Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance.

This study aimed to evaluate the utility of milk as a non-invasive sample type for the surveillance of foot-and-mouth disease (FMD), a highly contagious viral disease of cloven-hooved animals. Four milking Jersey cows were infected via direct-contact with two non-milking Jersey cows that had been previously inoculated with FMD virus (FMDV: isolate O/UKG/34/2001). Milk and blood were collected throughout the course of infection to compare two high-throughput real-time reverse transcription polymerase chain reaction (rRT-PCR) protocols with different RT-PCR chemistries. Using both methods, FMDV was detected in milk by rRT-PCR one to two days before the presentation of characteristic foot lesions, similar to detection by virus isolation. Furthermore, rRT-PCR detection from milk was extended, up to 28 days post contact (dpc), compared to detection by virus isolation (up to 14 dpc). Additionally, the detection of FMDV in milk by rRT-PCR was possible for 18 days longer than detection by the same method in serum samples. FMDV was also detected with both rRT-PCR methods in milk samples collected during the UK 2007 outbreak. Dilution studies were undertaken using milk from the field and experimentally-infected animals, where for one sample it was possible to detect FMDV at 10-7. Based on the peak CT values detected in this study, these findings indicate that it could be possible to identify one acutely-infected milking cow in a typical-sized dairy herd (100-1000 individuals) using milk from bulk tanks or milk tankers. These results motivate further studies using milk in FMD-endemic countries for FMD surveillance.

Interleukin-10 production at the early stage of infection with foot-and-mouth disease virus related to the likelihood of persistent infection in cattle.

The factors leading to persistent infection of foot-and-mouth disease (FMD) virus in ruminants are not well defined. This paper provides evidence of the presence of interleukin-10 (IL-10) early in the course of infection (1-4 days) as a factor in the development of persistence of FMD virus in cattle. Results showed that serum IL-10 in carrier cattle infected with FMD virus type O (n = 4) was detected and peaked at 1 or 2 days post infection and rapidly declined thereafter. In contract, serum IL-10 levels in non-carrier cattle (n = 21) were very low or undetectable during the same period.

Understanding foot-and-mouth disease virus transmission biology: identification of the indicators of infectiousness.

The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3-4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models.

Characterization of epitope-tagged foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals with an almost-worldwide distribution. Conventional FMD vaccines consisting of chemically inactivated viruses have aided in the eradication of FMD from Europe and remain the main tool for control in endemic countries. Although significant steps have been made to improve the quality of vaccines, such as improved methods of antigen concentration and purification, manufacturing processes are technically demanding and expensive. Consequently, there is large variation in the quality of vaccines distributed in FMD-endemic countries compared with those manufactured for emergency use in FMD-free countries. Here, we have used reverse genetics to introduce haemagglutinin (HA) and FLAG tags into the foot-and-mouth disease virus (FMDV) capsid. HA- and FLAG-tagged FMDVs were infectious, with a plaque morphology similar to the non-tagged parental infectious copy virus and the field virus. The tagged viruses utilized integrin-mediated cell entry and retained the tag epitopes over serial passages. In addition, infectious HA- and FLAG-tagged FMDVs were readily purified from small-scale cultures using commercial antibodies. Tagged FMDV offers a feasible alternative to the current methods of vaccine concentration and purification, a potential to develop FMD vaccine conjugates and a unique tool for FMDV research.

Airborne transmission of foot-and-mouth disease in pigs: evaluation and optimisation of instrumentation and techniques.

