Early Detection of Foot‐And‐Mouth Disease Virus from Infected Cattle Using A Dry Filter Air Sampling System

Foot‐and‐mouth disease (FMD) is a highly contagious livestock disease of high economic impact. Early detection of FMD virus (FMDV) is fundamental for rapid outbreak control. Air sampling collection has been demonstrated as a useful technique for detection of FMDV RNA in infected animals, related to the aerogenous nature of the virus. In the current study, air from rooms housing individual (n = 17) or two groups (n = 4) of cattle experimentally infected with FDMV A24 Cruzeiro of different virulence levels was sampled to assess the feasibility of applying air sampling as a non‐invasive, screening tool to identify sources of FMDV infection. Detection of FMDV RNA in air was compared with first detection of clinical signs and FMDV RNA levels in serum and oral fluid. FMDV RNA was detected in room air samples 1–3 days prior (seven animals) or on the same day (four animals) as the appearance of clinical signs in 11 of 12 individually housed cattle. Only in one case clinical signs preceded detection in air samples by one day. Overall, viral RNA in oral fluid or serum preceded detection in air samples by 1–2 days. Six individually housed animals inoculated with attenuated strains did not show clinical signs, but virus was detected in air in one of these cases 3 days prior to first detection in oral fluid. In groups of four cattle housed together, air detection always preceded appearance of clinical signs by 1–2 days and coincided more often with viral shedding in oral fluid than virus in blood. These data confirm that air sampling is an effective non‐invasive screening method for detecting FMDV infection in confined to enclosed spaces (e.g. auction barns, milking parlours). This technology could be a useful tool as part of a surveillance strategy during FMD prevention, control or eradication efforts.     

First detection of foot‐and‐mouth disease virus O/ME‐SA/ Ind2001e sublineage in Jordan

Foot‐and‐mouth disease (FMD) is a highly contagious vesicular disease that is caused by the FMD virus (FMDV). This disease affects both wild and domestic cloven‐hoofed animals, and the latter of which includes cattle, swine, sheep and goats. FMD is endemic to Jordan and has a severe impact on the productivity of domestic livestock. In January 2017, FMD outbreaks were detected in different animal species across Jordan, resulting in high mortality rates among young lamb and goat populations as well as causing classic FMD symptoms in cattle. In this study, clinical specimens were collected from animals affected by FMD. The results obtained from sequencing the VP1 gene place the studied FMDV isolate within the FMDV O/ME‐SA/ Ind2001e sublineage. Phylogenetic analysis of VP1 suggests that the O/JOR/1/2017 isolate is very similar to that of viruses isolated from Saudi Arabia in 2016. The possible introduction of this strain to Jordan might occur through transboundary animal movement or other transmission routes from Saudi Arabia, a neighbouring country.     

A Refined Guinea Pig Model of Foot‐and‐Mouth Disease Virus Infection for Assessing the Efficacy of Antiviral Compounds

An antiviral containment strategy for foot‐and‐mouth disease (FMD) outbreaks could support or replace current contingency plans in case of an outbreak in Europe and could spare many healthy animals from being pre‐emptively culled. Recently, substantial progress has been made towards the development of small molecule drugs that inhibit FMD virus (FMDV) replication in vitro. For the initial in vivo evaluation of antiviral lead molecules, a refined FMDV‐infection model in guinea pigs (GP) is herewith described. This GP model was validated by demonstrating the antiviral effect of T‐1105 (an influenza virus inhibitor with reported activity against FMDV). Sixteen animals were orally administered with T‐1105 twice daily (400 mg/kg/day) for five consecutive days and inoculated intraplantarly with 100 GPID₅₀ of the GP‐adapted FMDV strain O₁ Manisa 1 h after the first administration. The efficacy of T‐1105 was compared with that of prophylactic vaccination with a highly potent double‐oil emulsion‐inactivated O₁ Manisa vaccine. Ten animals received a single, full (2 ml) cattle vaccine dose and were inoculated 3 weeks later. Fourteen T‐1105‐treated and all vaccinated GP were completely protected from generalization of vesicular lesions. At 2 dpi, viral RNA was detected in serum of 9/16 T‐1105‐treated and of 6/10 vaccinated animals. At 4 dpi, viral RNA was detected in serum, organs and oral swabs of half of the T‐1105‐treated animals and only in the serum of 1/10 of the vaccinated animals. Mean viral RNA levels in serum and organs of T‐1105‐treated and vaccinated animals were reduced compared to untreated controls (P < 0.01). T‐1105 conferred a substantial clinical and virological protection against infection with O₁ Manisa, similar to the protection afforded by vaccination. These results validate the suitability of the enhanced GP model for the purpose of initial evaluation of inhibitors of FMDV replication and illustrate the potential of selective inhibitors of viral replication to control FMD outbreaks.     

