Title: Insects as potential vectors of African swine fever virus
in the Republic of Korea
Running head: Insects as vector of African swine fever virus
Authors : Hachung Yoon, Seong-Keun Hong, Ilseob Lee, and Eunesub
Lee
Veterinary Epidemiology Division, Animal and Plant Quarantine Agency
177 Hyeoksin 8-ro, Gimcheon, Gyeongsangbuk-do, 39660, Republic of Korea
Corresponding author : Hachung Yoon
e-mail: heleney@korea.kr
Summary
African swine fever (ASF) is a fatal disease in swine. Concentration of
cases over summer and outbreaks in farms with high levels of biosecurity
might support the hypothesis of ASF virus transmission via insects. A
total of 28,718 insects were collected from 14 pig farms affected by ASF
in the Republic of Korea. The insects collected were flies (N = 27,310,
95.1%), mosquitoes (N = 1,198, 4.2%), and cockroaches (N = 24, 0.1%).
The remaining 0.6% (N = 186) could not be taxonomically classified. All
the insects collected showed no trace of ASF Virus-DNA was detected by
laboratory analysis. It is premature to conclude on the role of insects
in the transmission of ASFV. However, the possibility of the virus
spreading via insects should not be overlooked.
Keywords: African swine fever, Insect, Fly, Mosquito, Republic
of Korea
Introduction
African swine fever (ASF) is a hemorrhagic fatal disease in swine.
Recently, there has been a drastic increase the affected areas,
worldwide especially in Europe and Asia. Since the first notification of
suspect cases at a pig farm in Paju, Gyeonggi-do Province, on September
16, 2019, the ASF outbreak had been confirmed in a total of 14 pig farms
by October 9, 2019, in Gyeonggi-do (Paju, Yeoncheon, and Gimpo) and
Incheon Metropolitan City (Ganghwa) in the Republic of Korea (South
Korea, hereafter Korea) [Kim et al., 2020]. On October 3, 2019, the
ASF virus (ASFV) was first detected in a wild boar found dead in the
demilitarized zone facing the border of the Democratic People’s Republic
of Korea (North Korea) [Jo & Gotyazar, 2020], and the documentation
of ASF cases in wild boars continued until April 2020 [Yoo et al.,
2020].
In Europe, the seasonality of ASF with a concentration of cases over
summer and outbreaks occurring even in farms with high levels of
biosecurity might support the hypothesis that ASFV is a vector-borne
disease [Petrasiunas et al., 2018]. The possibility of mechanical
transmission of ASFV by the stable fly (Stomoxys calcitrans ) and
the blowfly (Calliphoridae) has been reported in experimental studies
[Mellor et al., 1987; Forth et al., 2018]. More recently, it has
been found that ASFV could be detected in stable flies that fed on
ASFV-spiked blood 3 h and 12 h post-feeding. Moreover, pigs on which
these flies fed were infected with ASFV [Olesen et al., 2018a; Olesen
et al., 2018b]. Flies and mosquitoes exist all over the world, and the
possibility of ASFV transmission by insects cannot be completely ruled
out in Korea. Therefore, in this study, insects were collected and
identified in the ASF outbreak pig farms to determine the role of
insects as a potential vector of ASFV.
Materials and Methods
Insects were collected in the 14 ASF outbreak farms, inside and around
pig houses, both with and without virus-positive pigs, by the reverse
transcription polymerase chain reaction (RT-PCR) test. Sampling was
performed by installing collection tools, such as blacklight traps,
aerial nets, and sticky strips. These tools had been in place for at
least 24 h before being retrieved. The collection was performed jointly
by the Animal and Plant Quarantine Agency (APQA) and the Korea Centers
for Disease Control and Prevention (KCDC), APQA alone, or a private
company (Biogenoci Ltd.) under consignment by APQA for the collection
service. Laboratory tests for the detection of ASFV-DNA were conducted
by APQA using RT-PCR according to the method described in previous
studies by APQA [Kim et al., 2019; Kim et al., 2020]. Raw data on
the insect collection and laboratory tests were derived from the
government official document system of Korea. The data that support the
findings of this study are available in the Veterinary Epidemiology
Division of APQA.
