. canis gametocytes inside neutrophils of peripheral blood smears and bone marrow smear in Giemsa stain [28]. Previous studies used molecular and microscopic techniques to investigate the presence of H. Canis in Iran [14,15]. It should be noted that, the vector ticks of Hepatozoon species are completely understood in Iran. This study used serological and molecular survey based on PCR ampli cation and DNA sequencing to recognize the species of Hepatozoon in dogs and ticks feeding on the dogs. This is the rst report of H. canis in dogs and the ticks collected from dogs in northwest of Iran.
In this study, peripheral blood smears were used for identifying Hepatozoon spp. gamonts. H. canis was microscopically detected in only a small number of blood smears (2.03%) of dogs, which could be due to the low sensitivity of microscopic method. Based on the microscopic examination of blood smears, Hepatozoon spp. infection in sampled dogs in the present study (2.03%) is higher than the previous study conducted in Iran (1.6%) [29] which it may be related to the paraseitemia rate in sampled dogs [14]. There were some similar studies conducted in different countries which used microscopic examination of dog's blood smears and they showed Hepatozoon spp. infection in 2.7% of stray dogs in Thailand [30], 2.3% of stray dogs in India [31], 4.3% of dogs in Colombia [32], 1-10.6% of stray dogs in Turkey [19,33], 11.6% of pet and stray dogs in Pakistan [34], 23.7% of hunting dogs in Japan [35], 11.3% of rural dogs in Brazil [13], and 43.9% of young dogs that lived in a shelter located in Italy [36].
The seroprevalance of H. canis infection in this study was 12.6%. The study conducted by Karagenc et al. (2006) delineated that IFAT was able to detect more positive animals to Hepatozoon compared to microscopic methods and PCR. The existing evidence show lower sensitivity of direct observation compared to IFAT, because the investigation of H. canis prevalence by blood smear parasitology was only 1%, while IFAT yielded the result of 33.1% [37]. The absence of gamonts in the animals identi ed as positive with IFAT may be due to low or intermittent parasitemia or arrest of parasite development at the meront stage in visceral organs [3,37,38]. It should be noted that antibodies against Hepatozoon spp. might be present for several months after parasitemia can no longer be detected [21,39].
In the present study, H. canis was found in 9.34% of the dogs investigated with molecular analysis. The study conducted by Barati and Razmi (2018) [47], and 36.0% in Hungary [48]. The differences in reported prevalence might be related to several factors such as the population of tick vector, the methods used for animal management, and the climate [14].
This study is the rst in Iran to record H. canis in R. sanguineus (s.l) ticks from dogs. It should be noted that the previous studies have mentioned R. sanguineus s.l. as the most prevalent specie of tick to infest the dogs in Iran [29], but the vector competence of R. sanguineus s. l. for Hepatozoon protozoa from Iran has never been demonstrated. In the present study, R. sanguineus (s.l.) ticks was the only species found in sampled dogs of the study area which could be related to its huge capability to adapt [49][50][51][52]. The molecular screening of tick specimens collected in the study area reveals the importance of vector R. sanguineus (7/141 ticks positive to H. canis). The percentage of positive ticks in a previously conducted study by Aktas et al. [53] was higher (20.58%) compared to that of ours (4.96%; 7/141). In this study, fed males, partially and fully engorged adult females were the only life stages positive to H. canis. The results of this study are in line with that of Aktas et al. (2013), but it differs with the results reported by study conducted in Italy which showed the highest positivity to H. canis in unfed adults [51].
The frequency of Hepatazoon infection in dogs did not signi cantly vary regardless of age and gender in this study. The results of the study are in agreement with other studies done in the east of Iran [14], Turkey [33], in Japan [35,54], in Thailand [30], in Pakistan [34], and in India [55], but the results were not in line with two other studies. The study conducted by Aktas et al. (2015) in Turkey claimed that the frequency of Hepatozoon infection was signi cantly related to age of the sampled dogs. Another study in Brazil by de Miranda et al. (2014) showed the in uence of gender and age on frequency of Hepatozoon infection.

Conclusions
In conclusion, this study shows the predominance of R. sanguineus (s.l.) in infesting the domestic dogs living in the studied area and it also shows the existence of H. canis in these tick populations. As far as we know, our study is the rst molecular examination of tick vectors of H. canis which has been conducted in Iran. It should be noted that performing transmission evaluations is necessary to con rm the vector capacity of a given tick species. It is also necessary to conduct further studies to identify the epidemiology, and vector ticks of this infection in other parts of Iran.  Phylogenetic analysis of Hepatozoon canis isolates from dogs and ticks in present study and other isolates based on the partial sequence of the 18S rRNA gene in GenBank. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. Babesia canis was used as an outgroup. The phylogenetic tree was plotted by the neighbor joining method with bootstrap 1,000 (1,000 replicates). Evolutionary analyses were conducted in MEGA6.