J.R. Egerton

The type IV fimbrial subunit gene (fimA) of Dichelobacter nodosus is essential for virulence, protease secretion, and natural competence.

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.

Detection of Dichelobacter nodosus using species-specific oligonucleotides as PCR primers.

Dichelobacter nodosus is an essential causative agent of ovine footrot, a disease of major economic significance. Four oligonucleotides complementary to variable regions of the 16S rRNA of D. nodosus were identified, synthesized and tested for their specificity and sensitivity as probes for the detection of D. nodosus. In hybridization reactions using total RNA as the target nucleic acid, three probes were found to be both sensitive and species-specific. When these probes were used as primers in PCR reactions, on both purified D. nodosus DNA and whole cells, the sensitivity of detection was increased by several orders of magnitude. Using PCR, it was possible to detect the presence of D. nodosus by direct examination of lesion material from footrot infected sheep.

Protective antibody titres and antigenic competition in multivalent Dichelobacter nodosus fimbrial vaccines using characterised rDNA antigens

The relationship between K-agglutination antibody titres and protection against experimental challenge with Dichelobacter nodosus, the effect of increasing the number of D. nodosus fimbrial antigens, and the importance of the nature of additional antigens in multivalent vaccines on antibody response and protection against experimental challenge with D. nodosus were examined in Merino sheep. A total of 204 Merino sheep were allocated to one of 12 groups, and vaccinated with preparations containing a variable number of rDNA D. nodosus fimbrial antigens. The most complex vaccine contained ten fimbrial antigens from all major D. nodosus serogroups, while the least complex contained a single fimbrial antigen. In addition to D. nodosus fimbrial antigens, other bacterial rDNA fimbrial antigens (Moraxella bovis Da12d and Escherichia coli K99), and bovine serum albumin (BSA) were used in some vaccines. Antibody titres to fimbrial antigens and BSA were measured by agglutination and ELISA tests, respectively. Antibody titres were determined on five occasions (Weeks 0, 3, 6, 8, and 11 after primary vaccination). All sheep were exposed to an experimental challenge with virulent isolates of D. nodosus from either serogroup A or B, 8 weeks after primary vaccination. For D. nodosus K-agglutinating antibody titres, a strong negative correlation between antibody titre and footrot lesion score was observed. This relationship was influenced by the virulence of the challenge strain. Increasing the number of fimbrial antigens in experimental rDNA D. nodosus fimbrial vaccines resulted in a linear decrease in K-agglutinating antibody titres to individual D. nodosus serogroups. Similarly, a linear decrease in protection to challenge with homologous serogroups was observed as the number of D. nodosus fimbrial antigens represented in the vaccine increased. The reduction in antibody titres in multicomponent vaccines is thought to be due to antigenic competition. The level of competition between individual antigens is not constant and appears to be related to the immunodominance (nature) of the competing antigens. Both BSA ELISA, and M. bovis K-agglutinating antibody titres were adversely affected by the presence of two D. nodosus fimbrial preparations, whereas the antigenicity of E. coli K99 was unchanged by the presence of two additional D. nodosus antigens. Further studies are required to determine the step(s) in the immune response which are influenced by antigenic competition. Our results suggest that antigen presentation, particularly following primary vaccination, is the step most strongly influenced by antigenic competition.

Gene sequences and comparison of the fimbrial subunits representative of Bacteroides nodosus serotypes A to I: class I and class II strains

We have determined the nucleotide sequences of the genes encoding the fimbrial subunits representative of the known Bacteroides nodosus serogroups. All of the genes are preceded by a highly conserved region which includes the likely promoter and transcriptional regulator sites as well as the ribosome‐biding site, and are followed within a short but variable distance by a sequence with the characteristics of a transcription termination or attenuation signal. Based on sequence and organization, the subunits can be divided into two major classes called I (serogroups A, B, C., E, F, G, and I) and II (serogroups D and H). All contain the same seven‐amino‐acid positively charged leader sequence and conserved hydrophobic amino‐terminal sequence typical of type 4 fimbriae. Beyond this point the class II subunits are quite different from class I and share features more in common with those from other type 4 fimbriate bacteria, such as Moraxella bovis and Pseudomonas aeruginosa. The larger class I may be further subdivided into two subsets: (i) ((A, E, F)(B, I)) and (ii) (C, G). These proteins exhibit three major clusters of variation, at either end of the presumptive disulphide loop which spans the central third of the protein, and near the carboxy‐terimus, with dispersed changes in between. The length of the mature subunits varies from 152–156 amino acids, and the variation Includes small insertions or deletions in the variable clusters between more conserved domains. The class II subunits are 149 amino acids in length and contain two pairs of cysteine residues: one is at the end of the amino‐terminal conserved region, and the other is at the end of the protein. The major variation occurs in the central region of the molecule, and again small insertions or deletions are required to align adjacent conserved domains. There is also a striking absence of silent codon changes in the 5′ coding region of all of these genes, indicating that these sequences have a secondary genetic function, probably in recombi‐national exchange.

