H. canis has long been recognized to cause disease in dogs in all over the world [26, 27]. H. Canis infection was first 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 amplification and DNA sequencing to recognize the species of Hepatozoon in dogs and ticks feeding on the dogs. This is the first 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 identified 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) in northeast of Iran using molecular analysis found H. canis in 8% of the dogs investigated. Climatic difference can justify the difference in prevalence in the two regions. Different occurrence rates of Hepatozoon infection in dogs have also been reported in other countries. Using PCR, the prevalence of H. canis infection in dogs has been reported at 9.3% in Haiti [40], 0.7% in the United States [41], 31.8% in Colombia [32], 53.3–79.2% in Brazil [8, 13, 42], 3.6– 25.8% in Turkey [19, 33, 43], 5.5% in Palestine [44], 49.7% in India [31], 42.9% in Japan [35], 36.0% in Thailand [30], 20.3% in Nigeria [45], 57.8% in Italy [36], 11.8% in Croatia [46], 50.0% in Czech Republic [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 first 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 [49–52]. 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 significantly 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 significantly related to age of the sampled dogs. Another study in Brazil by de Miranda et al. (2014) showed the influence of gender and age on frequency of Hepatozoon infection.
The results of this study showed a correlation between presence of H. canis DNA with the presence of R. sanguineus in dogs (P < 0.05). The results obtained during this study are in agreement with the previous studies and all report that Hepatozoon infection has positive correlation with tick’s presence [53, 56, 57]. It was also shown that H. canis infection in stray dogs (15.53%) assessed by PCR was significantly higher compared to pet and shelter dogs which were 4.54% and 5.05%, respectively. This study proved the relationship between the prevalence of H. canis and the dogs’ lifestyle. This finding is in line with that of Soltani and Dalimi (2018).
A phylogenetic tree based on 18S rRNA gene in the present study showed that the H. canis sequences obtained in this study from both the ticks and dogs (with accession numbers MT810118 and MT810115) clustered with H. canis sequences of dogs from eastern Iran and other countries and it was different from the H. americanum and H. felis clades. This was the first study of the type conducted in ticks of Iran.