A Trifecta of New Insights into Ovine Footrot for Infection Drivers, Immune Response, and Host-Pathogen Interactions.

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry's largest welfare problems. The complex etiology of footrot makes in situ or in vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved and how they may interact with the host, ultimately providing a way to identify targets for future hypothesis-driven investigative work. Here, we present the first combined global analysis of bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intratissue and surface bacterial populations and the most abundant bacterial transcriptomes were analyzed, demonstrating that footrot-affected skin has reduced diversity and increased abundances of not only the causative bacterium Dichelobacter nodosus but also other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals the suppression of biological processes related to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot-affected interdigital skin compared to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome, which could lead to the identification of future therapeutic targets.

Characterization of two putative Dichelobacter nodosus footrot vaccine antigens identifies the first lysozyme inhibitor in the genus.

The Gram-negative anaerobic bacterium Dichelobacter nodosus (Dn) causes footrot in ruminants, a debilitating and highly contagious disease that results in necrotic hooves and significant economic losses in agriculture. Vaccination with crude whole-cell vaccine mixed with multiple recombinant fimbrial proteins can provide protection during species-specific outbreaks, but subunit vaccines containing broadly cross-protective antigens are desirable. We have investigated two D. nodosus candidate vaccine antigens. Macrophage Infectivity Potentiator Dn-MIP (DNO_0012, DNO_RS00050) and Adhesin Complex Protein Dn-ACP (DNO_0725, DNO_RS06795) are highly conserved amongst ~170 D. nodosus isolates in the https://pubmlst.org/dnodosus/ database. We describe the presence of two homologous ACP domains in Dn-ACP with potent C-type lysozyme inhibitor function, and homology of Dn-MIP to other putative cell-surface and membrane-anchored MIP virulence factors. Immunization of mice with recombinant proteins with a variety of adjuvants induced antibodies that recognised both proteins in D. nodosus. Notably, immunization with fimbrial-whole-cell Footvax vaccine induced anti-Dn-ACP and anti-Dn-MIP antibodies. Although all adjuvants induced high titre antibody responses, only antisera to rDn-ACP-QuilA and rDn-ACP-Al(OH)3 significantly prevented rDn-ACP protein from inhibiting lysozyme activity in vitro. Therefore, a vaccine incorporating rDn-ACP in particular could contribute to protection by enabling normal innate immune lysozyme function to aid bacterial clearance.

Investigation of immunity in sheep following footrot infection and vaccination.

Ovine footrot is a major disease affecting sheep welfare and production. The anaerobic Gram-negative bacterium Dichelobacter nodosus is the essential transmitting agent. Monovalent or bivalent vaccines induce high levels of D. nodosus antibodies and are the basis of several successful footrot treatment, control and eradication programs. Due to the rapid rate of disease transmission within a flock, the presence of therapeutic vaccination non-responders has major implications for a control program. The aim of this study was to assess the immunological basis of a therapeutic vaccination non-response. Sheep (n=120) were infected with D. nodosus in an artificial pen challenge. Once disease had established, animals were vaccinated with a serogroup specific D. nodosus fimbrial vaccine. Based on the response to therapeutic vaccination, animals were allocated into one of three groups: (i) TVNR where disease persisted despite vaccination (ii) non-diseased, where disease never established and (iii) TVR, where disease was established but resolved with vaccination. Factors related to both the innate and adaptive immune pathways were assessed. These included antigen-specific serum antibodies, interferon-γ, interleukin-10, proliferation of lymphocyte subsets and phagocytic activity of leukocytes. There was no significant difference between the three groups of sheep for any of these parameters. All three groups of sheep produced antibody in excess of a previously published minimum antibody titre required for protection. Opsonising activity in sera from the three groups of sheep was also not significantly different and phagocytic cells from sheep from all three groups were able to destroy D. nodosus intracellularly. These findings show that the measured systemic adaptive and innate immune responses were unlikely to be the cause of a therapeutic vaccination non-response. They also show that the accepted minimum protective titre may be incorrect and may need further examination.

Selenium supplementation restores innate and humoral immune responses in footrot-affected sheep.

