Antibody Prevalence to Influenza Type A in Wild Boar of Northern Ukraine

Introduction

Influenza viruses are segmented RNA viruses belonging to the Orthomyxoviridae, which includes three different influenza virus genera: A, B, and C (Acland et al. 1984). Among these, influenza A (IA) viruses (IAVs) are the most common and can infect people, domestic and wild birds, and mammals, including marine mammals and other animals (Webster et al. 1992, Fouchier et al. 2003, Wright et al. 2006). However, wild birds represent the natural hosts for these viruses and they are generally considered the major reservoir hosts for these viruses (Webster et al. 1992, Fouchier et al. 2003, Swayne and Halvorson 2003). Domestic swine have been viewed as important for the adaptation and spillover of type A influenza from birds into human populations as they are sensitive to both avian and mammalian (including human) influenza viruses (Runstadler et al. 2013). However, in much of Eurasia and North America, wild boar are geographically widespread, abundant, and often come in close contact with humans in rural and agricultural settings. The wild boar density was estimated as 0.15–0.18 head/km² in Belarus in 2008 by FAO reports. Regarding the density in Poland, there is a program to control the population of wild boar because of African swine fever, but recent population estimates are not available. The wild boar is a plentiful and widespread species in Ukraine and is frequently hunted. According to the latest data in Ukraine in 2015, the wild boar population density was estimated as 0.015–0.375 head/km² (Bagamian et al. 2014, asf.vet.ua. 2015). Until recently, little attention has been paid to this as an alternative route for IA transmission to human and domestic populations, and the significance of wild boar in the spread of IA is not clear.

In the past few years, IA infection in wild boar has been examined in several European countries. The reported prevalence has varied but ranged from 0.7% in Poland (Markowska-Daniel 2003), 6.4% in Spain (Closa-Sebastia et al. 2011), and 3.9% in Italy (Foni et al. 2013). A survey in Slovenia did not find any serological response for influenza virus in 178 swine that were examined (Vengust et al. 2006). In Germany, a survey was carried out for 2 consecutive years and showed 7.8% and 5.2% of positive samples, respectively (Kaden et al. 2008, 2009). In Croatia, a recent investigation revealed 9.7% positive response (Roic et al. 2012). Serological positive response was also shown in the United States with a baseline rate of 11.0% (Saliki et al. 1998). In France, no evidence of influenza virus circulation was found in nasal swabs (n = 315) and no influenza-specific antibodies were observed in serological samples (n = 20) collected from wild boar living in an area shared with migratory waterfowl (Vittecoq et al. 2012).

Numerous reports have been made of IAV infecting mammals that could transmit these viruses among other wild and domestic animals, posing a risk for virus spread and the emergence of mutant strains. Therefore, the monitoring of the exposure of wild mammals to IA was viewed as essential for identifying potential reservoir hosts impacting domestic animals and public health.

As part of a national level program, a preliminary serological survey was carried out to assess the likelihood of IA infection in wild boar and begin to characterize the role of wild boar in the epidemiology of the IAV in this region. The area selected for initial studies was in the northwest and north central region of Ukraine. This area is known to have substantial populations of wild boar (Biodiversity Monitoring in Ukraine 2008, Bagamian et al. 2014). In addition, in the northwest corner of the country, bordering Belarus and Poland, is an especially suitable area for waterfowl used during their migrations between Europe, Africa, and the Middle East (Shatsk Lake region).

Materials and Methods

From September to December 2014, wild boar sera were collected by professional hunters in four oblasts of Ukraine: Volyn, Rivne, Zhytomyr, and Chernihiv. Blood samples for serological survey were collected from the jugular vein immediately after shooting of the boar by hunters using medical syringes. The syringe plunger was pulled back and the syringes were kept upright in sample racks for transport. The samples were sent to the local veterinary diagnostic units where the sera were collected in tubes, separated from whole blood without centrifugation, and stored at −20°C until serologically tested. Later, the sera were transported to the Institute of Veterinary Medicine where the serological investigations were conducted. Before serologically testing, the additional sample preparation took place that included centrifugation (10 min/1308 × g). To detect antibodies to IA, a blocking the enzyme-linked immunosorbent assay (ELISA) was used. Serum samples were tested using commercial test kits “Influenza A Ab Test” (IDEXX). Specific antibodies in wild boar serum samples were detected based on manufacturer's instructions (IDEXX 2017). Absorbance was measured at 650 nm using an iMark Microplate Absorbance Reader and data were analyzed using Microsoft Excel. Based on the manufacturer's instructions, a serum sample was considered positive if the sample/negative control ratio (S/N) did not exceed a threshold of 0.60. Statistical analyses were performed using EpiTools (Sergeant, ESG 2017). Confidence intervals were calculated using Wilson's Score Interval.