Protection of sheep against footrot with a recombinant DNA-based fimbrial vaccine

Recombinant Pseudomonas aeruginosa cells containing the Bacteroides nodosus fimbrial subunit gene under the transcriptional control of a strong promoter produce large amounts of B. nodosus-type fimbriae. We have carried out vaccination trials which show that these fimbriae are just as effective as either natural fimbriae or whole cell preparations of B. nodosus in inducing protective immunity against homologous footrot challenge. The recombinant-produced fimbriae are also effective therapeutically in accelerating the rate of healing of pre-existing footrot lesions. These results confirm that the structural subunit of the fimbrial strand is a primary protective antigen against footrot, and demonstrate the practicality and potential of recombinant DNA approaches to the development of new vaccines against B. nodosus and other Type 4 fimbriate pathogens.

Eradication of virulent footrot from sheep and goats in an endemic area of Nepal and an evaluation of specific vaccination

Programmes based on the identification and treatment of cases and the culling of animals refractory to treatment had failed to eradicate virulent footrot from two districts in the western region of Nepal. From 1993 to 1996 vaccination against two endemic virulent strains of Dichelobader nodosus was tested for its potential to contribute to the eradication of footrot from the region. Only sheep and goats which had been free of signs of footrot at three inspections at monthly intervals before their annual migration to alpine pastures were eligible for inclusion. From November 1992, the treatment of cases identified during inspections included the injection of specific vaccine. Successfully treated cases migrated with their flocks but were excluded from the vaccine trial. Non-responding cases were culled. Forty combined flocks of sheep and goats (approximately 9500 animals) were used initially to compare three vaccination regimens. Eleven flocks (sheep and goats) were treated with two doses of specific vaccine (group A), nine (sheep and goats) were treated with commercial vaccine followed by specific vaccine (group B) and 10 (sheep and goats) were treated with two doses of commercial vaccine (group C) in March to April 1993 before the annual migration; 10 flocks (sheep and goats) remained unvaccinated (group D). Only sheep and goats free of signs of footrot were allowed to migrate. Nevertheless, virulent footrot recurred in many flocks three months later. However, its prevalence was significantly lower in group A than in the other three groups combined. Groups A, B and C then received the specific vaccine before their migrations in 1994 to 1996; group D remained unvaccinated. The annual programme of inspection and identification and treatment of cases continued for seven years, but the vaccinations ceased after four years. There was no recurrence of virulent footrot after November 1993. After the first season the virulent strains of D nodosus used in the specific vaccine could no longer be isolated, although antigenically distinct, benign strains of the organism persisted in cases of benign footrot.

The protective efficacy of cloned Moraxella bovis pili in monovalent and multivalent vaccine formulations against experimentally induced infectious bovine keratoconjunctivitis (IBK)

Calves were vaccinated with cloned Moraxella bovis pili of serogroup C (experiment 1) or B (experiment 2) either as a monovalent formulation or as part of a multivalent preparation with pili of six other serogroups. Within 4 weeks of the second vaccine dose vaccinated calves and non-vaccinated controls were challenged via the ocular route with either virulent M. bovis strain Dal2d (serogroup C) or M. bovis strain 3WO7 (serogroup B) in experiments 1 and 2, respectively. Calves vaccinated with multivalent vaccines had significantly lower antibody titres than those vaccinated with monovalent preparations. Nevertheless, the levels of protection against infectious bovine keratoconjunctivitis (IBK) achieved with multivalent vaccines were 72% and 83% for the groups challenged with M. bovis strains of serogroups B and C, respectively. The serogroup C monovalent vaccine gave 100% protection against experimentally induced IBK and M. bovis isolates cultured from the eyes 6 days post-challenge were identified as belonging solely to serogroup C. Unexpectedly, only 25% protection was achieved against homologous strain challenge of calves that received the monovalent serogroup B vaccine. Furthermore, the majority of M. bovis isolates recovered from calves in this group belonged to serogroup C, as did half of those isolates cultured from the multivalent vaccinates. The remaining bacterial isolates from the latter group, together with all isolates from the non-vaccinated controls, belonged to serogroup B. Results are consistent with the hypothesis that derivatives of the serogroup B challenge inoculum had expressed serogroup C pilus antigen within 6 days of the challenge, possibly as a result of pilus gene inversion occurring in response to the presence of specific antibody in eye tissues and tears.

