veterinary sciences Article Investigation on the Prevalence of Canine Microfilaremia in Thailand Using a Novel Microfluidic Device in Combination with Real-Time PCR Sumas Loymek 1, Achinya Phuakrod 2, Kati Zaelai 3, Witsaroot Sripumkhai 4, Prapakorn Vongjaroensanti 3 and Sirichit Wongkamchai 3,* 1 Office of Disease Prevention and Control Region 12 Songkhla, Department of Disease Control, Ministry of Public Health, Songkhla 90000, Thailand; [email protected] 2 Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; [email protected] 3 Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; [email protected] (K.Z.); [email protected] (P.V.) 4 Thai Microelectronic Center, National Electronics and Computer Technology Center, Thailand Science Park, Pathumthani 12120, Thailand; [email protected] * Correspondence: [email protected] Abstract: We conducted a survey of canine microfilaraemia in 768 dogs in Chanthaburi, Samut Sakhon, and Narathiwat provinces of Thailand using a novel semi-automated, microfluidic device that is easy and rapid to perform. Microfilariae species were identified using High Resolution Melting real-time PCR (HRM real-time PCR). The prevalence of canine microfilaremia was 16.2% (45/278) Citation: Loymek, S.; Phuakrod, A.; in Chanthaburi and 5.5% (12/217) in Samut Sakhon. The prevalence of canine microfilaremia Zaelai, K.; Sripumkhai, W.; in Narathiwat was 22.7% (67/273). Brugia pahangi and Dirofilaria immitis were the predominant Vongjaroensanti, P.; Wongkamchai, S. species of filariae found in the infected dogs from Chanthaburi and Narathiwat, respectively. The Investigation on the Prevalence of low prevalence of canine microfilaremia of Samut Sakhon may reflect the success of the Soi Dog Canine Microfilaremia in Thailand Using a Novel Microfluidic Device in foundation’s efforts and the establishment of veterinary control programs. An effective disease Combination with Real-Time PCR. control and prevention strategies is needed in Chanthaburi and Narathiwat to reduce the risks of Vet. Sci. 2021, 8, 39. https://doi.org/ zoonotic transmission of the parasites. An appropriate drug treatment should be given to infected 10.3390/vetsci8030039 dogs and prophylactic drugs are suggested to be given to dogs age ≤1-year-old to prevent filarial infection. The novel microfluidic device could be implemented for surveillance of filariae infection in Academic Editor: other animals. Antonio Frangipane Di Regalbono Keywords: Brugia malayi; Brugia pahangi; Dirofilaria immitis; HRM real time PCR; microfluidic Received: 14 February 2021 Accepted: 23 February 2021 Published: 28 February 2021 1. Introduction Publisher’s Note: MDPI stays neutral Filariasis is a disease caused by nematodes in the Filarioidea superfamily [1,2]. Human with regard to jurisdictional claims in published maps and institutional affil- infections with the filariae of animals, referred to as zoonotic filariasis, occur worldwide. iations. Dirofilaria immitis, Dirofilaria repens, Brugia malayi, and Brugia pahangi have been reported as important zoonotic filariae from dogs and cats [2–6]. In Thailand, human cases of zoonotic filariasis caused by Dirofilaria spp. and B. pahangi have also been reported [7–9]. In another report from Thailand, two cases of brugian filariasis were reported in Rayong province, with suspected zoonotic origin. Brugia pahangi was the infective agent and dogs Copyright: © 2021 by the authors. are suspected sources of the infection [10]. Licensee MDPI, Basel, Switzerland. Filarial infection in dogs in Thailand has been reported for the past 30 years. In 1987, This article is an open access article the high prevalence of canine filarial infection (45.67%) had been reported from Chiang distributed under the terms and conditions of the Creative Commons Mai [11]. A study in Bangkok found a 13.9% filarial infectivity rate in stray dogs [12] Attribution (CC BY) license (https:// whereas Kamyingkird et al. reported a prevalence of 24.1% of D. immitis infection in stray creativecommons.org/licenses/by/ dogs in Songkla and Satun [13]. All of these studies were performed in urban cities such 4.0/). as Bangkok, Chiang-Mai, and Songkhla where Culex spp., which is the main vector of Vet. Sci. 2021, 8, 39. https://doi.org/10.3390/vetsci8030039 https://www.mdpi.com/journal/vetsci Vet. Sci. 2021, 8, 39 2 of 9 Vet. Sci. 2021, 8, 39 2 of 9 dogs in Songkla and Satun [13]. All of these studies were performed in urban cities such as Bangkok, Chiang-Mai, and Songkhla where Culex spp., which is the main vector of Dirofilaria, is a common mosquito species in urban areas that “thrives and proliferates ex- Dirofilariacessively in, is crowed a common city areas” mosquito [14], species D. immitis in