Inferring transmission routes for foot-and-mouth disease virus within a cattle herd using approximate Bayesian computation.

To control an outbreak of an infectious disease it is essential to understand the different routes of transmission and how they contribute to the overall spread of the pathogen. With this information, policy makers can choose the most efficient methods of detection and control during an outbreak. Here we assess the contributions of direct contact and environmental contamination to the transmission of foot-and-mouth disease virus (FMDV) in a cattle herd using an individual-based model that includes both routes. Model parameters are inferred using approximate Bayesian computation with sequential Monte Carlo sampling (ABC-SMC) applied to data from transmission experiments and the 2007 epidemic in Great Britain. This demonstrates that the parameters derived from transmission experiments are applicable to outbreaks in the field, at least for closely related strains. Under the assumptions made in the model we show that environmental transmission likely contributes a majority of infections within a herd during an outbreak, although there is a lot of variation between simulated outbreaks. The accumulation of environmental contamination not only causes infections within a farm, but also has the potential to spread between farms via fomites. We also demonstrate the importance and effectiveness of rapid detection of infected farms in reducing transmission between farms, whether via direct contact or the environment.

Assessing the effectiveness of environmental sampling for surveillance of foot-and-mouth disease virus in a cattle herd.

The survival of foot-and-mouth disease virus (FMDV) in the environment provides an opportunity for indirect transmission, both within and between farms. However it also presents the possibility of surveillance and detection via environmental sampling. This study assesses the effectiveness of environmental sampling strategies in the event of an outbreak, using a previous model for transmission of FMDV in a cattle herd that had been parameterized using data from transmission experiments and outbreaks. We show that environmental sampling can be an effective means of detecting FMDV in a herd, but it requires multiple samples to be taken on multiple occasions. In addition, environmental sampling can potentially detect FMDV in a herd more quickly than clinical inspection. For example, taking 10 samples every 3 days results in a mean time to detection of 6 days, which is lower than the mean time to detection estimated for the 2001 UK epidemic (8 days). We also show how environmental sampling could be used in a herd considered to be at risk as an alternative to pre-emptive culling. However, because of the time taken for virus to accumulate at the start of an outbreak, a reasonable level of confidence (> 99%) that an at-risk herd is indeed free from infection is unlikely to be achieved in less than 1 week.

Airborne Transmission of Foot-and-Mouth Disease Virus: A Review of Past and Present Perspectives.

The primary transmission route for foot-and-mouth disease (FMD), a contagious viral disease of cloven-hoofed animals, is by direct contact with infected animals. Yet indirect methods of transmission, such as via the airborne route, have been shown to play an important role in the spread of the disease. Airborne transmission of FMD is referred to as a low probability- high consequence event as a specific set of factors need to coincide to facilitate airborne spread. When conditions are favourable, airborne virus may spread rapidly and cause disease beyond the imposed quarantine zones, thus complicating control measures. Therefore, it is important to understand the nature of foot-and-mouth disease virus (FMDV) within aerosols; how aerosols are generated, viral load, how far aerosols could travel and survive under different conditions. Various studies have investigated emissions from infected animals under laboratory conditions, while others have incorporated experimental data in mathematical models to predict and trace outbreaks of FMD. However, much of the existing literature focussing on FMDV in aerosols describe work which was undertaken over 40 years ago. The aim of this review is to revisit existing knowledge and investigate how modern instrumentation and modelling approaches can improve our understanding of airborne transmission of FMD.

Environmental sampling for the detection of foot-and-mouth disease virus and peste des petits ruminants virus in a live goat market, Nepal.

