All 86 wild boars (Sus scrofa), 170 hunting dogs, and 49 hunters sampled from three Brazilian regions were seronegative to Brucella spp. by the standard tube agglutination and 2-mercaptoethanol tests, suggesting a low circulation of Brucella spp. in wild boars, hunting dogs, and hunters in such areas.

Brucellosis is a zoonosis caused by gram-negative bacteria of the genus Brucella, which infects livestock, wildlife, and humans (Godfroid 2002; Ficht 2010). Wild boars (Sus scrofa), domestic pigs, and other wildlife species have been indicated as reservoirs of Brucella suis and Brucella abortus for both livestock and wildlife species (Godfroid 2002; Olsen and Tatum 2017).

In humans, hunting activities are a greater potential source of Brucella spp. exposure than are other working and recreational activities (Pereira et al. 2020). Additionally, wild boar hunting has been considered a source of B. suis infection in hunting dogs (Mor et al. 2016).

In Brazil, wild boars are classified as exotic invasive species, with nationwide hunting officially permitted for control, generally using hunting dogs for wild boar tracking (IBAMA 2013). Although B. abortus, B. suis, and Brucella melitensis have been recognized as the most prevalent Brucella spp. in Brazil and wild boar hunting has been associated with wildlife-dog-human transmission (Pereira et al. 2020), no study has simultaneously assessed Brucella spp. exposure in wild boars, hunting dogs, and hunters. Our study aimed to evaluate that.

Our study was conducted October 2016 through May 2018 in preserved and degraded areas in the Atlantic Forest biome of southern and southeastern Brazil, including the Vila Velha State Park in southern Brazil and the Vassununga State Park in southeastern Brazil, and degraded areas in the Cerrado biome of central-western Brazil at the Aporé municipality.

Blood samples were obtained from 86 free-ranging wild boars killed by legally registered hunters. These boars, from natural areas of the Vila Velha and Vassununga state parks, were baited, photo-monitored, trapped, and then killed by firearms; blood samples were collected by intracardiac puncture immediately after death. Blood was collected from 170 hunting dogs from various rural and urban areas using jugular venipuncture, with the approval from the Ethics Committee of Animal Use of the Federal University of Paraná (protocol no. 059/2017). Blood was collected from 49 hunters using cephalic venipuncture with the approval from the Ethical Appreciation at Ethics Committee in Human Health of the Brazilian Ministry of Health (protocol no. 97639017.7.0000.0102).

Testing for antibodies against Brucella spp. was performed at the Biological Institute, a National Livestock Reference Laboratory (São Paulo, Brazil). Sera were initially screened with the Rose Bengal test (RBT). Sera positive with the RBT were then tested using anti–B. abortus antibodies (which cross-react with B. suis and B. melitensis) in the standard tube agglutination test (STAT) and the 2-mercaptoethanol test (2MET; Paulin et al. 2002), performed in parallel.

Of the wild boar, only two individuals, one from southern and one from central-western Brazil, were seroreactive by the RBT; all samples from dogs and humans were negative in the RBT, so were not tested further. The two wild boar samples had negative results by both the STAT and 2MET; a sample must be positive in both the STAT and 2MET tests to be considered positive (to increase specificity of the final interpretation; OIE 2018). Thus, all 86 wild boars, 170 hunting dogs, and 49 hunters were considered seronegative to smooth pathogenic Brucella spp., suggesting a low circulation of Brucella spp. in the wild boars, hunting dogs, and hunters in this area.

The only other study conducted in Brazilian free-range feral pigs, in the central-western Brazil floodplains biome (Pantanal), also found all 105 animals to be seronegative to Brucella spp. (Zimmermann et al. 2018). Those authors concluded that, despite widespread brucellosis in bovine herds, sympatric feral pigs may have low environmental exposure to Brucella spp. because of low animal density and low cohabitation in such extensive areas.

In addition, a study of domestic pigs (Sus scrofa domesticus) from backyard pig farms in southeastern Brazil found only 1/346 (0.29%) pigs were seropositive to B. abortus, showing that 1/56 (1.79%) farms were infected (Ricardo et al. 2016), indicating a minor role of domestic pigs inn Brucella spp. transmission.

Our results contrast with those of previous studies, which have shown wild boar populations as the main reservoirs for B. suis biovar 1 in the US (Leiser et al. 2013). We hypothesize that the lack of brucellosis detection in free-ranging Brazilian wild boars may be due extensive natural and agricultural that do not predispose close contact with livestock.

In Australia, contact between hunting dogs and feral pigs has been associated with a 17-fold increase in canine brucellosis cases and a higher risk of B. suis transmission to hunters, household contacts, and livestock (Mor et al. 2016). In contrast, a study in Iran reported only 6/180 (3.3%) seropositive rural dogs, which was considered unrelated to hunting activities (Gharekhani and Sazmand 2019).

Although all hunters analyzed in our study tested negative to Brucella spp., hunters are reportedly more exposed to zoonotic pathogens, including Brucella spp., when compared with other risk groups, such as veterinarians, farmers, and slaughterhouse workers (Deutz et al. 2003), and particularly associated with feral pig population-control programs involving hunting dogs (Woldemeskel 2013). Our results contrasted with previous epidemiologic studies in the US and Europe, which found association between free-range wild boars and spreading of brucellosis, mostly to domestic pigs and hunters (Leiser et al. 2013). Because RBT is based on the Brucella smooth lipopolysaccharide, smooth strains detection of O-polysaccharide (O-PS), false-positive serologic reactions may occur, such as cross-reactions with O-PS bacteria in infected swine (Dieste-Pérez et al. 2015). Additionally, false-positives may occur if immunoglobulin M has been inadequately inactivated (OIE 2018). In contrast, false-negative reactions rarely occur with standard diagnostic methods for Brucella spp. (OIE 2018). The two false-positive RBT sera results may indicate Yersinia enterocolitica infection, given the similarity between the O-PS component of the smooth lipopolysaccharide of Brucella spp. and Y. enterocolitica, which have previously associated with false-positive RBT results in free-ranging and captive wild animals of southeastern Brazil (Antunes et al. 2010). Despite the known false-positives found with the RBT because of cross-reactivity with other pathogens or inadequately inactivated immunoglobulin M, the RBT is still useful as a screening test because of its excellent sensitivity, rapidity, feasibility, and low cost.

Because the serologic tests we used only tested smooth Brucella spp., further studies on Brazilian hunting dogs should focus on detection of Brucella canis, a rough Brucella sp. (Hensel et al. 2018).

We thank Ismail da Rocha Neto, Fabrício Pinheiro da Cunha, Fernanda Pistori Machado, and Osvaldir Hartmann for helping with in-park wild boar capture and sampling; João Henrique Perotta and Laís Giuliane Felipetto for veterinary assistance; the Environmental Institute of Paraná staff, particularly Mauro de Moura-Britto; and the Campos Gerais National Park for authorization and technical support. We also thank the City Secretary of Health in Ponta Grossa for human blood sampling. Louise Bach Kmetiuk has been supported by graduate fellowships from the Coordination for the Improvement of Higher Education Personnel (CAPES).

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