Entomological and Virological Methods for the Identification of Potential Vectors of Lumpy Skin Disease Virus in the South-Eastern Part of Northern Caucasus, Russia


 The article provides assessment of field and laboratory methods for the collection and evaluation of potential vectors of lumpy skin disease virus (LSDV) in one of the districts of Krasnodarskiy Kray in southern Russia. In this study, we tested several methods of vector collection and a PCR protocol for the detection of the LSDV genome in insects. Descriptive data on samples were collected using a free web-based application Epicollect5.
 Potential LSDV vectors are quite widely spread insects in this region. We identified 15 insect species, including Musca domestica, Musca autumnalis and Stomoxys calcitrans. Analysis of the insect population showed an increase in species diversity and a decrease in abundance of the insect population by the end of the flight season.
 PCR tests did not detect LSDV genome in the collected samples. All the methods tested were found suitable for large-scale monitoring of lumpy skin disease (LSD). Further studies on potential risk factors of LSD spread are necessary to improve measures on preventing and eliminating the disease.


INTRODUCTION
Lumpy skin disease (LSD) is one of the transboundary diseases of cattle that has emerged in Europe recently with a serious economic impact. It was introduced in the South of the Russian Federation several years ago, and despite taken measures it is still present in the country causing severe economic losses in livestock.
This viral infection causes fever, lymphatic dysfunction, visceral and hypodermal edema with specifi c skin nodules and lesions of the eyes and mucosa [1,2].
LSD is a transmissible disease, which requires restrictive measures [3]. The disease has high priority due to the spreading speed and economic losses associated with culling and cost of therapeutic and preventive measures.
The main sources of the virus are infected animals and animal skins affected by nodules. The virus has been isolated from the saliva, semen, milk, nasal and eye discharge, exudates, affected skin and mucosa [4,5]. However, direct contact rarely results in virus transmission. The virus spreads between animals mainly by mechanical vectors -blood-sucking insects. Their presence defi nes the speed and scale of LSD spread in the region.
At the same time, the introduction of the virus into previously free (remote) regions is generally caused by the movement (mainly illegal) of infected animals.  [4,5].
First LSD outbreaks in Russia were reported in 2015 in the North Caucasian federal district: in Dagestan, Chechnya, and the republic of North Ossetia-Alania, where the disease has been registered for several years [1]. In 2016, LSD spread north and eastward along the border with Kazakhstan.
The fi rst outbreak of the disease in the Krasnodarskiy Kray was offi cially registered in May 2016. However, LSD introduction routes and spreading ways in these outbreaks have not been studied.
These outbreaks were quickly eliminated, and every cattle was immunized with the heterologous dry vaccine produced by Federal Centre for Animal Health "Arriah" (Vladimir, Russia). Since then, preventive vaccination was annually carried out and the disease was not notifi ed in the Krasnodarskiy Kray.
Krasnodarskiy Kray is a region of developed animal husbandry, with an intensive movement of animals throughout the region and between neighboring regions. That makes study of LSDV risk factors important for risk assessment and preventive measures in case of its re-introduction.
One of the critical factors of LSDV spread are climate characteristics that affect number and diversity of vectors. The climate in Kray is similar to the Mediterranean, where LSD extensively spread in 2016.
However, there are no available entomological data on the presence and a number of potential vectors in the Krasnodarskiy Kray.
LSD outbreaks were also reported in 2016 in the neighboring regions (Republic of Adygea, Karachay-Cherkess Republic, Rostov region, Stavropol Territory), but monitoring of the virus circulation and its possible overwintering in adjacent and previously affected areas were not carried out.
This research was conducted as a part of the project, performed at Federal Research Center for Virology and Microbiology (FRCVM) to develop an integrated analysis of epizootic data on emerging animal diseases, based on monitoring studies.
The purpose of this work was to test different methods for collection and monitoring of potential LSDV vectors and detection of the LSDV genome in insects.
To achieve the goal the following tasks were assigned: • determination of climate characteristics during the insect fl ight period; • optimization of methods for collecting insects, determination of species composition and identifi cation of potential LSDV vectors and their seasonal dynamics in the Krasnodar region; • optimization of a PCR protocol for LSDV genome detection in insect samples.

