Current understanding of the aetiology and laboratory diagnosis of footrot

The Veterinary Journal The Veterinary Journal 171 (2006) 421–428 www.elsevier.com/locate/tvjl

Review

Current understanding of the aetiology and laboratory diagnosis of footrot S.A. Wani *, I. Samanta Division of Veterinary Microbiology and Immunology, S.K. University of Agricultural Sciences and Technology of Kashmir, Shuhama (Alusteng), Srinagar, Kashmir 190006, India Accepted 12 February 2005

Abstract Footrot is a highly contagious disease of the feet of ruminants caused by the synergistic action of certain bacterial species of which Dichelobacter nodosus (D. nodosus) is the main transmitting agent. The infection is specific to sheep and goats, although it has also been reported in cattle, horses, pigs, deer and mouflon. The antigenic diversity of D. nodosus is due to variations in the DNA sequence of its fimbrial subunit gene (fimA) and provides the basis for classification of the organism into at least 10 major serogroups (A–I and M), the distribution of which varies with different geographical locations. Host immune response to vaccination is serogroup specific. There are three different clinical forms of disease caused by virulent, intermediate and benign strains of D. nodosus, respectively. In order to facilitate rapid and reliable clinical diagnosis, virulence determination, strain differentiation and serogroup identification for effective control measures, immunological tests, DNA probes and PCR based techniques have been introduced. This review summarises the current understanding of the mechanisms of antigenic diversity of D. nodosus as well as advances made in its strain differentiation and diagnosis. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Footrot; Dichelobacter nodosus; Sheep; PCR; PCR-RFLP; PFGE

1. Introduction Footrot is a specific contagious disease of the feet of ruminants or, more properly, an infectious syndrome caused by the synergistic action of certain bacterial species of which Dichelobacter nodosus (D. nodosus) is the main transmitting agent. The infection is specific to sheep and goats, although it has also been reported in other species including cattle, horses, pigs, deer and mouflon (Beveridge, 1967; Ghimire et al., 2002). D. nodosus is an anaerobic, Gram negative, rod shaped bacteria, with characteristic knobs at each end, and is often heavily fimbriated.

*

Corresponding author. Tel./fax: +91 194 2262211. E-mail address: [email protected] (S.A. Wani).

1090-0233/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2005.02.017

Based on the surface (K) antigen, now found to be identical with the fimbriae, the D. nodosus strains have been classified into 10 serogroups (A–I, M) and there are 18 serotypes. Besides serving as basis for classification, the type IV fimbriae of D. nodosus are recognised as a major virulence factor and are highly immunogenic. The polymerase chain reaction (PCR) approach has vastly improved the accuracy in identification and grouping of D. nodosus from footrot lesions but the distribution of serogroups varies considerably from place to place and over different time periods. For the development of an effective vaccine, it is essential to understand which serogroup strains are prevalent in a particular geographical location. Footrot is characterised by an exudative inflammation with a strong odour, followed by necrosis of the epidermal tissues of the hoof (Hurtado et al., 1998). Three

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different clinical forms of the disease have been described: virulent, intermediate and benign (Stewart, 1989). Virulent footrot (VFR) is highly contagious and is economically one of the most important infections of sheep and goats because of its impact on livestock production and health; it is often the target of expensive control and/or eradication programmes. In contrast, benign footrot causes little or no production loss (Egerton and Raadsma, 1991), and does not warrant intervention. The difficulty in identifying the various forms of ovine footrot on the basis of clinical signs alone has provided the impetus for the development and use of laboratory diagnostic tests (Liu et al., 1994). Despite its worldwide distribution, footrot has a significant impact in those sheep-farming countries that have temperate climates and moderate to high rainfall, such as Australia and New Zealand (Stewart, 1989). In Australia, footrot has been estimated to cost $A20 million per year to the wool industry alone, through decreased production, time consuming treatment methods and management changes for the control or eradication of the disease (Barton, 1986). The disease has been found to cause a 10% production loss in body weight and wool growth in affected animals in addition to the increased treatment and control costs (Marshall et al., 1991; Glynn, 1993). A survey of lameness in sheep carried out in Britain in 1994 indicated that >3 million sheep become lame annually, with >1 million cases attributed to footrot (Grogono-Thomas and Johnston, 1997); UK farmers list the disease as a cause of concern second only to sheep scab (Wassink et al., 2003). Treatment and control has been achieved in various countries through improved management, foot bathing and vaccination. In recent years, there have been significant advances in the understanding of the aetiology of footrot, its immunogenecity, the development of improved diagnostic methods for isolation, virulence determination, serogroup identification and strain differentiation of D. nodosus. But the information is scattered and the aim of this review is to provide comprehensive source of information on the condition.

