Molecular Analysis of Canine Filaria and Its Wolbachia Endosymbionts in Domestic Dogs Collected from Two Animal University Hospitals in Bangkok Metropolitan Region, Thailand.

Hathaithip Satjawongvanit,Sonthaya Tiawsirisup,Padet Siriyasatien,Narisa Brownell,Kanok Preativatanyou,Sivapong Sungpradit,Atchara Phumee

doi:10.3390/pathogens8030114

Hathaithip Satjawongvanit, Sonthaya Tiawsirisup + Show 5 more

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https://doi.org/10.3390/pathogens8030114

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Journal: Pathogens	Publication Date: Jul 29, 2019
Citations: 34	License type: CC BY 4.0

Affiliation: Chulalongkorn University, Mahidol University

Abstract

Canine filariasis is caused by several nematode species, such as Dirofilaria immitis, Dirofilaria repens, Brugia pahangi, Brugia malayi, and Acanthocheilonema reconditum. Zoonotic filariasis is one of the world’s neglected tropical diseases. Since 2000, the World Health Organization (WHO) has promoted a global filarial eradication program to eliminate filariasis by 2020. Apart from vector control strategies, the infection control of reservoir hosts is necessary for more effective filariasis control. In addition, many studies have reported that Wolbachia is necessary for the development, reproduction, and survival of the filarial nematode. Consequently, the use of antibiotics to kill Wolbachia in nematodes has now become an alternative strategy to control filariasis. Previously, a case of subconjunctival dirofilariasis caused by Dirofilaria spp. has been reported in a woman who resides in the center of Bangkok, Thailand. Therefore, our study aimed to principally demonstrate the presence of filarial nematodes and Wolbachia bacteria in blood collected from domestic dogs from the Bangkok Metropolitan Region, Thailand. A total of 57 blood samples from dogs with suspected dirofilariasis who had visited veterinary clinics in Bangkok were collected. The investigations for the presence of microfilaria were carried out by using both microscopic and molecular examinations. PCR was used as the molecular detection method for the filarial nematodes based on the COI and ITS1 regions. The demonstration of Wolbachia was performed using PCR to amplify the FtsZ gene. All positive samples by PCR were then cloned and sequenced. The results showed that the filarial nematodes were detected in 16 samples (28.07%) using microscopic examinations. The molecular detection of filarial species using COI-PCR revealed that 50 samples (87.72%) were positive; these consisted of 33 (57.89%), 13 (22.81%), and 4 (7.02%) samples for D. immitis, B. pahangi, and B. malayi, respectively. While the ITS1-PCR showed that 41 samples (71.93%) were positive—30 samples (52.63%) were identified as containing D. immitis and 11 samples (19.30%) were identified to have B. pahangi, whereas B. malayi was not detected. Forty-seven samples (82.45%) were positive for Wolbachia DNA and the phylogenetic tree of all positive Wolbachia was classified into the supergroup C clade. This study has established fundamental data on filariasis associated with Wolbachia infection in domestic dogs in the Bangkok Metropolitan Region. An extensive survey of dog blood samples would provide valuable epidemiologic data on potential zoonotic filariasis in Thailand. In addition, this information could be used for the future development of more effective prevention and control strategies for canine filariasis in Thailand.

Highlights

Filariasis is a mosquito-borne parasitic disease, which is an important public health concern in tropical and subtropical areas
The gold standard method for the diagnosis of microfilariae in a blood smear is microscopic examination on glass slides stained with Giemsa or hematoxylin and eosin [29,30], this technique requires considerable expertise, has low sensitivity, and cannot clearly discriminate among closely related species of filarial nematodes, such as D. immitis, D. repens, and D. reconditum or B. malayi and B. pahangi [31]
We found that D. immitis is the most frequent species found in dog blood samples using both c oxidase subunit I (COI)-Polymerase chain reaction (PCR) (57.89%) and ITS1-PCR (52.63%)

Summary

Introduction

Filariasis is a mosquito-borne parasitic disease, which is an important public health concern in tropical and subtropical areas. It affects approximately 886 million people in 52 countries worldwide [1]. Several major canine filarial species have been documented in several localities around the world, including Dirofilaria immitis, Dirofilaria repens, Brugia malayi, Brugia pahangi, Brugia ceylonensis, Brugia patei, Cercopithifilaria grassii, Acanthocheilonema reconditum, and Acanthocheilonema dracunculoides [4,5,6]. In Thailand, the common filarial worms in dogs are D. immitis, B. malayi, B. pahangi, and A. reconditum [7,8,9]. D. immitis is the most common species; it causes canine heartworm disease or canine dirofilariasis and is widespread. Molecular analysis of the sample obtained from the eye of the patient showed that it was caused by an unknown Dirofilaria spp. closely related to Dirofilaria hongkongensis [17]

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