Livestock markets are considered vital parts of the agricultural economy, particularly in developing countries where livestock keeping contributes to both food security and economic stability. Animals from diverse sources are moved to markets, they mix while they are there and are subsequently redistributed over wide geographic areas. Consequently, markets provide an opportunity for targeted surveillance for circulating pathogens. This study investigated the use of environmental sampling at a live goat market in Nepal for the detection of foot-and-mouth disease virus (FMDV) and peste des petits ruminants virus (PPRV), both of which are endemic. Five visits to the market were carried out between November 2016 and April 2018, with FMDV RNA detected on four visits and PPRV RNA detected on all five visits. Overall, 4.1% of samples (nine out of 217) were positive for FMDV RNA and 60.8% (132 out of 217) were positive for PPRV RNA, though the proportion of positive samples varied amongst visits. These results demonstrate that non-invasive, environmental sampling methods have the potential to be used to detect circulation of high priority livestock diseases at a live animal market and, hence, to contribute to their surveillance and control.

Environmental and air sampling are efficient methods for the detection and quantification of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) can be found in all secretions and excretions and the breath of acutely infected animals. FMDV can survive in the environment, providing an opportunity for surveillance. The objective of this study was to assess the efficiency of sampling methods for the recovery and quantification of FMDV from a range of environmental surfaces and in aerosols. Selected surfaces, based on those likely to be found on farms, were spiked with a range of concentrations of FMDV, left to dry and then the surface was swabbed with an electrostatic dust cloth. For aerosol sampling, FMDV was nebulised at different concentrations and distances from the sampler. Recovery of viral RNA and infectious virus was measured by RT-qPCR and virus isolation respectively. FMDV RNA was detected from all surfaces at all concentrations except from glass. Infectious virus was recovered from all surfaces but only at higher concentrations. The higher the starting concentration of virus the more efficient the recovery was from surfaces and recovery was more consistent from non-porous surfaces than porous surfaces. FMDV was detected in aerosol samples and the amount of virus recovered decreased as the distance between the nebuliser and sampler increased. The higher the starting concentration of virus the more efficient the recovery was from sampled aerosols. The information provided in this study could be used to direct environmental and aerosol sampling approaches in the field and improve the detection efficiency of FMDV from an environment, thus extending the toolbox available for diagnosis and surveillance of this pathogen.

Quantifying the Transmission of Foot-and-Mouth Disease Virus in Cattle via a Contaminated Environment.

Indirect transmission via a contaminated environment can occur for a number of pathogens, even those typically thought of as being directly transmitted, such as influenza virus, norovirus, bovine tuberculosis, or foot-and-mouth disease virus (FMDV). Indirect transmission facilitates spread from multiple sources beyond the infectious host, complicating the epidemiology and control of these diseases. This study carried out a series of transmission experiments to determine the dose-response relationship between environmental contamination and transmission of FMDV in cattle from measurements of viral shedding and rates of environmental contamination and survival. Seven out of ten indirect exposures resulted in successful transmission. The basic reproduction number for environmental transmission of FMDV in this experimental setting was estimated at 1.65, indicating that environmental transmission alone could sustain an outbreak. Importantly, detection of virus in the environment prior to the appearance of clinical signs in infected cattle and successful transmission from these environments highlights there is a risk of environmental transmission even before foot-and-mouth disease (FMD) is clinically apparent in cattle. Estimated viral decay rates suggest that FMDV remained viable in this environment for up to 14 days, emphasizing the requirement for stringent biosecurity procedures following outbreaks of FMD and the design of control measures that reflect the biology of a pathogen.IMPORTANCE Effective control of a disease relies on comprehensive understanding of how transmission occurs, in order to design and apply effective control measures. Foot-and-mouth disease virus (FMDV) is primarily spread by direct contact between infected and naive individuals, although the high levels of virus shed by infected animals mean that virus can also be spread through contact with contaminated environments. Using a series of transmission experiments, we demonstrate that environmental transmission alone would be sufficient to sustain an outbreak. Key observations include that a risk of transmission exists before clinical signs of foot-and-mouth disease (FMD) are apparent in cattle and that survival of virus in the environment extends the transmission risk period. This study highlights the role a contaminated environment can play in the transmission of FMDV and presents approaches that can also be applied to study the transmission of other pathogens that are able to survive in the environment.

Environmental Sampling as a Low-Technology Method for Surveillance of Foot-and-Mouth Disease Virus in an Area of Endemicity.

