The results of entomological monitoring West Nile fever on the territory of the Crimean peninsula in the 2022 season

Introduction

The unique geographical location of the Crimean Peninsula, determining a significant separation of terrestrial fauna from the corresponding mainland populations and the diversity of natural and climatic conditions (air temperature, precipitation, vegetation, landscapes), served as a basis for the formation of natural foci of zoonotic infectious diseases. Despite a sufficient degree of study of such natural foci of infectious diseases as Crimean hemorrhagic fever, tularemia, tick-borne viral encephalitis, Ixodes tick-borne borreliosis, etc. [1], the data on the distribution of natural foci of zoonotic infectious diseases in Crimea are not available [1]. There were no data on the spread of West Nile fever (WNF) before 2013¹.

In the Republic of Crimea, the circulation of the West Nile virus (WNV) was first established in 2013, when a case of the disease was confirmed in an 11-year-old child, but this patient was not included in the statistical reporting. Official registration of WNF cases in the Republic of Crimea started in 2018 [2]. From that period to date, the incidence of WNF in the region was recorded at a sporadic level, with the exception of 2019, when 10 patients were identified [3]. At the same time, the hidden epidemic process is evidenced by the repeated registration of imported cases of WNF in other areas of Russia associated with the visit to the Republic of Crimea (2019 – Tula, Voronezh, and Kursk Regions, Republics of Adygea and Mordovia, 2022 – Rostov Region). The population immunity levels detected in single years (5.5–16% – 2019, 15.6% – 2022) are comparable with similar indicators in areas of high epidemiological risk, which confirms the intensive contact of the population with the WNF pathogen.

Entomological monitoring in the territory of the peninsula is conducted annually by specialists of the Federal State Budgetary Institution “Center of Hygiene and Epidemiology in the Republic of Crimea and the City of Federal Significance Sevastopol” and the Federal State Budgetary Institution “Anti-Plague Station of the Republic of Crimea” of Rospotrebnadzor. During monitoring studies, samples positive for WNV RNA were detected in 2018 from Culex pipiens larvae collected in the village of Orlovka in the Kachinsky municipal district of Sevastopol [4], and in 2019, from imago and larvae of Cx. pipiens collected in the city of Belogorsk [5]. Thus, until 2022, the circulation of WNV on the territory of the Crimean Peninsula was confirmed only in its southern part. The presence of favorable climatic conditions, a wide range of carriers of WNV, including the most effective ones, the location of the region at the intersection of global migration routes of migratory birds, and their concentration determine the conditions for active transmission of the pathogen.

Intensification of tourist flows to the region in summer, coinciding with the seasonal increase in the incidence rate of WNF and other natural focal zoonotic infections, as well as the movement and concentration of temporary contingents of the population in 2022, required reinforcement of epizootological monitoring activities. In order to ensure this task, specialists of the Volgograd Research Institute of Plague Control as part of the Rospotrebnadzor specialized anti-epidemic team (SAET) traveled to the territory of the Republic of Crimea. The results of their work in terms of WNF monitoring are presented below.

The study was aimed to assess the role of blood-sucking mosquitoes of different species inhabiting the northern and central regions of the Crimean Peninsula in the transmission of WNV.

Materials and Methods

Mosquitoes were collected with automatic traps Mosquito Magnet Executive (Woodstream, USA) using the attractant Octenol (Woodstream, USA) and a vertical hand vacuum cleaner Tefal X-PERT 3.60 Versatile Handstick TY6975WO (GROUPE SEB, France) in accordance with current SanPiN 3.3686-21 in 7 administrative formations of the Republic of Crimea: Krasnoperekopsk, Simferopol, Krasnogvardeysky, Saksky, Razdolnensky, and Bakhchisaraysky Districts, and the city of Simferopol. In urban biotopes, blood-sucking mosquitoes were collected in places of their potential aggregation (on homestead and dacha plots, in barns, poultry houses, and cemeteries), and in rural biotopes (around settlements, along the banks of water sources, on rice paddies, and in floodplain forests). During the period of July 1 – August 5, 2022, a total of 46 entomological surveys were conducted, which included 16 traps/nights and 45 traps/hours.