Dietary selenium (Se) alters whole-blood Se concentrations in sheep, dependent upon Se source and dosage administered, but little is known about effects on immune function. We used footrot (FR) as a disease model to test the effects of supranutritional Se supplementation on immune function. To determine the effect of Se-source (organic Se-yeast, inorganic Na-selenite or Na-selenate) and Se-dosage (1, 3, 5 times FDA-permitted level) on FR severity, 120 ewes with and 120 ewes without FR were drenched weekly for 62 weeks with different Se sources and dosages (30 ewes/treatment group). Innate immunity was evaluated after 62 weeks of supplementation by measuring neutrophil bacterial killing ability. Adaptive immune function was evaluated by immunizing sheep with keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. At baseline, FR-affected ewes had lower whole-blood and serum-Se concentrations; this difference was not observed after Se supplementation. Se supplementation increased neutrophil bacterial killing percentages in FR-affected sheep to percentages observed in supplemented and non-supplemented healthy sheep. Similarly, Se supplementation increased KLH antibody titers in FR-affected sheep to titers observed in healthy sheep. FR-affected sheep demonstrated suppressed cell-mediated immunity at 24 hours after intradermal KLH challenge, although there was no improvement with Se supplementation. We did not consistently prevent nor improve recovery from FR over the 62 week Se-treatment period. In conclusion, Se supplementation does not prevent FR, but does restore innate and humoral immune functions negatively affected by FR.

Higher whole-blood selenium is associated with improved immune responses in footrot-affected sheep.

We reported previously that sheep affected with footrot (FR) have lower whole-blood selenium (WB-Se) concentrations and that parenteral Se-supplementation in conjunction with routine control practices accelerates recovery from FR. The purpose of this follow-up study was to investigate the mechanisms by which Se facilitates recovery from FR. Sheep affected with FR (n = 38) were injected monthly for 15 months with either 5 mg Se (FR-Se) or saline (FR-Sal), whereas 19 healthy sheep received no treatment. Adaptive immune function was evaluated after 3 months of Se supplementation by immunizing all sheep with a novel protein, keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity (DTH) skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. Innate immunity was evaluated after 3 months of Se supplementation by measuring intradermal responses to histamine 30 min after injection compared to KLH and saline, and after 15 months of Se supplementation by isolating neutrophils and measuring their bacterial killing ability and relative abundance of mRNA for genes associated with neutrophil migration. Compared to healthy sheep, immune responses to a novel protein were suppressed in FR-affected sheep with smaller decreases in FR-affected sheep that received Se or had WB-Se concentrations above 250 ng/mL at the time of the immune assays. Neutrophil function was suppressed in FR-affected sheep, but was not changed by Se supplementation or WB-Se status. Sheep FR is associated with depressed immune responses to a novel protein, which may be partly restored by improving WB-Se status (> 250 ng/mL).

Evolution of oxidative/nitrosative stress biomarkers during an open-field vaccination procedure in sheep: effect of melatonin.

Melatonin has been shown to exert immunomodularory properties with broad application in veterinary medicine. In previous work we have described that subcutaneous coadministration of melatonin to seeps vaccinated against two stumps of A1 and C strains of Dichelobacter nodosus enhanced both the antibody titer and serum IgG levels to A1 and C strains of D. nodosus compared to vaccinated animals not treated with melatonin. Following a similar protocol here we have investigated the effect of a higher dose of melatonin (36mg/animal) in the improvement of the immune response and in the possible oxidative/nitrosative stress produced during the immunization protocol. Our results show that footrot vaccine application induced nitrosative but not oxidative stress at 42 days post-vaccination, which was neutralized by melatonin administration. On the other hand, melatonin improved the immune response with respect to our previous data increasing the time of permanence of antibodies in serum, opening new perspectives for melatonin as prophylactic drug.

Modulation of inter-vaccination interval to avoid antigenic competition in multivalent footrot (Dichelobacter nodosus) vaccines in sheep.

