Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 18, Number 4—April 2012

Serologic Evidence of Orthopoxvirus Infection in Buffaloes, Brazil

On This Page
Article Metrics
citations of this article
EID Journal Metrics on Scopus

Cite This Article

To the Editor: Since 1999, several exanthematous vaccinia virus (VACV) outbreaks affecting dairy cattle and rural workers have been reported in Brazil (1,2). VACV, the prototype of the genus Orthopoxvirus (OPV), exhibits serologic cross-reactivity with other OPV species and was used during the World Health Organization smallpox eradication campaign (3). The origin of VACV in Brazil is unknown, although some studies have suggested that VACV strains used during the campaign may be related to outbreaks of bovine vaccinia (BV) (2). In Brazil, BV affects the milk industry and public health services (1,2,4,5). During outbreaks, dairy cattle developed lesions on the teats and udders, causing a decrease in milk production (1,2,4,5).

Another VACV subspecies, buffalopox virus (BPXV), has been isolated from buffaloes (Bubalus bubalis) in rural areas in India and causes clinical signs that resemble those seen during BV outbreaks in Brazil (6). Recent genetic analysis of BPXV samples confirmed its close relationship to VACV-like viruses, although each virus has distinct genetic signatures (1,2,6). Until recently, buffalo herds have been almost exclusive to northern Brazil. However, the buffalo market has experienced great expansion in this country, and today, there are herds in all geographic regions of Brazil. These buffalo herds are hypothetically at risk for VACV infection, on the basis of the outbreaks caused by BPXV that have been described in India (6). To assess the risk for OPV infection in milk buffaloes in Brazil, we conducted a serosurvey of herds from southeastern Brazil, the region most affected by BV.

During October 2010, we screened milk buffalo herds in rural areas of Minas Gerais State, Brazil. Serum samples were collected from 48 female buffaloes used for milk production; these animals belonged to 3 neighboring properties in Carmo da Mata city (20°33′28′′S, 44°52′15′′W), which is in the same mesoregion where the VACV Passatempo virus strain was isolated during an outbreak in 2003 (5). Since then, several outbreaks have been reported in this area.

Serum samples were inactivated, and an OPV plaque-reduction neutralization test (PRNT) was performed (7). The serum titer was defined as the highest dilution that inhibited >70% of viral plaques relative to the level of inhibition of the negative controls. Samples also underwent ELISA for OPV IgG as described (4). Bovine serum samples were used as positive and negative controls (1,4). OPV-PRNT specificity (98.4%) and sensitivity (93.5%) were confirmed by using receiver-operating characteristic analysis as described (8). The tests were performed in duplicate.

Of the 48 buffalo serum samples, 15 (31.25%) contained neutralizing antibodies against OPV; of these, 6 (40%) had titers of 20, 5 (33.3%) had titers of 40, and 4 (26.6%) had titers >80 (Table). The ELISA yielded results similar to those of the PRNT; of the 48 serum samples, 17 (35.41) were IgG positive (Table). A total of 14 samples were coincident in the PRNT and the ELISA, including most of those with high titers by PRNT. To detect viral DNA, we conducted nested PCR to amplify the viral growth factor gene (9) and real-time PCR to amplify the A56R gene (10); results were negative for all 48 serum samples.

We detected antibodies against OPV in buffaloes in Brazil 10 years after the first reported VACV outbreak in cattle in southeastern Brazil (1). Because PRNT and ELISA indicate the presence of OPV antibodies in a nonspecific manner (OPV serologic cross-reaction), it was not possible to determine the species responsible for these results. However, seropositive buffaloes may have been exposed to VACV, the only OPV known to be circulating in Brazil (1,2,4,5,8).

The management of milk buffaloes in Brazil is similar to that of dairy cows, including manual milking (1,4,5). Cow milkers usually work on >2 farms, and the farm infrastructure commonly is unsophisticated (1,4,5). These conditions were shown to be favorable for the spread of VACV among cattle, which suggests that the same conditions could lead to the introduction of VACV into buffalo herds. Because some BV outbreaks are not reported by the farmers, it is not possible to know exactly how or when a buffalo herd in the study area was exposed to the virus. However, milkers who work with both cattle and buffalo may be a route of viral transmission, although other sources of exposure are possible (8). Although no exanthematous VACV outbreaks have been described in milk buffaloes in Brazil, our results suggest that buffalo herds may be exposed to VACV in BV-affected areas and therefore may be at risk for VACV infection. Further research is needed to determine routes of infection, including whether humans working as milkers contribute to virus transmission.



