Serosurvey of Eastern,Western,and Venezuelan Equine Encephalitis Viruses in Wild Boars ( Sus scrofa ),Hunting Dogs,and Hunters of Brazil

Introduction

Eastern equine encephalitis (EEE), western equine encephalitis (WEE), and Venezuelan equine encephalitis (VEE) are zoonotic arboviral diseases caused by arboviruses in the genus Alphavirus (Armstrong and Andreadis 2013). Birds and wild rodents are the main reservoir hosts for the causative agents EEE virus (EEEV), WEE virus (WEEV), and VEE virus (VEEV). Equids and humans are incidental hosts, and when infected, the disease often causes severe and usually fatal neurologic disease (Armstrong and Andreadis 2013). Domestic and wild animals worldwide, such as dogs (Canis familiaris), wild boars (Sus scrofa), and white-tailed deer (Odocoileus virginianus), are known to be susceptible to EEEV infections (Elvinger et al. 1996, Farrar et al. 2005, Tate et al. 2005).

The wild boar, S. scrofa, is not native to Brazil. It is a Eurasian domestic pig that was imported and then escaped into the wild >200 years ago (Desbiez et al. 2011, Brazil 2013a). Owing to its highly adaptative nature, the wild boar has invaded rural areas in all six Brazilian biomes, including the Atlantic Forest and the Cerrado. These regions include ecosystems that could sustain EEEV transmission (de Sousa et al. 2015). Because the wild boars are destructive and have a negative impact on natural and agricultural ecosystems, Brazil has permitted limited nationwide hunting of wild boar to control the population and limit expansion of the range (Normative Instruction 03/2013). The most popular wild boar hunting practices include using hunting dogs to track the wild boar (Brazil 2013a). This practice allows for potential exposure of the hunting dogs and the hunters to EEEV infections.

Although EEE outbreaks in equines and sporadic fatal human cases have been reported periodically in most regions of Brazil (de Novaes et al. 2014, de Sousa et al. 2015), no studies to date have evaluated EEEV infections in free-range wild boars, hunting dogs, or hunters. From 2016 through 2018, we screened sera from wild boars, hunting dogs, and hunters from southern, southeastern, and central-western Brazil for antibodies against EEEV, WEEV, and VEEV. In this report we describe our findings.

Materials and Methods

This study was conducted in rural areas in the Atlantic Forest and Cerrado biome of southern and southeastern Brazil, including the unit conservation Vila Velha State Park in southern Brazil and Vassununga State Park in southeastern Brazil, and in rural areas in the Cerrado biome of central-western Brazil, at the Aporé municipality (Fig. 1).

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FIG. 1.

Sampling locations of wild boars, hunting dogs, and hunters from southern, southeastern, and central-western Brazil.

Blood samples were obtained from free-range wild boars located in degraded areas after being slaughtered by firearm by legally registered hunters at the Brazilian Institute of the Environment and Renewable Natural Resources. Free-range wild boars from natural areas in the Vila Velha State Park and Vassununga State Park were baited, photo-monitored, trapped, and then slaughtered by firearm.

Serum samples of wild boars, hunting dogs, and hunters were conveniently sampled from October 2016 through May 2018. Blood samples were collected from 86 wild boars by intracardiac puncture immediately after death. In addition, blood samples of the 16 fetuses from 2 pregnant sows (8 each) were also collected after slaughter. Blood samples from 170 hunting dogs were collected by jugular puncture with approval from the ethics committee of animal use of the Federal University of Paraná (protocol 059/2017). Blood samples from 49 hunters were collected by cephalic puncture with approval from the Ethical Appreciation at Ethics Committee in Human Health of the Brazilian Ministry of Health (protocol 97639017.7.0000.0102).

Testing for the presence of antibodies against EEEV (Tatuí sample) was performed at the Biological Institute, a National Livestock Reference Laboratory located in São Paulo. All serum samples were tested in duplicate with a microplate serum neutralization test (Smart et al. 1975). In addition, the presence of antibodies against WEEV (TR 25717 sample) and VEEV (SP AN 15600 sample) was also done with a microplate serum neutralization test (Smart et al. 1975). Samples with titers of ≥5 were considered positive.

Results

Only antibodies for EEEV were detected in 3 of 102 (2.9%) free-range wild boars from the Cerrado biome of central-western Brazil, with titers 1:10 in one and 1:40 in two wild boars. None of the 170 hunting dogs or 49 hunters were seropositive for EEEV, WEEV, or VEEV.

Discussion

To our knowledge, this is the first study ever screening wild boar, hunting dogs, and hunters for EEEV antibodies. Our results indicate that ∼3% of the wild boars in central-western Brazil had been exposed to EEEV infections and are supported by the fact that there has been EEE outbreaks in horses within the same general area (Cunha et al. 2009). Taken together, our observation and those of Cunha et al. (2009) suggest that there is an EEEV activity focus in central-western Brazil. The EEEV antibody prevalence in wild boar found in this study was lower than the 16.5% detected in feral swine in Georgia, USA, which may suggest lower EEEV activity in central-western Brazil or that our sampling was on the periphery of the EEEV focus (Elvinger et al. 1996). Although we did not detect VEEV antibodies in wild boars, previous studies have reported high seropositive levels as high as 59.4% (Smart et al. 1975), which suggested that wild boars can be used as sentinels for VEEV activity.

All hunting dogs sampled in this study were seronegative for EEE, WEE, and VEE viruses, corroborating results or a previous study with rare seropositive dogs (Farrar et al. 2005). In a previous study of EEE endemic area, only 1 of 101 puppy serum samples was seropositive to EEEV (Farrar et al. 2005). Once dogs can produce antibody titers to EEEV, EEE should be considered as a differential diagnosis for other neurologic diseases, mainly in young dogs, such as toxoplasmosis, canine distemper, and rabies (Andrews et al. 2018).

In this study, none of the hunters sampled were seropositive for EEE, WEE, or VEE viruses. As with dogs, EEE in human beings have been infrequent but high-case fatality in Brazil (de Sousa et al. 2015). In another study, 28 of 337 (8.31%) students aged 6–14 years old, predominantly from rural areas, were seropositive for arbovirus antibodies, including EEEV (Iversson et al. 1982). In rural areas of the Cerrado biome, the EEEV seasonality may be the result of high seasonal rainfall leading to vector proliferation, as similar epidemiological findings observed worldwide in outbreak areas (Elvinger et al. 1996, de Sousa et al. 2015). All three seropositive wild boars were hunted at the central-western Brazil, within the same region of a previous report of EEEV circulation in horses (Cunha et al. 2009). Standard diagnostic tests herein were performed in duplicate, in compliance with the official mandatory communication of EEE, WEE, and VEE viruses in livestock animals at the Brazilian Ministries of Health, Agriculture and Livestock, as well as the World Organization for Animal Health (OIE) (Brazil 2013b, 2017, OIE 2018). In addition, tick interactions among hunters, hunting dogs, and wild boars have been associated with Brazilian spotted fever, another vector-borne disease (Kmetiuk et al. 2019). Finally, further studies are needed to evaluate the potential of incorporating serosurveys in routine arbovirus activity surveillance specifically to identify arbovirus activity foci and to help establish thresholds for epidemic transmission.

Conclusions

In summary, this is the first study ever screening wild boar, hunting dogs, and hunters for EEEV antibodies. Despite hunting dogs and hunters have been seronegative herein, free range wild boars from central-western Brazil may be exposed to EEEV, and further studies are needed to evaluate the potential of incorporating serosurveys in routine arbovirus activity surveillance.