Virulent footrot is a significant disease of sheep in most sheep farming countries; a strain/serogroup of the anaerobic bacterium Dichelobacter nodosus is the essential transmitting agent. Commercial multivalent footrot vaccines containing nine fimbrial serogroups (A through I) of D. nodosus produce relatively low and short term antibody responses due to antigenic competition, in contrast to higher and longer responses provided by monovalent or bivalent vaccines. The latter were important components of successful eradication programs for endemic footrot caused by either one or two serogroups of D. nodosus in Nepal, Bhutan, and several flocks in Australia. However, the presence of up to six serogroups in some Australian flocks and the use of an annual bivalent vaccination regime to progressively eradicate serogroups would require a long term program. In this study we report the results of a sequential vaccination trial testing different time intervals between different bivalent vaccinations. Intervals of 12, 9, 6, 3 and 0 months were tested. The 1st vaccination was with recombinant fimbrial antigens for serogroups A and B while the 2nd vaccination was with D and E. There were no significant differences between the antibody responses for time intervals of 3, 6, 9 and 12 months whereas there was a reduced response when sheep were vaccinated with two bivalent vaccines (four antigens) concurrently, indicating antigenic competition. Therefore an inter-vaccination interval of 3 months can be applied between two different bivalent vaccines without detrimental impact on the humoral immune responses to the various fimbrial antigens of D. nodosus. These results could have wider applications in vaccination against diseases caused by multivalent or multistrain microbes.

Identification of three immunodominant regions on leukotoxin protein of Fusobacterium necrophorum.

To analyze immunodominant regions of leukotoxin protein of Fusobacterium necrophorum strain H05, a series of truncated forms of leukotoxin gene were expressed in Escherichia coli using the vector pGEX-6p-1 or pPROEX HTa. The results of SDS-PAGE showed the truncated forms PL1, PL2, PL4, and PL5 were expressed in Escherichia coli using the vector pGEX-6p-1, and the truncated forms PL3 was expressed in Escherichia coli using the vector pPROEX HTa. These recombinant proteins were able to react with antisera against Fusobacterium necrophorum strain A25. In five recombinant proteins, the recombinant proteins PL1, PL3 and PL4 as vaccine were able to elicit formation of the better protective effects on mice against infection of Fusobacterium necrophorum strain A25.

Melatonin, as an adjuvant-like agent, enhances platelet responsiveness.

Melatonin exerts immunomodulatory actions that enhance the magnitude and quality of immune responses specific for certain antigens; this has raised the possibility of using melatonin to design novel vaccine adjuvant systems. The present study investigated the effect of subcutaneous slow-release melatonin implants and subcutaneous melatonin injections on the responsiveness of circulating platelets in sheep after vaccination against Dichelobacter nodosus (A1 and C serotypes), the bacterium that causes ovine footrot, a major cause of lameness in sheep. The experiments were carried out in sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by radioimmunoassay, sheep platelet aggregation was monitored using an aggregometer and Ca2+ mobilization was determined by spectrofluorimetry using fura-2. Administration of melatonin either by implants or subcutaneous injections increased plasma melatonin concentrations, an effect that was found to be greater and more sustained when melatonin was administered via implants. Vaccination per se, as well as melatonin, increased the percentage and rate of platelet aggregation and reduced the lag-time in response to the physiological agonist thrombin, an effect that was found to be significantly greater when melatonin was administered to vaccinated animals. Melatonin enhanced thrombin-evoked Ca2+ release and entry and further increased Ca2+ mobilization observed in platelets from vaccinated sheep. These observations suggest that the use of melatonin, as a novel adjuvant, induces beneficial effects on platelet function and haemostasis, and opens new perspectives for therapeutic manipulation of immune responses to vaccination.

Melatonin enhances the immune response to vaccination against A1 and C strains of Dichelobacter nodosus.

Melatonin has been shown to exert immunomodulatory properties with broad application in veterinary medicine. Here we have investigated the effect of exogenous melatonin in the improvement of the immune response to administration of an immune-preparation of two stumps of A1 and C strains of Dichelobacter nodosus in sheep. Subcutaneous administration of melatonin enhanced plasma levels of melatonin from days 42 to 120. Administration of melatonin to vaccinated animals enhanced both the titer of antibodies and serum IgG levels to A1 and C strains of D. nodosus compared to vaccinated animals not treated with melatonin. Our results suggest that melatonin increased the immune response to vaccination and open new perspectives in the design of prophylactic strategies.

[Structural and functional specificity of the Dichelobacter nodosus pili. Synthesis of the protein pilin of D. nodosus in various bacterial recombinant strains].