Environmental sampling enables disease surveillance beyond regular investigation of observed clinical cases, extending data on the circulation of a pathogen in a specific area. Developing straightforward, low-technology methods suitable for use under field conditions is key to the inclusion of such approaches alongside traditional surveillance techniques. Foot-and-mouth disease virus (FMDV) is an economically important livestock pathogen, affecting cloven-hoofed livestock in many countries. Countries with FMDV face severe trade restrictions, and infections can have long-term effects on the productivity of affected animals. Environmental contamination by the virus in excretions and secretions from infected individuals promotes transmission but also presents an opportunity for noninvasive sample collection, facilitating diagnostic and surveillance activities. We present environmental sampling methods that have been tested in the Kathmandu Valley, Nepal, where FMDV is endemic. A total of nine sites were visited and sampled between November 2016 and November 2017. Environmental swabs collected from sites with reported outbreaks of FMD were used to demonstrate successful detection of FMDV RNA from the environment. The development of methods that can reliably detect FMDV RNA in the environment is significant, since this possibility extends the toolbox available for surveillance for this disease. Similar methods have already been deployed in the effort to eradicate polio, and with FMDV, such methods could easily be deployed in the event of an outbreak to provide additional resources for detection that would relieve pressure on veterinary services. The development of low-technology, straightforward surveillance methods such as these can support a robust response to outbreaks.IMPORTANCE Prompt confirmation and diagnosis of disease are key factors in controlling outbreaks. The development of sampling techniques to detect FMDV RNA from the environment will extend the tool kit available for the surveillance of this pathogen. The methods presented in this article broaden surveillance opportunities using accessible techniques. Pairing these methods with existing and novel diagnostic tests will improve the capability for rapid detection of outbreaks and implementation of timely interventions to control outbreaks. In areas of endemicity, these methods can be implemented to extend surveillance beyond the investigation of clinical cases, providing additional data for the assessment of virus circulation in specific areas.

Efficacy of a high-potency multivalent foot-and-mouth disease virus vaccine in cattle against heterologous challenge with a field virus from the emerging A/ASIA/G-VII lineage.

In 2015, outbreaks of foot-and-mouth disease (FMD) in the Middle East were discovered to be caused by a viral lineage (A/ASIA/G-VII), which has recently emerged from the Indian sub-continent. In vitro vaccine matching data generated by the World Reference Laboratory (WRLFMD) indicated that A/ASIA/G-VII field viruses were poorly matched with vaccines (A-SAU-95, A22 IRQ and A-IRN-05) that are already used in the region. In order to assess the likely performance of one of these commercially available FMD vaccines, sixteen cattle were vaccinated with a polyvalent vaccine which contained two serotype A components (A-SAU-95 and A-IRN-05) with a homologous potency of at least 6PD50, and two cattle were left unvaccinated as controls. Twenty-one days later, all 18 cattle were challenged by tongue inoculation with an FMDV field isolate A/IRN/22/2015 from the A/ASIA/G-VII lineage, in line with the European Pharmacopeia PPG test conditions. The two control animals developed generalised FMD, and 7/16 vaccinated animals developed at least one foot lesion, thus only 56.3% were defined as protected. For the vaccine components, there was a significant increase in the probability of protection with increasing serological titres for A-SAU-95 (p = 0.03), but not for A-IRN-05 (p = 0.42). Analysis of FMDV in blood and nasal swabs suggested that vaccination reduced shedding and potential onward spread of FMD virus even if the animal developed foot lesions. In summary, the results from this study suggest that whilst this vaccine would not be appropriate for use in an emergency situation (in previously FMD-free countries), it may be partially effective in the field in endemic countries where repeat prophylactic vaccination is practiced. For emergency reactive vaccination, the findings from this study support the idea that a new vaccine strain should be developed that is tailored to the A/ASIA/G-VII lineage.

Predicting the Ability of Preclinical Diagnosis To Improve Control of Farm-to-Farm Foot-and-Mouth Disease Transmission in Cattle.