Detection of Foot‐and‐mouth Disease Virus RNA and Capsid Protein in Lymphoid Tissues of Convalescent Pigs Does Not Indicate Existence of a Carrier State

A systematic study was performed to investigate the potential of pigs to establish and maintain persistent foot‐and‐mouth disease virus (FMDV) infection. Infectious virus could not be recovered from sera, oral, nasal or oropharyngeal fluids obtained after resolution of clinical infection with any of five FMDV strains within serotypes A, O and Asia‐1. Furthermore, there was no isolation of live virus from tissue samples harvested at 28–100 days post‐infection from convalescent pigs recovered from clinical or subclinical FMD. Despite lack of detection of infectious FMDV, there was a high prevalence of FMDV RNA detection in lymph nodes draining lesion sites harvested at 35 days post‐infection, with the most frequent detection recorded in popliteal lymph nodes (positive detection in 88% of samples obtained from non‐vaccinated pigs). Likewise, at 35 dpi, FMDV capsid antigen was localized within follicles of draining lymph nodes, but without concurrent detection of FMDV non‐structural protein. There was a marked decline in the detection of FMDV RNA and antigen in tissue samples by 60 dpi, and no antigen or viral RNA could be detected in samples obtained at 100 dpi. The data presented herein provide the most extensive investigation of FMDV persistence in pigs. The overall conclusion is that domestic pigs are unlikely to be competent long‐term carriers of infectious FMDV; however, transient persistence of FMDV protein and RNA in lymphoid tissues is common following clinical or subclinical infection.     

Direct detection and characterization of foot‐and‐mouth disease virus in East Africa using a field‐ready real‐time PCR platform

Effective control and monitoring of foot‐and‐mouth disease (FMD ) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE ). However, the requirements for prompt and serotype‐specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD ‐endemic countries have motivated the development of simple diagnostic platforms to support local decision‐making. Using a portable thermocycler, the T‐COR ™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan‐serotype‐specific real‐time RT ‐PCR (rRT ‐PCR ) assay and a newly available FMD virus (FMDV) typing assay (East Africa‐specific for serotypes: O, A, Southern African Territories [SAT ] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan‐serotype‐specific lyophilized assay were comparable to that of an OIE ‐recommended laboratory‐based rRT ‐PCR (determined using a panel of 57 FMDV ‐positive samples and six non‐FMDV vesicular disease samples for differential diagnosis). The FMDV ‐typing assay was able to correctly identify the serotype of 33/36 FMDV ‐positive samples (no cross‐reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal–pharyngeal (OP ) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n  = 144) collected from pre‐clinical, clinical and clinically recovered cattle. These data support the use of field‐ready rRT ‐PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.     

Opportunities for enhanced surveillance of foot‐and‐mouth disease in endemic settings using milk samples