The ovine foot rot is a severe infectious disease of sheep. Dichelobacter nodosus is an essential pathogen of this disease. An obligatory anaerobic gram-negative rod-shaped microorganism has slow rate of accumulating bacterial density and fastidious growth requirements. This causes obstacles to vaccine production and makes it difficult to diagnose the disease. The diagnosis in this case is more expensive. Fimbriae (or pili) are one of the major factors of virulence of D. nodosus. Their antigenic and immunogenic properties make them good vaccine components for elevated immunogenicity. Since the nucleotide sequence of the fimA gene encoding fimbrial subunit was determined, attempts to produce recombinant pili were undertaken. The production of the genetic-engineering fimbriae would allow the price of the vaccines to be reduced and their manufacture to be simplified. The vaccine immunogenicity is increased in this case. At first, E. coli was selected as an expression system, but morphogenetic expression of the pili was not achieved on its surface because of some differences in the biogenesis and structure of fimbriae from D. nodosus. Successful morphogenesis of the pili was achieved in Pseudomonas aeruoginosa, which had closest similarity in the structure of pili. The level of the immunity obtained after immunization of the sheep with recombinant pili was similar to the level of the immunity after native pili or whole cells of D. nodosus had been used. This review contains information regarding the recombinant strains of Pseudomonas aeruoginosa obtained using fimbriae of D. nodosus and expression of pilin genes in different bacterial systems.

Genome sequence and identification of candidate vaccine antigens from the animal pathogen Dichelobacter nodosus.

Dichelobacter nodosus causes ovine footrot, a disease that leads to severe economic losses in the wool and meat industries. We sequenced its 1.4-Mb genome, the smallest known genome of an anaerobe. It differs markedly from small genomes of intracellular bacteria, retaining greater biosynthetic capabilities and lacking any evidence of extensive ongoing genome reduction. Comparative genomic microarray studies and bioinformatic analysis suggested that, despite its small size, almost 20% of the genome is derived from lateral gene transfer. Most of these regions seem to be associated with virulence. Metabolic reconstruction indicated unsuspected capabilities, including carbohydrate utilization, electron transfer and several aerobic pathways. Global transcriptional profiling and bioinformatic analysis enabled the prediction of virulence factors and cell surface proteins. Screening of these proteins against ovine antisera identified eight immunogenic proteins that are candidate antigens for a cross-protective vaccine.

Distribution and prevalence of footrot in Bhutan.

The first cases of footrot in Bhutan were reported in sheep in 1990 at the National Sheep Breeding Centre (NSBC), which supplies breeding animals to village sheep flocks throughout Bhutan. Despite the presence of footrot at the Centre the distribution of apparently disease-free sheep continued. Cases of footrot were reported in village flocks soon after the disease was diagnosed at NSBC. A national survey was designed to establish the distribution and prevalence of footrot in Bhutan. This detected footrot in 19/94 village sheep flocks surveyed. The 19 affected flocks were distributed among nine different administrative districts whereas the villages selected were in 13 of a total of 16 sheep growing districts. The highest within-flock prevalences were among the seven flocks sampled in Bumthang district (mean 20.4%). The prevalence of the disease within flocks was generally much lower in other affected districts and in three districts a single affected animal was identified in the sample of 14 sheep examined in each village. Nationally, footrot prevalence was estimated to be 3.1% (95% CI 2.16-4.04%). There was a positive association between the receipt of animals from NSBC and the presence of footrot. The prevalence of the disease was higher in flocks with a migratory system of management than in those using a sedentary system. The relative risk of there being footrot in a migratory flock was nine-times higher than in a non-migratory flock. Only one strain of Dichelobacter nodosus (serogroup B) was identified among the 234 isolates obtained from the 19 affected flocks. Sheep with footrot healed quickly when treated with a vaccine made from this strain.

The use of an autogenous Dichelobacter nodosus vaccine to eliminate clinical signs of virulent footrot in a sheep flock in Bhutan.

