Draft genome sequence of Dichelobacter nodosus JKS-07 serogroup E from India.

OBJECTIVES: Dichelobacter nodosus is an anaerobic bacterium with fastidious growth requirements that is the principal cause of footrot associated with lameness in sheep and goats. In India, D. nodosus serogroups B and E have been recorded as major causes of footrot. Here we report the draft genome sequence of a D. nodosus serogroup E strain (JKS-07) from a case of virulent footrot in India. METHODS: The whole genome of the D. nodosus JKS-07 serogroup E was sequenced using an Illumina HiSeq 2500 platform and was annotated according to functional gene categories. De novo genome assembly and annotation were performed using Perl scripts developed in-house using the Nr/Nt and UniProt databases. RESULTS: The assembled genome is 1389350bp and contains 1301 genes. The genome has 45 tRNAs and 9 rRNAs. The draft genome sequence will provide insight into the various genes and regulators involved in D. nodosus growth and survival. CONCLUSION: Information on the genome of the D. nodosus serogroup E strain is important bearing in mind the fact that both serogroups B and E are associated with virulent footrot, either alone or frequently together. In order to develop an efficacious vaccine against virulent footrot, it is essential to know the serological diversity as well as the virulence status of the D. nodosus strains. Serogroups B and E are potential vaccine candidates to mitigate ovine footrot in India.

Determination of prevalence, serological diversity, and virulence of Dichelobacter nodosus in ovine footrot with identification of its predominant serotype as a potential vaccine candidate in J&K, India.

The aim of this study was to determine the prevalence, serological diversity, and virulence of Dichelobacter nodosus in footrot lesions of sheep and identification of its predominant serotype as a potential vaccine candidate. The overall prevalence of footrot in sheep was 16.19%, and ranged from 13.69 to 19.71%, respectively. A total of 759 flocks with 22,698 sheep were investigated for footrot and 2374 clinical samples were collected from naturally infected sheep exhibiting footrot lesions. Of the 2374 samples collected, 1446 (60.90%) were positive for D. nodosus by polymerase chain reaction (PCR). These positive samples when subjected to serogroup-specific multiplex PCR, 1337 (92.46%) samples carried serogroup B, 247 (17.08%) possessed serogroup E, 86 (5.94%) serogroup I, and one (0.069%) serogroup G of D. nodosus. While mixed infection of serogroups B and E was detected in 127 (8.78%), B and I in 46 (3.18%) and B, E, and I in 26 (1.79%) samples, respectively. The serogroup B of D. nodosus was the predominant (92.47%) serogroup affecting sheep population with footrot followed by serogroup E (19.91%) and serogroup I (4.57%), respectively. Virulent status of D. nodosus strains were confirmed by presence of virulence-specific integrase A (intA) gene and the production of thermostable proteases. The intA gene was detected in 709 (72.79%) samples while gelatin gel test carried out on 246 representative isolates all positive for intA gene produced thermostable proteases, confirming their virulence nature. The PCR-restriction fragment length polymorphism (PCR-RFLP) of whole fimA gene of serogroup B revealed the predominance of serotype B5 (82.97%) of serogroup B. This information suggests that serotype B5 is the predominant serotype of D. nodosus associated with severe footrot lesions in sheep in Jammu & Kashmir (J&K), India. Hence, this serotype can be a potential vaccine candidate for the effective control and treatment of ovine footrot.

Melatonin enhances responsiveness to Dichelobacter nodosus vaccine in sheep and increases peripheral blood CD4 T lymphocytes and IgG-expressing B lymphocytes.

The immunomodulatory functions mediated by melatonin support its use as vaccine adjuvant. Previously, we have demonstrated that melatonin enhances antibody responses in sheep vaccinated against Dichelobacter nodosus. Here, we analyze the effect of melatonin on T and B lymphocyte subsets in peripheral blood of sheep vaccinated against D. nodosus. We also compare the use of melatonin in implants and in injections. Melatonin administration either as implants or by injection produced higher antibody titers against A1 and C serotypes compared to those animals that received only the vaccine. These results support the use of melatonin as an adjuvant in vaccination against D. nodosus. Firstly, melatonin induces higher antibody titer than the vaccine alone, secondly, melatonin increase IgG+ B lymphocytes and CD4+ T lymphocytes in vaccinated sheep. These results suggest that melatonin enhances T CD4 cell activation and subsequently secondary humoral immune responses. Further studies are required to determine the mechanism underlining the immunomodulatory role of melatonin in the context of vaccination.

Comparison of footbathing and vaccination to control ovine footrot in an experimentally infected flock.

