the outbreak was reported in july 2010 in bom jesus do tocantins county ( 52 60s , 483636w ) , par state , in the amazon region of brazil ( figure 1 , appendix ) .
bom jesus do tocantins county ( black ) in par state ( gray ) , brazil , where an outbreak of bovine vaccinia occurred in 2010 .  
dairy cattle and workers were affected . however , the source ( index case ) of this outbreak was not identified . at the study site ,
44 lactating dairy cows became sick and had painful vesicular lesions on teats , udders , and inner thighs that rapidly progressed to ulcerative lesions and scabs ( figure 2 , panels b and d ) .
two animals had extensive necrosis because of secondary infections , which led to loss of teats .
lesions were also observed on lips , muzzles , oral cavities , and tongues of calves ( figure 2 , panel d ) .
three dairy workers became sick during the outbreak after direct contact with sick animals and had typical orthopoxvirus ( opv ) lesions on their hands , forearms , and abdomen ( figure 2 , panel a ) .
pain in the lesion region , fever , and fatigue were also reported by sick patients .
b and d ) typical lesions on teats and udder of a dairy cow infected by vacv at different stages , ranging from ulceration to scabs .
c ) lesions on a calf s muzzle probably caused by vacv infection during suckling .
we collected 4 scabs and 44 serum samples from the 44 sick animals and 3 serum samples from the 3 dairy workers .
serum samples were tested by using 50% plaque reduction neutralization tests as described ( 8) . neutralizing antibodies were detected in 40 ( 90.0% ) bovine and in 3 ( 100% ) human samples , and titers ranged from 20 to 640 neutralizing units / ml .
supernatants were diluted 1:100 in phosphate - buffered saline and used in a nested pcr specific for the c11r viral growth factor gene as described ( 9,10 ) .
we isolated virus from 1 of the samples that was positive for viral growth factor by nested pcr .
negative results for vacv by pcr and virus isolation might have been caused by loss of virus titers and dna degradation during sample transportation .
after a typical poxvirus cytopathic effect was observed , virus was plaque purified and placed on vero cell monolayers for viral amplification .
this new vacv isolate was named par virus ( parv ) . to investigate the relationship between parv and bav , virus gene a56r ( hemagglutinin )
was amplified and sequenced ( 11 ) . the a56r gene is traditionally used for phylogenetic analysis . in addition , parv a26l ( a - type inclusion body ) was also sequenced ( 12 ) .
the pcr fragments obtained were directly sequenced in both orientations and in triplicate by using a mega - bace 1000 sequencer ) ( ge healthcare , little chalfont , uk ) .
sequences were aligned with opv sequences from genbank by using clustalw ( www.ncbi.nlm.nih.gov/pmc/articles/pmc308517 ) , and alignments were manually checked with mega version 4.0 software ( arizona state university , phoenix , az , usa ) .
optimal alignment of the a56r gene showed high identity among parv and several group 1 vacv - br isolates ( average identity 99.8% ) , including vacv - to ca ( gu322359 ) ( identity 99.9% ) , an amplicon obtained from blood of an amazon monkey in tocantins state , brazil , in 2002 ( figure 3 ) .
parv also showed a signature deletion of 18 nt that was also present in a56r sequences of other group 1 vacv - br isolates   phylogenetic trees based on orthopoxvirus nucleotide sequences of a56r ( a ) and a26l ( b ) genes of vaccinia virus ( vacv ) , par state , brazil .
phylogenetic analysis showed that parv ( black dots ) clustered in the vacv - br - g1 clade and that bean58058 virus ( bav ) clustered in the vacv - br - g2 clade .
trees were constructed by using the neighbor - joining method , the tamura - nei model of nucleotide substitutions , and bootstrap of 1,000 replicates in mega version 4.0 software ( arizona state university , phoenix , az , usa ) . in panel
gp2v , guarani p2 virus ; murv , muriae virus ; ctgv , cantagalo virus ; carv , carangola virus ; marv , mariana virus ; arav , araatuba virus ; hspv , horsepox virus ; gp1v , guarani p1 virus ; mpxv , monkeypox virus ; pstv , passatempo ; cpxv , cowpox virus ; varv , variola virus .
phylogenetic trees of the a56r ( figure 3 , panel a ) or a26l ( figure 3 , panel b ) genes were constructed by using the neighbor - joining method , 1,000 bootstrap replicates , and the tamura 3-parameter model ( mega version 4 ) .
parv sequences clustered with several group 1 vacv - brs isolated during several bovine vaccinia outbreaks in brazil . in both trees , parv clustered in group 1 vacv - br , whereas bav clustered in group 2 .
our results confirm circulation of a new group 1 vacv - br isolate in par state in the amazon region of brazil in the same location where bav ( group 2 vacv - br ) was isolated ( 6,7 ) . in recent years
, bovine vaccinia outbreaks in southeastern brazil rapidly spread to neighboring states ( 1 ) .
epidemiologic studies suggest that movement of sick humans and the animal trade are the main causes of this circulation ( 1 ) .
the relevance of vacv circulation in the context of bovine vaccinia outbreaks has been discussed ( 13 ) .
several isolates belong to group 1 , which is most commonly isolated from sick cattle or cow milkers ; some isolates were detected in peridomestic rodents and wild monkeys ( 8,13 ) .
therefore , although our data demonstrated that parv does not cluster with bav , it is not possible to phylogenetically define which group 1 isolate specifically generated parv or caused the outbreak because of limited number of available gene sequences from vacv - br isolates .
nevertheless , we believe the presence of this new isolate in par state likely resulted from virus spread from tocantins , maranho , or mato grosso , 3 neighboring states of par state , which had bovine vaccinia outbreaks in recent years , and not from reemergence of bav ( 1 ) .
although group 2 vacv - br isolates , including bav , have not been detected in the amazon region of brazil in recent years , we believe that these viruses may be silently circulating or associated with bovine vaccinia outbreaks . as in other regions in brazil , vacv outbreaks are underreported in the amazon region because of its large size and the natural complexity involved in surveillance of zoonotic diseases .
our results reinforce the need for studies on vacv diversity and its transmission chain , which would be useful for the amazon region in brazil .
 in 2010 , vaccinia virus caused an outbreak of bovine vaccinia that affected dairy cattle and rural workers in par state , brazil . 
 genetic analyses identified the virus as distinct from bean58058 vaccinia virus ( identified in 1960s ) and from smallpox vaccine virus strains . 
 these findings suggest spread of autochthonous group 1 vaccinia virus in this region . 
The Study
Conclusions