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.     

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.     

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.     

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.     

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 .     

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.     

Detection of Capripoxvirus DNA Using a Field‐Ready Nucleic Acid Extraction and Real‐Time PCR Platform

Capripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real‐time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory‐based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log₁₀ more sensitive than the laboratory‐based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory‐based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings.     

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.     

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.     

Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot‐and‐mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user‐developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user‐intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT‐PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn‐around time of approximately 6 hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross‐reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.     

Generation of monoclonal antibodies against foot‐and‐mouth disease virus SAT 2 and the development of a lateral flow strip test for virus detection

Foot‐and‐mouth disease (FMD) remains a major economic concern for the livestock productivity in many developing countries and a continued threat to countries that are disease free because of its potential devastating impact on agricultural, food chain and tourism sectors. FMD virus (FMDV) is recognized as having seven serotypes: O, A, C, Asia 1, South African Territories (SAT) 1, 2, 3 and multiple subtypes within each serotype. FMD outbreaks due to SAT 2 have been reported in many African countries. The development of a rapid and easily performed test for FMD detection is critical for controlling FMD outbreaks and containing its spread. The present project developed a lateral flow immunochromatographic (LFI) strip test for the rapid detection of FMDV SAT 2. A panel of monoclonal antibodies (mAbs) against FMDV serotype SAT 2 was produced and characterized. One mAb (#10) was selected as the capture mAb because it reacted to all 23 SAT 2 isolates archived at the National Center for Foreign Animal Disease. The LFI strip test was developed using biotin‐conjugated mAb #10, and the colloid gold‐conjugated FMDV serotype‐independent mAb as the detection mAb. A generic Rapid Assay Device (gRAD) with one test line and a control line was used for the test. The LFI strip test detected all 23 tested SAT 2 isolates and recent outbreak strains. The results indicated that the diagnostic specificity and sensitivity of the LFI strip test were greater than the double antibody sandwich (DAS) DAS ELISA. The ability of the LFI strip test to produce rapid diagnostic results will be useful for early on‐site diagnosis during FMD outbreaks.     

Emergence and Distribution of Foot‐and‐Mouth Disease Virus Serotype A and O in Bangladesh

Foot‐and‐mouth disease (FMD) is endemic in Bangladesh and is predominantly due to FMDV serotype O. In 2012, FMD outbreaks were identified in five different districts of Bangladesh. Of 56 symptomatic cattle epithelial tissue samples, diagnostic PCR assay based on 5′‐URT detected 38 FMDV infections. Viral genotyping targeting VP1‐encoding region confirmed emergence of two distinct serotypes, A and O with an abundance of serotype A in Chittagong and Gazipur districts and serotype O in Pabna and Faridpur. Only single lineage of both A and O was retrieved from samples of five different regions. Sequencing and phylogenetic analysis of VP1 sequences revealed that serotype O sequences were closely related to the Ind 2001 sublineage of Middle East–South Asia (ME‐SA) topotype that was previously circulating in Bangladesh, and serotype A sequences belonging to the genotype VII that was dominant in India during the last decade. The results suggest that extensive cross‐border animal movement from neighbouring countries is the most likely source of FMDV serotypes in Bangladesh.