OBJECTIVE: Compare footbathing and vaccination for control of footrot during a transmission period in a sheep flock deliberately infected with multiple strains of Dichelobacter nodosus. METHODS: The strains included a known virulent strain, a benign strain and several intermediate strains. The resulting footrot was clinically intermediate. A total of 1450 Polwarth sheep aged 1-3 years were allocated to one of five treatment groups: untreated, weekly walkthrough zinc sulfate footbathing, 1-hour stand-in Footrite® footbathing every 3 weeks, vaccination with a commercial multivalent whole-cell vaccine and vaccination with a novel recombinant DNA fimbrial vaccine. There were four replicates, in four paddocks. RESULTS: Of the untreated animals, 76% had footrot. Footbathing, either weekly or every 3 weeks, restricted the prevalence to 6/283 (2%; 97% effective) and 18/275 (6.5%; 91% effective), respectively. This was significantly lower than the prevalence in either the untreated or vaccinated group (P < 0.001). Weekly footbathing resulted in significantly fewer affected sheep than footbathing for 1 h every 3 weeks (P < 0.05). Vaccination with either whole-cell or recombinant vaccines significantly (P < 0.001) reduced the prevalence ((142/280 (51%; 33% effective), 114/278 (41%; 46% effective) respectively), with the recombinant vaccine superior (P < 0.05) to whole-cell vaccination. Significantly (P < 0.05) fewer 1-year-old sheep had footrot than older sheep. A single Footrite treatment reduced the prevalence to 12% (53/445) compared with a prevalence of 57% (27/47) for untreated sheep (79% effective). CONCLUSION: In this study footbathing was more effective than vaccination at controlling and treating multistrain footrot.

Characterisation of Dichelobacter nodosus and detection of Fusobacterium necrophorum and Treponema spp. in sheep with different clinical manifestations of footrot.

The aim of this study was to determine the proportion of Dichelobacter nodosus, Fusobacterium necrophorum and Treponema spp. in sheep with different clinical manifestations of footrot compared to healthy sheep both at flock and individual level. The second aim was to characterise D. nodosus with respect to virulence, presence of intA gene and the serogroups. Swab samples (n=1000) from footrot-affected (n=10) and healthy flocks (n=10) were analysed for the presence of D. nodosus, F. necrophorum and Treponema spp. by real-time PCR and culturing (D. nodosus only). Dichelobacter nodosus isolates (n=78) and positive swabs (n=474) were analysed by real-time PCR for the aprV2/B2 and the intA genes and by PCR for the fimA gene (isolates only). D. nodosus was more commonly found in flocks affected with footrot than in clinically healthy flocks. A significant association was found between feet with severe footrot lesions and the aprV2 gene and between feet with moderate or no lesions and the aprB2 gene, respectively. F. necrophorum was more commonly found in flocks with footrot lesions than in flocks without lesions. No significant association was found between sheep flocks affected with footrot and findings of Treponema spp. or the intA gene. Benign D. nodosus of six different serogroups was detected in twelve flocks and virulent D. nodosus of serogroup G in one. In conclusion, D. nodosus and F. necrophorum were more commonly found in feet with footrot than in healthy feet. The majority of D. nodosus detected was benign, while virulent D. nodosus was only detected in a single flock.

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.

Outbreak-specific monovalent/bivalent vaccination to control and eradicate virulent ovine footrot.

Footrot is a contagious disease of small ruminants which is caused by the bacterium Dichelobacter nodosus. In its virulent form there are severe economic losses and a very significant animal welfare issue. Sheep and goats can be vaccinated for treatment and prevention of the disease. There are 10 different serogroups of D. nodosus (A-I and M) and immunity is serogroup-specific. When all 10 serogroups are presented together in a vaccine, protection persists for only a few months due to "antigenic competition". Consequently we evaluated the use of sequential monovalent or bivalent vaccines to control/eliminate/eradicate virulent footrot in a longitudinal intervention study on 12 commercial farms in southeast Australia with flock sizes of approximately 1200-4200 sheep. Overall, virulent footrot was eradicated from 4 of the flocks, 2 of which had 2 serogroups, and the others 4 or 5 serogroups. Where there were only 1 or 2 serogroups (3 farms) the clinical response was rapid and dramatic; prevalence was reduced from 45 to 50% before vaccination to 0% (2 farms) or 0.4% (1 farm) after one round of vaccination. In the remaining 9 flocks there were more than 2 serogroups and successive bivalent vaccines were administered leading to eradication of virulent footrot on 2 farms over 4 years and control of the disease on all but 3 of the others. Of the latter farms, 1 discontinued, and 2 initially had poor response to vaccine due to misdiagnosis of serogroup 'M', which was previously unknown in Australia. Control was achieved after administration of a serogroup M vaccine. These results provide clear evidence for control, elimination and eradication of virulent footrot by outbreak-specific vaccination in Australia.

Diagnostic sampling strategies for virulent ovine footrot: simulating detection of Dichelobacter nodosus serogroups for bivalent vaccine formulation.

