Footrot is a disease of ruminants caused by infection of Dichelobacter nodosus (D. nodosus), a Gram negative, anaerobic rod [1]. Footrot is predisposed by the colonisation of Fusobacterium necrophorum, a coliform bacteria, that can cause ovine interdigital dermatitis and optimal tissue conditions for D. nodosus invasion [2]. Disease in sheep has been classified into three forms; benign, intermediate and virulent, which are distinguished through the prevalence of severe lesions [3]. Lesions are characterised by interdigital dermatitis which can progress to severe necrotic separation of soft and hard horn from underlying stroma [4]. This progression to the more severe “underrunning” of the heel is dependent on bacterial virulence and environmental factors [5]. Moisture and warmth are key environmental factors for disease transmission with unfavourable conditions significantly limiting spread and severity of footrot [5, 6]. In NSW, the optimal transmission period is generally restricted to a spring time 4-10 week window [6], however in other parts of the world, including the southern states of Australia, footrot can have a significant economic and animal welfare impact year round [7].
The key immunogen and virulence factor of D. nodosus is the type IV fimbriae which also provides the basis for differentiation of D. nodosus into 10 major serogroups (A-I, M) and 18 serotypes [8-10]. The type IV fimbrial subunit gene (fimA) facilitates adhesion and penetration of the stratum corneum [11] with further research indicating protease secretion, adhesion and tissue invasion are all associated with type IV fimbrial biogenesis [12, 13]. Twitching motility of fimbriae and the secretion of subtilisin-like serine proteases have been shown to be essential virulence factors for D. nodosus in sheep [14, 15].
Footrot has been studied for almost a century and remains a debilitating disease causing lameness, weight loss and poor wool growth, leading to significant impacts on animal welfare and poor economic outcomes [3, 5]. Virulent footrot is a notifiable disease in NSW, resulting in property quarantine and the instalment of mandated eradication programs [16]. Although the prevalence of virulent footrot is estimated at <1% in NSW [16], the cost of treatment, prevention and the economic impact on stock is estimated at $32 million annually in Australia [17], while footrot remains significant in most other wool and sheep-meat producing countries [18-22].
Traditional treatment methods for ovine footrot include extensive foot paring to remove overgrown hoof and subsequent foot-bathing in antiseptic solutions such as zinc sulphate or formalin [1, 3, 23, 24]. Treatment of individual animals with antimicrobials is also known to be effective [25, 26] and will reduce the prevalence of footrot on farms, but remains cost prohibitive [27]. Ideal management protocols for animal welfare and economic outcomes would consist of preventative measures employed at a flock/farm level [28]. The first vaccines used for footrot control were tested in 1969 and consisted of killed D. nodosus whole cells emulsified in an oil adjuvant [29]. These vaccines proved both prophylactic and therapeutic in homologous infections [30], however vaccine resistance was encountered when different serotypes were present in a footrot infection [31]. Slide agglutination testing allowed for the identification of different serogroups of D. nodosus [8], with surface antigen K (pilus antigen) deemed the principle antigen to elicit serological antibody response [32, 33]. This lead to the development of monovalent vaccinations with purified pilus antigen subunits which proved capable of provoking an equivalent immune response [34, 35]. With aims to reduce the cost of producing a vaccine, a recombinant fimbrial vaccine was developed by overexpression of fimbriae producing genes in recombinant Pseudomonas aeruginosa strains, which later became the preventative gold standard for homologous infection [36, 37]. As multiple D. nodosus serogroups have been identified within a single naturally occurring field infection [8] multivalent vaccinations have also been trialled [38-40]. However the efficacy of multivalent vaccination is limited by antigenic competition, resulting in a shorter duration of immunity and less overall protection when compared to monovalent vaccines [39-41].
Identification of serogroups present within a flock/population and then development of a specific vaccine, has been shown to be effective in the eradication of virulent footrot in Nepal, Bhutan and also facilitate elimination or control in selected Australian flocks [7, 42, 43]. Flock specific vaccination is a time intensive process reliant on culture, purification and typing taking up to 3-4 weeks before a vaccine can then be produced [44]. The development of serogroup specific and multiplex PCR has allowed for more accurate and rapid identification of D. nodosus serogroups, however this process can still take up to 2-3 weeks [44]. Serogroup specific vaccination still has reduced efficacy when flocks are infected with more than two serogroups of D. nodosus, due to antigenic competition [45]. Although challenging, this can be overcome by sequential bivalent vaccinations with an inter-vaccination window of 3-12 months [45, 46]. Laboratory investigations have also demonstrated the potential for serogroup transformation with implications for serogroup specific vaccine resistance [47]. Therefore, the development of a cross-protective vaccination with the capability to prevent virulent footrot without farm-specific serogroup testing could provide means for disease eradication worldwide.
The previous generation of vaccines had relied on the identification of causative pathogens and subsequent inoculation with a killed or attenuated whole cell vaccine. This process was updated after realisation that subunits of pathogens could elicit sufficient immune response. Once the entire genome of different pathogens could be sequenced, reverse vaccinology arose as a means to identify potential immunogenic proteins and new vaccine candidates previously hidden by dominant immunogens or deemed too difficult to isolate/purify [48]. The D. nodosus genome was sequenced in 2007 and reverse vaccinology facilitated the identification of 99 proteins that were either surface located or excreted as potential immunogens [49]. A field-based vaccination trial was designed as one stage in a larger study undertaken to test potential cross-protective vaccine candidates for the prevention of virulent footrot. Three distinct groups of proteins were tested in this trial and the methodology, results and implications are discussed in this report.