The World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals describes a diverse array of assays that can be used to detect, characterise and monitor the presence of infectious agents of farmed livestock. These methods have been developed in different laboratories, at different times, and often include tests or kits provided by the commercial sector. Reference panels are essential tools that can be used during assay development and in validation exercises to compare the performance of these varied (and sometimes competing) diagnostic technologies. World Organisation for Animal Health Reference Laboratories already provide approved international standard reagents to help calibrate diagnostic tests for a range of diseases, but there remain important gaps in their availability for comparative purposes and the calibration of test results across different laboratories. Using foot and mouth disease (FMD) as an example, this review highlights four specific areas where new reference reagents are required. These are to: reduce bias in estimates of the diagnostic sensitivity and inter-serotypic specificity of tests used to detect diverse strains of FMD virus (FMDV), provide bio-safe positive controls for new point-of-care test formats that can be deployed outside high containment, harmonise FMDV antigens for post-vaccination serology, and address inter-laboratory differences in serological assays used to measure virus-specific FMD antibody responses. Since there are often limited resources to prepare and distribute these materials, sustainable progress in this arena will only be achievable if there is consensus and coordination of these activities among OIE Reference Laboratories.

African swine fever virus (ASFV) is one of the most threatening infectious diseases of pigs. There are not sufficient data to indicate the importance of the sylvatic cycle in the spread and maintenance of the disease locally and potentially, globally. To assess the capacity to maintain ASF in the environment, we investigated the presence of soft tickreservoirs of ASFV in Gorongosa National Park (GNP) and its surrounding villages. A total of 1,658 soft ticks were recovered from warthog burrows and pig pens at the wildlife/livestock interface of the GNP and viral DNA was confirmed by nested PCR in 19% of Ornithodoros porcinus porcinus and 15% of O.p.domesticus. However, isolation of ASFV was only achieved in approximately 50% of the PCR-positive samples with nineteen haemadsorbing virus isolates recovered. These were genotyped using a combination of partial sequencing of the B646L gene (p72) and analysis of the central variable region (CVR) of the B602L gene. Eleven isolates were classified as belonging to genotype II and homologous to contemporary isolates from southern Africa, the Indian Ocean and eastern Europe. Three isolates grouped within genotype V and were similar to previous isolates from Mozambique and Malawi. The remaining five isolates constituted a new, previously unidentified genotype, designated genotype XXIV. This work confirms for the first time that the virus currently circulating in eastern Europe is likely to have a wildlife origin, and that the large diversity of ASFV maintained in wildlife areas can act as a permanent sources of different strains for the domestic pig value chain in Mozambique and beyond its boundaries. Their genetic similarity to ASFV strains currently spreading across Europe justifies the need to continue studying the sylvatic cycle in this African country and other parts of southern Africa in order to identify potential hot spots of ASF emergence and target surveillance and control efforts.

Potency tests for commercial oil-adjuvanted foot-and-mouth disease (FMD) vaccines are usually carried out in cattle, using a full dose (2 ml) of vaccine and homologous virus challenge. However, in sheep the recommended vaccine dose is half of the cattle dose (1 ml) and most vaccines have not been potency tested for this species, especially with heterologous viruses. To determine the efficacy of a high potency (>6PD50) FMD virus (FMDV) O1Manisa vaccine in sheep, we carried out a study using a heterologous FMDV (FMDV O/SKR/2010 - Mya-98 strain) challenge. Groups of seven animals each were vaccinated with 2×, 1×, 1/2× or 1/4× dose (2 ml, 1 ml, 0.5 ml or 0.25 ml respectively) and challenged at 7 days post vaccination (dpv). Only 3 of the 7 sheep in the group vaccinated with 2 ml were protected. With 2 additional groups, receiving double or single doses and challenged at 14 dpv, 4 of 7 sheep were protected in each group. None of the sheep had measurable neutralising antibodies against the vaccine or challenge virus at 7 dpv. However, all vaccinated animals challenged at 14 dpv had a homologous neutralising response against FMDV O1 Manisa on the day of challenge and all but one animal also had a heterologous response to FMDV O/SKR/2010. Infectious FMDV and viral RNA could be found in nasal swabs between 1 and 6 days post challenge (dpc) in most vaccinated sheep, but those vaccinated with higher doses or challenged at 14 dpv showed significant decreases in the level of FMDV detection. Intermittent virus shedding was noticed between 1 and 35 dpc in all vaccinated groups, but persistent infection could be demonstrated only in 4 sheep (20%). This study showed that at the recommended dose, a high potency (>6 PD50) FMDV O1Manisa vaccine does not protect sheep against a heterologous challenge at 7 dpv. However, partial protection was observed when a double dose was used at 7 dpv or when double or single dose vaccinated sheep were challenged at 14 dpv.

