Foot-and-Mouth Disease in Red Deer - Experimental Infection and Test Methods Performance.

The aim of this study was to evaluate a number of foot-and-mouth disease (FMD) test methods for use in red deer. Ten animals were intranasally inoculated with the FMD virus (FMDV) O UKG 11/2001, monitored for clinical signs, and samples taken regularly (blood, serum, oral swabs, nasal swabs, probang samples and lesion swabs, if present) over a 4-week period. Only one animal, deer 1103, developed clinical signs (lesions under the tongue and at the coronary band of the right hind hoof). It tested positive by 3D and IRES real-time reverse transcription polymerase chain reaction (rRT-PCR) in various swabs, lesion materials and serum. In a non-structural protein (NSP) in-house ELISA (NSP-ELISA-IH), one commercial ELISA (NSP-ELISA-PR) and a commercial antibody NSP pen side test, only deer 1103 showed positive results from day post-inoculation (dpi) 14 onwards. Two other NSP-ELISAs detected anti-NSP serum antibodies with lower sensitivity. It also showed rising antibody levels in the virus neutralization test (VNT), the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR at dpi 9, and in another two commercial SPO-ELISAs at dpi 12 (SPO-ELISA-IV) and dpi 19 (SPO-ELISA-IZ), respectively. Six of the red deer that had been rRT-PCR and antibody negative were re-inoculated intramuscularly with the same O-serotype FMDV at dpi 14. None of these animals became rRT-PCR or NSP-ELISA positive, but all six animals became positive in the VNT, the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR. Two other commercial SPO-ELISAs were less sensitive or failed to detect animals as positive. The rRT-PCRs and the four most sensitive commercial ELISAs that had been used for the experimentally inoculated deer were further evaluated for diagnostic specificity (DSP) using 950 serum samples and 200 nasal swabs from non-infected animals. DSPs were 100% for the rRT-PCRs and between 99.8 and 100% for the ELISAs.

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems.

Bacterial infections in pigs experimentally infected with Nipah virus.

Nipah virus (NiV; Paramyxoviridae) caused fatal encephalitis in humans during an outbreak in Malaysia in 1998/1999 after transmission from infected pigs. Our previous study demonstrated that the respiratory, lymphatic and central nervous systems are targets for virus replication in experimentally infected pigs. To continue the studies on pathogenesis of NiV in swine, six piglets were inoculated oronasally with 2.5 x 10(5) PFU per animal. Four pigs developed mild clinical signs, one exudative epidermitis, and one neurologic signs due to suppurative meningoencephalitis, and was euthanized at 11 days post-inoculation (dpi). Neutralizing antibodies reached in surviving animals titers around 1280 at 16 dpi. Nasal and oro-pharyngeal shedding of the NiV was detected between 2 and 17 dpi. Virus appeared to be cleared from the tissues of the infected animals by 23 dpi, with low amount of RNA detected in submandibular and bronchial lymph nodes of three pigs, and olfactory bulb of one animal. Despite the presence of neutralizing antibodies, virus was isolated from serum at 24 dpi, and the viral RNA was still detected in serum at 29 dpi. Our results indicate slower clearance of NiV from some of the infected pigs. Bacteria were detected in the cerebrospinal fluid of five NiV inoculated animals, with isolation of Streptococcus suis and Enterococcus faecalis. Staphylococcus hyicus was isolated from the skin lesions of the animal with exudative epidermitis. Along with the observed lymphoid depletion in the lymph nodes of all NiV-infected animals, and the demonstrated ability of NiV to infect porcine peripheral blood mononuclear cells in vitro, this finding warrants further investigation into a possible NiV-induced immunosuppression of the swine host.