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.     

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.     

Serological and molecular epidemiology of foot‐and‐mouth disease viruses in agro‐pastoralist livestock herds in the kachia grazing reserve,Nigeria

The Kachia Grazing Reserve (KGR) is located in Kaduna state in north‐western Nigeria and consists of 6 contiguous blocks housing 744 defined households (HH), all engaged in livestock keeping. It is considered as a homogenous epidemiological unit and a defined study area. In 2012, all cattle and sheep of 40 selected HH were sampled to determine sero‐prevalence of antibodies to foot‐and‐mouth disease virus (FMDV) and of FMDV. The overall sero‐prevalence of antibodies to the non‐structural 3ABC protein (NSP‐3ABC ELISA) was 28.9% (380/1,315) (30.6% cattle; 16.3% sheep), and in 4.5% (62/1,380) (5% cattle; 0.6% sheep) of the examined sera FMD viral RNA could be detected by real‐time RT‐PCR (rRT‐PCR). Additionally, in 2012 and 2014 serum, epithelium and probang samples were collected from cattle in reported FMD outbreaks and the causative FMDVs were molecularly characterized. Approximately half (28/59) of the outbreak sera reacted positive in NSP‐3ABC ELISA, and 88% (52/59) of the outbreak sera contained detectable viral RNA. Overall, antibodies against five FMDV serotypes (O, A, SAT1, SAT2 and SAT3) were detected by solid phase competitive ELISA with combinations of two or more serotypes being common. Of the 21 FMDVs that could be isolated 19 were sequenced and 18 were confirmed as SAT2 (lineage VII) while one was characterized as serotype O (EA‐3 topotype). Phylogenetic analysis revealed a close relationship between Nigerian FMDV strains and strains in this region and even with strains in North‐Africa. Our findings indicate that FMD constitutes an endemic health problem to cattle rearing in the agro‐pastoralist community in the KGR and that the KGR is not a closed epidemiological unit. Insight into the local FMDV epidemiology and in the circulating FMDV serotypes/strains is of support to the relevant authorities in Nigeria when considering the need for an FMD control policy to improve animal production in grazing reserves.     

Rapid and simple detection of foot‐and‐mouth disease virus: Evaluation of a cartridge‐based molecular detection system for use in basic laboratories

Highly contagious transboundary animal diseases such as foot‐and‐mouth disease (FMD ) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen‐side. Here, we report on a study to evaluate a fully automated cartridge‐based real‐time RT ‐PCR diagnostic system (Enigma MiniLab^®) for the detection of FMD virus (FMDV ). The modular system integrates both nucleic acid extraction and downstream real‐time RT ‐PCR (rRT ‐PCR ). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV , and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter‐laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge‐based automated real‐time RT ‐PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.     

Molecular Characterisation of Foot‐and‐Mouth Disease Viruses from Pakistan, 2005–2008

Foot‐and‐mouth disease (FMD), an economically important disease of cloven‐hoofed animals, is endemic in Pakistan where three virus serotypes are present (O, A and Asia 1). Fifty‐eight clinical samples collected between 2005 and 2008 from animals with suspected FMD in various locations in Pakistan were subjected to virus isolation on primary cell culture, antigen ELISA and real‐time RT‐PCR (rRT‐PCR). Viruses were isolated from 32 of these samples and identified as FMDV type O (n = 31) or type A (n = 1). Foot‐and‐mouth disease virus (FMDV) genome was detected in a further 11 samples by real‐time RT‐PCR. Phylogenetic analyses of the VP1 nucleotide sequences showed that all of the type O viruses belonged to the MIDDLE EAST–SOUTH ASIA topotype with the majority belonging to the PanAsia‐2 lineage; a single example of the older PanAsia lineage was identified. The single FMDV type A virus belonged to the ASIA topotype, but did not cluster with known strains that are currently circulating (such as Iran‐05) and was not closely related to other type A viruses from the region. These findings demonstrate the widespread distribution of O‐PanAsia‐2 in Pakistan and the presence of undisclosed novel type A lineages in the region.     