Foot-and-mouth disease (FMD) can be transmitted in a variety of ways, one of which is through virus exhaled into the air by infected livestock. It is clear that where there is close contact there will be a range of possible mechanisms for the transmission of disease from animal to animal, including the airborne route if simple barriers between livestock exist. In transmission of FMD over longer distances, airborne transmission represents a significant challenge to the veterinary services in that the mechanism is essentially uncontrollable if the primary source of the disease is not contained. In the event of an epidemic of FMD, such as the one experienced in the United Kingdom in 2001, it is important for disease control purposes to understand the contribution made to the overall spread of disease by aerosolised virus. This assessment is based on a combination of measurements made in the laboratory and through clinical observations in the field. To date, laboratory measurements have used a number of instruments that were not specifically designed for working with FMD virus or whose performance have not been fully compared and documented. This paper compares four samplers and describes the method by which samples are processed. Overall it is concluded that there is no optimum air sampling instrument which could be successfully employed for all situations but the work provides guidance to those wishing to make measurements in the future and establishes a baseline against which any new samplers can be compared.

WITHDRAWN: Foot-and-mouth disease: How much airborne virus do animals exhale?

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Foot-and-mouth disease virus (O/UKG/2001) is poorly transmitted between sheep by the airborne route.

Foot-and-mouth disease virus (FMDV) can be spread by the airborne route and therefore atmospheric dispersion models have been developed to predict where the virus might spread during a disease outbreak. Airborne transmission between sheep of the FMDV strain involved in the outbreak in Europe in 2001 (O/UKG/2001) was studied experimentally. Recipient animals were exposed to two donor sheep excreting virus for 2, 4, 6, 8 or 24 h. Although FMDV was detected in air samples collected during challenge, none of the recipient sheep became infected. These data suggest that O/UKG/2001 is not efficiently transmitted by the airborne route between sheep.

Foot-and-mouth disease: measurements of aerosol emission from pigs as a function of virus strain and initial dose.

Measurements of airborne foot-and-mouth disease virus have been made using 20 pigs that had either O UKG or C Noville injected into their heel-pads to determine if the kinetics of virus emission are related to the virus strain and dose administered in the challenge inoculum. Viable virus was detected in aerosol emissions for 3 days regardless of the strain or dose of virus given. No correlation was found between the peak level of virus emission and dose, but pigs infected with a lower dose of virus had a delayed onset of aerosol emission and emitted a greater total amount of aerosolised virus. Irrespective of the dose, both the total amount and the peak level of virus emission were higher from pigs infected with C Noville compared to those infected with O UKG. The results suggest that care should be taken when extrapolating from laboratory derived data to the field; this is particularly the case in the early days of an outbreak when the aerosol characteristics of the virus involved may be unknown and the amount of virus that an individual animal has been challenged with remains uncertain.

Foot-and-mouth disease - quantification and size distribution of airborne particles emitted by healthy and infected pigs.

There is strong evidence to suggest that foot-and-mouth disease (FMD) can be transmitted by airborne virus up to many kilometres from a virus source. Atmospheric dispersion models are often used to predict where this disease might spread. This study investigated whether FMD virus (FMDV) aerosol has specific characteristics which need to be taken into consideration in these models. The characteristics and infectiousness of particles emitted by 12 pigs have been studied pre- and post-infection with O UKG 2001 FMDV. Aerosol generated by individual pigs was found log normally distributed in the range 0.015-20.0microm with concentrations between 1000 and 10000cm(-3) at the smallest size and <1cm(-3) above 10microm. No differences in either the total number of particles produced or their size distribution were detected between uninfected and infected pigs. However, a correlation between aerosol concentration and animal activity was found with a more active pig producing significantly greater concentrations than those that were less active. Viable virus was found up to a maximum of 6.3 log TCID(50)/24h/animal. The virus was distributed almost equally across the three size ranges; <3, 3-6 and >6microm. No correlation could be established between the production of virus and animal activity. In general the production of airborne virus closely followed the detection of viraemia in the blood and the presence of clinical symptoms. However, in one instance a pig excreted as much airborne virus as the other animals in the study, but with less virus detected in its blood. The results suggest that there is little merit in including a sophisticated virus release pattern based on physical activity periods or FMDV aerosol size spectrum, together with the appropriate dry deposition calculations, in models used to predict airborne spread of FMD. An estimate of the total daily virus production based on the clinical assessment of disease and virus strain is sufficient as input.