Disease resistance in Merino sheep. III. Genetic variation in resistance to footrot following challenge and subsequent vaccination with an homologous rDNA pilus vaccine under both induced and natural conditions

SUMMARY Eight traits representing clinical indicators of resistance to footrot were examined in 1562 Merino sheep, representing the progeny from 162 sires in four major bloodlines. Over a 4-year period, sheep were exposed to virulent isolates of Dicbelobacter nodosus under both an experimental challenge in which footrot was induced, and a separate natural challenge involving a different isolate of D. nodosus. Five footrot traits and three healing traits were each recorded on seven occasions following induced challenge, and on five occasions following natural challenge. All sheep were vaccinated with a primary and booster injection of an homologous rDNA pilus vaccine, 9 and 6 weeks after initiation of the induced and natural challenge respectively. The major fixed effects which influenced variation in resistance were (in order of importance) time of inspection after challenge, year and group in which sheep were challenged, and sex of the animal. Date of birth, birth-rearing type and age or dam were unimportant in the expression of footrot. Half-sib heritability estimates of resistance to footrot were low to moderate for single observations recorded pre-vaccination (0.07-0.22), and slightly lower for inspections made after vaccination (0.07-0.15). Repeatability estimates for footrot traits during a challenge ranged from 0.31 to 0.70 for inspections pre-vaccination, and 0.19 to 0.35 for inspections post-vaccination. Genetic correlations among footrot traits recorded at repeat inspections were high for observations pre-vaccination (range 0.87-1.00) and slightly lower for observations made after vaccination (0.52-1.00). Heritability estimates derived from repeat measurements approached 0.30 for most traits, except for traits describing healing, which had a heritability of almost zero. Heritability estimates of liability to footrot ranged between 0.09 and 0.41 depending on the time after challenge when the inspections were made. The genetic correlation between induced and natural footrot ranged from 0.14 to 0.95, depending on the period over which inspections were made, with an average of 0.67. In addition to within-flock genetic variation in resistance to footrot, significant differences were observed between different bloodlines within the experimental flock. It was concluded that there is substantial genetic variation in resistance to challenge with virulent isolates of D. nodosus. However, practical restrictions of exploiting available genetic variation may limit the widespread adoption of direct selection. ZUSAMMENFASSUNG: Krankheitsresistenz in Merinos III. Genetische Variabilität in Moderhinke Resistenz nach Infektion und folgender Impfung mit homologer rDNA pilus Vakzine unter induzierten und natürlichen Bedingungen Acht Merkmale, die als klinische Hinweise auf Moderhinkeresistenz betrachtet werden, wurden in 1562 Merino Schafen aus 162 Vatertieren von vier wichtigen Linien untersucht. Über eine 4-Jahresperiode wurden die Schafe virulenten Isolaten von Dichelobacter nodosus unter Versuchsbedingungen ausgesetzt und eine getrennte natürliche Infektion mit verschiedenen Isolaten von D. nodosus durchgeführt. Fünf Moderhinkemerkmale und drei Gesundungsmerkmale wurden nach Infektion bei sieben Gelegenheiten festgehalten und an fünf nach natürlicher Infektion. Alle Schafe wurden mit einer primären und einer booster Injektion homologer rDNA pilus Vakzine geimpft, 9 und 6 Wochen nach der induzierten und natiirlichen Infektion. Die wichtigsten fixen Effekte, welche die Variabilität der Resistenz beeinflussen, waren, nach Wichtigkeit gereint, Zeit der Prüfung nach Impfung, Jahr und Gruppe in welcher Schafe geimpft wurden und Geschlecht. Geburtsdatum, Aufzuchttyp und Mutterschaf-alter waren im Hinblick auf Moderhinke unwichtig. Halbgeschwister-Heritabilitätsschätzungen ihrer Resistenz waren niedrig bis mittel für Einzelbeobachtungen vor der Impfung (0,07-0,22) und geringfügig geringer für Beurteilung nach Impfung (0,07-0,15). Wiederholbarkeitsschätzungen für Moderhinkemerkmale bewegten sich von 0,31 bis 0,70 für Inspektionen vor und 0,19-0,35 für Inspektionen nach Impfung. Genetische Korrelationen zwischen Moderhinkemerkmalen bei verschiedenen Untersuchungen waren fur Beobachtungen vor der Impfung hoch (0,87-1) und geringfügig niedriger nachher (0,52-1). Heritabilitätsschätzungen von wiederholten Messungen erreichten 0,30 für die meisten Merkmale außer für jene, welche Heilung beschreiben, die nahezu keine Heritabilität zeigen. Heritabilitätsschätzungen für Moderhinkeempfindlichkeit variierten zwischen 0,09 und 0,41 in Abhängigkeit von der Untersuchungszeit nach den Impfungen. Die genetische Korrelation zwischen induzierter und natürlicher Moderhinke schwankte von 0,14 bis 0,95 in Abhängikeit von der Dauer der Beobachtungsperiode, durschnittlich 0,67. Zusätzlich zur genetischen Variabilität innerhalb der Herde wurden signifikante Unterschiede zwischen verschiedenen Linien innerhalb der Versuchsherde gefunden. Darauf wird es geschlossen, daß substantielle genetische Variabilität für Resistenz gegenüber virulenten Isolaten von D. nodosus existiert. Allerdings können praktische Hindernisse die Ausnutzung der vorhandenen genetischen Variabilität durch direkte Selektion einschränken.