2. Antigenic variation D. nodosus has been known to have antigenic diversity since it was first identified by Beveridge (1941). Egerton (1973) was the first to detect surface (K) antigens using a slide agglutination test. Based on this antigen, which is now found to be identical with fimbriae (pili), Egerton (1973) and Stewart (1978) classified D. nodosus into three serogroups, namely A, B and C. Simultaneously, the host response to vaccination was found to be serogroup specific (Egerton et al., 1972;

Stewart, 1978; Thorley and Egerton, 1981). Extending this system, Claxton et al. (1983) and Claxton (1986) classified D. nodosus isolates from Australian sheep into nine (A–I) serogroups and 18 serotypes. Strains within a serogroup were further subdivided into serotypes if sufficient antigenic variation was observed between isolates. An alternate serogrouping system, known as the British system, was proposed by Day et al. (1986) to accommodate the further nine serotypes J–R. However, three British serotypes (Chetwin et al., 1991) and three American serotypes (Gradin et al., 1993) seemed to be antigenically unrelated with any of the nine Australian serogroups. Subsequently, Ghimire et al. (1998) reported that 66/1063 strains of D. nodosus, isolated from lesions of footrot in sheep and goats in Nepal, could not be classified into any of the previously known nine serogroups. These isolates were agglutinated by antiserum against serotype M of the British classification system. The distinct antigenic character of these isolates was further confirmed by DNA sequence analysis. Thus, it was proposed that these isolates together with isolates previously classified as serotype M (Chetwin et al., 1991) be classified as serogroup M. On the basis of morphology and haemagglutinating properties, bacterial fimbriae are classified into five types, I–V. Guinea-pig, fowl and horse erythrocytes are strongly agglutinated by type-I fimbriae and, since this interaction is inhibited by the sugar D-mannose, these adhesins are termed as mannose sensitive (MS). On the other hand, type IV fimbriae are extremely thin, peritrichously arranged and confer mannose resistance (MR), haemagglutinating and adhesive properties on the cells. Fimbriae are composed of a single protein, pilin, encoded by a chromosomal gene (Hammond et al., 1984). The fimbriae of D. nodosus are classified as type IV because of their highly conserved amino-terminal region, polar location, association with twitching motility, and the presence of an N-methylphenylalanine residue as the N-terminal amino acid (Strom and Lory, 1993). Elleman (1988) showed that antigenic diversity of D. nodosus was due to variation in the DNA sequence of its fimbrial subunit gene (fimA). Sequence analysis of the fimbrial subunit protein of the major serogroups shows that there are generally more than 35 differences at the amino acid sequence level between serogroups and up to 15 differences between serotypes (Mattick et al., 1991; Kennan et al., 2003). Based on the general sequence homology, Mattick et al. (1991) and Hobbs et al. (1991) classified D. nodosus fimbriae into two distinct fimbrial classes namely class I and class II. In class I strains (Serogroup A, B, C, E, F, G and I), fimA gene is followed by fimB, which encodes a potential 29.5 kDa membrane protein of unknown function but which is postulated to play a role in the ex-

