The present study is the first comprehensive study on the detection of TBH in ticks and dogs in Malaysia. The present work investigated the relationship of the tick-borne pathogens in dogs and ticks, the co-infection presence, and phylogenetic analysis of both sequences from dogs and ticks. In general, the present study showed E. canis as predominated TBH in dogs, followed by A. platys, B. gibsoni and B. vogeli. In ticks, there is detection of A. platys, E. canis, B. vogeli and no detection of B. gibsoni.
Previous work on TBH in dogs in Malaysia utilizing microscopical examination of the blood smear, serology, however, recent work have changed the method of detection to molecular method. The used of microscopical examination of the blood smear and serology methods are both lacking in terms of sensitivity. The use of serology method, notably, lacking in differentiating past from on-going infection after antibiotic treatment due to the continued existence of antibodies, thus resulting in false-positive results [1, 48-50]. The availability of these works on TBH provide little epidemiological data in Malaysia as most of the work were limited to small areas in the country. Our study included eight states in Peninsular Malaysia as well as the capital region of Malaysia where the cities in this states have high density of human as well as pets and stray dogs. Stray dogs has an important role of maintaining the TBH disease as a result of being neglected [1, 6]. Animal shelters is a place where strays dogs being rescued and able to represent the bigger picture of the condition of stray dogs and what diseases they carry. Therefore, the current work use molecular methods and a large scale of sampling area in the present study were expected to give a better picture of TBH detection in dogs in Peninsular Malaysia.
E. canis infection was reported to be generally higher than the other TBH observed in current work and Malaysian dogs, which also agree with those reported from other Southeast Asian countries [1, 51 - 52]. For other TBH, previous work have reported varying detection of E. canis (0-55.6%), A. platys (3.3-13.3%), B. gibsoni (0-17.7%) and B. vogeli (0-10%) in dogs in Peninsular Malaysia [16, 32, 34 - 36,39, 40, 54]. Meanwhile, a higher detection of TBH, E. canis (33-56.7%), A. platys (27-38.5%), and Babesia (65.4%) was reported in dogs from East Malaysia, [21,37]. The differences in the prevalence of TBH obtained were most likely due to the number of dogs, geographical area of sampling, selection criteria and target gene [1].
Male dogs more likely to encounter A. platys infection based on our findings and young dogs have higher infection rate of B. vogeli. Some previous studies reported significantly higher haemopathogen infection in young dogs compared to adult dogs [1, 54-55]. The best explanation are young dogs normally prone to have tick infestation with higher burden compared to adult dogs [56]. Other works showed higher infection rates of TBH in female as a result of they are less active than male, which allows high chances of getting infested by ticks and being infected with TBH [57]. However the higher detection in male in current study warrant further investigation or could be due to the population of study were different from other study. Higher detection of TBH were observed from northern region while previous work reported higher detection in the central and eastern regions [35-36, 38]. The previous work were mainly focusing on the central region in capital city of Malaysia where other region were understudied. In relation to management practices, most of the infected samples were from animal shelters that do not practice giving preventive medicine to the dogs residing in their shelter. Treatment was only given to dogs with clinical signs, which as far as tick-borne haemopathogens is concerned; the infection can occur subclinically [16]. Inadequate funding and lack of staff are the reasons that prevent these animal shelters from giving the dogs the medication they need. The southern region animal shelter recorded low detection of TBH as they provides good preventive measures and treatments for their dogs. The animal shelter practices application of dipping bath and doxycycline treatment (10mg/kg) once a day for a month, orally, to new dogs. In addition, dipping bath was conducted once every three weeks to all dogs residing in the shelter. Therefore in our work, management does play a significant role which variation in tick-borne haemopathogen prevalence is related to differences in management practices of different shelters [36].
The low detection of TBH in ticks in present works were alligned with other reports where low detection of the pathogens in the tick vector were found compared to the detection in dogs [1, 58-63]. Climatic conditions play roles in the detection, activity, and survival of the tick vector [38, 64-66]. The climate was considered as one of the leading causes of these tick-borne haemopathogens prevalence changes between regions due to its capability to alter the disease transmission dynamics and their geographical distribution, although other factors such as ecological changes and importation of animals in an area can also contribute to the changes[67-69]. Furthermore, the chance of the pathogen transmission is also relatively high through co-feeding, where the previously uninfected ticks can be infected by blood-feeding in close proximity to an infected tick [70]. The other possible reasons for the low prevalence of TBH in ticks due to the other ticks infected with TBH might already fall off the dog host prior to sample collection. The attachment period of ticks was different in terms of their life stages. Female R. sanguineus (sensu lato) in its adult form, for example, usually blood-feeding ranging from five to 21 days, while nymph blood-feeding for three to 11 days before they drop off to the ground [71]. Approximately 5% of the tick vectors could be found on the dog host, while the rest can be found in the environment [10]. Future studies are recommended to work on the detection of TBH in ticks, which were not only attached to the dog host but also off-host ticks to understand further the true prevalence of the pathogens they could carried.
The co-infection of TBH has been reported in Malaysian dogs [35,37,38]. To the best of our knowledge, there has been no report of A. platys and B. gibsoni co-infection in the literature in Malaysia, thus indicates that this is the first report of the co-infection in the country. The most common co-infection seen in dogs in the present work was E. canis and A. platys co-infection, which agree with the report from other previous studies elsewhere [52, 72-74]. The co-infection of TBH in dogs is common and has been reported in countries worldwide. Co-infection of pathogens in the host warrant proper surveillance due to the possibility of pathophysiological alteration by multiple pathogens, and the difficulty in giving proper treatment to the co-infected dogs [75-77]. Although co-infection of these TBH is possible in ticks [38-39,78], no co-infection of these haemopathogens were observed in ticks in the present study. From the current work, ticks positive for TBH were all the ticks removed from infected dogs which showed the high possibility of the ticks obtaining the infection from the infected dogs, although the ticks could obtained the infection from their previous blood meal [38].
In general, the phylogenetic analysis of the 16S rRNA and 18S rRNA gene of the tick-borne haemopathogens showed that the Malaysian isolates shared high similarity and clustered together with isolates from other Asian countries (Philippines, China, Thailand, Myanmar, Taiwan, Japan, India, and Vietnam.) as well as countries outside Asia (Brazil, Cape Verde, Panama, Italy, Portugal, Venezuela, and Nigeria). These results suggested that the 16S rRNA and 18S rRNA gene of the tick-borne haemopathogens are conserved regions, which have high similarity within species. Although it is only for one sample, the similarity of sequence in A. platys in dogs and ticks most likely suggesting the tick could be a source of infection for A. platys in the dog, and this could be a good indicator in tick-borne disease diagnosis and tracking the source of infection. However, there is no specific pattern or separation between isolates of tick-borne haemopathogens in dogs and ticks might be due to the conserved region of the gene of interest used in present work. A more variable region can be used for detailed phylogenetic analysis, for example, mitochondrial gene.