MATERIAL AND METHODS
The study area was the Vyselkovsky district of the Krasnodarskiy Kray, since LSD outbreaks were reported in the adjacent territories (Tbilissky and Gulkevichsky districts) in 2016.
Affected districts of Krasnodarskiy Kray and the LSD epizootic situation in 2016 are presented in Figure 1.
The Vyselkovsky district is located in the center of the Krasnodar Territory, the central part of the Prikubanskaya Plain, and borders with Pavlovsky, Tikhoretsky, Tbilissky, Ust-Labinsky, Korenovsky, and Bryukhovetsky districts. The hydrographic network of the region consists of Zhuravka and Beysug rivers with their confl uents. Beysug fl ows into the Sea of Azov, which connects to the Black Sea via the Kerch Strait [6].
The climate in the region is similar to the Mediterranean, with a mild winter and torrid summer. Eastern and northeastern winds dominate. The total number of strong wind days is up to 25 per year. The winds cause drought in the summer and strong cooling, soil and seed shifting in the winter and spring. Forest belts protect some territories [6]. The summary of average climatic data in the Vyselkovsky district is presented in Table 1.
Climate characteristics at the site of insect collection were obtained from public sources, particularly Gismeteo website (https://www.gismeteo.ru), and were set into Epicollect5 form along with other information. Entomological studies were carried out in two stages: the insects were collected at the beginning (May, 2017) and at the end (September, 2017) of the fl ight period in a cattle holding in the Vyselkovsky district (with 300 heifers housed during the period of the study).
According to the guidelines [7], 3 types of traps were used to collect insects. 1. Fly strips were placed at the loafi ng area and several points in the barn, 2 strips per point. 2. UV traps were placed in the barn above the animals, opposite to the entrance. 3. Liquid traps for horsefl ies were placed near the loafi ng area. The time of exposition for these traps was 12 hours.
We also used sweep net for capturing fl ies and midges on the fl y; the net was periodically inverted with the removal of its content into a killing jar.
We used Epicollect5 (© 2019 Imperial College London, https://fi ve.epicollect.net) to collect entomologic data. This application allows downloading images and linking data to geographic location, making it very useful for quick map visualization. The following information was recorded: insect collection date, district name, holding name and type, coordinates of collection site, air temperature, humidity and pressure, type and exposure time for traps.
Identifi cation of insects was performed using a 5-fold magnifying glass and a Key to Insects [8] on the base of Samara Research Veterinary Institute -branch of FRCVM.
Data was statistically evaluated with the MS Offi ce Excel 2010 software.
We used real-time PCR to detect LSD genome in insect samples. All insects were pooled according to their taxonomical group with 5-7 species in each pool. For each species at least 2 pools were formed, with additional pools in prevalent species. Each pool was analyzed as a separate sample. Overall, we tested 36 samples.
DNA was amplifi ed on a CFX96 Touch Real-Time PCR Detection System (BioRad, USA). The following program for PCR was used: pre-denaturation for 3 min at 95 °С, followed by 45 amplifi cation cycles, each consisting of 15 s at 95 °С and 30 s at 60 °С.
LSDV DNA isolated from an infected culture with 10 2 TCID50/cm3 activity was used as the positive control. Negative PCR control consisted of redistilled water.
Ethical approval: The conducted research is not related to animal use.

DISCUSSION
Analysis of climatic parameters during the insect's fl ight period showed that the temperature in September 2018 was 3-4 °С higher than average values. It led to the extension of the breeding season for insects and an increase in their population.
According to the purpose of this study, we evaluated the effectiveness of different types of insect traps. Fly strips and UV traps were effi cient for insect collection and provided approximately 93% of all insects caught. The sweep net was less effi cient. It was effi cient for catching fl ies but not Simuliidae. The liquid trap was ineffi cient due to being attractive for the cattle as a source of water. According to this, sweep net and liquid traps were found ineffective for the purpose of this study. Epicollect5 notably facilitated data collection, storage, transfer, and processing of epizootological data.
Analysis of the insect composition has shown an increase in species diversity and a decrease in populations of most species by the end of the fl ight period ( Figure  3). Species composition and quantitative analysis showed that Musca domestica was the predominant species throughout the entire study period, while Musca autumnalis was more common at the beginning and Stomoxys calcitrans at the end of the fl ight period. Role of Stomoxys calcitrans as a vector of lumpy skin disease was investigated in several studies, which showed high potential of S.calcitrans for virus transmission [11][12][13]. The role of Musca domestica as a vector of LSDV is not yet clear, but case of detection of virus in M.domestica samples indicates its potential for mechanical transmission [14].
PCR protocol was effective for detection of LSDV genome in infected tissue cultures. LSDV genome was not detected in the insects pools.
These results show a signifi cant risk of LSD spreading through vectors in the case of re-introduction to this region. Annual control of density and species composition of pathogen vectors is important for comprehensive analysis of risk factors conducive for LSDV.
The study will be continued to provide further information on the LSD related risk factors critical for the prevention of LSD outbreaks.