A Moraxella bovis pili vaccine produced by recombinant DNA technology for the prevention of infectious bovine keratoconjunctivitis

Pili (fimbriae) were prepared from Moraxella bovis strain Dalton 2d (Dal2d) and from a derivative of Pseudomonas aeruginosa K/2PfS that contained a plasmid-borne Dal2d pilin gene and produced pili having serogroup-specific identity to Dal2d. Nine calves were vaccinated with two doses each of 30 micrograms authentic M. bovis Dal2d pili in oil adjuvant and 10 calves were vaccinated with a similar dose of P. aeruginosa-derived Dal2d pili in the same formulation. All 19 calves and 10 non-vaccinated controls were challenged by instillation of 1 x 10(9) virulent M. bovis Dal2d cells into both conjunctival sacs 19 days after the second vaccine dose. The serological response to vaccination and the degree of protection against experimentally induced infectious bovine keratoconjunctivitis (IBK) were assessed. None of the nine calves vaccinated with authentic M. bovis Dal2d pili developed IBK while two of those vaccinated with P. aeruginosa-derived Dal2d pili developed lesions which accounted for a mean group lesion score of 0.3. In contrast, 9 of the 10 non-vaccinated calves developed IBK lesions, the majority of which were progressive, required early treatment and accounted for a mean group lesion score of 1.5. These results demonstrate the potential of a relatively low dose of pili produced by recombinant DNA technology for development of an effective vaccine against IBK.

Characterisation of Dichelobacter nodosus isolated from footrot in sheep and goats in Nepal

Abstract Cases of footrot from sheep and goats in 45 village flocks in Nepal were cultured for Dichelobacter nodosus . Six hundred and eighty-two isolates were obtained from 25 flocks, of which 527 were derived from field isolation, and 155 from a vaccination trial. Of the 527 field isolates, 477 (90.5%) were provisionally classified as serogroup E. The remainder of those classified were either serogroup B (20) or serogroup C (14). Sixteen isolates were not classifiable by slide agglutination tests with antisera to any of the nine recognised serogroups of D. nodosus . Representative serogroup E isolates were all elastase positive and produced thermostable proteases. Those of serogroup B were either elastase positive and thermostable or elastase negative and thermolabile while those of serogroup C and the untypable isolates were elastase negative and thermolabile. Initially, all D. nodosus isolates from the field were of serogroup E. To increase the likelihood of isolating other serogroups, 21 Baruwal sheep affected with footrot were treated twice 30 days apart with a vaccine based on the Nepalese variant of serogroup E. Thirty days after receiving a second dose of vaccine, 17 of 20 affected animals were free of clinical footrot compared with 5 of 17 unvaccinated animals. Whereas at the beginning of this experiment only serogroup E could be recovered, at its conclusion although serogroup E persisted in the unvaccinated animals, isolates of serogroups B and C, and one untypable isolate were obtained from vaccinates. The demonstration of two virulent strains of D. nodosus (serogroup B and E) in these investigations provided an incentive to proceed with specific vaccination as a method of footrot management in these flocks.