This study assessed research knowledge gaps in the field of FMDV (foot-and-mouth disease virus) vaccines. The study took the form of a literature review (2011-15) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD vaccine research. Vaccines play a vital role in FMD control, used both to limit the spread of the virus during epidemics in FMD-free countries and as the mainstay of disease management in endemic regions, particularly where sanitary controls are difficult to apply. Improvements in the performance or cost-effectiveness of FMD vaccines will allow more widespread and efficient disease control. FMD vaccines have changed little in recent decades, typically produced by inactivation of whole virus, the quantity and stability of the intact viral capsids in the final preparation being key for immunogenicity. However, these are exciting times and several promising novel FMD vaccine candidates have recently been developed. This includes the first FMD vaccine licensed for manufacture and use in the USA; this adenovirus-vectored FMD vaccine causes invivo expression of viral capsids in vaccinated animals. Another promising vaccine candidate comprises stabilized empty FMDV capsids produced invitro in a baculovirus expression system. Recombinant technologies are also being developed to improve otherwise conventionally produced inactivated vaccines, for example, by creating a chimeric vaccine virus to increase capsid stability and by inserting sequences into the vaccine virus for desired antigen expression. Other important areas of ongoing research include enhanced adjuvants, vaccine quality control procedures and predicting vaccine protection from immune correlates, thus reducing dependency on animal challenge studies. Globally, the degree of independent vaccine evaluation is highly variable, and this is essential for vaccine quality. Previously neglected, the importance of evaluating vaccination programme effectiveness and impact is increasingly being recognized.

We assessed knowledge gaps in foot-and-mouth disease (FMD) research, and in this study, we consider (i) epidemiology, (ii) wildlife and (iii) economics. The study took the form of a literature review (2011-2015) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD research. During 2011-2015, modelling studies were dominant in the broad field of epidemiology; however, continued efforts are required to develop robust models for use during outbreaks in FMD-free countries, linking epidemiologic and economics models. More guidance is needed for both the evaluation and the setting of targets for vaccine coverage, population immunity and vaccine field efficacy. Similarly, methods for seroprevalence studies need to be improved to obtain more meaningful outputs that allow comparison across studies. To inform control programmes in endemic countries, field trials assessing the effectiveness of vaccination in extensive smallholder systems should be performed to determine whether FMD can be controlled with quality vaccines in settings where implementing effective biosecurity is challenging. Studies need to go beyond measuring only vaccine effects and should extend our knowledge of the impact of FMD and increase our understanding of how to maximize farmer participation in disease control. Where wildlife reservoirs of virus exist, particularly African Buffalo, we need to better understand when and under what circumstances transmission to domestic animals occurs in order to manage this risk appropriately, considering the impact of control measures on livelihoods and wildlife. For settings where FMD eradication is unfeasible, further ground testing of commodity-based trade is recommended. A thorough review of global FMD control programmes, covering successes and failures, would be extremely valuable and could be used to guide other control programmes.

This study assessed gaps and priorities for FMDV (foot-and-mouth disease virus) research in the field of immunology. The study took the form of a literature review (2011-15) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD research. Improved understanding of FMDV immunology facilitates the development of vaccines, adjuvants and diagnostic tests, and will allow better assessment and prediction of vaccine potency and match, with reduced use of animals, particularly large animals, in experimental studies. Continued characterization of the immune systems of several FMD host species has underpinned substantial advances in knowledge of their interaction with FMDV. Recent studies have shed light on the mechanisms underlying formation of the bovine B- and T-cell response; there is also a greater understanding of the significance of non-neutralizing antibodies during FMDV infection and the interactions of antibody-bound virus with immune cells. This knowledge is directly relevant to vaccine development, as well as understanding protection and cross-protection. Despite ongoing research, significant knowledge gaps remain in the areas of neonatal and mucosal immunity. The impact of maternally derived antibody upon the neonate's ability to respond to FMD vaccination has received some attention, but few firm conclusions can be drawn at this stage, and little is known of the cellular response of young animals in general. The mucosal immune system of FMDV-susceptible species requires continued characterization, especially if the potential of mucosal vaccine-delivery systems is to be realized for FMD immunization.