An outbreak of virulent footrot was investigated in a flock of 605 Merino cross-bred sheep in Bhutan. Conventional control methods in the preceding eight years had reduced its prevalence from 36-79% in different components of the flock to about 15% overall. Only one serogroup (B) of Dichelobacter nodosus was identified among 40 isolates cultured from affected sheep. A vaccine prepared from this strain was used in a pilot trial to compare the response of 14 treated and 14 untreated sheep. All affected, vaccinated animals in this trial healed quickly and were protected against re-infection while additional cases developed among untreated sheep during a period favourable for the spread of footrot. The serogroup B vaccine was administered to the whole flock for two successive years. No other footrot treatment was given during these or subsequent years. The whole flock was examined three times, foot by foot, for two years and twice yearly for another two years. When vaccination began there were 88 affected sheep in the flock, an affected sheep being defined as an animal with a foot-score of 2 or greater in one or more feet. There were neither affected sheep in the flock 30 days after the first dose of vaccine nor were any identified in later inspections. Virulent footrot, originating from the farm under investigation, persisted in neighbouring village flocks during this period. It was concluded that whole flock specific D. nodosus vaccination made a major contribution to the elimination of all clinical signs of footrot from the flock of 605 sheep where the condition had previously persisted for 10 years.

The use of melatonin as a vaccine agent.

Molecules with immunomodulatory properties determine the magnitude and quality of immune responses specific for the coadministered antigen. Melatonin is considered a biological-response modifier of the immune system with broad application in veterinary medicine. In seasonally-breeding animals, the indolamine is able to improve reproductive performance. With the purpose of expanding new advantageous roles for melatonin, we investigated the effect of subcutaneous slow-release melatonin implants in the humoral response after a vaccination. We reported here a new feature of melatonin as an adjuvant-like system towards Dichelobacter nodosus (A1 and C serotypes)--the bacterium which cause ovine footrot--the most important cause of lameness in sheep. Antibody titres determined by both agglutination and ELISA techniques were substantially higher and were sustained for a longer duration than non-implanted animals. Remarkably, the effect of melatonin was completely dependent on the presence of aluminium hydroxide. The finding that melatonin enhances a defined immune response in vivo opens new perspectives for the improvement of Th2-biased immune responses by alum adjuvants.

[Selection for disease and epidemic resistance in domestic ruminants and swine by indicator traits, marker and causal genes--a review. Part 2: Special immunogenetics of sheep and goats with particular regard for endoparasitoses, scrapie, foot rot and maedi-visna virus infection]. / Selektion beim domestizierten Wiederkäuer und Schwein auf Krankheits- und Seuchenresistenz mit Hilfe von Indikatormerkmalen, Marker- und Kausalgenen--ein Uberblick. 2. Mitteilung: Spezielle Immungenetik bei Schaf und Ziege unter besonderer Berücksichtigung der Endoparasitosen, Scrapie, Moderhinke und der Maedi-Visna-Virusinfektion.

The introduction of the first part deals with immunogenetic investigations on the field of life-stock. The main chapter is outlined as a tabular overview of current opportunities of the application of indicator traits as well as marker and causal genes in breeding for disease resistance in cattle, sheep, goats and swine. In the discussion of the second part, emphasis was laied on diseases of small ruminants in central and western Europe with special respect of endoparasitoses, scrapie, foot-rot and maedi-visna virus infection. Indicator traits are discussed with respect of their advantages and disadvantages. The rigorous selection on specific traits is connected with an increase of the number of homozygotes. In contrary, pathogens do undergo mutations, thus escaping the host's immune system. Out of this point of view it is advisable, to set on selection very cautiously. The role of technologies of modern immunogenetics is pointed out in respect of constructing disease resistant animals.

Diagnosis of footrot in goats: application of ELISA tests for response to antigens of Dichelobacter nodosus.

Goats are an important natural host for footrot and are infected with Dichelobacter nodosus that have virulence characteristics similar to those of sheep strains. However, the humoral response of goats to D. nodosus antigens and the possibility of a serological diagnosis of footrot in goats have not been studied. With the aim of evaluating a diagnostic ELISA test, we investigated the primary immune response of goats to experimental and natural infection, the memory response in recovered animals, and the transfer and persistence of colostral antibodies in kids. Footrot stimulated the goat's immune system and, as in sheep, under-running lesions were the primary stimulus for production of anti-D. nodosus antibodies. The immune response could be detected in ELISA using either fimbrial or outer membrane protein (KSCN) antigens of D. nodosus. Antibody titres resulting from infection declined quickly after recovery and reached pre-infection levels within 3-4 months. Previously affected animals, however, mounted a memory response when injected with purified D. nodosus antigens. Antibody levels attained after anamnestic challenge were correlated with the maximum levels attained during infection, and were therefore indicative of the infection status. Anti-D. nodosus antibodies were also transferred to kids via colostrum, but these antibodies did not persist and therefore were unlikely to interfere with the diagnostic ELISA after 3 months of age. Though these ELISA tests were highly specific, their sensitivity was rather low. Therefore, they are only suitable for a herd diagnosis of footrot in goats and are dependent on the development of advanced under-running infections in a proportion of affected goats.

