july 2014 , serum and exanthematous lesion samples were collected from 6 patients in the bordering municipalities of valparaso ( patients 15 ) and solita ( patient 6 ) in the department of caquet ( figure 1 ; table ) in the amazon region of colombia . archived serum samples collected in december 2012april 2013 from 11 patients with exanthematous lesions ( from several farms ) in the municipality of valparaso who consulted the local hospital were also analyzed ( table ) . in all patients , the incubation period ranged from 47 days , after which nodules on the hands or forearms appeared , along with fever , lymphadenopathy , and localized pain . lesions increased in size and progressed from erythematous macules to papules , vesicles , and pustules that subsequently exhibited bacterial infection by the fourth week of symptom onset . by the fifth and sixth week lesions appeared mainly in hands with preexisting cuts , abrasions , or other skin barrier defects . direct contact with lesions from the udders of cattle during milking was reported by all but 1 patient . patient 5 was a 24-year - old woman who reported no recent contact with infected animals , but her husband was a milker who had exanthematous lesions on his hands 2 weeks before she displayed symptoms . location of 6 patients with poxvirus infections and photographs of lesions from 3 patients , colombia , 2014 . a ) the municipalities of valparaso ( residence of patients 15 ) and solita ( residence of patient 6 ) are 36 km apart in the southwestern region of the department of caquet . yellow , white , and blue stars denote locations of patients 2 , 3 , and 4 , respectively . d ) exanthematous lesions on the hands of dairy farm workers depicting the varying sizes of lesions and typical evolution of ulcers . patient 2 ( b ) tested positive for vaccinia virus , patient 3 ( c ) had inconclusive results , and patient 4 ( d ) was positive for parapoxvirus . * ct , cycle threshold ; eqi , equivocal ; inc , inconclusive ; neg , negative ; pos , positive ; unk , unknown . all serum samples were tested by elisa for opxv igg and igm as described ( 11 ) ( technical appendix ) . fifteen serum samples were positive for opxv igg ( table ) ; 9 were also positive for opxv igm , which can persist for up to 6 months after primary infection or vaccination ( 11 ) . reliable vaccination data was not available for these patients ; however , on the basis of age , only patients 3 , 8 , and 11 could have been vaccinated . these 3 patients exhibited detectable opxv igg levels , and patients 3 and 11 were also positive for opxv igm . these results suggest that these patients had not been vaccinated and that their lesions were caused by recent opxv exposure . patients 4 , 10 , and 14 exhibited detectable opxv igg without detectable opxv igm . these results suggest the patients had previous opxv infections , given that an active parapoxvirus infection was demonstrated in patient 4 and vaccination could be ruled out for patients 10 ( a 20-year - old ) and 14 ( a 24-year - old ) based on their age . dna was extracted from lesion and serum samples by using the purelink viral rna / dna extraction kit ( invitrogen inc . , carlsbad , ca , usa ) according to the manufacturer s instructions and amplified by using pcr with generic and specific poxvirus primers as previously described ( technical appendix ) ( 12 ) . opxv dna was detected in lesion samples from patients 1 , 2 , and 5 ( table ) . for further genetic characterization , the a56r ( hemagglutinin , 1,134 bp ) gene from patients 2 and 5 were amplified by pcr , sequenced ( technical appendix ) , and compared with reference orthopoxvirus a56r sequences ( technical appendix table 1 ) . our phylogenetic analysis confirmed vacv to be the etiologic agent of infection in these patients ( figure 2 , panel a ) . the opxv isolates detected in our samples were related to group 1 ( passatempo , aracatuba , muriae , and other strains from brazil ) ; however , bootstrap score and branch positioning suggest the strains from brazil and colombia diverged long ago or independently arose . phylogenetic characterization of orthopoxvirus gene a56r and parapoxvirus gene p37k of viruses obtained from patient lesion samples from an outbreak in colombia , 2014 . a ) nucleotide sequences of the a56r gene ( 829 bp ) of reference orthopoxvirus strains were aligned and used for phylogenetic inference . the evolutionary distances were computed by using the t92+g model ( shape : 0.69 ) . b ) nucleotide sequences of the partial ( 445 bp ) p37k gene of reference parapoxvirus strains were aligned and used for phylogenetic inference . the evolutionary distances were computed by using the t92+g model ( shape : 0.39 ) . the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 1,000 replicates ) are shown at the nodes . genbank accession numbers and further information on the sequences included in the analyses are provided in technical appendix tables 1 , 2 . bpsv , bovine papular stomatitis virus ; cpxv , cowpox virus ; rpxv , rabbitpox virus ; mpxv , monkeypox virus ; orfv , orf virus ; pcpxv , pseudocowpox virus ; pvnz , parapoxvirus of red deer in new zealand ; spv , sealpox virus ; varv , variola virus . for parapoxvirus detection and characterization , we designed generic and degenerate primers to amplify a partial sequence of the p37k ( envelope protein b2l , 489 bp ) gene by pcr ( technical appendix ) . parapoxvirus dna was detected in lesion samples from patients 4 and 6 ( table ) . nucleotide sequencing and phylogenetic analysis of the sample from patient 6 showed a close relationship with previously reported pcpv strains circulating in brazil and worldwide ( technical appendix table 2 ; figure 2 , panel b ) . we found opxv igg and igm in serum samples collected from patients with exanthematous lesions and from those who had such lesions in the past . molecular methods allowed for the detection and characterization of vacv ( 3 patients ) and pcpv ( 2 patients ) . lesions caused by poxviruses affected the hands and forearms and disappeared within weeks without treatment . however , by limiting the patients daily activities , the infections had substantial negative impacts on the economies of dairy farmworkers and their local communities . the disease burden of poxvirus infections in colombia has not been estimated . however , anecdotal communications and data suggest it might be a serious and increasing health problem because farmworkers and healthcare personnel are not trained to recognize the disease and prevent subsequent transmission . the rapid spread of poxviruses could be facilitated by farmworkers performing daily milking activities at several farms and trading cattle . also , factors such as human - to - human and fomite transmission , a growing susceptible population , and an unknown animal reservoir might increase the potential risk for infection at the community level . the serious consequences that poxvirus infections could have on immunocompromised persons ( 13 ) , the increased risk for transmission related to the anatomic site of the lesions ( 14 ) , the potential for long - lasting sequelae , and the possibility of the virus evolving into a more virulent strain ( 15 ) could pose a great threat to individual persons , populations , and public health systems in the near future . studies focused on determining the prevalence of poxvirus infections , risk factors for disease , and the geographic distribution of poxvirus strains are needed to understand the disease burden and guide effective prevention and control measures and educational outreach . analysis and characterization of vacv and pcpv complete genomes could provide clues to explain their emergence and recent evolutionary histories , and research aimed at identifying the domestic animal hosts and wildlife reservoirs of poxviruses could further our understanding of their natural transmission cycles . description of methods and information on the a56r and p37k reference genes used in phylogenetic analyses .