Title: Arthropods as potential vectors of African swine fever virus
outbreaks on pig farms in the Republic of Korea
Running head: Arthropods, potential vector of ASFV
Authors: Hachung Yoon, Seong-Keun Hong, Ilseob Lee, Eunesub Lee
Affiliation: Veterinary Epidemiology Division,
Animal and Plant Quarantine Agency,
Address: 177 Hyeoksin 8-ro, Gimcheon, Gyeongsangbuk-do, 39660 Republic
of Korea
Corresponding author: Hachung Yoon
e-mail: heleney@korea.kr
Abstract
The seasonality of African swine fever (ASF), with cases concentrated
over the summer in Europe, in addition to outbreaks on farms with high
levels of biosecurity, suggest that ASF virus (ASFV) may be transmitted
by arthropod vectors. In this study, arthropods were collected from
Korean pig farms with ASF outbreaks to determine the role of arthropods
as a potential vector of ASFV. Arthropods were collected from 14 farms
with ASF outbreaks, from September 27 to October 31, 2019. A total of
28,729 arthropods, including 28,508 (99.2%) Diptera, were collected
using blacklight traps, insect nets, and yellow sticky strips. All
arthropods samples were negative for ASFV genomic DNA according to
laboratory tests using real-time polymerase chain reaction.
Nevertheless, it is premature to conclude that arthropods do not play
any role in ASFV transmission.
Keywords: African swine fever, arthropod, Diptera, transmission,
Republic of Korea
Introduction
African swine fever (ASF) is a hemorrhagic fatal disease for both
domestic pigs and wild boars. Recently, there has been a drastic
increase in the areas affected by ASF in Asia, Europe, and Africa. Since
the first suspected case on a pig farm in Paju, Gyeonggi-do Province on
September 16, 2019, ASF outbreaks were confirmed on a total of 14 pig
farms in the Republic of Korea (hereafter Korea) by October 9, 2019.
Farms with ASF outbreaks were confirmed in four counties in Gyeonggi-do
(Paju, Yeoncheon, and Gimpo) and Incheon Metropolitan City (Ganghwa)
(Yoon et al., 2020). On October 3, 2019, the ASF virus (ASFV) was first
detected in a wild boar carcass found in the demilitarized zone on the
border of the Democratic People’s Republic of Korea (North Korea) (Jo &
Gortazar, 2020); reports of ASF cases in wild boars have continued until
October 2020 (Kim et al., 2020; Yoo, Kim, Lee, & Yoo, 2020).
The seasonality of ASF, with cases concentrated over the summer in
Europe, combined with outbreaks occurring on farms with high levels of
biosecurity, suggests that ASFV may be transmitted by arthropod vectors
(Petrasiunas, Bernotiene, & Turcinaviciene, 2018). Soft ticks of the
genus Ornithodoros are known to be biological vectors (Sur,
2019), but there has only been one report of this in Korea, on an
uninhabited island (Han et al., 2019). Experimental studies have
reported the possibility of the mechanical transmission of ASFV by
stable flies (Stomoxys calcitrans ) and blowflies (Calliphoridae)
(Forth, Amendt, Blome, Depner, & Kampen, 2018; Mellor, Kitching, &
Wilkinson, 1987). More recently, stable flies that fed on ASFV-spiked
blood had detectable levels of ASFV 3 and 12 hours after feeding;
moreover, the pigs upon which these flies fed became infected with ASFV
(Olesen, Hansen, et al., 2018; Olesen, Lohse, et al., 2018). Arthropods,
especially flies and mosquitoes, exist all over the world. Therefore,
in this study, arthropods were
collected from pig farms with ASF outbreaks to determine their role as a
potential vector of ASFV.
Materials and Methods
Arthropods were collected from 14 farms with ASF
outbreaks, located in 4 regions
(counties): 5 farms in Paju, 2 in Yeoncheon, 2 in Gimpo, and 5 in
Ganghwa. Arthropod specimens were collected using blacklight traps
(UV-LED Blackholeplus®, Biotrap Ltd.), insect nets, and yellow sticky
traps. These traps were installed inside and around the pig houses, and
were not targeted to catch specific arthropods. These tools were left in
place for at least 24 hours before the trapped arthropods were
retrieved. Arthropod collection was performed in three ways: via
collaboration between the Animal and Plant Quarantine Agency (APQA) and
the Korea Centers for Disease Control and Prevention (KCDC), via APQA
alone, or via a private company (Biogenoci Ltd.) under contract by APQA.
Arthropods collected using blacklight traps and insect nets were
classified in the field by researchers from the KCDC or Biogenoci Ltd.,
then confirmed in the APQA laboratory using Optinity KS-200® with 0.8 to
5× magnification. Yellow sticky strips were sent directly to the
laboratory without on-site classification. Specimens were pooled
according to location and insect type to enable the detection of ASFV
genomic DNA using real-time polymerase chain reaction (PCR). Laboratory
tests were conducted by APQA and results were released through the
government official document system of Korea.
Results
A total of 28,729 arthropod specimens were collected through 111
collecting operations carried out between September 27 and October 31,
2019. The median number of insects collected per farm was 1,047
(minimum: 341, maximum: 11,359). The time interval between the ASF
outbreak and arthropod collection varied from 1 to 44 days. On one farm
in Paju and another farm in Gangwha, collection was performed during the
on-farm outbreak investigation.
The most common (99.2%) arthropod collected belonged to the order
Diptera: 28,508 individuals from 10 families were collected (Table 1).
