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.     

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.     

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.     

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.     

Outbreak investigations and molecular characterization of foot‐and‐mouth disease viruses circulating in south‐west Niger

In Niger, the epidemiological situation regarding foot‐and‐mouth disease is unclear as many outbreaks are unreported. This study aimed (i) to identify Foot‐and‐mouth disease virus (FMDV ) strains currently circulating in cattle herds, and (ii) to identify risk factors associated with Foot‐and‐mouth disease (FMD )‐seropositive animals in clinical outbreaks. Epithelial tissues (n  = 25) and sera (n  = 227) were collected from cattle in eight districts of the south‐western part of Niger. Testing of clinical material revealed the presence of FMDV serotype O that was characterized within the O/WEST AFRICA topotype. The antigenic relationship between one of the FMDV isolates from Niger (O/NGR /4/2015) and three reference vaccine strains was determined by the two‐dimensional virus neutralization test (2dmVNT ), revealing a close antigenic match between the field isolate from Niger and three FMDV serotype O vaccine strains. Serological analyses using a non‐structural protein (NSP ) test provided evidence for previous FMDV infection in 70% (158/227) of the sera tested. Multivariate logistic regression analysis revealed that only the herd composition (presence of both cattle and small ruminants) was significantly associated with FMDV seropositivity as defined by NSP ‐positive results (p ‐value = .006). Of these positive sera, subsequent testing by liquid‐phase blocking ELISA (LPBE ) showed that 86% (136/158) were positive for one (or more) of four FMDV serotypes (A, O, Southern African Territories (SAT ) 1 and SAT 2). This study provides epidemiological information about FMD in the south‐western part of Niger and highlights the complex transboundary nature of FMD in Africa. These findings may help to develop effective control and preventive strategies for FMD in Niger as well, as other countries in West Africa.     

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.     

Foot‐and‐Mouth Disease and Its Effect on Milk Yield: An Economic Analysis on Livestock Holders in Pakistan

A longitudinal study has been conducted in the provinces of Sindh, Punjab and Islamabad Capital Territory area, Pakistan, to evaluate the impact of foot‐and‐mouth disease on milk yield in a sample of farmers owning cattle and buffaloes. The sample consisted of 50 farms where the presence of foot‐and‐mouth disease (FMD) virus was initially suspected on the basis of clinical signs and subsequently confirmed through either a field test or laboratory confirmation. In each farm, the total number of clinical cases was registered, and clinically diseased milking cattle and buffaloes were followed up for the next 60 days from the onset of clinical signs and the amount of milk yield measured. The average milk yield, estimated to be around 10 l per animal before the onset of FMD, decreased significantly in the 2 months following the onset of acute clinical disease. The loss of milk production in the 60 days following the onset of clinical signs was estimated to be around 220 and 201 l for cattle and buffaloes, respectively. Under the assumption that the administration of a good‐quality vaccine matching circulating FMD strains could protect against clinical disease, the benefit/cost ratio for having all animals vaccinated in all 50 farms was estimated to be 5.7.     

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.     

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 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.     

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 .     

Diagnosis and Control Measures of the 2010 Outbreak of Foot‐and‐Mouth Disease A Type in the Republic of Korea

In January 2010, foot‐and‐mouth disease (FMD) occurred for the first time in 8 years in Korea. The outbreaks were because of A serotype, different from the O type, which had occurred previously in 2000 and 2002. The FMD outbreaks were identified in seven farms, consisting of six cattle farms where viruses were detected and one deer farm where only FMDV antibody was detected. The seven farms were within 9.3 km of each other. All susceptible animals within 10 km radius of the outbreak farms were placed under movement restrictions for 3–11 weeks. No vaccination took place to facilitate the clinical observation of infected animals and virus detection. After clinical observations and serological tests within the control zones showed no evidence of FMD infection, the movement restrictions were lifted, followed by FMD‐free declaration (23 March) at 80 days after the first outbreak on 2 January. This communication describes the outbreak of FMD A serotype, and control measures applied to eradicate the disease in Korea.     

Absence of Heat Intolerance (Panting) Syndrome in Foot‐and‐Mouth Disease‐Affected Indian Cattle (Bos indicus) is Associated with Intact Thyroid Gland Function

Foot‐and‐mouth disease (FMD) is a highly contagious and economically important viral disease with high morbidity and reduced productivity of affected animals. We studied the heat intolerance (HI) (panting) syndrome and the effect of FMD virus (FMDV) infection on thyroid gland function in Indian cattle (Bos indicus). Experimental infection with FMDV Asia 1 resulted in a mild form of disease with superficial lesions. Heat intolerance syndrome and its signs were not observed among the recovered animals. Subtle changes in the serum level of thyroid hormones, triiodothyronine (T₃) and thyroxine (T₄) were observed. However, there were no distinct histological changes in the thyroid gland, and FMDV antigens were not detected in the thyroid tissues. Our results thus suggest that the absence of panting syndrome in FMD‐affected Bos indicus cattle may be associated with intact thyroid gland function.     

