Foot-and-mouth disease virus infection in young lambs: Pathogenesis and tissue tropism

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Abstract

Foot-and-mouth disease (FMD) in adult sheep usually causes milder clinical signs than in cattle or pigs, and is often subtle enough to go undiagnosed. In contrast, FMD in lambs has been reported to cause high mortality during field outbreaks. In order to investigate the pathogenesis of FMD in lambs, two groups, aged 10–14 days, were infected with foot-and-mouth disease virus (FMDV) type O UKG. One group of lambs (n = 8) was inoculated with FMDV in the coronary band, while the other (n = 4) was infected by direct contact with FMDV-inoculated ewes. Daily serum samples and temperature measurements were taken. Lambs were killed sequentially and tissue samples taken for analysis. Using real-time RT-PCR, viral RNA levels in tissue samples and serum were measured, and a novel strand-specific real-time RT-PCR assay was used to quantify viral replication levels in tissues. Tissue sections were examined for histopathological lesions, and in situ hybridisation (ISH) was used to localise viral RNA within histological sections. The contact-infected lambs became infected approximately 24 h after the ewes were inoculated. Vesicular lesions developed on the feet of all lambs and on the caudo-lateral part of the tongues of six of the eight inoculated lambs and three of the four contact-infected lambs. Although no lambs developed severe clinical signs, one of the contact-infected lambs died acutely at 5 days post-exposure. Histological examination of the heart from this lamb showed multi-focal areas of lymphocytic-plasmacytic myocarditis; similar lesions were also observed in the hearts of three of the inoculated lambs. Using ISH, viral RNA was localised within cardiac and skeletal muscle cells from the lamb which had died, and also from vesicular lesions on the coronary band and tongue of inoculated lambs. These results provide a detailed description of the pathogenesis of the disease in lambs.

Introduction

Foot-and-mouth disease (FMD) is a highly contagious acute vesicular disease of cloven-hoofed animals. The aetiological agent, foot-and-mouth disease virus (FMDV), is classified with the Aphthovirus genus as a member of the Picornaviridae family (Belsham, 1993). FMD is an economically devastating disease, causing significant production losses in infected domestic livestock. As a result, it is a major hindrance to international trade in animals and animal products. The cost of an outbreak can be enormous; the 2001 outbreak in the UK is estimated to have cost the agricultural sector £3.1 billion, with similar losses to the tourism sector (Thompson et al., 2002).

Clinical signs are generally milder in sheep than in cattle or pigs. Viraemia may be present for up to 3 days before the appearance of vesicular lesions (Alexandersen et al., 2003). During this time the sheep may be pyrexic and distressed with lameness spreading through the flock. Agalactia may occur in ewes. Vesicular lesions occur in the interdigital cleft, along the coronary bands and on the bulb of the heels. Oral lesions are less common but can occur on the dental pad, tongue and gums (Hughes et al., 2002). In pregnant sheep, transplacental infection of the foetal lamb may occur, causing abortion (Ryan et al., 2007).

Young lambs may die acutely following infection; associated clinical signs include collapse, fever, tachycardia and marked abdominal respiration (Garcia-Mata et al., 1954, Garcia-Mata et al., 1955, Geering, 1967, Pay, 1988). Deaths in lambs start to occur 2–3 days after the appearance of clinical signs in the ewes, and are usually reported to be the result of heart failure or starvation; post-mortem lesions include myocarditis, septicaemia, abomasitis and enteritis (Littlejohn, 1970, Salyi, 1939). Reported mortality rates vary widely, from 4.7% in an outbreak in India (Panisup et al., 1979) to 94.5% in lambs of 2–25 days old in a Russian outbreak (Khankishiev et al., 1958). Neonatal lamb deaths were reported in Dumfries during the 2001 outbreak in the UK (Reid, 2002). A PanAsia type O virus strain was reported to have caused large-scale lamb mortality in Iraq (Knowles and Samuel, 2003).

Mortality among FMD-infected calves and pigs has also been described. Death may occur without vesicular lesions due to the peracute onset of FMD in these animals. Pale foci of myocardial necrosis is seen in the ventricular wall and papillary muscle, referred to as “tiger heart” due to striping effect (Jubb et al., 1993). Donaldson et al. (1984) reported that the majority of piglets infected with an O1 Lausanne strain died or became moribund and were killed without developing vesicles. The deaths of the piglets started before clinical disease was evident in the sows. Macroscopic “tiger heart” lesions were not observed in any piglet hearts, although in all but one, myocarditis was present on histopathological examination.

Despite the numerous reports of FMD-related deaths in lambs, there is little data available on FMDV tropism or quantitative aspects of viral RNA kinetics in neonatal lambs, which would help understand the reasons for this high mortality and the influence of maternal infection on young lambs. In this study, viral distribution, tissue tropism and associated pathology during the acute stages of FMD were investigated in neonatal lambs experimentally infected with FMDV O UKG.

Section snippets

Inoculum

The FMDV strain used was O UKG 34/2001, isolated from a pig at Cheale Meats Abattoir, Essex on 20 February 2001 (Alexandersen and Donaldson, 2002). The inoculum was the second pig passage of original pig epithelial tissue suspension.

Animal experiments

Animals were housed in a category four bio-containment animal unit (Specified Animal Pathogens Order, DEFRA 1998). Animal experimentation was carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. Two separate

Kinetics of viral RNA in ewes experimentally infected with FMDV

All inoculated ewes developed signs of clinical signs of FMD within 1–2 dpi. The kinetics of viral RNA in serum samples are shown in Fig. 1 with daily temperature data. All inoculated ewes developed viraemia at 1 dpi (average 9.6 log10 copies/ml). Viral RNA levels then peaked at 2 dpi (average 10 log10 copies/ml). The tissue distribution of viral RNA in samples taken at post-mortem examination is summarised in Table 1.

Ewes infected by direct contact with inoculated lambs developed viraemia from 2 to 3

Discussion

The experiments described here characterised the pathogenesis and development of FMD in neonatal lambs infected with FMDV. The viral RNA levels in serum and in tissues samples were quantified, and viral replication levels in various tissues was analysed by a novel negative strand RT-PCR assay. Importantly, viral RNA was localised in cardiac and skeletal muscle cells from a lamb which died of FMD, confirming the tropism of the virus for these tissues. The results provide a detailed description

Acknowledgements

We would like to thank Yanmin Li, Pip Hamblin and Caroline Wright for performing the antibody ELISAs and Nicholas Juleff for help with the ISH. We thank Colin Randall, Bev Standish and Malcolm Turner for their assistance with and handling of the animals. This work was funded by Defra, UK.

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