African swine fever (ASF) is a mostly fatal viral infection of domestic pigs for which there is no vaccine available. The disease is endemic to most of sub-Saharan Africa, causes severe losses and threatens food security in large parts of the continent. Naturally occurring attenuated ASF viruses have been tested as vaccine candidates, but protection was variable depending on the challenge virus. In this study, the virulence of two African isolates, one from a tick vector and the other from an indigenous pig, was determined in domestic pigs to identify a potential vaccine strain for southern Africa. Neither isolate was suitable as the tick isolate was moderately virulent and the indigenous pig virus was highly virulent. The latter was subsequently used as heterologous challenge in pigs first vaccinated with a naturally attenuated isolate previously isolated in Portugal. Although a statistically significant reduction in death rate and virus load was observed compared with unvaccinated pigs post-challenge, all pigs succumbed to infection and died.

An epidemiological study of African swine fever (ASF) was conducted between March 2006 and September 2007 in a rural area adjacent to the Gorongosa National park (GNP) located in the Central Mozambique. Domestic pigs and warthogs were sampled to determine the prevalence of antibodies against ASF virus and the salivary antigens of Ornithodoros spp. ticks, while ticks collected from pig pens were tested for the presence of ASFV. In addition, 310 framers were interviewed to gain a better understanding of the pig value chain and potential practices that could impact on the spread of the virus. The sero-prevalence to ASFV was 12.6% on farms and 9.1% in pigs, while it reached 75% in warthogs. Approximately 33% of pigs and 78% of warthogs showed antibodies against salivary antigens of ticks. The differences in sero-prevalence between farms close to the GNP, where there is greater chance for the sylvatic cycle to cause outbreaks, and farms located in the rest of the district, where pig to pig transmission is more likely to occur, were marginally significant. Ornithodoros spp. ticks were found in only 2 of 20 pig pens outside the GNP, and both pens had ticks testing positive for ASFV DNA. Interviews carried out among farmers indicated that biosecurity measures were mostly absent. Herd sizes were small with pigs kept in a free-ranging husbandry system (65%). Only 1.6% of farmers slaughtered on their premises, but 51% acknowledged allowing visitors into their farms to purchase pigs. ASF outbreaks seemed to have a severe economic impact with nearly 36% of farmers ceasing pig farming for at least 1year after a suspected ASF outbreak. This study provides the first evidence of the existence of a sylvatic cycle in Mozambique and confirms the presence of a permanent source of virus for the domestic pig value chain.

The Global Foot-and-mouth disease (FMD) Research Alliance periodically reviews the state of FMD research to assess progress and to identify new priorities. In this supplement we provide an update of global FMD research, comprising (i) this overview paper, which includes background information with key findings, and papers covering (ii) epidemiology, wildlife and economics, (iii) vaccines, (iv) diagnostics, (v) biotherapeutics and disinfectants, (vi) immunology and (vii) pathogenesis and molecular biology. FMD research publications were reviewed (2011-2015) and activity updates were obtained from 33 FMD research institutes from around the world. Although a continual threat, FMD has been effectively controlled in much of the world using existing tools. However, control remains a challenge in most developing countries, where little has been done to understand the ongoing burden of FMD. More research is needed to support control in endemically infected countries, particularly robust field studies. Traditional FMD vaccines have several limitations including short duration and spectrum of protection, cold chain requirements, and the costs and biosecurity risks associated with vaccine production. Significant progress has been made in the development of novel vaccine candidates, particularly in the use of recombinant vaccines and virus-like particles as an alternative to traditional inactivated whole virus vaccines. Continued investment is needed to turn these developments into improved vaccines produced at scale. Increased knowledge of cellular and mucosal immunity would benefit vaccine development, as would further advances in our ability to enhance vaccine capsid stability. Developments in molecular biology and phylogenetics underlie many of the recent advances in FMD research, including improved vaccines and diagnostics, and improved understanding of FMD epidemiology. Tools for genetic analyses continue to become both more powerful and more affordable enabling them to be used to address an ever-expanding range of questions. This rapidly advancing field potentiates many areas of FMD research and should be prioritized.