Delivery to the laboratory of the mobile complex of Rospotrebnadzor SAET, determination of species affiliation, and packing of mosquito pools for research was carried out in compliance with the requirements of the “cold chain”. Blood-sucking mosquitoes were identified to species using standard keys^2,3 with the help of a stereomicroscope MSP-1, variant 22 (LOMO, Russia).

The dominance index was evaluated according to the system proposed by Raisky: eudominants ≥ 15%; dominants – 5.1 to 15%; subdominants – 2.1 to 5%; rare – 1.1 to 2%; extremely rare ≤ 1.1%⁴.

Detection of WNV RNA in samples was performed by RT-PCR using the AmpliSense WNV-FL reagent kit (Central Research Institute of Epidemiology, Rospotrebnadzor, Russia) according to the manufacturer’s instructions. Infection was determined by calculating the specific weight of positive samples from the total number of tested samples.

Results

A total of 18,593 mosquito specimens of 12 species were collected and identified, and 693 samples were prepared for the study. Biocenoses of all the territories surveyed by the authors were conditionally divided into urban and rural (Table 1).

In urban biotopes, a total of 4,966 specimens of mosquitoes of 9 species were captured. The invasive species Aedes albopictus was found for the first time in the central part of the peninsula in Bogdanovskoye cemetery in Simferopol. In rural biotopes and their vicinities, a total of 13,627 specimens of 11 species were collected.

WNV RNA was detected in 14 mosquito samples collected in the Krasnoperekopsk District: 9 – Ae. caspius, 4 – Cx. modestus, 1 – Cx. pipiens (Fig. 1). Positive samples were detected only in mosquitoes collected in the homestead plots of Krasnoperekopsk (45.959814 N, 33.806703 E) (urban biotope) and in the vicinity of the village of Ilyinka (45.81714 N, 33.806703 E) (rural biotope). All carriers positive for WNV RNA were sampled in the first half of July, which can be regarded as a precursor of a possible complication of the epidemiologic situation.

Specialists of the Volgograd Research Anti-Plague Institute of Rospotrebnadzor isolated 3 isolates of WNV (2 – Cx. modestus and 1 – Ae. caspius) from mosquito samples positive for WNV RNA. There is no information on the isolation of WNV strains in the territory of the Crimean Peninsula. Phylogenetic analysis of the complete genomes of these isolates showed that they belonged to the clade of WNV of the 2nd genotype formed in 2021, which included isolates from Volgograd, Astrakhan, Rostov, Voronezh Regions, Republics of Dagestan, Kalmykia, and Karachayevo-Chircassian Republic.

Discussion

Studied areas. According to the existing schemes of physico-geographic zoning, the entire Crimean Peninsula is divided into the Crimean steppe province, which includes among the surveyed territories the Krasnoperekopsk, Razdolnensky, Pervomaysky, Krasnogvardeysky and Saksky Districts, the Mountain-Crimean province formed by foothill forest-steppe (Simferopol District) and the main mountain-meadow-forest ridge (Bakhchisarai District). In general, the climate here varies from moderately hot and semi-moist in the lowlands to cool and excessively humid in the western yailas. The moderately continental climate prevailing in the steppe province of Crimea with average summer temperatures ranging from +22 °C in the north to +24.8 °C in the south provides favorable conditions for the formation of local foci of WNF regardless of temperature fluctuations in the season of possible transmission of the pathogen.

The comparatively small amount of precipitation (not more than 300–400 per year)⁵ and long hot summers lead to scarce hydrography of the peninsula. There are no large freshwater lakes in the territory, but there are more than 50 lakes-estuaries, separated from the sea by spits and sand bars and having various degrees of mineralization. Most rivers have the status of small rivers, which dry up in summer and fill with water during heavy rains⁶. Small standing water bodies are formed along the banks of small rivers and temporary watercourses, which are potential breeding grounds for preimaginal stages of blood-sucking mosquitoes.