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port of fimbrial subunits to the cell surface. Strains of serogroups D and H belong to class II and have three additional fimA genes downstream, namely fimC, fimD and fimZ. The fimD gene is postulated to be a functional homologue of fimB; fimC has sequence similarity to traX from the F-plasmid (Firth and Skurray, 1995), and fimZ may represent a redundant fimbrial subunit. Not much is known about the pathogenesis of ovine footrot, but the type IV fimbriae (Elleman, 1988) and extracellular proteases (Kortt et al., 1993) of D. nodosus have been traditionally considered to be virulence factors. In addition, the virulent associated protein (vap) and virulence related locus (vrl) genomic islands have been shown to be preferentially associated with virulent strains (Billington et al., 1996). The vap locus is present in multiple copies whereas, vrl is present in single copy (Billington et al., 1999). By contrast, the vrl gene appears to be more specifically associated with D. nodosus isolates of greater virulence, as 87% and 6% of virulent and benign strains, respectively, hybridised with probes specific to this locus. Since vap and vrl are not exclusively associated with strains of D. nodosus with greater virulence, it is plausible to assume that these sequences are able to affect virulence either directly, by encoding a virulence factor, or indirectly, by regulating the expression of genes involved in D. nodosus pathogenesis. Both the virulence-associated regions, vap and vrl, appear to have arisen from similar site-specific insertion events, resulting in the integration of a sequence of probable phage or plasmid origin (Billington et al., 1999). Rood et al. (1996) examined 800 D. nodosus isolates and failed to detect a single strain, which carried the vrl locus alone. These findings are consistent with the hypothesis that the vap-encoded integrase is required for the integration of the vrl locus into the D. nodosus chromosome. Previously, serogrouping required the isolation of the organisms and purification by subculture followed by antigenic analysis by serological methods (slide and tube agglutination test) using rabbit antisera against fimbriae or pili antigens of D. nodosus. This would usually take 3–4 weeks and the specificity of the tests was influenced by cross reactivity of fimbrial antibodies and other external factors. This led to the development of PCRsequencing based methods for D. nodosus typing (John et al., 1999; Zhou and Hickford, 2000a). But, like serological methods, DNA sequencing methods in the absence of isolation were unable to type mixtures of D. nodosus strains. Since multiple strains of D. nodosus are frequently found on a single hoof, there was a need to develop a technique that was able to genotype mixtures of D. nodosus strains directly from footrot samples. Accordingly, Zhou et al. (2001) developed a more rapid and accurate typing method for D. nodosus using PCR amplification and reverse dot-blot hybridisation. Currently, a simple and rapid serogroup specific PCR test

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developed by Dhungyel et al. (2002) is being used which differentiates the D. nodosus strains into nine (A–I) serogroups using group specific primers. Availability of more sequence data will enable the detection of unknown serogroups of D. nodosus. Serogrouping, although useful for epidemiological investigation of ovine footrot (Claxton et al., 1983), does not differentiate D. nodosus strains as virulent, intermediate or benign. The only advantage of this classification system is the direct relationship between serogroups and protection following vaccination (Stewart, 1978; Thorley and Egerton, 1981). Although the diversity of D. nodosus fimbriae is well documented, little is known of the molecular mechanisms that lead to this diversity. Serogroup conversion may possibly occur in D. nodosus strains as a means of evading immune response, but this was not conclusively demonstrated until 2003 either experimentally or in nature. In other bacteria possessing type IV fimbriae, such as Neisseria gonorrhoeae and Moraxella bovis, the variation may result from DNA recombination between different silent and expressed fimbrial genes (Seifert, 1996) or site-specific recombination within the coding region of the fimbrial gene (Marrs et al., 1988). However, as D. nodosus possesses neither silent fimbrial loci nor multiple copies of subunit genes, there is no chance of recombination to generate antigenic variation. One possibility arising from the antigenic variation in D. nodosus could be natural transformation (Moore et al., 1990). Accordingly, Kennan et al. (2001) showed that many strains of D. nodosus are naturally competent for transformation. Subsequently, Kennan et al. (2003) conclusively demonstrated that serogroup conversion could occur by natural transformation and homologous recombination. The authors actually demonstrated this when they used a suicide plasmid containing the fimbrial subunit gene fimA of a serogroup G for successful conversion of a strain of serogroup I to serogroup G. These observations explained the failure of certain fimbrial vaccines to control footrot and it was suggested that benign strains of D. nodosus could play an important role as a reservoir of alternative fimbrial antigens. The other mechanisms for gene transfer in D. nodosus could be transduction mediated by bacteriophages, as has been observed in a number of microorganisms including Pseudomonas aeruginosa (Trevors et al., 1987), and phage like structures have been observed with D. nodosus (Gradin et al., 1991). Or, the gene transfer could occur by some unknown transposons or RNAs, as RNA recombination is a widespread phenomenon (Lai, 1992; Zhou and Hickford, 2000a). Further work in this direction would be informative. Serogroups as described by Claxton (1986) have now been reported from other countries including Nepal (Ghimire et al., 1996), Spain (Hurtado et al., 1998), USA (John et al., 1999) and New Zealand (Zhou and

