The use of milk for the surveillance of foot-and-mouth disease

Effective surveillance of foot-and-mouth disease (FMD) is of the utmost importance in order to understand the disease risks and implement effective control strategies. Epidemiological data obtained for FMD is mostly obtained through recognition and reporting of clinical cases by farmers which has several limitations. For example, under-reporting of disease is common, due to deficiencies in veterinary infrastructure, the effort involved for sample collection, or the repercussions of control measures for farmers. Diagnostic sample types, usually vesicular epithelium and fluid, are invasive and labour intensive to obtain, and can only be collected from acutely infected animals. Therefore, animals with sub-clinical FMD infection (particularly those in vaccinated herds) may not be identified but may still contribute to disease transmission. It is likely, therefore, that the true prevalence of FMD is not accurately known in parts of the world where the disease is endemic. Consequently, the requirement exists for a simple approach for FMD surveillance that does not rely on farmer reporting. Milk is a non-invasive sample type routinely collected from dairy farms and has been utilised for the surveillance of a number of other diseases. Despite numerous publications suggesting the potential of milk as a valuable sample type for foot-and-mouth disease (FMD) surveillance, empirical studies have mainly focused on the risk of transmission via milk, or the detection of FMD virus (FMDV) in milk from individual animals. This thesis aimed to expand on previous studies to determine the utility of milk and its limitations for the surveillance of FMD at the individual and herd level, using data collected from experimental and field studies. A highly sensitive and specific high-throughput RNA extraction and real-time rRT-PCR was optimised and utilised for FMDV RNA genome identification throughout the project. Using this method, it was demonstrated that FMDV RNA genome could be detected up to 28 days post infection using milk samples collected from individual cows. Further analysis using serotyping or lineage-specific rRT-PCR assays and VP1 sequencing of milk samples collected from individual cows in northern Tanzania highlighted the use of milk as a suitable alternative to more invasive sample types such as epithelium. Additionally, storage and shipment condition simulations performed demonstrated good stability of FMDV RNA genome within milk samples. Following these experiments, the potential use of pooled milk for herd–level FMD surveillance was investigated. Two proof-of-concept pilot studies were performed comparing the rRT-PCR results of pooled milk samples collected from both a large-scale dairy farm in Saudi Arabia and milk pooling facilities supplied by smallholder dairy farms in Kenya, with farmer reports of clinical disease. Results supported laboratory limit of detection studies, demonstrating that FMDV could be detected from milk pools of up 10,000 litres, even when there were low numbers of clinical cases. Furthermore, both studies suggested the detection of subclinical infection in milk samples, where disease was not reported. Data from pilot studies performed in this thesis therefore support the use of milk as a simple, non-invasive approach for herd-level FMD surveillance. Further field studies are required to determine the full utility of this method before it may be implemented for targeted/risk-based surveillance alongside existing surveillance systems to facilitate improved knowledge of FMD epidemiology, or for use in FMD contingency plans

The development of two field‐ready reverse transcription loop‐mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1

Seneca Valley virus 1 (SVV‐1) has been associated with vesicular disease in swine, with clinical signs indistinguishable from those of other notifiable vesicular diseases such as foot‐and‐mouth disease. Rapid and accurate detection of SVV‐1 is central to confirm the disease causing agent, and to initiate the implementation of control processes. The development of rapid, cost‐effective diagnostic assays that can be used at the point of sample collection has been identified as a gap in preparedness for the control of SVV‐1. This study describes the development and bench validation of two reverse transcription loop‐mediated amplification (RT‐LAMP) assays targeting the 5′‐untranslated region (5′‐UTR) and the VP3‐1 region for the detection of SVV‐1 that may be performed at the point of sample collection. Both assays were able to demonstrate amplification of all neat samples diluted 1/100 in negative pig epithelium tissue suspension within 8 min, when RNA was extracted prior to the RT‐LAMP assay, and no amplification was observed for the other viruses tested. Simple sample preparation methods using lyophilized reagents were investigated, to negate the requirement for RNA extraction. Only a small delay in the time to amplification was observed for these lyophilized reagents, with a time from sample receipt to amplification achieved within 12 min. Although diagnostic validation is recommended, these RT‐LAMP assays are highly sensitive and specific, with the potential to be a useful tool in the rapid diagnosis of SVV‐1 in the field

