we analyzed serum samples that were collected in may 2009 from 125 dairy cows in durazno county ( 33230s , 56310w ) , durazno state , uruguay ( figure 2 ) .
the cattle herds had no clinical sings of disease at the time of serum collection . to determine the presence of neutralizing antibodies in the serum samples , we used an orthopoxvirus plaque - reduction neutralization test as previously described ( 9 ) .
the serum titer was defined as the highest dilution that inhibited > 70% of virus plaques compared with negative controls ( 4 ) .
chronologic detection of vaccinia virus in south america . zoomed - in map shows location of durazno county , uruguay , where serum samples were collected from dairy cattle in 2009 to test for the presence of vaccinia virus .
brazil states : ac , acre ; am , amazonas ; al , alagoas ; ap , amap ; ba , bahia ; ce , cear ; es , esprito santo ; go , gois ; ma : maranho ; mg , minas gerais ; ms , mato grosso do sul ; mt , mato grosso ; pa , para ; pb , paraba ; pe , pernambuco ; pi , piau ; pr , paran ; rj , rio de janeiro ; rn , rio grande do norte ; ro , rondnia ; rr , roraima ; rs , rio grande do sul ; sc , santa catarina ; se , sergipe ; sp , so paulo ; to , tocantins .   because previous studies have detected viral dna in the serum of animals and humans with and without clinical manifestations ( 4,10,11 ) , we performed a molecular investigation to identify orthopoxvirus .
we used quantitative pcr ( qpcr ) to amplify vacv growth factor gene ( c11r ) dna .
this qpcr tool has high sensitivity and specificity and , thus , has been routinely used as an orthopoxvirus diagnostic tool by our group ( 12 ) . for molecular characterization
the pcr a56r products obtained from c11r pcr  positive samples were sequenced in both orientations and subjected to capillary electrophoresis ( 3130 genetic analyzer , bigdye terminator cycle sequencing kit v3.1 ; applied biosystems , foster city , ca , usa ) .
we used the clustalw ( http://www.clustal.org/ ) method to align sequences with previously published orthopoxvirus sequences from genbank ; alignments were manually checked with mega6 ( http://www.megasoftware.net/ ) .
we constructed phylogenetic trees using the neighbor - joining method with 1,000 bootstrap replicates and the tamura 3-parameter model in mega6 .
we detected neutralizing antibodies against orthopoxvirus in 28 ( 22.4% ) of 125 serum samples from cattle in uruguay ; titers were 100 neutralizing units ( nu)/ml in 10 ( 35.7% ) samples , 200 nu / ml in 11 ( 39.3% ) samples , 400 nu / ml in 5 ( 17.9% ) samples , and 800 nu / ml in 2 ( 7.1% ) samples ( technical appendix figure 1 , panel a ) . to confirm that the orthopoxvirus seropositivity represented seropositivity to vacv
, we used the c11r gene to molecularly test dna in the serum samples ; 2 ( 1.6% ) samples were positive by qpcr , and 1 of those was also positive by the plaque - reduction neutralization test .  
one of the 2 c11r pcr  positive isolates was also positive for the a56r gene ; we sequenced the gene and named the strain vacv uruguay .
alignment of the a56r nucleotide sequence showed the presence of an 18-nt signature deletion , which is also present in sequences of brazilian - vacv group i , but not group ii , viruses .
unlike sequences for other vacvs , the sequence for vacv uruguay had an at polymorphism ( technical appendix figure 2 , panel a ) .
vacv uruguay exhibited higher identity with group i ( 98.6% identity ) viruses from brazil and argentina than to group ii ( 97.3% identity ) viruses from brazil ( technical appendix figure 1 , panel b ) .
furthermore , in the phylogenetic tree based on a56r nucleotide sequences , the vacvs from uruguay clustered with group i vacvs that had been detected during outbreaks in brazil and with viruses from argentina ( technical appendix figure 2 , panel b ) .
since 1999 , vacv has been isolated from symptomatic and asymptomatic cattle , humans , and wildlife from the north to the extreme south of brazil ( 5,6,8 ) , and in 2014 , vacv was described in bovine serum samples from argentina ( 4 ) . although no exanthematous vacv outbreaks have been reported among cattle in uruguay , we detected orthopoxvirus antibodies and vacv dna in serum samples from dairy cattle in the country , indicating they have been exposed to vacv .
uruguay shares a border with brazil , and its western border is shared with entre rios province in argentina , where vacv dna has been detected .
in addition , uruguay shares its northern and eastern borders with rio grande do sul state , brazil , where pelotas vacv has been isolated from horses ( 4,5 ) .  
our finding of orthopoxvirus antibodies and vacv dna indicates a possible undetected or silent circulation of vacv in uruguay . considering the importance of the livestock sector in all countries of south america , concern exists about the possible spread of vacv beyond brazil , argentina , and uruguay ( 46,14 ) . despite surveillance by veterinarians ,
efforts to stop the spread of vacv at borders may be hampered by the movement of infected rural workers , the marketing of asymptomatic live animals , and the misdiagnosis of vacv infection .
furthermore , vacv has been shown to circulate in wild environments , and it has been hypothesized that rodents may serve as vacv hosts , as they do for other orthopoxviruses , and facilitate the spread of vacv in border areas ( 15 ) .   bovine vaccinia outbreaks in south america were first reported in brazil , but we can not rule out the possibility of autochthonous circulation of vacv in uruguay and other countries in south america .
additional studies are needed to elucidate vacv seroprevalence in other countries in south america , and further research is needed to clarify the transmission pathways related to the spread of vacv in south america .
orthopoxvirus antibody titers in serum samples from 125 cattle in durazno , uruguay , and molecular characterization of a newly isolated virus , vacv uruguay .
 we detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in uruguay . 
 two samples were pcr - positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in brazil . 
 autochthonous circulation of vaccinia virus in uruguay and other south american countries can not be ruled out . 
The Study
Conclusions
None