Prevalence of Dirofilaria immitis infection in dogs in Henan province, central China

The heartworm Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariosis in dogs and cats, and also infects humans. However, there has been no study on dirofilariasis in dogs in central China. From March 2015 to February 2016, sera from 1176 randomly selected household dogs from Henan province, central China were examined for D. immitis antigen using the Canine Heartworm Antigen Test Kit. The overall seroprevalence of D. immitis in dogs in Henan province was 13% (155/1176). The prevalence was significantly higher in older dogs and dogs kept outdoors, compared to the younger ones and those sheltered indoors. No significant difference of prevalence was observed between sexes. The results suggest that the risk of exposure to D. immitis in dogs is high in Henan, and prophylaxis against the parasite is advisable to decrease the incidence of canine dirofilariosis in this region.


Introduction
The causative agent of cardiopulmonary dirofilariasis, Dirofilaria immitis (heartworm), affects domestic dogs, cats, and various wild mammals, with increasing incidence in temperate and tropical areas [9,12,16]. As mosquito-borne zoonotic pathogens, heartworms can also be transmitted to humans, where they cause diseases such as pulmonary dirofilariasis and subcutaneous dirofilariasis [5,19]. Adult D. immitis worms can survive for seven years or more in dogs, usually producing chronic inflammatory vascular disease [19].
In addition, the simultaneous death of groups of adult worms can trigger an acute disorder characterized by the exacerbation of inflammatory reactions and the occurrence of serious thromboembolism that put the life of the infected dogs at immediate risk [14].
There are many reports on the prevalence of D. immitis infection in dogs from various countries, including Korea [3], Iran [10], Poland [11], Portugal [1], Costa Rica [18], and Hungary [2]. In recent years, studies of D. immitis infection in dogs have been undertaken in different regions of China (Table 1 [6-9, 13, 20-22] Adult worms of D. immitis reside in pulmonary arteries and the right ventricles, resulting in production of blood-circulating microfilariae in dogs as natural hosts [12]. Because dogs with a low worm burden are usually asymptomatic, primary diagnostic screening by detecting blood microfilariae or circulating heartworm antigens is necessary prior to treatment [17]. However, due to occult infection in some cases, antigen testing is considered the most sensitive diagnostic method [17]. Therefore, in the present study, we studied the seroprevalence of D. immitis infection in domestic dogs in central China for the first time and evaluated the main risk factors associated with exposure to D. immitis in this area.

Ethical statement
The study was reviewed and approved by the Ethics Review Committee of the Xinxiang Medical University (Reference No. 2015016).

Study site
The study was conducted in Henan province, located in the central part of mainland China (Fig. 1), and covering an area of 167,000 km 2 and a population of approximately 106.01 million. Its geographical position is at east longitude 110°21 0 -116°39 0 and at north latitude 31°23 0 -36°22 0 . The Yellow River passes through central Henan. The area has a continental monsoon climate, with four distinctive seasons. The average annual temperature is 12.1-15.7°C, with a mean annual rainfall of 532.5-1380.6 mm. As shown in Figure 1, there are 17 provincial cities distributed in Henan province, with the city of Zhengzhou as its capital. Five cities , located in the northern, western, central, southern, and eastern parts of Henan province, were selected for sample collection.

Sample collection
A total of 1176 blood samples of domestic dogs were collected from these five cities in Henan province between March 2015 and February 2016. Dog owners were asked for details of the animals' age, sex, rearing condition, and medical history using a structured questionnaire. Blood samples were centrifuged and sera were recovered and transferred to 1.5 mL Eppendorf tubes. All sera were then stored at À80°C until testing for circulating D. immitis antigens.

Test for D. immitis antigens
In order to identify D. immitis infection, all serum samples were analyzed for the D. immitis antigens using the Canine Heartworm Antigen Test Kit (IDEXX Laboratories, Westbrook, ME, USA) according to the manufacturer's instructions. When testing samples from dogs with more than two adult female worms, sensitivities and specificities of this kit are 94% and 98% for D. immitis, respectively [4].

Statistical analysis
Statistical analysis was performed using SPSS 20 software for Windows (SPSS Inc, Chicago, IL, USA). Statistical analyses of D. immitis prevalence in different variables were performed by the v 2 -test. The differences were considered statistically significant if p < 0.05.
A significant difference was observed in prevalence of D. immitis between dogs sheltered in different conditions [9,13]. In this study, the seroprevalence of D. immitis infection in outdoor dogs (16.32%) was significantly higher than that in indoor dogs (9.54%; p < 0.01). A similar higher prevalence was also observed in outdoor dogs by Hou et al. [9] and Liu et al. [13]. The possible reason is that dogs outdoors had a greater chance of being bitten by mosquitoes [9,13]. In the present study, the highest prevalence of infection (22.11%) was detected in six-year-old or older dogs, followed by intermediate prevalence (12.22%) in the 3-6 year age group, while the prevalence found in dogs in the 3 year age group was 6.92% ( Table 2). The prevalence of D. immitis infection in dogs increased significantly (p < 0.05) with the increase in age. The difference in prevalence with respect to age coincides with other studies where higher prevalence was observed in the adult group than in the juvenile one [10,13,15]. These findings suggest that the risk of exposure to D. immitis increases with age.

Conclusions
In conclusion, a high prevalence of D. immitis infection (13%) was found in domestic dogs in Henan, central China. Therefore, lifelong chemoprophylaxis is needed to prevent canine dirofilariosis. Monitoring the prevalence of this particular nematode among domesticated dogs is also important because it poses a serious health risk to humans. Values bearing a different superscript letter (a-c) within a column differ significantly from one another (p < 0.05).