The OPV/PPV multiplex PCR was designed based on computer simulation of different combinations of several published primer pairs, using software available online 19. Two exclusive and highly conserved genes were targeted by nested-multiplex PCR: the OPV viral growth factor (vgf) and the PPV major viral glycoprotein (b2l); these genes have been widely used in OPV and PPV diagnosis and phylogenetic analysis (Table 1). The nested-multiplex PCR was carried out in a two-step reaction protocol. In the first step, the OPV primers vgfF and vgfR 20 were used in association with the PPV primers OVB2LF1 and OVB2LR1 21. In the nested step, a pair of internal OPV primers (vgfF2: ACACGGTGACTGTATCCA and vgfR2: CTAATACAAGCATAATAC) were designed from alignment of the vgf sequences of Brazilian VACV strains (Drumond and others, data not published) and other available OPV sequences (GenBank accession nos. [AY243312.1 (VACV-WR); AY678276.1 (VACV-LISTER); DQ792504.1 (Horsepox virus - HSPV); AY484669.1 (Rabbitpox virus - RPV); DQ437590.1 (VARV); AF482758.2 (CPXV)]); these primers were then used in association with the PPV primers PPP-1 and PPP-4 7. Several chemical and thermal conditions were evaluated. The best conditions were established based on amplicon yield and specificity [corresponding to the expected fragments of 170 bp (OPV) and 592 pb (PPV)], described as follows. In the first step, 2 μL of template were added to 18 μL of the PCR reaction mixture containing 0.4 mM of OPV primers (VGF-F and VGF-R), 0.8 mM of PPV primers (OVB2LF1 and OVB2LR1), 10 mM dNTPs, 2.0 mM MgCl₂, 500 ng Bovine Serum Albumin (BSA) and 2 U of Taq DNA polymerase (Promega, Madison, USA), using the manufacturer's supplied 10× buffer. Reactions were performed with a DNA Mastercycler Epgradient (Eppendorf, Hamburg, Germany) using the following protocol: incubation at 95°C for 9 min; 30 cycles of denaturation (94°C, 1 min), annealing (45°C, 1 min) and extension (72°C, 1 min); final extension (72°C, 10 min). The nested PCR step was carried out using 1 μL of undiluted first PCR product as template. The same chemical and thermal conditions were used, but using internal OPV (vgfF2 and vgfR2 - 0.4 mM) and PPV (PPP-1 and PPP-4 - 0.8 mM) primers. The PCR products were electrophoresed on 8% PAGE gels and silver stained 22. These same conditions were used in sensitivity tests. Some reactions were performed with the addition of both PPV and OPV scabs, with the purpose of simulating a possible co-infection. In order to confirm the OPV/PPV specificity, other exanthematic infectious agents were submitted to PCR: (i) a Bovine herpes virus positive scab kindly provided by Dr. Z. Lobato (Minas Gerais Federal University, Brazil), and (ii) a Brazilian Sthaphylococcus aureus strain, isolated from a hospital infection, kindly provided by Dr. L. Parucker (Santa Catarina Federal University, Brazil).

Vesicle contents and dried scabs from cattle udders and milkers' hands were collected during Brazilian BV outbreaks or from sheep and goats during CE outbreaks. This collection was accomplished using 1-ml insulin syringes, 0.45 mm×13 mm needles, and cotton swabs or a pair of forceps. Collected samples were chilled, transported to the laboratory, and stored at -70°C until processed. Vesicular liquid swabs were added to 200 μL of PBS and centrifuged at 2000 × g for 3 min. Scabs were macerated by a homogenizer (Politron, Littau, Switzerland) in PBS (0.1 g scab/0.9 mL PBS) and clarified by centrifugation at 2000 × g for 3 min. Two microliters of the supernatants were used in the nested-multiplex PCR. Some expected PCR products were directly sequenced (ET Dynamic Terminator for MegaBACE - GE Healthcare, Fairfield, USA) and compared with available GenBank sequences using an online blast program http://www.ncbi.nlm.nih.gov/blast. To avoid any possibility of laboratory cross-contamination, the different samples were manipulated separately.

A total of 64 clinical samples were collected and then submitted to OPV/PPV nested-multiplex PCR (Table 2). Of these samples, 56 were collected during BV outbreaks (36 from bovines and 20 from humans) and 8 samples were collected during CE outbreaks (3 from caprines and 5 from ovines). All collected BV and CE clinical samples were previously tested by other molecular methods (Fonseca et al., 1998; Inoshima et al., 2000) and were confirmed VACV and ORFV infections, respectively. Among the BV clinical samples, the OPV/PPV nested-multiplex PCR detected OPV DNA in 53 scabs/vesicles (94.4%). The multiplex was able to detect PPV DNA in all analyzed CE clinical samples. Considering all bovine, human, ovine and caprine samples, the nested-multiplex PCR presented a positivity of 95.3%. The sequences of the amplified fragments again confirmed the PCR specificity, showing high identity with the VACV vgf gene or the ORFV b2l gene sequences. No co-infection case was detected in this molecular screening.