urban was the areas only that species “thrives of filariae and proliferates reported in excessivelythese studies. in crowed city areas” [14], D. immitis was the only species of filariae reported in theseClimate studies. change, extended ranges for vectors and infected animal reservoirs, increas- ing urbanization,Climate change, and extended outdoor ranges recreational for vectors activities and infected are among animal the reservoirs, factors increasingthat have urbanization,broadened the and profile outdoor of filariasis recreational-affected activities individuals are among [15]. the The factors awareness that have of the broadened potential thezoonotic profile filarial of filariasis-affected infections, the individualsknowledge [of15 ].the The incidence, awareness and of the the impact potential of zoonoticzoonotic filarialparasite infections,s in animal the populations knowledge are of thecrucial incidence, points andto prevent the impact the ofinfection zoonotic in parasitespeople and in animalother animals populations and to are control crucial the points spread to preventof the filariae. the infection in people and other animals and toIn controlthe present the spread study, of we the conducted filariae. a survey of canine microfilaremia in three rural provincesIn the located present on study, the shore we conducted of the Gulf a surveyof Thailand of canine using microfilaremia a novel semi-automated in three rural mi- provincescrofluidic device located that on is the easy shore and of rapid the Gulfto perform of Thailand in combination using a novel with semi-automatedHigh Resolution microfluidicMelting real- devicetime PCR that (HRM is easy real and-time rapid PCR). to perform in combination with High Resolution Melting real-time PCR (HRM real-time PCR). 2. Materials and Methods 2. Materials and Methods 2.1. Ethics Approval Approval number 008/2562008/2562 was was acquired from the Animal Ethics CommitteeCommittee ofof thethe Faculty of Medicine at Siriraj Hospital, Mahidol University, basedbased onon thethe EthicsEthics ofof AnimalAnimal Experimentation ofof thethe National ResearchResearch CouncilCouncil ofof Thailand.Thailand. DogsDogs werewere examinedexamined withwith the consent ofof theirtheir owner.owner. 2.2. Study Area The study was performed in Chanthaburi, Samut Sakhon,Sakhon, andand NarathiwatNarathiwat provincesprovinces on the shore of thethe GulfGulf ofof ThailandThailand (Figure(Figure1 1)) from from April April to to December December 2019. 2019. ForFor Chan-Chan- thaburi, blood samples werewere obtainedobtained fromfrom dogsdogs presentedpresented forfor medicalmedical consultationconsultation atat thethe veterinary clinic of of one one animal animal hospital hospital in in the the capital capital district, district, as aswell well as asdogs dogs presented presented for fora sterilization a sterilization/anti-rabies/anti-rabies vaccination vaccination,, which which was was conducted conducted at the at district the district hall hallof each of each dis- districttrict by bythe the Chanthaburi Chanthaburi Provincial Provincial Livestock Livestock Office Office.. For For Samut Samut Sakhon, blood samplessamples were obtainedobtained fromfrom dogsdogs presentedpresented forfor sterilization/vaccinationsterilization/vaccination at SoiSoi DogDog foundation’sfoundation’s mobile veterinaryveterinary clinic.clinic. ForFor NarathiwatNarathiwat province,province, the the survey survey was was organized organized by by the the Filari- Fila- asisriasis Project, Project, Pikhunthong Pikhunthong Royal Royal Development Development Study Study Center,Center, inin thethe Su-ngaiSu-ngai PadiPadi district,district, which isis anan endemicendemic areaarea ofof thethe nocturnallynocturnally sub-periodicsub-periodic B.B. malayimalayi.. Figure 1. The map of Samut Sakhon, Chanthaburi,Chanthaburi, and Narathiwat provinces showing the location of the study sites. Vet. Sci. 2021, 8, 39 3 of 9 2.3. Study Population Vet. Sci. 2021, 8, 39 The study population consists of 278 dogs from Chantaburi, 217 from Samut3 Sakhon, of 9 and 273 dogs from Narathiwat. Dogs less than 6-months of age, pregnant dogs, and dogs whose owners did not give permission were excluded from the study. The dogs have their ages recorded2.3. Study fromPopulation the owners’ accounts or from estimation based on body size, appearance, and dentition.The study population The samples—one consists of 278 milliliterdogs from Chantaburi, of blood—were 217 from collected Samut Sakhon, from the cephalic veinand and 273 then dogs transported from Narathiwat. to theDogs Laboratory less than 6-months of the of Departmentage, pregnant dogs, of Parasitology, and dogs whose owners did not give permission