Genetic diversity and comparison of diagnostic tests for characterization of foot‐and‐mouth disease virus strains from Pakistan 2008–2012

We report the laboratory analysis of 125 clinical samples from suspected cases of foot‐and‐mouth disease (FMD ) in cattle and Asian buffalo collected in Pakistan between 2008 and 2012. Of these samples, 89 were found to contain viral RNA by rRT ‐PCR , of which 88 were also found to contain infectious FMD virus (FMDV ) by virus isolation (VI ), with strong correlation between these tests (κ = 0.96). Samples that were VI ‐positive were serotyped by antigen detection ELISA (Ag‐ELISA ) and VP 1 sequence acquisition and analysis. Sequence data identified FMDV serotypes A (n  = 13), O (n  = 36) and Asia‐1 (n  = 41), including three samples from which both serotypes Asia‐1 and O were detected. Serotype A viruses were classified within three different Iran‐05 sublineages: HER ‐10, FAR ‐11 and ESF ‐10. All serotype Asia‐1 were within Group VII (Sindh‐08 lineage), in a genetic clade that differs from viruses isolated prior to 2010. All serotypes O were classified as PanAsia‐2 within two different sublineages: ANT ‐10 and BAL ‐09. Using VP 1 sequencing as the gold standard for serotype determination, the overall sensitivity of Ag‐ELISA to correctly determine serotype was 74%, and serotype‐specific sensitivity was 8% for serotype A, 88% for Asia‐1 and 89% for O. Serotype‐specific specificity was 100% for serotype A, 93% for Asia‐1 and 94% for O. Interestingly, 12 of 13 serotype A viruses were not detected by Ag‐ELISA . This study confirms earlier accounts of regional genetic diversity of FMDV in Pakistan and highlights the importance of continued validation of diagnostic tests for rapidly evolving pathogens such as FMDV .     

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.     

Serological Evidence Indicates that Foot‐and‐Mouth Disease Virus Serotype O,C and SAT1 are most Dominant in Eritrea

Foot‐and‐mouth disease (FMD) is endemic in Eritrea and in most parts of Africa. To be able to control FMD using vaccination, information on the occurrence of various foot‐and‐mouth disease serotypes in Eritrea is needed. In this cross‐sectional study, 212 sera samples were collected from FMD infected and recovered animals in Eritrea. These samples were tested for the presence of antibodies against FMD non‐structural proteins (NSP) and neutralizing antibodies against six of the seven (all but SAT 3) serotypes of FMD virus (FMDV). Of these, 67.0% tested positive to non‐structural protein antibodies in the FMD NS ELISA. By virus neutralization, FMDV serotype O antibodies were shown to be the most dominant (approximately 50%). Virus neutralization test results indicate that infection with serotype C and SAT 1 might have occurred, although there are no reports of isolation of these two serotypes. Because the samples were not randomly selected, further random serological surveillance in all age group animals is necessary both to estimate the prevalence of FMD in the country and to confirm the serological results with serotype C and SAT 1.     

A Survey on the Frequency of Foot‐and‐Mouth Disease Virus Carriers in Cattle in North–East of Iran by RT‐PCR: Implications for Revising Disease Control Strategy

Foot‐and‐mouth disease (FMD) is endemic in Iran. It is essential to timely evaluate the current disease control programme in Iran. Here, we report the frequency of FMD virus (FMDV) carrier state in cattle slaughtered in Mashhad abattoir, Mashhad, Khorasan Razavi, north–east of Iran, which contains long common borders with Afghanistan and Turkmenistan. Soft palate samples were collected immediately after slaughter for the detection of FMDV by RT‐PCR. The results show that 37.7% of cattle (96 of 255) were carriers of the virus. Among positive samples (96), 58 (60.4%) belonged to serotype O. No evidence was detected for the presence of Asia 1 and A serotypes. Nucleotide sequencing and phylogenic dendogram showed close similarity and common lineage between our samples and viruses isolated in Pakistan. With an approximate more than 80% of cattle population vaccination coverage such a high rate of carrier state may show an extensive FMDV exposure. Therefore, limiting control programmes to timely prophylactic vaccination may be insufficient. This is also true when meat market instabilities act as a temptation to import livestock, legally or illegally, through the eastern frontiers. It is recommended to change the current prophylactic vaccination strategy to a well‐developed regional control programme, with close monitoring of animal movement through eastern frontiers, supported by government commitment and educational programmes. Timely estimation of the frequency of carrier state both in cattle and small ruminants is also advocated as a gauge to monitor the virus status in the region.     