Heterogeneity in the Antibody Response to Foot‐and‐Mouth Disease Primo‐vaccinated Calves

Foot‐and‐mouth disease (FMD) vaccines are routinely used as effective control tools in large regions worldwide and to limit outbreaks during epidemics. Vaccine‐induced protection in cattle has been largely correlated with the FMD virus (FMDV)‐specific antibodies. Genetic control of cattle immune adaptive responses has been demonstrated only for peptide antigens derived from FMDV structural proteins. Here, we quantify the heterogeneity in the antibody response of cattle primo‐vaccinated against FMD and study its association with the genetic background in Holstein and Jersey sires. A total of 377 FMDV‐seronegative calves (122 and 255 calves from 16 and 15 Holstein and Jersey sires, respectively) were included in the study. Samples were taken the day prior to primo‐vaccination and 45 days post‐vaccination (dpv). Animals received commercial tetravalent FMD single emulsion oil vaccines formulated with inactivated FMDV. Total FMDV‐specific antibody responses were studied against three viral strains included in the vaccine, and antibody titres were determined by liquid‐phase blocking ELISA. Three linear hierarchical mixed regression models, one for each strain, were formulated to assess the heterogeneity in the immune responses to vaccination. The dependent variables were the antibody titres induced against each FMDV strain at 45 dpv, whereas sire's ‘breed’ was included as a fixed effect, ‘sire’ was included as a random effect, and ‘farm’ was considered as a hierarchical factor to account for lack of independence of within herd measurements. A significant association was found between anti‐FMDV antibody responses and sire's breed, with lower immune responses found in the Jersey sires’ offspring compared with those from Holstein sires. No significant intrabreed variation was detected. In addition, farm management practices were similar in this study, and results of the serological assays were shown to be repeatable. It therefore seems plausible that differences in the immune response may be expected in the event of a mass vaccination campaigns.     

Collection of Oral Fluids Using Cotton Ropes as a Sampling Method to Detect Foot‐and‐Mouth Disease Virus Infection in Pigs

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

Challenges for Serology‐Based Characterization of Foot‐and‐Mouth Disease Outbreaks in Endemic Areas; Identification of Two Separate Lineages of Serotype O FMDV in Uganda in 2011

Control of foot‐and‐mouth disease (FMD) in Uganda by ring vaccination largely depends on costly trivalent vaccines, and use of monovalent vaccines could improve the cost effectiveness. This, however, requires application of highly specific diagnostic tests. This study investigated outbreaks of FMD in seven Ugandan districts, during 2011, using the PrioCHECK^® FMDV NS ELISA, solid‐phase blocking ELISAs (SPBEs) and virus neutralization tests (VNTs), together with virological analyses for characterization of the responsible viruses. Two hundred and eighteen (218) cattle and 23 goat sera as well as 82 oropharyngeal fluid/epithelial tissue samples were collected. Some 50% of the cattle and 17% of the goat sera were positive by the PrioCHECK^® FMDV NS ELISA, while SPBEs identified titres ≥80 for antibodies against serotype O FMD virus (FMDV) in 51% of the anti‐NSP positive cattle sera. However, 35% of the anti‐NSP positive cattle sera had SPBE titres ≥80 against multiple serotypes, primarily against serotypes O, SAT 1 and SAT 3. Comparison of SPBEs and VNTs for the detection of antibodies against serotypes O, SAT 1 and SAT 3 in 72 NSP positive cattle sera showed comparable results against serotype O (P = 0.181), while VNTs detected significantly fewer samples positive for antibodies against SAT 1 and SAT 3 than the SPBEs (P < 0.001). Detection of antibodies against serotype O was consistent with the isolation of serotype O FMDVs from 13 samples. Four of these viruses were sequenced and belonged to two distinct lineages within the East Africa‐2 (EA‐2) topotype, each differing from the currently used vaccine strain (EA‐1 topotype). The relationships of these lineages to other serotype O viruses in the Eastern Africa region are discussed. To enhance the control of FMD in Uganda, there is need to improve the specificity of the SAT‐SPBEs, perform vaccine matching and implement improved regional FMD control.     

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.     

Development of a chemiluminescence immunoassay using recombinant non‐structural epitope‐based proteins to accurately differentiate foot‐and‐mouth disease virus‐infected and vaccinated bovines

The contamination of inactivated vaccine with non‐structural proteins (NSP s) leads to a high false‐positive rate, which is a substantial barrier to accurately differentiate foot‐and‐mouth disease virus (FMDV )‐infected animals from vaccinated animals. To address this problem, a new chemiluminescence immunoassay (CLIA ) method was developed to detect antibodies targeting the two recombinant epitope‐based proteins located in 3A and 3B. The 3Aepitp‐3Bepitp CLIA exhibited a diagnostic sensitivity of 94.0% and a diagnostic specificity of 97.5% for the detection of serum samples (naïve bovines, n  = 52, vaccinated bovines, n  = 422, infected bovines, n  = 116) from animals with known status. The CLIA method also had a concordance rate of 88.1% with the PrioCHECK FMDV NSP ELISA based on the detection of 270 serum samples from the field. Importantly, the 3Aepitp‐3Bepitp CLIA produced no false‐positives when used to detect FMDV in samples from bovines that had been vaccinated up to five times, and it was demonstrated a low false‐positive rate when the bovines had been vaccinated up to ten (2.15%) and fifteen times (5.93%). Therefore, the 3Aepitp‐3Bepitp CLIA detects FMDV in samples from frequently vaccinated bovines with high accuracy and represents an alternative method to differentiate FMDV ‐infected and vaccinated bovines.