Isolation,identification and retrospective study of foot‐and‐mouth disease virus from affected Mithun (Bos frontalis) in north‐eastern India

Foot‐and‐mouth disease (FMD ) is a contagious disease of cloven‐hoofed animals that causes substantial and perpetual economic loss. Apart from the contagious nature of the disease, the FMD virus can establish in a “carrier state” among all cloven‐hoofed animals. The Mithun (Bos frontalis ), popularly called the “Cattle of Mountain,” is found in the geographically isolated, hilly region of north‐east India: Arunachal Pradesh, Nagaland, Manipur and Mizoram. Despite the geographical inaccessibility, infection by FMD virus has emerged as the single most devastating disease among Mithun after the eradication of rinderpest from this region. Samples from outbreaks of FMD in Mithun were analysed by sandwich ELISA , multiplex RT ‐PCR (MRT ‐PCR ) and liquid‐phase blocking enzyme‐linked immunosorbent assay and isolated in the BHK ‐21 cell line. The results indicate the presence of FMDV serotype “O.” The sequencing and molecular phylogenies have revealed close relationships in the lineage of type “O” isolates from Bangladesh. The findings will provide useful information for further research and development of a sustainable programme for the progressive control of FMD in the Mithun population.     

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.     

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.     

Development and evaluation of multiplex real‐time RT‐PCR assays for the detection and differentiation of foot‐and‐mouth disease virus and Seneca Valley virus 1

Foot‐and‐mouth disease virus (FMDV) causes a highly contagious and economically important vesicular disease in cloven‐hoofed animals that is clinically indistinguishable from symptoms caused by Seneca Valley virus 1 (SVV‐1). To differentiate SVV‐1 from FMDV infections, we developed a SVV‐1 real‐time RT‐PCR (RT‐qPCR) assay and multiplexed with published FMDV assays. Two published FMDV assays (Journal of the American Veterinary Medical Association, 220, 2002, 1636; Journal of Virological Methods, 236, 2016, 258) targeting the 3D polymerase (3D) region were selected and multiplexed with the SVV‐1 assay that has two targets, one in the 5′ untranslated region (5′ UTR, this study) and the other in the 3D region (Journal of Virological Methods, 239, 2017, 34). In silico analysis showed that the primers and probes of SVV‐1 assay matched 98.3% of the strain sequences (113/115). The primer and probe sequences of the Shi FMDV assay matched 85.4% (806/944), and that of the Callahan FMDV assay matched 62.7% (592/944) of the sequences. The limit of detection (LOD) for the two multiplex RT‐qPCR assays for SVV‐1 was both 9 copies per reaction by cloned positive plasmids and 0.16 TCID50 per reaction by cell culture. The LOD for FMDV by both multiplex assays was 11 copies per reaction using cloned positive plasmids. With cell cultures of the seven serotypes of FMDV, the Shi assay (Journal of Virological Methods, 236, 2016, 258) had LODs between 0.04 and 0.18 TCID50 per reaction that were either the same or lower than the Callahan assay. Interestingly, multiplexing with SVV‐1 increased the amplification efficiencies of the Callahan assay (Journal of the American Veterinary Medical Association, 220, 2002, 1636) from 51.5%–66.7% to 89.5%–96.6%. Both assays specifically detected the target viruses without cross‐reacting to SVV‐1 or to other common porcine viruses. An 18S rRNA housekeeping gene that was amplified from multiple cloven‐hoofed animal species was used as an internal control. The prevalence study did not detect any FMDV, but SVV‐1 was detected from multiple types of swine samples with an overall positive rate of 10.5% for non‐serum samples.     

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.     

Foot‐and‐Mouth Disease Virus Concentrations in Products of Animal Origin

Foot‐and‐mouth disease (FMD) is a highly contagious disease of cloven‐hoofed animals which can have devastating economic consequences. Maintaining an FMD‐free status is a priority for non‐endemic countries, which restrict importation of animals and animal products from countries in which the disease is present or sporadic, thus presenting a considerable barrier to international trade. This review examines the concentration of FMD virus in animal tissues during the viraemic stage of disease and in animal products derived from infected animals.     

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.     

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.     