According to the analysis of earlier entomological surveys of the territory of the Crimean Peninsula, the species composition of the fauna of bloodsucking mosquito family includes 40 species, of which 38 (95%) are found in the mountain ecosystem, and 19 (47.5%) – in the plain ecosystem [3]. The most abundant species of p. Culex mosquitoes in the plain and mountainous parts of Crimea is Cx. pipiens. Cx. modestus occupies mainly the territory of the North Crimean lowland steppe, and in the mountainous landscape-climatic zone of Crimea, a high density of Cx. hortensis is observed [6]. The main representative of the genus Anopheles is An. maculipennis, of the genus Aedes – Ae. caspius. In addition to endemic species, imported mosquito species were found in Crimea: Ae. koreicus – in 2018 in Simferopol and Sevastopol [7], Ae. albopictus – in 2019 in the cemetery of Yalta [8].

The structure of the mosquito species composition of the Crimean Peninsula and the quantitative ratios of representatives of different ecological-faunal complexes changed significantly under the influence of anthropogenic factors. The implementation of large irrigation systems, reservoirs and irrigation systems on agricultural lands in the steppe part of Crimea in the second half of the 20th century led to an increase in the moisture content of the territory. The abundance of typical gallophiles (An. atroparvus and Ae. caspius) decreased in the first years after the construction of the North Crimean Canal systems, and then, due to the gradual salinization of water bodies, began to recover, and by 2000, had sharply increased.

The development of rice cultivation led to a significant increase in the abundance of An. maculipennis, An. atroparvus, An. messeae, and Cx. modestus [9]. In recent years, in the conditions of insufficient water supply in the Crimean Peninsula, the areas of active irrigated agriculture and rice paddies areas have reduced, which could affect the biodiversity and territorial distribution of bloodsucking mosquitoes, but there were no comprehensive monitoring studies on the fauna and ecology of Culicidae.

Mosquito fauna of urban biotopes. A total of 4,966 mosquito specimens of 9 species were captured in biotopes. The mosquito fauna in barns is represented by 3 species: An. maculipennis – the most numerous species in collections, absolute eudominant; Ae. caspius – dominant; An. claviger – extremely rare (Table 1). In the territory of homestead plots, Ae. caspius was the most abundant species in collections, preferring temporary or permanent water bodies with a well-defined drainage zone for breeding sites. The dominant species included Cx. pipiens and Cx. modestus. The other encountered species here were classified as extremely rare.

Only 3 species of mosquitoes were found in cemeteries. The low faunal diversity is explained by the absence of habitual breeding grounds for most species of mosquitoes in the area. The most abundant species was Ae. albopictus. It should be noted that during entomological monitoring, previously conducted by specialists of the Federal State Committee on Plague Control Station of the Republic of Crimea of Rospotrebnadzor, a significant expansion of the habitat of this carrier species throughout the southern coast of Crimea was revealed [10] with “displacement” of other local mosquito species.

The authors found the presence of Ae. albopictus mosquitoes in the central part of the Crimean Peninsula (Simferopol, Bogdanovskoye cemetery), which is not mentioned in the available literature. This carrier is of particular interest due to the experimentally established high efficiency of WNV transmission, which is much higher than that of Culex mosquitoes. Another invasive mosquito new to this region, Ae. koreicus, which showed in laboratory conditions its ability to transmit Japanese encephalitis and dengue viruses, was not detected.

Ae. caspius was the absolute eudominant in urban biotopes on homestead plots, with a dominance index of 72.42%. Probably, its high abundance here is maintained due to breeding in estuaries located in close proximity to the surveyed settlements. This species tolerates well the conditions of slightly saline water bodies.

It should be noted that the fauna of closed biotopes is much poorer than that of open biotopes due to the absence of exophilic species. The high abundance of Cx. pipiens in urban biotopes is explained by the presence of artificial containers with water reserves for irrigation on homestead plots due to the aridity of the area. Such containers are the optimal place for larvae of this species. Development of aquatic stages of Cx. modestus occurs only in water bodies with reed beds along the banks. As a rule, mosquitoes of this species do not disperse from the brood sites, but concentrate along the water edge. They are probably brought to the territory of settlements in the surveyed areas by wind from estuaries and other water bodies.

Mosquito fauna of rural biotopes. Eleven species of blood-sucking mosquitoes (13,627 specimens) were found in the biotopes. The eudominant species were Culex modestus and Aedes caspius (Table 1). Their high abundance is due to the ability of mosquito larvae of these species to tolerate increased salinity of water bodies located near rural settlements.