Results
A total of 28,718 insects were collected from 14 pig farms affected by
the ASF outbreak in 4 regions: Paju (5 farms), Yeoncheon (2 farms),
Gimpo (2 farms), and Ganghwa (5 farms). The first two collections were
performed on September 27, 2019 in Ganghwa and October 2, 2019 in Paju
during a field epidemiological investigation. Only one fly was caught on
each farm. All other samplings were conducted between October 10, 2019
and October 31, 2019. In the three outbreak farms, insect collection was
performed in pig houses the day after the virus has been detected in one
or more of the pigs. The interval between the outbreak and the insect
sampling varied from 1 to 44 days.
The most common type of insect was flies (nine species, N = 27,310,
95.1%), followed by mosquitoes (four or more species, N = 1,198,
4.2%). Cockroaches (N = 24, 0.1%) were also collected in all regions,
except for Paju. The remaining 0.6% (N = 186) of all collected insects
could not be taxonomically classified. The largest number of insects was
collected in Yeoncheon (N = 13,672), most (N = 13,545, 99.1%) of which
were flies. The proportion of mosquitoes was higher in Paju (12.8%)
than in other three regions (0.7% in Yeoncheon, 5.6% in Ganghwa, and
6.6% in Gimpo, respectively) (Supplementary Table 1).
The flies collected were mostly houseflies (Musca domestica , N =
21,553) and blowflies (or blue bottle flies, Calliphora
vomitoria , N = 5,285), which together accounted for 98.3% of all
collected flies. The number of yellow dung flies (Scatophaga
stercoraria , N = 144) and flesh flies (Sarcophaga spp., N = 108)
was small; however, the two species were found in all four regions. In
Gimpo and its neighboring region, Ganghwa, crane flies (Tipulidae, N =
200) were collected. Blood-feeding flies, including non-biting midges
(Chironomidae, N = 10 in Gimpo), stable flies (S. calcitrans , N =
6 in Paju and Yeoncheon), and drain flies (Psychodidae spp., N =
2 in Yeoncheon), were found only in some regions. One each black soldier
fly (Hermetia illucens ) was found in Paju and Yeoncheon,
respectively (Supplementary Table 2).
The most common type of classified mosquitoes was the biting midge
(Culicoides spp., N = 324, 27.0%). The second most common type
was yellow fever mosquitoes (Aedes aegypti spp., N = 77), the
number of which was not even one fourth that of biting midges
(Supplementary Table 3). Aedes vexans was found in Paju (N = 8)
and Yeoncheon (N = 3), and the Korean yellow fever mosquito (Aedes
koreicus , N = 1) was found only in Paju. Anopheles mosquitoes
(Anopheles spp., N = 7) were found in regions other than Paju,
whereas Culex pipiens (Culex spp. , N = 4) was found
in regions other than Yeoncheon. The Culex orientalis (N = 1) was
found only in Paju. More than half (65.6%) of the collected mosquitoes
could not be classified.
All 28,718 insects (flies, mosquitoes, and cockroaches) collected from
ASF outbreak farms in four regions were proved to be ASFV-DNA-negative
by RT-PCR.