In high-density farming practices, it is important to constantly monitor for infectious diseases, especially diseases that have the potential to spread rapidly between holdings. Pigs are known to amplify foot-and-mouth disease (FMD) by excreting large amounts of virus, and it is therefore important to detect the virus quickly and accurately to minimize the spread of disease. Ropes were used to collect oral fluid samples from pigs, and each sample was compared to saliva samples collected from individual animals by detecting FMD virus RNA using real-time PCR. Two different experiments are described where groups of pigs were infected with different serotypes of FMD virus, either with or without vaccination, and unvaccinated pigs were kept in aerosol contact. The sensitivity of the rope sampling varied between 0.67 and 0.92, and the statistical agreement between this method and individual sampling ranged from substantial to moderate for the two different serotypes. The ease of collecting oral fluids using ropes together with the high sensitivity of subsequent FMD detection through PCR indicates that this could be a useful method to monitor pig populations for FMD virus infection. With further validation of the sensitivity of detection of FMD virus RNA, this can be a cost-effective, non-invasive diagnostic tool.

The non-structural proteins of foot-and-mouth disease virus (FMDV) are responsible for RNA replication, proteolytic processing of the viral polyprotein precursor, folding and assembly of the structural proteins and modification of the cellular translation apparatus. Investigation of the amino acid heterogeneity of the non-structural proteins of seventy-nine FMDV isolates of SAT1, SAT2, SAT3, A and O serotypes revealed between 29 and 62% amino acid variability. The Leader protease (L(pro)) and 3A proteins were the most variable whilst the RNA-dependent RNA polymerase (3D(pol)) the most conserved. Phylogeny based on the non-structural protein-coding regions showed separate clusters for southern African viruses for both the L(pro) and 3C protease (3C(pro)) and sequences unique to this group of viruses, e.g. in the 2C and 3C(pro) proteins. These groupings were unlike serotype groupings based on structural protein-coding regions. The amino acid substitutions and the nature of the naturally occurring substitutions provide insight into the functional domains and regions of the non-structural proteins that are critical for structure-function. The L(pro) of southern African SAT type isolates differed from A, O and SAT isolates in northern Africa, particularly in the auto-processing region. Three-dimensional structures of the 3C protease (3C(pro)) and 3D(pol) showed that the observed variation does not affect the enzymatic active sites or substrate binding sites. Variation in the 3C(pro) cleavage sites demonstrates broad substrate specificity.

Genetic information regarding the leader (L) and complete capsid-coding (P1) region of FMD serotype A and O viruses prevalent on the African continent is lacking. Here, we present the complete L-P1 sequences for eight serotype A and nine serotype O viruses recovered from FMDV outbreaks in East and West Africa over the last 33 years. Phylogenetic analysis of the P1 and capsid-coding regions revealed that the African isolates grouped according to serotype, and certain clusters were indicative of transboundary as well as intra-regional spread of the virus. However, similar analysis of the L region revealed random groupings of isolates from serotypes O and A. Comparisons between the phylogenetic trees derived from the structural coding regions and the L region pointed to a possibility of genetic recombination. The intertypic nucleotide and amino acid variation of all the isolates in this study supported results from previous studies where the externally located 1D was the most variable whilst the internally located 1A was the most conserved, which likely reflects the selective pressures on these proteins. Amino acids identified previously as important for FMDV structure and functioning were found to be highly conserved. The information gained from this study will contribute to the construction of structurally designed FMDV vaccines in Africa.

To eliminate incursions of foot-and-mouth disease (FMD) quickly, a combination of measures, including emergency vaccination, can help block the spread of infection. For the earliest recovery of the FMD-free status for trade, without the slaughter of uninfected vaccinated animals, a serosurvey for antibodies to FMD virus non-structural proteins (NSP) must be used to substantiate absence of occult virus infections. Areas of doubt over requirements for post-vaccination serosurveillance and its feasibility include the required and achievable confidence, the amount of sampling necessary, and the appropriate responses to and consequences of different seropositive findings. This derives largely from uncertainty over the extent of localised pockets of virus infection that may remain within vaccinated populations and the circumstances that permit this. The question therefore remains whether tests are sufficiently sensitive and specific to detect and eliminate infected animals, without excessive culling of uninfected animals, before vaccinated animals mix with non-vaccinated livestock when movement restrictions are lifted. It is recommended to change the rationale for serosurveillance after emergency vaccination. Only when emergency vaccination is used in limited outbreaks is it possible to test and cull comprehensively, an approach compatible with a three-month minimum period to recover the FMD-free status. In other situations, where emergency vaccination is used, such as dealing with large outbreaks in animal-dense regions and where the onset of vaccination has been delayed, post-vaccination serosurveys should be targeted and focus on providing an assurance to detect higher levels of infection, in case of inadequate control measures. As this provides less assurance of absence of infection, the approach would be compatible with a six-month waiting period for free-status recovery and should be complemented by other methods to provide evidence that vaccination and control measures have been effectively implemented, as these are the best guarantee against continuing virus transmission.