The mass appearance of Ae. caspius occurs in late spring, but, since this species is polycyclic, in especially rainy seasons, it is possible that several generations develop during the entomological season, which implies that gonotrophic cycles are repeated by the end of summer, the epidemiologically dangerous period, and therefore, this species is more likely to be infected with WNV. Cx. modestus poses the greatest threat as a carrier of WNV due to its high abundance and active attack on humans during daylight hours in close proximity to water bodies.

The dominance index of Cx. pipiens was also significantly reduced in rural habitats due to the presence of a small number of water bodies suitable for larval brood-rearing. However, Coquillettidia richiardii, a water-loving species that prefers natural water bodies with lineid thickets for brooding, joins the list of dominant species.

Such species as An. algeriensis and Ae. flavescens are subdominants in rural habitats. Mosquitoes of the species Culiseta annulata and An. claviger are polyphagous and actively attack humans. These two species were not numerous in the authors’ collections, but they should also be considered as potential carriers of WNV due to the detection of markers of the pathogen in the endemic areas. An. hyrcanus and Ae. vexans, which are rare or extremely rare in rural biotopes, were not detected in the collections in urban biotopes, probably, due to the insufficient number of surveyed sites and the biological peculiarities of these blood-sucking mosquito species.

The authors established the presence of 12 mosquito species in the northern and central part of Crimea. The obtained faunistic list is not complete and does not fully reflect the species composition of mosquitoes for this territory due to the limited time period of research, as well as ethological features and specific trophic relationships of blood-sucking mosquitoes.

The highest level of WNV infection (6.9%) in the present studies was found for Ae. caspius mosquitoes in urban biotopes. Summarizing the data on the infestation of this carrier, its dominance in urban and rural biotopes, and peculiarities of trophic relationships (polyphagous, showing a significant level of anthropophilia), it can be assumed that in the territory of Crimea, this species plays an important role in the circulation of WNV.

For Cx. modestus, the rate of WNV infection in rural habitats was 1.8%, and for Cx. pipiens – 2.63%. In populated areas, no WNV-infected mosquitoes of these species were detected, which is probably due to the small volume of material examined.

The overall infection rate of blood-sucking mosquitoes was 2.02%. In urban biotopes, this indicator was 2 times higher and reached 4.32%, determining a high risk of WNV infection among urban population.

The data and the generalization of the results of the monitoring of the WNV pathogen for the period of 2014–2022, conducted by Rospotrebnadzor bodies and institutions, allowed the authors to assess the zoning of the territory of the Crimean Peninsula (Fig. 2). It was found that in 13 administrative entities, the circulation of WNV was not confirmed so far. Thus, a list of territories subject to priority monitoring for the causative agent of WNV for the purpose of zoning the territory of the Republic of Crimea by the degree of risk of WNV infection was proposed.

Conclusion

According to the results of the study, the species composition of potential transmitters of WNV was clarified, and the presence of the invasive mosquito species Ae. albopictus in the central part of the Crimean Peninsula was established for the first time, which indicates the expansion of its habitat. The high level of infection and dominance of Aedes caspius mosquitoes in urban areas indicates their active participation in the circulation of WNV and the risk of infection of the population in these biotopes. In rural areas, Culex modestus and Aedes caspius mosquitoes were identified as the dominant species, but Culex pipiens had the highest level of WNV infection, which confirms the role of these three species in the transmission of the pathogen in natural anthropurgic foci.

For the first time, the circulation of the WNF pathogen was confirmed in the north of the Crimean Peninsula (Krasnoperekopsk District) and WNV was isolated, which is the first report on the isolate of WNF pathogen in the Republic of Crimea.

Taking into account the obtained evidence of intensive circulation of WNV in the epizootologic cycle, the specialists of the Volgograd Research Anti-Plague Institute of Rospotrebnadzor, which is the base of the Reference Center for monitoring WNF pathogen of Rospotrebnadzor, recommendations were provided for Rospotrebnadzor agencies and institutions to strengthen epidemiological surveillance and preventive measures, which allowed them to prevent complication of the epidemiological situation.