Foot-and-mouth disease surveillance using pooled milk on a large-scale dairy farm in an endemic setting

Pooled milk is used for the surveillance of several diseases of livestock. Previous studies demonstrated the detection of foot-and-mouth disease virus (FMDV) in the milk of infected animals at high dilutions, and consequently, the collection of pooled milk samples could be used to enhance FMD surveillance. This study evaluated pooled milk for FMDV surveillance on a large-scale dairy farm that experienced two FMD outbreaks caused by the A/ASIA/G-VII and O/ME-SA/Ind-2001d lineages, despite regular vaccination and strict biosecurity practices. FMDV RNA was detected in 42 (5.7%) of the 732 pooled milk samples, and typing information was concordant with diagnostic reports of clinical disease. The FMDV positive milk samples were temporally clustered around reports of new clinical cases, but with a wider distribution. For further investigation, a model was established to predict real-time RT-PCR (rRT-PCR) C values using individual cattle movement data, clinical disease records and virus excretion data from previous experimental studies. The model explained some of the instances where there were positive results by rRT-PCR, but no new clinical cases and suggested that subclinical infection occurred during the study period. Further studies are required to investigate the effect of vaccination on FMDV excretion in milk, and to evaluate more representative sampling methods. However, the results from this pilot study indicate that testing pooled milk by rRT-PCR may be valuable for FMD surveillance and has provided evidence of subclinical virus infection in vaccinated herds that could be important in the epidemiology of FMD in endemic countries where vaccination is used. [Abstract copyright: Copyright © 2020 Armson, Gubbins, Mioulet, Qasim, King and Lyons.

Knowledge and risk factors for foot-and-mouth disease among small-scale dairy farmers in an endemic setting

Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven-hoofed animals. In Kenya, the disease is endemic with outbreaks typically occurring throughout the year. A cross-sectional study was undertaken in Nakuru County to investigate farmer knowledge and risk factors for clinical disease. Semi-structured interviews were conducted on 220 smallholder farmers, selected using random spatial sampling. The majority of respondents (207/220 [94.1%]) knew of FMD and 166/207 (80.2%) of them could correctly identify the disease based on their knowledge of the clinical signs. Forty-five out of 220 farmers (20.4%) vaccinated their livestock against FMD in the previous 6 months, although of those who knew of FMD only 96/207 (46.4%) perceived it as a preventive measure undertaken to reduce the risk of disease in their farm. FMD had occurred in 5.9% of the surveyed farms within the previous 6 months (from May to November 2016). Using multivariate analysis, the use of a shared bull (OR = 9.7; p = 0.014) and the number of sheep owned (for each additional sheep owned OR = 1.1; p = 0.066) were associated with an increased likelihood of a farm experiencing a case of FMD in the previous 6 months, although the evidence for the latter was weak. This study reports risk factors associated with clinical FMD at the farm level in a densely populated smallholder farming area of Kenya. These results can be used to inform the development of risk-based strategic plans for FMD control and as a baseline for evaluating interventions and control strategies

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance

This study aimed to evaluate the utility of milk as a non-invasive sample type for the surveillance of foot-and-mouth disease (FMD), a highly contagious viral disease of cloven-hooved animals. Four milking Jersey cows were infected via direct-contact with two non-milking Jersey cows that had been previously inoculated with FMD virus (FMDV: isolate O/UKG/34/2001). Milk and blood were collected throughout the course of infection to compare two high-throughput real-time reverse transcription polymerase chain reaction (rRT-PCR) protocols with different RT-PCR chemistries. Using both methods, FMDV was detected in milk by rRT-PCR one to two days before the presentation of characteristic foot lesions, similar to detection by virus isolation. Furthermore, rRT-PCR detection from milk was extended, up to 28 days post contact (dpc), compared to detection by virus isolation (up to 14 dpc). Additionally, the detection of FMDV in milk by rRT-PCR was possible for 18 days longer than detection by the same method in serum samples. FMDV was also detected with both rRT-PCR methods in milk samples collected during the UK 2007 outbreak. Dilution studies were undertaken using milk from the field and experimentally-infected animals, where for one sample it was possible to detect FMDV at 10 . Based on the peak C values detected in this study, these findings indicate that it could be possible to identify one acutely-infected milking cow in a typical-sized dairy herd (100-1000 individuals) using milk from bulk tanks or milk tankers. These results motivate further studies using milk in FMD-endemic countries for FMD surveillance