Discussion
Flies, mosquitoes, and cockroaches are hygiene pests that may harm
animals, both directly and indirectly. The hygiene pests may bite
animals to injure the skin, spread pathogens through the wound, and take
blood and nutrients from the animals. Even when they do not bite, they
may transmit pathogens mechanically or biologically. Several cases have
been documented where hygiene pests have spread pathogens in pig farms
[Baldacchino et al., 2013; Fila et al., 2020]; however, no clear
evidence of the spread of ASFV has been reported, and there is only
anecdotal support for the hypothesis. In a pig farm in Estonia in 2016,
clinical symptoms suggestive of ASF were firstly recognized in
mid-August, followed by the confirmation of the outbreak in 8 days. At
the time of the epidemiological investigation conducted on the following
day, 13 flies (M. domestica , N = 9, and Drosophilaspp. , N = 4) and 2 mosquitoes in close contact with pigs in the
affected unit were randomly caught using an aerial net. ASFV-DNA was
detected in two flies (one was M. domestica and the other wasDrosophila ) and two pooled mosquitoes; however, high Ct
(threshold cycles) values indicated only very small quantities of the
virus [Herm et al., 2019]. In Poland, ASFV-DNA was detected in
stable flies collected during the ASF outbreak in pigs; however, the
exact sampling period was not reported [Mazur-Panasiuk et al.,
2019]. In Estonia, ASFV-DNA was detected in farm insects in August
during summer [Herm et al., 2019]. In Lithuania, a study was
conducted to collect blood-feeding insects (Diptera: Tabanidae and
Muscidae [Stomoxys ]) using the NZI traps; these insects are
likely to play a role in the ASFV transmission. Most insects were
collected in July and August (summer). Contrarily, the specimens ofStomoxys , the most well-known species of blood-feeding flies,
were mostly collected in October (fall) [Petrasiunas et al., 2018].
The aforementioned East European countries, including Poland, Lithuania,
and Estonia, are located at higher latitudes (46°–54° North) than
Korea; however, the average temperature in July–August when the insect
collection was performed in these countries was higher than 20 °C
[AccuWeather, 2020; Korea Meteorological Administration, 2020]. The
farms affected by the ASF outbreak in Korea were located at latitudes of
37°–38° North and the average daily temperatures in the affected
regions from October 10 to 31, which is the collection period, were
7.4°C–16.1°C (in Paju and Yeoncheon) and 8.6°C–17.7°C (in Gimpo and
Ganghwa) [Korea Meteorological Administration, 2020]. Due to the low
temperatures in Korea in the fall, the environment where the insects
were collected may be critically different from that of the Eastern
European countries at the time of the sampling. Nevertheless, the
species of collected flies in Korea were similar to those collected from
the farms in Germany in June–September [Forster et al., 2007]. The
housefly (M. domestica ) was the most common species among the
collected insects in the present study and in the study conducted in
Germany. However, there is little evidence to support the hypothesis
that these insects are spreaders of ASFV, although they have the
potential owing to their ecological characteristics, in addition to the
reported evidence of transmitting other diseases [Fila et al.,
2020]. The blowfly, which is a type of necrophagous, was the second
most common species of the collected flies in the present study. In an
experimental study of the larvae of Lucilia sericata andCalliphora vicina , which are species of blowflies, ASFV genomic
DNA was detected inside and on the body surface of the larvae that fed
on ASFV-infected tissues. However, the study reported that when the
larvae became pupae 10 days later, very small quantities of virus DNA
were detected in only a few individuals. This observation suggested that
the virus failed to replicate in the bodies of the larvae [Forth et
al., 2018]. Although an experimental study has shown that ASFV can
survive for 2 days in S. calcitrans (stable flies) without losing
titers or the ability to spread [Mellor et al., 1987], the number of
collected specimens in this study was too small to assess its role in
the transmission of ASFV in Korea. Small species, such as stable flies
and blowflies, do not move around for long distances and may thus be
involved in the spread of the virus within a farm. Although missing in
this study, the Tabanidae, a large species called “horsefly,” travels
farther and could therefore be involved in the spread of the virus
between farms or in the wild boar/domestic pig transmission interface
[Fila et al., 2020].
Conclusions
In this study, a total of 28,718 insects were collected in ASF outbreak
farms, and no trace of ASFV-DNA was detected by laboratory analysis.
This may reflect the effects of cleaning and disinfection, following the
detection of ASF outbreak in the early phase of infection [Yoon et
al., 2020]. The results of the present study and the experimental
research and fieldwork previously conducted in other countries suggest
that it is premature to conclude on the role of insects in the
transmission of ASFV. However, the possibility of the virus spreadingvia insects 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.: N-1543068-2015-99-01].
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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