In the order Diptera, the majority belonged to Muscidae (N = 21,559,
75.6%) and Calliphoridae (n = 5,285, 18.5%; Table 2). Most Muscidae
flies were Musca domestica , (houseflies, n = 21,553), butStomoxy calcitrans (stable flies, n = 6) were also collected from
3 farms (Supplementary Table 1). In addition, small numbers of Blattaria
(n = 24), Ixodidae (n = 11), Lepidoptera (n = 5), and Coleoptera (n = 1)
were collected, but only in some regions. One hundred-eighty (0.6%)
specimens could not be classified (Table 1). All 28,729 collected
arthropods were negative for ASFV genomic DNA.
Discussion
Flies, mosquitoes (Diptera), and cockroaches (Blattaria) are hygiene
pests that are capable of harming animals, both directly and indirectly.
They can be hematophagous or necrophagous. Hygiene pests may bite
animals to injure the skin, spread pathogens through the wound, and take
blood and nutrients. Even when they do not bite, these pests may
transmit pathogens mechanically or biologically. ASFV is known to
persist for extended periods in wild boar carcasses with the
colonization of necrophagous pests, mainly belonging to the orders
Diptera and Coleoptera (Bonnet et al., 2020); however, to date, there
have been no reports of the spread of ASFV via these pests, and there is
only anecdotal support for this hypothesis.
Similar research studies have been conducted in other countries. In
August 2016, during an epidemiological investigation of an ASF-positive
pig farm in Estonia, 13 flies (Musca domestica , n = 9, andDrosophila spp. , n = 4) and 2 mosquitoes found in close
contact with pigs in the affected unit were randomly caught using an
aerial net. ASFV DNA was detected in two of the flies (one Musca
domestica and the other Drosophila ) and the pooled mosquitoes;
however, high Ct (threshold cycles) values indicated that only very
small quantities of the virus were present (Herm, Tummeleht, Jurison,
Vilem, & Viltrop, 2020). In Poland, ASFV DNA was detected in stable
flies collected on a pig farm with an ASF outbreak; however, the exact
sampling period was not reported (Mazur-Panasiuk, Zmudzki, &
Wozniakowski, 2019). In Lithuania, a study collected blood-feeding
insects, including Tabanidae and Muscidae Stomoxy spp. Most
insects were collected during the summer (July and August), but theStomoxys specimens, the most well-known blood-feeding fly
species, were mostly collected in the autumn (October) (Petrasiunas et
al., 2018). The aforementioned Eastern European countries are located at
higher latitudes (46°–54° North) than Korea; however, their average
temperatures in July and August, when insect collection was performed,
were higher than 20°C (AccuWeather, 2020;
KoreaMeteorologicalAdministration, 2020). Korean farms with ASF
outbreaks were located at latitudes 37°–38° North, with average daily
temperatures of 7.4°C–16.1°C (in Paju and Yeoncheon) and 8.6°C–17.7°C
(in Gimpo and Ganghwa) over the main collection period
(KoreaMeteorologicalAdministration, 2020). Due to the low fall
temperatures in Korea, the environment in which the insects were
collected may be critically different from that of the Eastern European
countries at the time of insect collection. Nevertheless, the species of
collected flies in Korea were similar to those collected from farms in
Germany over a study period from June to September, with Musca
domestica being the most commonly collected insects in both studies
(Forster et al., 2007).
The number of insects collected in this study was especially high on a
farm in Yeoncheon (Supplementary Table 1), likely because collection was
conducted while pigs were still present in the barns. Calliphoridae, a
necrophagous insect, was the second most common family of Diptera (Table
2). In an experimental study of Calliphoridae larvae (Lucilia
sericata and Calliphora vicina ), ASFV genomic DNA was detected
inside and on the body surface of larvae that fed on ASFV-infected
tissues; however, when the larvae became pupae 10 days later, very small
quantities of virus DNA were detected in only a few individuals,
suggesting that the virus failed to replicate within the bodies of the
larvae (Forth et al., 2018). Although ASFV can survive for 2 days inStomoxy calcitrans without a reduction in titer or ability to
spread (Mellor et al., 1987), the number of specimens collected in this
study was too small (2 of each, for a total of 6 over 3 farms) to assess
its role in the transmission of ASFV in Korea. Small species, such as
stable flies and blowflies, do not travel long distances and may thus be
involved in the spread of the virus within a single farm. Although not
collected in this study, Tabanidae (horsefly) travel farther distances
and could therefore be involved in the spread of ASFV between farms or
in the wild boar/domestic pig transmission interface (Fila &
Wozniakowski, 2020).
Conclusions
In this study, a total of 28,729 arthropods were collected from farms
with ASF outbreaks. No trace of ASFV genomic DNA was detected by
laboratory analysis. This result may reflect the effects of cleaning and
disinfection in the early phase of infection following the detection of
an ASF outbreak (Yoon et al., 2020). The results of the present study,
combined with previously conducted experimental research and fieldwork
in other countries, suggest that it is premature to conclude that
arthropods play a role in the transmission of ASFV; however, the
possibility of the virus spreading via arthropods should not be
overlooked.
Acknowledgments
This study was supported by the Animal and Plant Quarantine Agency of
the Republic of Korea [R&D Project No.: B-1543068-2020-21-010101].
Ethics Statement
The authors confirm that the ethical policies of the journal, as noted
on the journal’s author guidelines page, have been adhered to. No
ethical approval was required as this article is based on field work not
involving any experiment.
Conflict of Interest Statement
None
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Table 1.
Number of arthropods collected on pig farms with ASF outbreak, by order
and region