Chemotaxis, phagocytosis, and oxidative metabolism in bovine macrophages exposed to a novel interdigital phlegmon (foot rot) lesion isolate, Porphyromonas levii.

OBJECTIVE: To examine the host response toward Porphyromonas levii, by evaluating chemotaxis, phagocytosis, and oxidative burst of bovine macrophages in vitro. SAMPLE POPULATION: Cultured bovine macrophages obtained from monocytes harvested from blood samples of 15 Holstein steers. Porphyromonas levii was isolated from the foot rot lesion of an acutely affected feedlot steer. PROCEDURE: Monocytes were cultured for macrophage differentiation over 7 days. Porphyromonas levii was cultured in strict anaerobic conditions for experimentation. Chemotaxis was evaluated by quantifying macrophage migration toward P. levii in Boyden chambers. Phagocytosis was assessed by quantification of macrophages engulfing P. levii following incubation with or without anti-P. levii serum or purified IgG. Oxidative burst was measured by use of the nitroblue tetrazolium reduction assay. RESULTS: Chemotaxis toward P. levii was not significantly different from control values at any of the tested bacterial concentrations. Phagocytosis of P. levii was approximately 10% at a 10:1 bacterium to macrophage ratio and did not change significantly over time. When higher proportions of P. levii were tested for phagocytosis, the 1,000:1 bacterium to macrophage ratio had a significant increase, compared with the 10:1 test group. Opsonization of P. levii with high-titeranti-P. levii serum or anti-P. levii IgG produced a significant increase in macrophage phagocytosis. Oxidative production significantly increased compared with control in the 1,000:1 test group only. CONCLUSIONS AND CLINICAL RELEVANCE: Porphyromonas levii may evade host detection by decreased chemotaxis, phagocytosis, and oxidative burst by macrophages. Acquired immunity may be beneficial for clearance of P. levii in foot rot lesions in cattle.

Pilus ELISA and an anamnestic test for the diagnosis of virulent ovine footrot and its application in a disease control program in Nepal.

The immunological memory (anamnestic) responses in sheep recovered from virulent footrot (VFR) can be aroused by subcutaneous injection of outer membrane protein (OMP) antigens of Dichelobacter nodosus. The magnitude of this response is directly correlated to the highest antibody response attained during infection and memory lasts at least a year after recovery from VFR. However, some older animals show non-specific responses to OMP antigens. In this study an evaluation of D. nodosus pilus antigen for the anamnestic diagnosis of footrot in sheep was undertaken. The results indicated that the primary and anamnestic responses to pilus were similar in character to OMP antigen but were highly specific. The sensitivity of the procedure for detection of sheep with a history of VFR was approximately 80%. A low proportion of sheep with mild lesions due to virulent strains of D. nodosus reacted to anamnestic challenge. Anamnestic challenge with 10 microg pilus was used in a VFR surveillance program in migratory sheep flocks in Nepal. Conventional diagnostic methods could not be applied during the disease transmission periods in these flocks because of their migration to alpine pastures far away from human habitation. The results supported clinical and bacteriological findings suggesting that virulent strains of D. nodosus have apparently been eliminated from these flocks in Nepal.

Vaccination against ovine footrot using a live bacterial vector to deliver basic protease antigen.

A strain of Corynebacterium pseudotuberculosis, designated Toxminus, that has been rationally attenuated by deletion of the phospholipase D gene, is being developed as a live vaccine vector for the delivery of veterinary vaccine antigens. In the present study a recombinant form of the basic protease gene of the ovine footrot causative bacterium, Dichelobacter nodosus, was introduced into the vector strain using the high copy number plasmid pEP2. This strain secreted the basic protease protein. Vaccination trials in sheep with the recombinant strain demonstrated that, although an IgG immune response was elicited, the animals were not protected from footrot following artificial challenge under pen conditions. Although the sheep were not protected there was evidence that the progression of the disease was slowed.