Korean Journal of
Veterinary Service

African swine fever (ASF) is a highly infectious viral disease with various pathogenicity, with a mortality rate of 100 % in the acute form (Beltran-Alcrudo et al, 2019). ASF is a disease of social and economic importance that seriously affects the entire farming industry and threatens the stable availability of food from the country of origin (Sánchez-Cordón et al, 2018). It is asymptomatic in indigenous African wild pigs such as warthogs. Still, it causes fatal diseases in domestic pigs and wild boars (Sus scrofa) of all ages (Thomson et al, 1980; Zani et al, 2020). However, ASF has limited public health impact as it is not a zoonosis of the argument (Sanchez-Vizcaino and Arias, 2012; Arias et al, 2018).

In Korea, the first case was confirmed in Paju on September 17, 2019. After the first outbreak, pigs in Ganghwa, Gimpo, Paju, and Yeoncheon were culled (Yoo et al, 2020). At the beginning of the ASF outbreaks, carcasses of infected wild boars were found only in northern Gyeonggi and Gangwon-do, bordering North Korea areas. But recently, the carcasses have also been reported far from the relevant area (Sangju, and Uljin, Gyeongsangbuk-do) (Yoo et al, 2020; Ministry of Environment, 2022). It was concluded that there is a possibility that ASF inflow and main transmission route in Korea are caused by infected wild boars (Yoo et al, 2020). Although direct contact is the main transmission route, several studies have emphasized that indirect transmission cannot be ignored (Guinat et al, 2016; Timothée et al, 2021). In Africa, indirect transmission through soft ticks (Ornithodoros spp.) as competent vectors is well known. However, soft ticks are known to hardly exist on Paleo-European ecosystems, suggesting that ASF outbreak patterns in Africa, Europe, and Asia are different (Plowright et al, 1969; Penrith et al, 2019; Timothée et al, 2021). In particular, a strong seasonality was observed in which ASF occurrence in domestic pigs peaked in summer compared to winter (Miteva et al, 2020). Thus, questions have been raised about the potential role of hematophagous arthropods, which exhibit similar seasonal activity and bite other hosts. Studies have been conducted suggesting a high possibility of interspecies transmission by a stable fly (Stomoxys calcitrans) (Fila and Woźniakowski, 2020; Timothée et al, 2021).

Few reports have shown that soft ticks were found in migratory seabirds living on uninhabited islands. Still, studies on the possibility of transmission by hematopoietic arthropods such as soft ticks, ticks, or stable flies have not been actively conducted in Korea (Kim et al, 2016; Han et al, 2020). ASF Virus is also detected in water puddles and soil around the detection point, capture and search equipment, and vehicles (Wales and Davies, 2021). Therefore, continuous monitoring is necessary not only for wild boars but also for wild mammals, birds, and various hematopoietic arthropods (ticks and insects including mosquitoes and flies) that have the potential to become infected with viruses by accessing infected wild boar carcasses or habitats. In this study, to trace the path of ASF virus infection by the vector, we intend to collect samples of vector animals from the ASF outbreak area to investigate the presence of ASF infection and utilize it to help in the epidemiologic investigation and disease prevention.

Survey areas

A total of 4 wild boar habitats were investigated using unmanned cameras (BOAN-HUNTER EYE, Boancom, Korea) in Cheorwon and Yanggu, Gangwon-do, where the incidence of ASF is high (Fig. 1). In addition, nine farms were selected from six regions (Cheorwon, Chuncheon, Hongcheon, Hwacheon, Inje, and Yanggu), considering the ASF outbreak areas and areas of concern for disease transmission (Fig. 2).

Figure 1. Investigate wild boar habitats using unmanned cameras (A) Cheorwon-gun and (B) Yanggu-gun.

Figure 2. Nine farms were selected from six regions (Cheorwon, Chuncheon, Hongcheon, Hwacheon, Inje, and Yanggu) considering the ASF outbreak areas.

