Source of samples
A total of 460 oral swabs from adult cattle of both sexes were used in this study. These samples originated from different regions according to the following groups. Group 1 consisted of 200 healthy cattle from an area free of FMD with vaccination (101 samples from Pedro Leopoldo, State of Minas Gerais; 99 samples from Igarapé, State of Minas Gerais). Group 2 consisted of 60 cattle from Carero da Várzea, State of Amazonas (region classified as "unknown risk for FMD"), which were sampled during an outbreak of FMD (type C virus) in 2004 . These cattle did not have any clinical signs and were sampled more than 30 days after the identification of the index case. Group 3 consisted of 200 cattle suspected of FMD (some with clinical changes compatible with FMD from Eldorado and Japorã, State of Mato Grosso do Sul). These swabs were obtained during an outbreak of FMD (type O virus) in 2005 .
Dacron-tipped swabs were obtained from the oral cavity by swabbing the oral mucosa and tongue, saturating the swab with saliva while avoiding contamination with ingested material. Immediately after sampling, the swabs were placed into a 2-ml sterile cryogenic tube containing 1.5 ml of Dulbecco's Minimal Essential Medium [DMEM] (Gibco®, Invitrogen Corp., Carlsbad, CA) with antibiotic and antimycotic (Gibco®). The tubes containing the swabs from cattle from an area free of FMD (State of MinasGerais) were immediately frozen on dry ice and stored at -70°C until further processing. However, due to the unavailability of dry ice in the areas of the outbreaks (Eldorado and Japorã in the State of Mato Grosso do Sul, and Carero da Várzea in the State of Amazonas) some samples were kept at 4°C for up to 7 days prior to freezing and storage at -70°C.
RNA samples were extracted using the Mini RNeasy kit (Qiagen Inc., Valencia, CA) following the manufacturer's instructions. Briefly, the tubes containing the swabs were thawed and thoroughly homogenized by vortexing, and 140 μl of the medium were added to 540 μl of the RLT buffer that is included in the kit, followed by 700 μl of 70% ethanol, and then transferred to a Mini RNeasy column previously inserted into a 2-ml collecting tube. RNA was immobilized in the column by centrifugation, sequentially washed, and eluted in 40 μl of RNase-free water.
In addition to the RNA samples obtained from oral swabs as described above, RNA samples were purified using Trizol (Invitrogen Corp., Carlsbad, CA.) from 3 reference strains (O1 Campos, A24 Cruzeiro, and C3 Indaial) representing the 3 serotypes that occur in Brazil (A, O, and C), and 2 previously characterized field isolates (FMDV serotypes A and O, isolated from outbreaks in the states of Roraima in 1999 and Mato Grosso do Sul in 2005). These RNA samples were used as positive controls.
Real-time reverse transcription polymerase chain reaction
The rRT-PCR amplification protocol used in the current study was previously described . This method amplifies a conserved segment of the FMDV RNA polymerase gene (3D; GenBank AF189157). The following primers were used 5'-ACTGGGTTTTACAAACCTGTGA-3' and 5'-GCGAGTCCTGCCACGGA-3' along with the probe 5'-TCCTTTGCACGCCGTGGGAC-3' labeled with 6-carboxyfluorescein at the 5' end, and the quencher tetramethyl rhodamine at the 3' end. This method amplifies the RNA from all serotypes of FMDV, but does not amplify nucleic acids from other viruses that cause vesicular diseases . Reactions were performed using 25 μl Cepheid tubes (Cepheid Inc., Sunnyvale, CA) containing all the dehydrated reagents required for amplification of the FMDV RNA (Vet-Alert, Foot-and-Mouth Disease, Tetracore Inc., Gaithersburg, MD) that were rehydrated with 22.5 μl of rehydration buffer (Vet-Alert). As controls, 2.5 μl of RNA samples or an oligonucleotide used as positive control (Vet-Alert) or TE buffer (Tris-ethylenediamine tetra-acetic acid [EDTA]) instead of RNA as a negative control, were added to the respective reaction tubes. The real-time RT-PCR reactions were carried out in a Smart Cycler II thermocycler (Cepheid Inc.). The one-step real-time RT-PCR amplification started with reverse transcription for 1 hr at 60°C, followed by PCR with the following parameters: 55 cycles of 2 sec at 95°C and 30 sec at 60°C. One positive and 1 negative control were included in each reaction.
The samples (medium containing the swabs) were filtered using a 0.2-μm filter, and 500 μl was then inoculated onto a monolayer of IBRS (Instituto Biologico Rim Suino)-2 cells grown in 25-cm2 flasks and kept without washing for the duration of the assay (up to 72 hr). After inoculation, the cultures were checked for cytopathic effects (CPE) every 24 hr for 72 hr. Cultures that did not develop CPE were lysed by freezing at -70°C and reinoculated (500 μl) onto a new monolayer of IBRS-2 cells, which was then evaluated for an additional 72 hr. Cultures with CPE were stored at -70°C until processing for indirect sandwich ELISA.
Indirect sandwich enzyme-linked immunosorbent assay
Samples from cultures that had CPE were tested by indirect sandwich ELISA, according to the protocol recommended by the Pan American Foot-and-Mouth Disease Center (PANAFTOSA). Briefly, anti-FMDV serotypes A, O, and C rabbit antiserum was incubated in ELISA plates for 18 hr at 4°C. Antiserum was generated by using a pool of inactivated virus subtypes O1 Campos, A24 Cruzeiro, and C3 Indaial as antigen. The plate was washed and incubated with 1% ovalbumin for 1 hr at room temperature. After washing, the reference antigens and testing samples were added to the wells and incubated for 1 hr at 37°C under agitation. The plate was then washed again and incubated with Guinea pig anti-FMDV (serotypes A, O, and C) antiserum for 30 min at 37°C under agitation, followed by washing and incubation with an anti-Guinea pig antibody conjugated with peroxidase for 30 min at 37°C under agitation. The plate was washed and incubated with a solution of hydrogen peroxide (0.012% H2O2 in 0.1 M of citric acid, 0.2 M of sodium phosphate, and 0.04% orthophenyldiamine) as a substrate for 15 min at room temperature, and read in an ELISA reader at 492 nm.