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.     

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.     

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.     

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.     

Options for Decentralized Testing of Suspected Secondary Outbreaks of Foot‐and‐mouth Disease

This article reviews the options for use of virus detection techniques for decentralized testing of samples from suspected secondary outbreaks of foot‐and‐mouth disease (FMD). These options have been expanded by the advent of new tests including disposable lateral flow devices (LFDs) that detect viral proteins and portable RT‐PCR equipment that detects viral RNA. LFDs have been developed with similar sensitivity to antigen detection ELISA but with the ability to provide a result 1–30 min after the addition of epithelium or vesicular fluid. Portable RT‐PCR platforms are being developed that can detect FMD viral RNA in blood, epithelium or other materials with minimal sample processing and with high sensitivity, in as little as 60 min in some cases. These devices may be used on infected farms as pen‐side tests, in regional, local or mobile laboratories, or in National Reference Laboratories (NRL). Advantages and disadvantages of different testing options are considered to inform decisions on the optimal strategies for different national circumstances. Issues include validation and quality control, containment needs, availability of test devices and reagents, the decision tree for declaring an outbreak, training issues and provision of samples for subsequent viral characterization. Tests to confirm the diagnosis of the index case of an outbreak of FMD should continue to be carried out in the NRL.     

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.     

Horizontal transmission of foot‐and‐mouth disease virus O/JPN/2010 among different animal species by direct contact

Foot‐and‐mouth disease (FMD) is highly contagious and easily transmitted among species of cloven‐hoofed animals. To investigate the transmission of FMD virus (FMDV) among different animal species, experimental infections using the O/JPN/2010 strain were performed in cows, goats and pigs. One cow or two goats/pigs were housed with a different species of inoculated animals, and clinical observations, virus shedding and antibody responses were analysed daily. Whilst all cows and goats were infected horizontally by contact with inoculated pigs, transmission from cows to goats/pigs and from goats to cows/pigs was not observed in all in‐contact animals. In particular, no pigs were infected horizontally by contact with inoculated goats. Comparison with our previous study on experimental infections among animals of the same species indicates that horizontal transmission occurred more easily between animals of the same species than between those of the different species. These findings will be useful for establishing and performing species‐specific countermeasures in farms and regions where multiple species of animals coexist in potential future outbreaks.     

Genetic Characterization of Serotypes A and Asia‐1 Foot‐and‐mouth Disease Viruses in Balochistan,Pakistan, in 2011

This study reports characterization of foot‐and‐mouth disease virus (FMDV) in samples collected from Balochistan, Pakistan. FMDV was detected by pan‐FMDV real‐time RT‐PCR in 31 samples (epithelial and oral swabs) collected in 2011 from clinical suspect cases. Of these, 29 samples were serotyped by serotype‐specific real‐time RT‐PCR assays and were confirmed by sequencing the VP1 coding region. Sixteen samples were found positive for serotype A and eight for serotype Asia‐1, whereas five samples were found positive for both serotypes A and Asia‐1. Two serotype A positive samples were found positive for two different strains of serotype A FMDV each. Phylogenetic analyses of serotype A FMDVs showed circulation of at least three different sublineages within the A‐Iran05 lineage. These included two earlier reported sublineages, A‐Iran05^HER−10 and A‐Iran05^FAR−11, and a new sublineage, designated here as A‐Iran05^BAL−11. This shows that viruses belonging to the A‐Iran05 lineage are continuously evolving in the region. Viruses belonging to the A‐Iran05^FAR−11 sublineage showed close identity with the viruses circulating in 2009 in Pakistan and Afghanistan. However, viruses belonging to the A‐Iran05^HER−10 detected in Balochistan, Pakistan, showed close identity with the viruses circulating in Kyrgyzstan, Iran and Kazakhstan in 2011 and 2012, showing that viruses responsible for outbreak in these countries have a common origin. Serotype Asia‐1 FMDVs reported in this study all belonged to the earlier reported Group‐VII (Sindh‐08), which is currently a dominant strain in the West Eurasian region. Detection of two different serotypes of FMDV or/and two different strains of the same serotype in one animal/sample shows complexity in occurrence of FMD in the region.