Clin Infect Dis. serosurvey Thirty-four years ago, the world celebrated the eradication of smallpox, a lethal disease caused by Variola virus infection. The massive antismallpox vaccination campaign was promoted by the World Health Organization (WHO) during the 1960s and 1970s (Fenner et al. 1988, Damon 2013). Vaccinia virus (VACV), a species belonging to the Orthopoxvirus (OPV) genus that demonstrates serological cross-reactivity with other OPV species, was used as the vaccine antigen during the WHO campaign. Following smallpox eradication in the late 1970s, vaccination was suspended due to several instances of adverse reactions to the vaccine (Cono et al. 2003). The natural circulation of VACV began to be reported in Brazil in 1999 and has been associated with several exanthematic VACV outbreaks that have been described in PU 02 Brazilian rural areas (da Fonseca et al. 2011,Kroon et al. 2011, Singh et al. 2012, Shchelkunov 2013). VACV infection causes lesions on the teats and udders of dairy cattle, leading to a decrease in milk production. VACV is the cause of a zoonotic disease known as bovine vaccinia PU 02 (BV) and can be transmitted to humans by direct contact with infected animals during milking, resulting in lesions on the hands and arms (Damaso et al. 2000, Trindade et al. 2003, 2007, 2009, Leite et al. 2005, Lobato et al. 2005, Megid et al. 2008, Silva-Fernandes et al. 2009, Abrah?o et al. 2010a, Schatzmayr et al. 2011, deAssis et al. 2013, de SantAna et al. 2013). The lesions evolve from macules to papules to vesicles to pustules, which ulcerate and result in scar formation. Nonspecific symptoms such as fever and lymphadenopathy can also be observed in most infected individuals (Silva-Fernandes et al. 2009, Trindade et al. 2009). The transmission of VACV is associated with unprotected contact between BV-affected cattle and milkers. Although BV outbreaks associated with vaccine strains were reported during the smallpox eradication campaigns in Latin America and Asia (Fenner et al. 1988), these notifications ceased after vaccination suspension, with only a few cases reported in the 1980s in Southeast Brazil related to contact with cows during milking (Silva et al. 1986). It remains unclear why BV outbreaks have re-emerged after 20 years of absence. Possible explanations for the lack of reported cases for decades include the effective immune response generated by massive smallpox vaccination during the 1970s, significant under-reporting leading to misdiagnoses and the absence of a specific government-enforced surveillance policy (Trindade et al. 2009, da Fonseca et al. 2011). Despite the fact that these outbreaks, as well as the individuals affected by each case, seem to be systematically increasing PU 02 from year to year both in quantity and in geographic distribution, there remains no officially reported number of human cases across the country. Theories that propose VACV circulation and maintenance in Brazilian forests have gained attention in recent years, mainly after the detection of VACV in wild and peridomestic animals (Abrah?o et TMOD3 al. 2009, 2010b, Peres et al. 2013). Indeed, VACV strains were previously detected in wild and sentinel rodents from the Brazilian Amazon and southeastern forests in the 1960s and 1970s PU 02 (Lopes et al. 1965, Fonseca et al. 1998). Thus, human exposure to VACV could be related to activities distinct from milking, as suggested by Mota et al. (2010). Although there are numerous studies related to the occurrence of VACV in Brazil, little is known about anti-OPV immunity in vulnerable populations. A recent study performed by our research group identified a low prevalence of OPV immunity in laboratory workers (Costa et al. 2013). However, most studies have concentrated their efforts on the analysis of humoral responses in patients affected by BV outbreaks or in rural areas where the occurrence of BV has never been reported (Silva-Fernandes et al. 2009, Mota et al. 2010). Indeed, there are no data thus far regarding humoral immunity to OPV in rural populations at high risk of VACV infection. The present study retrospectively analysed serological protection against OPV in two Brazilian populations from the Amazon and Southeast Regions, where BV cases have not been observed since.