Under‐reporting of foot‐and‐mouth disease (FMD) masks the true prevalence in parts of the world where the disease is endemic. Laboratory testing for the detection of FMD virus (FMDV) is usually reliant upon the collection of vesicular epithelium and fluid samples that can only be collected from acutely infected animals, and therefore animals with sub‐clinical infection may not be identified. Milk is a non‐invasive sample type routinely collected from dairy farms that has been utilized for surveillance of a number of other diseases. The aim of this study was to examine the application of milk as an alternative sample type for FMDV detection and typing, and to evaluate milk as a novel approach for targeted surveillance of FMD in East Africa. FMDV RNA was detected in 73/190 (38%) individual milk samples collected from naturally infected cattle in northern Tanzania. Furthermore, typing information by lineage‐specific rRT‐PCR assays was obtained for 58% of positive samples, and corresponded with the virus types identified during outbreak investigations in the study area. The VP1‐coding sequence data obtained from milk samples corresponded with the sequence data generated from paired epithelial samples collected from the same animal. This study demonstrates that milk represents a potentially valuable sample type for FMDV surveillance and might be used to overcome some of the existing biases of traditional surveillance methods. However, it is recommended that care is taken during sample collection and testing to minimize the likelihood of cross‐contamination. Such approaches could strengthen FMDV surveillance capabilities in East Africa, both at the individual animal and herd level.     

Foot‐and‐mouth disease virus transmission dynamics and persistence in a herd of vaccinated dairy cattle in India

Foot‐and‐mouth disease (FMD ) is an important transboundary disease with substantial economic impacts. Although between‐herd transmission of the disease has been well studied, studies focusing on within‐herd transmission using farm‐level outbreak data are rare. The aim of this study was to estimate parameters associated with within‐herd transmission, host physiological factors and FMD virus (FMDV ) persistence using data collected from an outbreak that occurred at a large, organized dairy farm in India. Of 1,836 regularly vaccinated, adult dairy cattle, 222 had clinical signs of FMD over a 39‐day period. Assuming homogenous mixing, a frequency‐dependent compartmental model of disease transmission was built. The transmission coefficient and basic reproductive number were estimated to be between 16.2–18.4 and 67–88, respectively. Non‐pregnant animals were more likely to manifest clinical signs of FMD as compared to pregnant cattle. Based on oropharyngeal fluid (probang) sampling and FMDV ‐specific RT ‐PCR , four of 36 longitudinally sampled animals (14%) were persistently infected carriers 10.5 months post‐outbreak. There was no statistical difference between subclinical and clinically infected animals in the duration of the carrier state. However, prevalence of NSP ‐ELISA antibodies differed significantly between subclinical and clinically infected animals 12 months after the outbreak with 83% seroprevalence amongst clinically infected cattle compared to 69% of subclinical animals. This study further elucidates within‐herd FMD transmission dynamics during the acute‐phase and characterizes duration of FMDV persistence and seroprevalence of FMD under natural conditions in an endemic setting.     

Serotype Specificity of Antibodies against Foot‐and‐Mouth Disease Virus in Cattle in Selected Districts in Uganda

Uganda had an unusually large number of foot‐and‐mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot‐and‐mouth disease virus (FMDV) by ELISA for antibodies against non‐structural proteins and structural proteins. Three hundred and forty‐nine cattle sera were collected from seven districts in Uganda, and 65% of these were found positive for antibodies against the non‐structural proteins of FMDV. A subset of these samples were analysed for serotype specificity of the identified antibodies. High prevalences of antibodies against non‐structural proteins and structural proteins of FMDV serotype O were demonstrated in herds with typical visible clinical signs of FMD, while prevalences were low in herds without clinical signs of FMD. Antibody titres were higher against serotype O than against serotypes SAT 1, SAT 2 and SAT 3 in the sera investigated for serotype‐specific antibodies. Only FMDV serotype O virus was isolated from one probang sample. This study shows that the majority of the FMD outbreaks in 2006 in the region studied were caused by FMDV serotype O; however, there was also evidence of antibodies to both SAT 1 and SAT 3 in one outbreak in a herd inside Queen Elizabeth national park area.     