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 utilised 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. Further, 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 minimise the likelihood of cross‐contamination. Such approaches could strengthen FMDV surveillance capabilities in East Africa, both at the individual animal and herd level

Utilising 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. 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, i.e. 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

PRAGMATIST: A tool to prioritize foot-and-mouth disease virus antigens held in vaccine banks

Antigen banks have been established to supply foot-and-mouth disease virus (FMDV) vaccines at short notice to respond to incursions or upsurges in cases of FMDV infection. Multiple vaccine strains are needed to protect against specific FMDV lineages that circulate within six viral serotypes that are unevenly distributed across the world. The optimal selection of distinct antigens held in a bank must carefully balance the desire to cover these risks with the costs of purchasing and maintaining vaccine antigens. PRAGMATIST is a semi-quantitative FMD vaccine strain selection tool combining three strands of evidence: (1) estimates of the risk of incursion from specific areas (source area score); (2) estimates of the relative prevalence of FMD viral lineages in each specific area (lineage distribution score); and (3) effectiveness of each vaccine against specific FMDV lineages based on laboratory vaccine matching tests (vaccine coverage score). The output is a vaccine score, which identifies vaccine strains that best address the threats, and consequently which are the highest priority for inclusion in vaccine antigen banks. In this paper, data used to populate PRAGMATIST are described, including the results from expert elicitations regarding FMD risk and viral lineage circulation, while vaccine coverage data is provided from vaccine matching tests performed at the WRLFMD between 2011 and 2021 (n = 2,150). These data were tailored to working examples for three hypothetical vaccine antigen bank perspectives (Europe, North America, and Australia). The results highlight the variation in the vaccine antigens required for storage in these different regions, dependent on risk. While the tool outputs are largely robust to uncertainty in the input parameters, variation in vaccine coverage score had the most noticeable impact on the estimated risk covered by each vaccine, particularly for vaccines that provide substantial risk coverage across several lineages

PRAGMATIST: a tool to prioritize foot-and-mouth disease virus antigens held in vaccine banks

Antigen banks have been established to supply foot-and-mouth disease virus (FMDV) vaccines at short notice to respond to incursions or upsurges in cases of FMDV infection. Multiple vaccine strains are needed to protect against specific FMDV lineages that circulate within six viral serotypes that are unevenly distributed across the world. The optimal selection of distinct antigens held in a bank must carefully balance the desire to cover these risks with the costs of purchasing and maintaining vaccine antigens. PRAGMATIST is a semi-quantitative FMD vaccine strain selection tool combining three strands of evidence: (1) estimates of the risk of incursion from specific areas (source area score); (2) estimates of the relative prevalence of FMD viral lineages in each specific area (lineage distribution score); and (3) effectiveness of each vaccine against specific FMDV lineages based on laboratory vaccine matching tests (vaccine coverage score). The output is a vaccine score, which identifies vaccine strains that best address the threats, and consequently which are the highest priority for inclusion in vaccine antigen banks. In this paper, data used to populate PRAGMATIST are described, including the results from expert elicitations regarding FMD risk and viral lineage circulation, while vaccine coverage data is provided from vaccine matching tests performed at the WRLFMD between 2011 and 2021 ( = 2,150). These data were tailored to working examples for three hypothetical vaccine antigen bank perspectives (Europe, North America, and Australia). The results highlight the variation in the vaccine antigens required for storage in these different regions, dependent on risk. While the tool outputs are largely robust to uncertainty in the input parameters, variation in vaccine coverage score had the most noticeable impact on the estimated risk covered by each vaccine, particularly for vaccines that provide substantial risk coverage across several lineages