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Hickford, 2000b). For a distribution of D. nodosus serogroups, see Table 1. Claxton et al. (1983) showed that the highest proportion of D. nodosus strains isolated and identified in Australia belonged to serogroup B (28.2%). Hindmarsh and Fraser (1985) reported a predominant prevalence of serogroup B (40.4%) in Great Britain. Thorley and Day (1986) while investigating the distribution of D. nodosus serogroups in France, The Netherlands, Belgium, Italy, Sicily and Sardinia, reported that majority of the strains belonged to serogroup H (32.7%), followed by D (14.4%) and C (13.5%); only 4–5% of the strains belonged to serogroup A. Kingsley et al. (1986) studied the distribution of D. nodosus serogroups in New Zealand and Great Britain and found that serogroup B predominated in New Zealand (56.2%), while in Great Britain the following percentages were recorded: B 40.3%, H 26.5%, and D 10.9%. Only 2% of D. nodosus strains belonged to serogroups A and C. In the western hills of Nepal, footrot has become endemic in sheep and goats (Karki, 1983). D. nodosus from cases of footrot was extensively isolated and characterised (Ghimire et al., 1996) and during these studies, a few isolates could not be classified into any of the nine Australian serogroups. So a new ÔMÕ serogroup was proposed for these isolates. Ghimire et al. (1996) reported that only three (B, C and E) of the known serogroups were present in sheep and goats in Nepal and in an initial survey, they collected lesion materials from 78 sheep and 11 goats from 15 flocks representative of the endemic area. Serogroup E was identifiable in most flocks whereas serogroups B and C were present only in a restricted number of flocks. Zakaria et al. (1998) were the first workers to isolate and characterise D. nodosus by Pulsed Field Gel Electrophoresis (PFGE) from sheep suffering from footrot in three Government farms in Malaysia. Strains with their origin in different farms were shown to have different PFGE patterns. On the basis of their PFGE, all field strains used in the study differed from the reference Table 1 Country distribution of predominant serogroups of Dichelobacter nodosus Country/Place

Predominant serogroups of D. nodosus

References

Australia Great Britain New Zealand France The Netherlands Belgium Italy Sicily Sardinia Nepal India

B B B H H H H H H E B

Claxton et al. (1983) Hindmarsh and Fraser (1985) Kingsley et al. (1986) Thorley and Day (1986) Thorley and Day (1986) Thorley and Day (1986) Thorley and Day (1986) Thorley and Day (1986) Thorley and Day (1986) Ghimire et al. (1996) Wani et al. (2004)

strain. The group further reported that there were several clonal types of D. nodosus isolates and indicated there was probably more than one source of pathogen in the farms studied. In India, a preliminary investigation on the distribution of serogroups of D. nodosus revealed the presence of serogroup B in three clinical samples of ovine footrot (Wani et al., 2004). Screening a further 10 samples, originating from eight sheep and two goats using multiplex PCR, also showed the presence of serogroup B only (unpublished data). Recently the authors have isolated the D. nodosus for the first time in India and observed the presence of serogroup E also in Kashmir, India (unpublished data). These observations indicated that a wide range of D. nodosus serogroups were responsible for ovine footrot in different parts of the world.

3. Virulence testing Virulence of bacteria can be determined by their ability to digest insoluble elastin, as was originally demonstrated for oral Staphylococci (Murphy, 1974). To classify D. nodosus strains as virulent, intermediate or benign pathovarieties, an elastase test was developed by Stewart (1979). This classification was carried out in Australia, with a view to eliminate chronic carriers of virulent pathogens. Over the years, this test has been refined, and the time taken from inoculation of the isolate onto an elastin agar plate to the detection of elastase activity is now used to classify the virulence of that isolate. It was observed that the isolates that produced the elastase within 7–11 days were mainly responsible for severe VFR while, the isolates with delayed elastase activity (21–28 days) were causing intermediate or benign footrot (see review by Liu and Yong, 1997). Williams et al. (1988) provided a simple and sensitive method by using soluble elastin for the detection of bacterial elastase produced during the growth of aerobic and anaerobic bacteria. Piriz et al. (1991) compared the efficiency and sensitivity of insoluble and soluble elastin substrates (using D. nodosus culture) and showed that latter provided greater sensitivity, speed and objectivity than the former. Palmer (1993) developed a gelatin gel test to detect the virulence of D. nodosus strains. Extensive studies on the proteases of D. nodosus isolates have resulted in the identification of a number of extracellular proteases including the acidic and basic proteases (Kortt et al., 1993). Monoclonal antibodies against several of these proteases were produced and incorporated into an enzyme linked immunosorbent assay (ELISA). This was promising for the improved differentiation of strains of D. nodosus causing virulent, intermediate and benign footrot (Stewart et al., 1990) as it reduced the test time and was amenable to identification of the D. nodosus