Results

Sera from 120 wild boar that were hunted in autumn/winter 2014, with a distribution of 30 boar collected from each of four oblasts in the north central and northwestern regions of Ukraine, were tested (Fig. 1). Antibodies against IAV were detected using ELISA in 27 samples (22.5%; Table 1). Antibodies to IAV were detected in at least some of the wild boar from all of the four oblasts. The highest percentages of seropositive samples were detected in wild boar from Volyn and Zhytomyr oblasts, 36.6% and 23.3%, respectively, whereas the lowest seroprevalence of 13.3% was found in Chernihiv oblast (Fig. 1). Overall, there was no statistically significant difference among oblasts in the prevalence of infection (χ² = 5.50; 3 df, p < 0.14), although the geographically extreme locations differed most in prevalences (Volyn and Chernhiv oblasts; Table 1). Power calculations under simple assumptions of independence indicate up to 75 individuals from each oblast would have been needed to detect differences as statistically significant.

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FIG. 1.

Locations of sampled oblasts in northern Ukraine and seroprevalence in wild boar.

Results expressed as S/N values showed substantial differences in the average S/N values of the positive serum samples (0.36 ± 0.03) compared with the negative samples (0.78 + 0.04). The weakest S/N values were found among the seven positive sera samples of wild boar from Zhytomyr oblast (0.45 ± 0.041).

Discussion

This preliminary survey of IA antibodies in wild boar populations of northern Ukraine indicates a substantial presence of exposure to IAV throughout the region. The overall prevalence (22.5%) was substantially higher than reported in other European nations that have been surveyed to date (3, 5, 7–10, 12–14). If the S/N values are used as an indicator of antibody responses in the samples, the data also suggest that boar from Zhytomyr oblast had lower concentrations of specific antibodies. This may suggest that the viruses infected the wild boar in this region earlier than in the other oblasts. Natural infection of IAV in swine (e.g., swine influenza virus: H3N2) has shown that antibodies to this virus could still be detected 28 months postinfection (Desrosiers et al. 2004). It has been noted that a detectable antibody response in naturally exposed humans was long lasting, up to nearly 5 years in patients followed that long (Kitphati et al. 2009). The scientific literature is surprisingly limited on the long-term persistence of select IAV antibodies after natural infections (Desrosiers et al. 2004), especially in wildlife species. Other reasons also may include variation in sample maintenance or differences in the age structures of local boar populations across regions, which could not be assessed in the current analysis.

Infection of wild boar populations throughout the region could provide an alternative, or additional, route for spillover from wild populations to domestic animals and humans. This potential has received relatively little attention until recently, likely, in part, because wild boar populations have not been viewed as a serious source of infection in most regions of the world where the natural history of IA has been studied. However, more recently, One Health studies and more integrated studies have yielded a clearer understanding of the potential interplay of these populations. For example, regional estimates of wild boar densities in eastern Europe and the western Russian Federation show the region of our study was among the highest in Ukraine but remained substantially below (one-third to one quarter) the estimated densities in neighboring countries (Beltrán-Alcrudo et al. 2010).

The highest observed prevalence of IA in wild boar in this study was from Volyn oblast, in northwestern Ukraine on the Polish–Belarus borders. In this area, more than one-third of tested animals were seropositive (Table 1; Fig. 1). The relatively limited sample sizes make it challenging to determine whether the geographic variation in prevalence reflects simple random variation or requires further explanation. If the latter, an obvious aspect within the study region is that Volyn oblast is home to the Shatsky National Natural Park, which serves as a stopover for migratory birds, including waterfowl. These conditions might allow for greater contact between the wildlife species involved in transmission. Data on AIV prevalence in migratory birds in Volyn oblast are limited due to the lack of previous studies, but their presence is likely based on known parameters. For example, in Poland that borders on this oblast, AIV was registered both in wild and domestic birds. Also, wild birds migrate through the forest zone of Ukraine (northwestern regions) in the latitudinal direction, forming the “Polis'kyi migration path.” Migratory birds using this flyway path winter in western and central Europe, including several well-known mass wintering sites in the Netherlands, England, Belgium, Denmark, France, and Germany.

All four oblasts included in the study are predominantly rural, relying on agriculture and forestry activities for much of the economic development in the region. Even the urban areas within the region are relatively small and closely abut the rural areas. In addition, collection of wild fruits and other food is often performed by residents of the region, allowing for greater contact with wild animals than might otherwise be expected. Farm holdings tend to be small and owned by single families—again allowing for substantial contact between people, their animals, and wildlife.

Conclusions

These results indicate that further, more extensive and detailed analyses of the risk of transmission of AIV from various wildlife sources into domestic animal and associated human populations should be considered. For example, as wild boar are omnivores, they could eat diseased or dead migratory birds, from the areas where subtype H5N1 viruses have been reported, and then transfer the virus as they migrate. Special attention should focus on those areas where contact from wildlife sources to urban centers that could allow risk of pandemic transmission through intermediary regional human populations draws first attention. Further investigation and surveillance of influenza virus infections in wild birds and interactions with mammals such as wild boar are needed to better understand influenza ecology.