Wild African Suidae, the common warthog (Phacochoerus africanus) and bushpig (Potamochoerus larvatus), were experimentally infected with classical swine fever (CSF) virus following the diagnosis of CSF subtype 2.1 in domestic pigs in South Africa in 2005. No data regarding the susceptibility or potential lesions of these African wild suids are available. Seven subadult warthogs and six bushpigs were captured and infected intranasally with the South African isolate. Two in-contact control animals of the same species in each experiment verified intra-species transmission. Surviving animals were euthanized after 44 days. Formalin-fixed tissue samples collected from them as well as animals euthanized during the trial were evaluated for histological lesions. The warthogs, which were clinically normal throughout the study, developed histological lesions that were inconsistently present and sometimes subtle. Three individuals, including one in-contact control, developed distinct lympho-plasmacytic cuffing in their brains. Subtle lesions included scant lympho-plasmacytic infiltration of various organs, occasionally accompanied by perivascular cuffing. In contrast, the bushpigs developed overt clinical signs similar to CSF in domestic pigs. Four of six animals, including two in-contact controls, died or were euthanized during the trial. On postmortem examination, intestinal necrosis and ulceration, purulent rhinitis and pneumonia were present. Affected animals developed lymphoid necrosis and depletion whilst surviving individuals showed perivascular cuffing in multiple organs. From the present work, we conclude that these wild Suidae are susceptible to CSF virus and intra-species transmission under experimental conditions can occur.

An incursion of classical swine fever virus (CSFV) into the domestic pig population in South Africa, identified in 2005, raised the concern that infection might spread to wildlife species and be maintained in these hosts. This study sought to determine whether two wildlife Suidae species present in South Africa, the bushpig (Potamochoerus larvatus) and the common warthog (Phacochoerus africanus), could support productive CSFV infection. Both species could be infected with CSFV and transmitted infection to in-contact animals of the same species. Viral antigen and RNA genome were detected in blood/serum and animals that survived initial infection seroconverted approximately 10-14 days post-inoculation. Viral RNA remained detectable in nasal and saliva secretions for prolonged periods until monitoring ended at 42-44 days after initial challenge. These data suggest that both Suidae species could serve to spread circulating CSFV within wild populations, with implications for disease control.

W. VOSLOO, S.P. SWANEPOEL, M. BAUMAN, B. BOTHA, J.J. ESTERHUYSEN, C.I. BOSHOFF, D.F. KEET, A. DEKKER Transboundary Animal Diseases Programme, ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110, South Africa Department of Tropical Veterinary Diseases, Faculty of Veterinary Medicine, University of Pretoria, South Africa Deltamune, PO Box 14167, Lyttelton, 0140, South Africa Department of Biomedical Sciences, Tshwane University of Technology Private Bag X680, Pretoria, 0001, South Africa Office of the State Veterinarian, Kruger National Park, P.O. Box 12, Skukuza, 1350, South Africa

The potential role of giraffe (Giraffa camelopardalis) in the epidemiology and spread of foot-and-mouth disease (FMD) SAT types was investigated by experimental infection and detection of virus in excretions using virus isolation on primary pig kidney cell cultures. In two experiments separated by a period of 24 months, groups of four animals were needle infected with a SAT-1 or SAT-2 virus, respectively and two in-contact controls were kept with each group. Viraemia was detected 3-9 days post-infection and virus isolated from mouth washes and faeces only occasionally up to day 13. The SAT-1 virus was transmitted to only one in-contact control animal, probably via saliva that contained virus from vesicles in the mouth of a needle-infected animal. None of the animals infected with the SAT-2 virus had any vesicles in the mouth, and there was no evidence of transmission to the in-contact controls. No virus was detected in probang samples for the duration of the experiments (60 days post-infection), indicating that persistent infection probably did not establish with either of these isolates. Giraffe most likely do not play an important role in FMD dissemination. Transmission of infection would possibly occur only during close contact with other animals when mouth vesicles are evident.