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from lesion materials. An ELISA using a potassium thiocyanate extract of outer membrane protein (OMP) antigen was extensively evaluated for diagnostic purpose particularly in relation to the severity of foot lesions (Whittington and Nicholas, 1995). The development of a dot-blot hybridization test using virulence specific gene probes by Katz et al. (1991) resulted in the differentiation of D. nodosus strains into virulent and benign. However, benign isolates did not hybridize with these probes making them incompetent reagents for diagnosis of footrot. Selection of additional gene probes based on screening of nine virulent and nine benign strains of serogroups A–I, enabled precise differentiation of D. nodosus strains into virulent, intermediate and benign (Liu, 1994) and turning the probes into competent reagents for footrot diagnosis. The gene probes, being non-serogroup specific, were effective with isolates of all serogroups. Liu (1994) and Liu et al. (1994) found general agreement among gene probe based dot-blot hybridization, elastase and gelatin gel tests and found these correlated well with clinical manifestation of footrot. Using virulence specific primers (Vf2 and Vr2) derived from pV470-13, and a pair of benign specific primers (Bf and Br) derived from pB645-335, a PCR assay was developed with improved differentiation of D. nodosus strains causing virulent, intermediate and benign footrot (Liu and Webber, 1995). Virulence and benign specific primers used in combination in a PCR assay would make it a rapid, sensitive and specific test to differentiate the D. nodosus into virulent, intermediate and benign strains and should make the results available in 5–6 h after arrival of field samples in the laboratory. The diagnosis of footrot is a tedious and time-consuming process, complicated by the fastidious growth requirements and slow growing nature of D. nodosus. The organism has nutritional requirements that are not satisfied by the normal laboratory media. Beveridge (1941) was the first to incorporate horse serum into a medium for the growth of D. nodosus. Subsequently, Thomas (1958) used powdered hoof in the medium for the isolation of D. nodosus. However, even under these conditions the organism was found to grow slowly. Skerman (1975) investigated the growth response of D. nodosus to a variety of nutritional substances and showed that a particle free medium containing trypticase, peptone, arginine, lab-lemco, yeast extract, serine and certain salts improved growth and facilitated the isolation of the organism. But still the work on D. nodosus remained hampered by the lack of methods by which the organism could be consistently isolated and handled in the laboratory. Thorley (1976) was first to provide the details of a simplified method, which was used with consistent success for the isolation of D. nodosus strains from sheep. He observed that hoof agar (4% for primary isolation and

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2% for subculture) could be successfully used for isolation of D. nodosus using an anaerobic jar with proper gas arrangement at 37 °C for 7 days. Rapid progress has been made in the diagnosis of footrot over the past few years using new generation molecular technologies such as gene probes, PCR and monoclonal antibodies enabling improved determination of the virulence of D. nodosus strains (Katz et al., 1991). Fontaine et al. (1993) developed oligonucleotides complementary to variable regions of the 16S rRNA of D. nodosus as probes for the detection of D. nodosus in hybridization tests. Among them, three probes were found to be sensitive and species specific. Fontaine et al. (1993) developed a highly sensitive PCR assay for the direct identification of D. nodosus from lesion materials and showed its potential in eliminating the need to culture the organism. The sensitivity of detection of D. nodosus was improved by several orders of magnitude. The test could detect even a single cell of D. nodosus by direct examination of lesion material from footrotinfected animals. John et al. (1999) carried out identification and grouping of . D. nodosus using PCR and found that PCR approach vastly improved the accuracy in the identification and grouping of D. nodosus from footrot lesions. However, all of these methods had limitations in tracing the source of infection as they lacked the strain–specific differentiation. To overcome this, Zakaria et al. (1998), in Malaysia, used PFGE for strain differentiation of D. nodosus with high resolution. PFGE banding profiles enabled them to differentiate 12 strains of D. nodosus into eight genome types and all of the field strains were shown to differ from the reference strains. Because of its potential for strain differentiation, this technique could be used for monitoring the pathogenic capability of D. nodosus strains for efficient diagnosis and control. Subsequently, Ghimire and Egerton (1999) used PCR-restriction fragment length polymorphism (PCRRFLP) of outer membrane proteins of D. nodosus as a new tool for strain differentiation of D. nodosus in Nepal and demonstrated its potential in tracing the source of infection (Ghimire and Egerton, 1999). These tools are useful in effective surveillance programmes as they define the identity of the strains causing the disease and so facilitate the eradication of target strains. Such tests become impracticable under certain field conditions, for example when flocks are grazing on alpine pastures away from human habitation. Also a high proportion of lesions in affected animals heal naturally before they arrive back in the villages after downward migration in winter, making the bacteriological examination of feet unrewarding. A test was required that identified VFR independent of clinical signs and bacteriological examination. For this reason, several antigens of D. nodosus were evaluated (Whittington et al., 1997)