Utilizing milk from pooling facilities as a novel approach for foot‐and‐mouth disease surveillance

This study investigated the potential of pooled milk as an alternative sample type for foot‐and‐mouth disease (FMD) surveillance. Real‐time RT‐PCR (rRT‐PCR) results of pooled milk samples collected weekly from five pooling facilities in Nakuru County, Kenya, were compared with half‐month reports of household‐level incidence of FMD. These periodic cross‐sectional surveys of smallholder farmers were powered to detect a threshold household‐level FMD incidence of 2.5% and collected information on trends in milk production and sales. FMD virus (FMDV) RNA was detected in 9/219 milk samples, and using a type‐specific rRT‐PCR, serotype SAT 1 was identified in 3/9 of these positive samples, concurrent with confirmed outbreaks in the study area. Four milk samples were FMDV RNA‐positive during the half‐months when at least one farmer reported FMD; that is, the household‐level clinical incidence was above a threshold of 2.5%. Additionally, some milk samples were FMDV RNA‐positive when there were no reports of FMD by farmers. These results indicate that the pooled milk surveillance system can detect FMD household‐level incidence at a 2.5% threshold when up to 26% of farmers contributed milk to pooling facilities, but perhaps even at lower levels of infection (i.e., below 2.5%), or when conventional disease reporting systems fail. Further studies are required to establish a more precise correlation with estimates of household‐level clinical incidence, to fully evaluate the reliability of this approach. However, this pilot study highlights the potential use of this non‐invasive, routinely collected, cost‐effective surveillance tool, to address some of the existing limitations of traditional surveillance methods.     

Review of the Global Distribution of Foot‐and‐Mouth Disease Virus from 2007 to 2014

Foot‐and‐mouth disease (FMD) virus affects livestock worldwide. There are seven different serotypes, each with a diversity of topotypes, genetic lineages and strains. Some lineages have different properties that may contribute to sporadic spread beyond their recognized endemic areas. The objective of this study was to review the most significant FMD epidemiological events that took place worldwide between 2007 and 2014. Severe epidemics were caused by FMD virus (FMDV) lineage O/Asia/Mya‐98 in Japan and South Korea in 2010, both previously free of disease. In India, where FMD is endemic, the most important event was the re‐emergence of lineage O/ME‐SA/Ind‐2001 in 2008. Notably, this lineage, normally restricted to India, Bangladesh, Nepal and Bhutan, was also found in Saudi Arabia and Libya in 2013 and has caused several outbreaks in Tunisia and Algeria in 2014–2015. In January 2011, FMDV‐positive wild boars were found in Bulgaria, where the disease last occurred in 1996, followed by 12 outbreaks in livestock infected with FMDV O/ME‐SA/PanAsia2. In 2012, FMDV SAT2 caused outbreaks in Egypt and the Palestinian Autonomous Territories. Another significant event was the emergence of FMDV Asia1 Sindh‐08 in the Middle East. In South America, one outbreak of FMDV serotype O, topotype Euro‐SA was reported in Paraguay in 2011, which was recognized as FMD‐free with vaccination at the time. Lessons learned from past events, point out the need for an integrated strategy that comprises coordinated global and regional efforts for FMDV control and surveillance. Specific local characteristics related to host, environment and virus that condition FMD occurrence should be carefully considered and incorporated to adapt appropriate strategies into local plans. In this review, we compiled relevant epidemiological FMD events to provide a global overview of the current situation. We further discussed current challenges present in different FMD areas.     

Proof of Concept for the Inhibition of Foot‐and‐Mouth Disease Virus Replication by the Anti‐Viral Drug 2′‐C‐Methylcytidine in Severe Combined Immunodeficient Mice

Recent European contingency plans envisage emergency vaccination as an animal‐friendly control strategy for foot‐and‐mouth disease (FMD). Anti‐viral drugs may be used as an alternative or complementary measure. We here demonstrate that the nucleoside analogue 2′‐C‐methylcytidine (2′CMC) protects severe combined immunodeficient (SCID) mice against lethal FMD virus infection. In brief, SCID mice were inoculated with serotype A FMD virus and treated for five consecutive days with 2′CMC. All 15 treated mice remained healthy until the end of the study at 14 days post‐infection (dpi). At that time, viral RNA was no longer detected in 13 of 15 treated mice. All eight untreated mice suffered from an acute generalized disease and were euthanized for ethical reasons on average at 4 dpi. These results illustrate the potential of small molecules to control FMD.     