Foot-and-mouth disease (FMD) can cause large disruptive epidemics in livestock. Current eradication measures rely on the rapid clinical detection and removal of infected herds. Here, we evaluated the potential for preclinical diagnosis during reactive surveillance to reduce the risk of between-farm transmission. We used data from transmission experiments in cattle where both samples from individual animals, such as blood, probang samples, and saliva and nasal swabs, and herd-level samples, such as air samples, were taken daily during the course of infection. The sensitivity of each of these sample types for the detection of infected cattle during different phases of the early infection period was quantified. The results were incorporated into a mathematical model for FMD, in a cattle herd, to evaluate the impact of the early detection and culling of an infected herd on the infectious output. The latter was expressed as the between-herd reproduction ratio, Rh , where an effective surveillance approach would lead to a reduction in the Rh value to <1. Applying weekly surveillance, clinical inspection alone was found to be ineffective at blocking transmission. This was in contrast to the impact of weekly random sampling (i.e., using saliva swabs) of at least 10 animals per farm or daily air sampling (housed cattle), both of which were shown to reduce the Rh to <1. In conclusion, preclinical detection during outbreaks has the potential to allow earlier culling of infected herds and thereby reduce transmission and aid the control of epidemics.

Use of Bacillus subtilis PXN21 spores for suppression of Clostridium difficile infection symptoms in a murine model.

Clostridium difficile is the primary cause of nosocomial diarrhoea in healthcare centres of the developed world. Only a few antibiotics are available for treatment, and relapses are common in patients undergoing antibiotic therapy. New approaches are required to reduce reliance on antibiotics, the use of which represents a primary risk factor for development of C. difficile infections. Supplementation of the gut flora with probiotics represents a key area for producing more successful treatment options for C. difficile infection (CDI). In this study, spores of B. subtilis have been evaluated as a potential probiotic treatment against CDI. Using a murine model of infection, we demonstrate that oral administration of B. subtilis spores can attenuate the symptoms of infection. We further show that (1) suppression of symptoms was better if spores were administered post infection, and (2) germination of the spore to a vegetative cell may be an integral part of how CDI is suppressed. The results of this study highlight the potential of this bacterium as a probiotic treatment for CDI.

The spore-associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile.

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild-type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID50 ) of spores was significantly higher (2-logs) in the bclA1(-) mutant compared to the isogenic wild-type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild-type spores. bclA1(-) spores germinated faster than wild-type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre-spore germination) stages of infection. We also show that the ID50 was higher in mice infected with R20291, a 'hypervirulent' 027 strain, that carries a truncated BclA1 protein.

Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine.

Heat killed spores of the Gram-positive bacterium Bacillus subtilis have been evaluated as a vaccine delivery system with mucosal adjuvant properties for influenza. Killed spores were able to bind H5N1 virions (NIBRG-14; clade 1) and, when intra-nasally administered to mice, resulting immune responses, both humoral and cell mediated, were enhanced compared to immunization with the virion alone. Levels of both systemic IgG and mucosal sIgA specific to the virion were elevated. Levels of IgG2a (a Th(1) antibody type) were strongly enhanced when the virion was co-administered with killed spores. Cytokine induction in stimulated splenocytes was also apparent indicating balanced T(h)1 and T(h)2 responses. Evidence of cross-neutralization of clade 2.2 viruses was shown. In a challenge experiment mice dosed two times with spores adsorbed with just 20 ng HA (hemagglutinin) of inactivated NIBRG-14 were fully protected against challenge with 20 LD(50) of H5N2 virus. Interestingly, partial protection (60%) was observed in animals dosed only with killed spores. Mice dosed only with killed spores were shown to be fully protected against H5N2 (5 LD(50)) infection indicating that innate immunity and its stimulation by spores may play an important role in protection. Supporting this killed spores were (i) shown to stimulate TLR-mediated expression of NF-κB, and (ii) able to recruit NK cells into lungs and induce maturation of DCs. This work demonstrates the potential and underlying mechanism for the use of bacterial spores as an adjuvant for H5N1 vaccination.