Collection of wildlife and environmental samples

A total of 317 samples were collected for wildlife and environment samples (Table 1). Wildlife samples (243 cases) were collected from 238 individuals of 36 species of wildlife animals that the Gangwon Wildlife Medical Rescue Center rescued from all regions of Gangwon-do, which are believed to have access to the carcasses or habitats of wild boars such as raccoon dogs, water deer, and wild birds. One hundred sixty-five swab samples were collected from presumed contamination sites around the mouth and feet and gathering them into one sample. In addition, 56 whole blood samples were collected. Also, 22 samples of hematopoietic arthropods (ticks, louse flies, lice, and fleas) that suck the blood of rescued wildlife animals were collected. Insect nets were installed in 9 sites of selected farms to collect 48 insects (flies and mosquitoes) samples, and 12 tick samples were collected by flagging and dragging methods (collecting cloth; 1×1 m) from grassland near the selected farm. And then, 14 soil samples were collected from mud and rubbing trees in wild boar habitats investigated using an unmanned camera.

Table 1 . A total of 317 wildlife and environmental samples were used to detect the ASF virus.

Sample	Class	Family	Common name	Scientific name	Swab	Whole blood	Hematopoietic arthropods	Total
Wildlife samples	Mammalia	Bovidae	Long-tailed goral	Naemorhedus caudatus	1	2	-	3
		Canidae	Raccoon dog	Nyctereutes procyonoides	39	15	5	59
		Cervidae	Korean water deer	Hydropotes inermis	62	17	1	80
			Roe deer	Capreolus pygargus	12	4	-	16
		Erinaceidae	Korean hedgehog	Erinaceus amurensis	1	-	2	3
		Felidae	Leopard cat	Prionailurus bengalensis	1	1	-	2
		Leporidae	Domestic rabbit	Oryctolagus cuniculus	-	2	-	2
		Mustelidae	Eurasian badger	Meles leucurus	3	4	-	7
			Weasel	Mustela sibirica coreana	-	1	-	1
			Yellow-throated marten	Martes flavigula koreana	-	2	1	3
		Sciuridae	Korean squirrel	Sciurus vulgaris	1	-	-	1
			Squirrel	Pteromys volans	1	-	-	1
			Asiatic chipmunk	Tamias sibiricus	1	-	-	1
		Suidae	Wild boar	Sus scrofa	1	-	2	3
		Vespertilionidae	Oriental-discoloured bat	Vespertilio sinensis	2	-	-	2
	Aves	Accipitridae	Goshawk	Accipiter gentilis	1	2	1	4
			Sparrow hawk	Accipiter nisus	2	-	1	3
		Ardeidae	Grey heron	Ardea cinerea	1	2	-	3
			Intermediate egret	Mesophoyx intermedia	1	-	-	1
			Green-backed heron	Butorides striata	1	-	-	1
		Columbidae	Rufous turtle dove	Streptopelia orientalis	-	1	1	2
			Domestic pigeon	Columba livia	-	1	-	1
		Corvidae	Azure-winged magpie	Cyanopica cyanus	3	-	-	3
			Carrion crow	Corvus corone orientalis	3	1	-	4
			Eurasian Jay	Garrulus glandarius	5	-	-	5
			Jungle crow	Corvus macrorhynchos	2	-	-	2
			Oriental Magpie	Pica sericea	11	-	-	11
		Coraciidae	Broad-billed roller	Eurystomus orientalis	1	-	-	1
		Falconidae	Hobby	Falco subbuteo	-	-	1	1
			Kestrel	Falco tinnunculus interstinctus	4	-	2	6
		Laridae	Black-tailed gull	Larus crassirostris	1	-	-	1
		Oriolidae	Black-naped oriole	Oriolus chinensis	1	-	-	1
		Scolopacidae	Eastern curlew	Numenius madagascariensis	-	-	1	1
		Strigidae	Brown hawk-owl	Ninox scutulata	-	1	1	2
			Eurasian eagle-owl	Bubo bubo	3	-	2	5
			Scops owl	Otus scops	-	-	1	1
Environ­mental samples		Insects	Eight areas (Cheorwon, Chuncheon, Hongcheon, Hwacheon, Inje, Yanggu)					48
		Ticks	12 areas (Cheorwon, Gangneung, Goseong, Hoengseong, Hwacheon, Sokcho, Wonju, Yangyang)					12
		Soil	4 areas (Cheorwon, Yanggu)					14
Total								317