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and pilus antigen was found to evoke more specific anamnestic immune response than OMP (Dhungyel et al., 2001). Based on these findings, Dhungyel et al. (2001) developed a pilus anamnestic ELISA, a valuable complementary diagnostic tool for confirming the outbreaks of footrot in absence of lesions and where bacteriological examination is unrewarding. Thus it became possible to detect otherwise unnoticed carriers under extensive management practices. The immune response of goats against D. nodosus was also analysed by ELISA using either fimbrial or OMP antigens of D. nodosus (Ghimire et al., 2002). These workers investigated the primary and secondary responses of goats to both antigens of D. nodosus separately in response to infection and subsequent stimulation by injection of purified antigens. It was reported that the magnitude of humoral immune responses against D. nodosus was much lower in goats than sheep. The authors reported that a low magnitude immune response could be caused as D. nodosus infection is less invasive in goats. Furthermore, the authors observed that maternal antibodies against D. nodosus, transferred to kids via colostrum, were unlikely to interfere with a diagnostic ELISA and concluded that these tests were: (1) only suitable for herd diagnosis of footrot in goats and (2) dependent on the development of advanced under-running infections in a proportion of affected goats.

4. Conclusion and future perspective D. nodosus has been the focus of extensive investigation in many parts of world and especially in Australia, New Zealand, the United Kingdom, Nepal, Malaysia and India. Isolation from VFR cases of a novel spirochaete, in the absence of D. nodosus, has added another dimension to the aetiology of footrot (Demirkan et al., 2001). In recent years, with the availability of sequence information of the fimA subunit gene of type IV fimbriae of D. nodosus, traditional serogrouping methods for D. nodosus have been replaced by more rapid, sensitive and specific diagnostic assays such as sequencing and PCR using serogroup specific primers. The main advantage of these developments has been rapidity, specificity and elimination of time-consuming isolation procedures. At present, 9/10 major serogroups have been recognised by PCR assay and the availability of more sequence data will enable the detection of further unknown serogroups. Serogroups reported from Australia have also been reported from other countries including Nepal, Spain, USA, New Zealand, UK and India. Similarly, the availability of monoclonal antibody-based ELISAs, gene probes and PCR assays have proved very useful for the rapid and reliable diagnosis of D. nodosus into virulent, intermediate and benign strains. The develop-

ment of a PCR assay for the amplification of a species-specific 16 S rDNA fragment has made isolation procedures unnecessary for the detection and identification of D. nodosus, reducing laboratory time from 3 to 4 weeks to one day. Pilus anamnestic ELISA has facilitated the diagnosis of footrot in the absence of active lesions after recovery enabling the detection of otherwise unnoticed carriers, such as can arise in flocks during migration to alpine pastures. Use of PFGE and PCR-RFLP of OMP with improved power of strain differentiation has overcome the limitation of other diagnostic methods, which lacked the ability to trace the routes of infections of outbreaks, and explains the failure of certain specific vaccines under typical field conditions. Demonstration that D. nodosus strains transform in vitro from one serogroup to another should explain the failure of fimbrial vaccines under certain conditions. However, more studies are required to demonstrate that serogroup conversion by natural transformation can occur in footrot lesions in the field. Such investigations would have significant implications for the control and treatment of VFR outbreaks. Virulence mechanisms of D. nodosus have been elucidated further because of the ever-increasing information about the molecular characteristics of the fimbriae of D. nodosus. Recent improvements in the strain differentiation of D. nodosus should help further in understanding the epidemiology and selection of appropriate strains for the development of suitable vaccines for the efficient control of footrot.

Acknowledgement The authors sincerely thank Professor Anwar Alam, Vice-Chancellor, SKUAST-Kashmir, for his inspiration and encouragement for preparation of the manuscript.

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