Quantitative characteristics of the foot‐and‐mouth disease carrier state under natural conditions in India

The goal of this study was to characterize the properties and duration of the foot‐and‐mouth disease (FMD ) carrier state and associated serological responses subsequent to vaccination and naturally occurring infection at two farms in northern India. Despite previous vaccination of cattle in these herds, clinical signs of FMD occurred in October 2013 within a subset of animals at the farms containing juvenile‐yearling heifers and steers (Farm A) and adult dairy cattle (Farm B). Subsequent to the outbreak, FMD virus (FMDV ) asymptomatic carriers were identified in both herds by seroreactivity to FMDV non‐structural proteins and detection of FMDV genomic RNA in oropharyngeal fluid. Carriers’ seroreactivity and FMDV genome detection status were subsequently monitored monthly for 23 months. The mean extinction time of the carrier state was 13.1 ± 0.2 months, with extinction having occurred significantly faster amongst adult dairy cattle at Farm B compared to younger animals at Farm A. The rate of decrease in the proportion of carrier animals was calculated to be 0.07 per month. Seroprevalence against FMDV non‐structural proteins decreased over the course of the study period, but was found to increase transiently following repeated vaccinations. These data provide novel insights into viral and host factors associated with the FMDV carrier state under natural conditions. The findings reported herein may be relevant to field veterinarians and governmental regulatory entities engaged in FMD response and control measures.     

Safe and cost‐effective protocol for shipment of samples from Foot‐and‐Mouth Disease suspected cases for laboratory diagnostic

An essential step towards the global control and eradication of foot‐and‐mouth disease (FMD ) is the identification of circulating virus strains in endemic regions to implement adequate outbreak control measures. However, due to the high biological risk and the requirement for biological samples to be shipped frozen, the cost of shipping samples becomes one of major obstacles hindering submission of suspected samples to reference laboratories for virus identification. In this study, we report the development of a cost‐effective and safe method for shipment of FMD samples. The protocol is based on the inactivation of FMD virus (FMDV ) on lateral flow device (LFD , penside test routinely used in the field for rapid immunodetection of FMDV ), allowing its subsequent detection and typing by RT ‐PCR and recovery of live virus upon RNA transfection into permissive cells. After live FMDV collection onto LFD strip and soaking in 0.2% citric acid solution, the virus is totally inactivated. Viral RNA is still detectable by real‐time RT ‐PCR following inactivation, and the virus strain can be characterized by sequencing of the VP 1 coding region. In addition, live virus can be rescued by transfecting RNA extract from treated LFD into cells. This protocol should help promoting submission of FMD suspected samples to reference laboratories (by reducing the cost of sample shipping) and thus characterization of FMDV strains circulating in endemic regions.     

Safety profile of a replication‐deficient human adenovirus‐vectored foot‐and‐mouth disease virus serotype A24 subunit vaccine in cattle

The safety of a replication‐deficient, human adenovirus‐vectored foot‐and‐mouth disease virus (FMDV ) serotype A24 Cruzeiro capsid‐based subunit vaccine (AdtA24) was evaluated in five independent safety studies. The target animal safety studies were designed in compliance with United States (U.S.) regulatory requirements (Title 9, U.S. Code of Federal Regulation [9CFR ]) and international standard guidelines (VICH Topic GL ‐44) for veterinary live vaccines. The first three studies were conducted in a total of 22 vaccinees and demonstrated that the AdtA24 master seed virus (MSV ) was safe, did not revert to virulence and was not shed or spread from vaccinees to susceptible cattle or pigs. The fourth safety study conducted in 10 lactating cows using an AdtA24 vaccine serial showed that the vaccine was completely absent from milk. The fifth safety study was conducted under typical U.S. production field conditions in 500 healthy beef and dairy cattle using two AdtA24 vaccine serials. These results demonstrated that the vaccine was safe when used per the product label recommendations. Additional data collected during these five studies confirmed that AdtA24 vaccinees developed FMDV A24 and the HA d5 vaccine vector serum neutralization antibodies that test negative in a FMDV non‐structural protein antibody test, confirming AdtA24 vaccine's capability to differentiate infected from vaccinated animals (DIVA ). In conclusion, results from this comprehensive set of cattle studies demonstrated the safety of the replication‐deficient AdtA24 vaccine and fulfilled safety‐related requirements for U.S. regulatory requirements.