ASF viral DNA detection of all samples

ASF infection was investigated using real-time polymerase chain reaction (RT-PCR) with collected 317 samples. DNA extraction from each sample was performed using a QIAamp DNA Mini kit (QIAGEN, USA). DNA was extracted using 150 μL of each sample, lysis buffer, 70% ethanol, washing buffer, and distilled buffer. Biorad CFX96 (Bio-rad Laboratories Inc., California, USA) and VDx^® ASFV qPCR (Median diagnostics Inc., Korea) were used for the RT-PCR procedure. 5 μL of the DNA sample was dispensed into 15 μL of the premix containing the enzyme and primer, 5 μL of the provided control DNA was used as a positive control, and 5 μL of H₂O was used as a negative control. RT-PCR assays were performed in a 20 μL reaction mixture at 94℃ for 10 min, followed by 40 cycles for 15 sec at 95℃, 60 sec at 58℃. RT-PCR for ASF viral detection was performed twice or more.

Finally, the negative ASF viral DNA detection for all 317 test samples (Fig. 3). In this study, 12 out of 317 samples showed a false positive. Still, the final negative was confirmed through retesting, and the cause of false positives is pointed out as cross-contamination during the experiment. The extreme sensitivity of PCR makes it susceptible to cross-contamination, so this risk must be minimized and controlled (Scherczinger et al, 1999; Czurda et al, 2016).

Figure 3. ASF viral DNA detection for all 317 test samples was confirmed as negative (*FAM_ASFV, 6-carboxy fluorescein_African Swine Fever virus; HEX_IPC, N-HEX-6-aminohexano_Internal Positive Control).

At this point, when borders and regional boundaries are collapsing due to the development of transportation and various ecological changes due to climate change, livestock is threatened by the outbreak and spread of new infectious diseases, and everyone is very interested. ASF has been continuously occurring since the first report on September 2019 in Korea (2,120 cumulative confirmed cases as of February 13^th, 2022) (Ministry of Environment, 2022). ASF is transmitted directly through the secretions of infected domestic pigs or wild boars (Guinat et al, 2016; Timothée et al, 2021). In addition, the ASF virus is known to be very stable in the environment. The carcasses of infected domestic pigs and wild boars can act as important sources of disease transmission, and hematopoietic arthropods and wildlife animals can help maintain and spread the disease (Mazur-Panasiuk et al, 2019; Zani et al, 2019). Thus, it is necessary to examine ASF infection in environmental samples of different time points and locations to confirm that viruses from major ASF outbreak regions remain in the environment.

Finally, all the RT-PCR results from ASF infection in various wildlife animals and environmental samples were negative. However, ASF continues to occur in the wild boars and is moving to the southern regions of the Korean peninsula. So far, quarantine has been carried out well, but there is a possibility that ASF will occur again in domestic pigs. There are few studies on wildlife animals except wild boars, including stable fly and soft ticks are known as ASF vectors in Korea (Kim et al, 2016, Han et al, 2020). Therefore, it should be used as primary data to take measures to effectively prevent the occurrence and spread of ASF disease through the long-term and continuous investigation into various wildlife animals and environmental samples and the management of vectors.

This work was supported by the Ministry of Environment in 2020.

No potential conflict of interest relevant to this article was reported.