this study was approved by the ethical committee of animals uses in veterinary medicine and animal production of so paulo state university . the capture of wild animals was authorized by the brazilian institute of environment and natural resources renewable . in october 2012 , the veterinary service of itatinga county ( 2367s , 483657w ) in so paulo state , brazil , reported an outbreak similar to that of bovine smallpox that affected 2 small dairy farms . the veterinary medical and animal husbandry school of so paulo state university were contacted , and a team of veterinarians visited the 2 dairy farms to collect samples for diagnosis and relevant information by using an epidemiologic questionnaire . both farms ( 1.5 km apart ) used manual milking systems and had similar sanitary management of herds . animals were raised in pastures , and there were no changes in management or introduction of new animals on both farms . blood and scab samples from udders of cows or nostrils of calves were collected from 31 affected animals . serum and whole blood were collected from 2 humans who worked as milkers and had lesions on their hands and arms ( figure 1 ) . blood samples were also collected from 6 dogs , 6 pigs , 2 horses , 2 rams , 3 opossums , 1 coati ( nasua nasua ) , and 2 wild rodents ( akodon montensis and nectomys squamipes ) . both domestic and wild mammals were evaluated at the time of collection for characteristic clinical signs of vacv , such as vesicles , scabs , or crusts . lesion on arm of a human who worked as a milker , so paulo state , brazil . opossums and the coati were captured by using a trap ( tomahawk live trap , hazelhurst , wi , usa ) containing thigh or drumstick chicken as bait . these animals were anesthetized with tiletamine and zolazepam by using the recommended dose for each species ( 7 ) . tallahassee , fl , usa ) containing peanuts , cornmeal , oats , and canned sardines as bait . these animals were anesthetized in an autoclavable bag containing gauze soaked in ethyl ether and then euthanized by using deep anesthesia . five trap nights were required to capture animals in the native forest areas surrounding both farms . virus dna was extracted from scabs and total blood samples by using the invisorb spin tissue mini kit and the invisorb spin blood mini kit ( stratec molecular , berlin , germany ) , respectively . a seminested pcr was conducted for amplification of the a56r gene of vacv ( 8) , and positive samples were submitted for gene sequencing at the federal university of minas gerais state ( belo horizonte , brazil ) . responses to the epidemiologic questionnaire showed that no factors could be correlated with the 2 outbreaks . no domestic or wild mammals had clinical signs at the time of blood sample collection . however , 3 dogs and 3 opossums were positive by pcr for vacv . kj741387.1 , kj741388.1 , kj741389.1 , kj741390.1 , kj741391.1 , and kj741392.1 ) showed that vacv isolated in this study had the same deletion of six amino acids at position 251 that is found in group i vacvs from brazil , such as cantagalo virus , araatuba virus , passatempo virus , and guarani p2 virus ( 4,9,10 ) ( figure 2 ) . tree was constructed by using the neighbor - joining method , the tamura-3 model of nucleotide substitutions , and 1,000 bootstrap replicates in mega 4.0 ( http://www.megasoftware.net/mega4/mega.html ) . i d 5 ( kj741390.1 ) , i d 7 ( kj741391.1 ) , and i d 13 ( kj741392.1 ) are from opossum ( didelphis albiventris ) blood samples ; i d 5 and i d 7 are from the first farm sampled ; i d 13 is from the second farm sampled ; i d 4 ( kj741387.1 ) , i d 9 ( kj741388.1 ) , and i d 10 ( kj741389.1 ) are from canine blood samples ; and i d 4 , i d 9 , and i d 10 are from the first farm sampled . murv , muria virus ; ctgv , cantagalo virus ; arav , araatuba bat virus ; marv , mariana virus ; pstv , passatempo virus ; gp2v , guarani p2 vaccinia virus ; hspv , horsepox virus ; mpxv , monkeypox virus ; varv , variola virus ; cpxv , cowpox virus . the fact that the dogs and opossums positive by pcr for vacv did not have clinical signs of infection might indicate only a subclinical infection or may be related to the dichotomy of vacvs from brazil , which is not only genetic but also biologic . ferreira et al . ( 4 ) showed that although there was no difference in severity of lesions in humans and cows affected by group i and ii vacvs , balb / c mice that were intranasally inoculated with group i vacvs did not have clinical signs . in contrast , mice inoculated with group ii vacs , such as belo horizonte virus and gurani p1 virus , had acute respiratory illness , followed by death . more than 1 mammalian species , either wild or domestic , might be acting as a reservoir of group i vacvs from brazil , possibly acquiring and transmitting the virus without showing clinical signs . ( 6 ) , which showed a high seroprevalence in dogs without clinical signs , questioning their possible role as a reservoir and suggesting more studies to confirm these findings . genetic and epidemiologic analysis showed that vacv circulating in this region are members of group i vacvs in brazil . these findings might support the absence of clinical signs and raise major questions about the potential for > 1 mammalian species , either wild or domestic , to act as a reservoir .
during a vaccinia virus ( vacv ) outbreak in so paulo state , brazil , blood samples were collected from cows , humans , other domestic animals , and wild mammals . 
 samples from 3 dogs and 3 opossums were positive for vacv by pcr . 
 results of gene sequencing yielded major questions regarding other mammalian species acting as reservoirs of vacv .