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.     

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.     

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.     

Foot‐and‐Mouth Disease in Feral Swine: Susceptibility and Transmission

Experimental studies of foot‐and‐mouth disease (FMD) in feral swine are limited, and data for clinical manifestations and disease transmissibility are lacking. In this report, feral and domestic swine were experimentally infected with FMDV (A24‐Cruzeiro), and susceptibility and virus transmission were studied. Feral swine were proved to be highly susceptible to A‐24 Cruzeiro FMD virus by intradermal inoculation and by contact with infected domestic and feral swine. Typical clinical signs in feral swine included transient fever, lameness and vesicular lesions in the coronary bands, heel bulbs, tip of the tongue and snout. Domestic swine exhibited clinical signs of the disease within 24 h after contact with feral swine, whereas feral swine did not show clinical signs of FMD until 48 h after contact with infected domestic and feral swine. Clinical scores of feral and domestic swine were comparable. However, feral swine exhibited a higher tolerance for the disease, and their thicker, darker skin made vesicular lesions difficult to detect. Virus titration of oral swabs showed that both feral and domestic swine shed similar amounts of virus, with levels peaking between 2 to 4 dpi/dpc (days post‐inoculation/days post‐contact). FMDV RNA was intermittently detectable in the oral swabs by real‐time RT‐PCR of both feral and domestic swine between 1 and 8 dpi/dpc and in some instances until 14 dpi/12 dpc. Both feral and domestic swine seroconverted 6–8 dpi/dpc as measured by 3ABC antibody ELISA and VIAA assays. FMDV RNA levels in animal room air filters were similar in feral and domestic swine animal rooms, and were last detected at 22 dpi, while none were detectable at 28 or 35 dpi. The FMDV RNA persisted in domestic and feral swine tonsils up to 33–36 dpi/dpc, whereas virus isolation was negative. Results from this study will help understand the role feral swine may play in sustaining an FMD outbreak, and may be utilized in guiding surveillance, epidemiologic and economic models.     

Detection and Molecular Characterization of Foot and Mouth Disease Viruses from Outbreaks in Some States of Northern Nigeria 2013–2015

Control measures for foot and mouth disease (FMD) in Nigeria have not been implemented due to the absence of locally produced vaccines and risk‐based analysis resulting from insufficient data on the circulating FMD virus (FMDV) serotypes/strains. In 2013‐2015, blood and epithelial samples were collected from reported FMD outbreaks in four states (Kaduna, Kwara, Plateau and Bauchi) in northern Nigeria. FMDV non‐structural protein (NSP) seroprevalence for the outbreaks was estimated at 80% (72 of 90) and 70% (131 of 188) post‐outbreak. Antibodies against FMDV serotypes O, A, SAT1, SAT2 and SAT3 were detected across the states using solid‐phase competitive ELISA. FMDV genome was detected in 99% (73 of 74) of the samples from FMD‐affected animals using rRT‐PCR, and cytopathic effect was found in cell culture by 59% (44 of 74) of these samples. Three FMDV serotypes O, A and SAT2 were isolated and characterized. The phylogenetic assessments of the virus isolates showed that two topotypes of FMDV serotype O, East Africa‐3 (EA‐3) and West Africa (WA) topotypes were circulating, as well as FMDV strains belonging to the Africa genotype (G‐IV) of serotype A and FMDV SAT2 topotype VII strains. While the serotype O (EA‐3) strains from Nigeria were most closely related to a 1999 virus strain from Sudan, the WA strain in Nigeria shares genetic relationship with three 1988 viruses in Niger. The FMDV serotype A strains were closely related to a known virus from Cameroon, and the SAT2 strains were most closely related to virus subtypes in Libya. This study provides evidence of co‐occurrence of FMDV serotypes and topotypes in West, Central, East and North Africa, and this has implication for control. The findings help filling the knowledge gap of FMDV dynamics in Nigeria and West Africa subregion to support local and regional development of vaccination‐based control plans and international risk assessment.