We thank João Rodrigues dos Santos, Ângela Sana Lopes, Ilda Gama, and colleagues from the Laboratório de Vírus for their excellent technical support.

Financial support was provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundação de Amparo à Pesquisa do Estado de Minas Gerais, and Ministério da Agricultura, Pecuária e Abastecimento. F.L.A. received fellowships from CNPq; E.G.K., C.A.B., G.S.T., and P.C.P.F. are researchers supported by CNPq.


Felipe Lopes de Assis1, Graziele Pereira1, Cairo Oliveira, Gisele Olinto Libânio Rodrigues, Marcela Menezas Gomes Cotta, Andre Tavares Silva-Fernandes, Paulo Cesar Peregrino Ferreira, Cláudio Antônio Bonjardim, Giliane de Souza Trindade, Erna Geessien Kroon, and Jônatas Santos AbrahãoComments to Author 
Author affiliations: Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil



  1. de Souza Trindade  G, da Fonseca  FG, Marques  JT, Nogueira  ML, Mendes  LC, Borges  AS, Araçatuba virus: a vaccinialike virus associated with infection in humans and cattle. Emerg Infect Dis. 2003;9:15560.PubMedGoogle Scholar
  2. Damaso  CR, Esposito  JJ, Condit  RC, Moussatche  N. An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine. Virology. 2000;277:43949. DOIPubMedGoogle Scholar
  3. Damon  IK. Poxviruses. In: Knipe DM, Howley PM, editors. Fields virology, 5th ed., vol. II. Philadelphia: Lippincott Williams & Wilkins; 2007. p. 2947.
  4. Silva-Fernandes  AT, Travassos  CE, Ferreira  JM, Abrahão  JS, Rocha  ES, Viana-Ferreira  F, Natural human infections with vaccinia virus during bovine vaccinia outbreaks. J Clin Virol. 2009;44:30813. DOIPubMedGoogle Scholar
  5. Leite  JA, Drumond  BP, Trindade  GS, Lobato  ZI, da Fonseca  FG, dos Santos  JR, Passatempo virus, a vaccinia virus strain, Brazil. Emerg Infect Dis. 2005;11:19358.PubMedGoogle Scholar
  6. Bhanuprakash  V, Venkatesan  G, Balamurugan  V, Hosamani  M, Yogisharadhya  R, Gandhale  P, Zoonotic infections of buffalopox in India. Zoonoses Public Health. 2010;57:e14955. DOIPubMedGoogle Scholar
  7. Newman  FK, Frey  SE, Blevins  TP, Mandava  M, Bonifacio  A Jr, Yan  L, Improved assay to detect neutralizing-antibody following vaccination with diluted or undiluted vaccinia (Dryvax) vaccine. J Clin Microbiol. 2003;41:31547. DOIPubMedGoogle Scholar
  8. Abrahão  JS, Silva-Fernandes  AT, Lima  LS, Campos  RK, Guedes  MI, Cota  MM, Vaccinia virus infection in monkeys, Brazilian Amazon. Emerg Infect Dis. 2010;16:9769.PubMedGoogle Scholar
  9. Abrahão  JS, Lima  LS, Assis  FL, Alves  PA, Silva-Fernandes  AT, Cota  MM, Nested-multiplex PCR detection of Orthopoxvirus and Parapoxvirus directly from exanthematic clinical samples. Virol J. 2009;6:140. DOIPubMedGoogle Scholar
  10. de Souza Trindade  G, Li  Y, Olson  VA, Emerson  G, Regnery  RL, da Fonseca  FG, Real-time PCR assay to identify variants of Vaccinia virus: implications for the diagnosis of bovine vaccinia in Brazil. J Virol Methods. 2008;152:6371. DOIPubMedGoogle Scholar




Cite This Article

DOI: 10.3201/eid1804.111800

1These authors contributed equally to this article.

Related Links


Table of Contents – Volume 18, Number 4—April 2012

EID Search Options
presentation_01 Advanced Article Search – Search articles by author and/or keyword.
presentation_01 Articles by Country Search – Search articles by the topic country.
presentation_01 Article Type Search – Search articles by article type and issue.



Please use the form below to submit correspondence to the authors or contact them at the following address:

Jônatas Abrahão, Laboratório de Vírus, ICB, UFMG, Brazil

Send To

10000 character(s) remaining.


Page created: March 16, 2012
Page updated: March 16, 2012
Page reviewed: March 16, 2012
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.