Dichelobacter nodosus is a slow-growing anaerobic bacterium that is the causative agent of virulent ovine footrot. Vaccination targeted at up to two specific serogroups can eliminate those serogroups from infected flocks, but requires identification of serogroups present in infected flocks. Serogroups can be identified using slide agglutination or polymerase chain reaction (PCR) methods. The objectives of this project were to use stochastic simulation modeling to estimate the efficacy of sampling strategies encompassing 5-40 sheep per flock and 2-4 colonies per sheep, and to compare efficacies based on slide agglutination or multiplex PCR test results. Foot swabs collected from sheep in 12 flocks were used as the basis for a sampling strategy simulation model. None of the evaluated sampling strategies identified the two most common serogroups in the flock, or all serogroups present in the flock, in 95% of iterations. However, a simulated sample of 22 sheep/flock and 2 colonies/sheep resulted in a simulated vaccine that protected 95% of the sheep that could be protected by a single bivalent vaccine, while a sample of 24 sheep/flock and 2 colonies/sheep resulted in a series of simulated bivalent vaccines that protected 95% of diseased infected sheep. The difference in outcome was due to the distribution and frequency of serogroups within certain flocks where some serogroups were uncommon and others dominant. A sampling strategy (>40 sheep/flock, 4 colonies/sheep) that will identify the two most common serogroups in a flock 95% of the time may not be cost effective. Evaluating efficacy based on the expected effect on the flock may be more useful than one which seeks to determine the most common serogroups. These findings are broadly applicable to diseases where more than one strain or type of pathogen may be present and must be represented in a vaccine.

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.

Proteomic profiling of ovine serum by SELDI-TOF MS: optimisation, reproducibility and feasibility of biomarker discovery using routinely collected samples.

The diagnosis of infectious diseases in animals may be enhanced by study of the serum proteome in which myriad components are influenced by physiological and pathological processes. Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) has the capacity to detect known and unknown immunologically relevant molecules in the serum proteome. Optimum combinations of ProteinChip array surfaces, energy absorbing molecules, sample dilutions and instrument settings were determined for spectral generation from whole ovine sera. The coefficient of variation for within and between chip mass/charge and peak intensity were <0.03% and <23%, respectively. There were minor alterations in spectra associated with storage of chips or machine drift. Clotting times of 30 min to 3h did not greatly alter protein spectra although storage of sera at -20 degrees C led to alterations. However, routinely collected serum samples stored at -20 degrees C were useful for identification of biomarkers associated with vaccination with a bacterial antigen. This information will inform future studies on serum proteome profiling in livestock, but independent assessments are recommended for each species.

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.

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.

Effectiveness of different adjuvants in stimulating Dichelobacter nodosus antibody in sheep vaccinated against ovine footrot.

This research consists of an evaluation of the effectiveness of different substances administered as adjuvants in the stimulation of humoral immune response induced by the vaccine composed of strains A1, A2 and C of Dichelobacter nodosus. To do this, a total of 120 Merino sheep were vaccinated and revaccinated. These sheep were selected from a farm located in the region of Extremadura (Spain), and they were divided into 12 groups of 10 animals each. An additional group with 10 sheep was used as control. The immune response (titre of antibodies) was determined by agglutination tests and ELISA. The most pronounced immune response was obtained by the use of Freund's incomplete adjuvant and aluminium hydroxide as adjuvants.

Ovine periodontitis as a potential model for periodontal studies. Cross-sectional analysis of clinical, microbiological, and serum immunological parameters.

OBJECTIVES: : To investigate infection and host immunity patterns in sheep with naturally occurring "broken-mouth" periodontitis. MATERIALS AND METHODS: : Eight periodontally healthy (HS) and eight periodontally diseased ewes (PDS) were selected. Subgingival plaque and sera were collected and examined for evidence of human periodontitis-associated pathogens. Serum IgG titers were measured by ELISA to multiple strains of Porphyromonas gingivalis, Bacteroides forsythus, Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum as well as several purified antigens (cysteine proteases, LPS, K, and fimbriae). RESULTS: : Neither the organism Aa nor antigens to Aa were found in any animal. Most animals were positive for Pg, Bf, and Pi, but DNA probes detected no difference between HS and PDS relative to amounts of pathogens in subgingival plaque. PDS had significantly higher serum IgG titers to all Pg strains, to 50% of Bf strains, to the Pi and Fn strains, and to fimbriae and the two cysteine proteases (p-values ranging from 0.05 to 0.001). Regression analysis demonstrated a significant association between number of teeth lost and serum IgG antibody titers to whole-cell sonicate antigens of P. gingivalis strains (p<0.01) and body weight (p<0.01). CONCLUSIONS: : The presence of pathogens associated with periodontitis was reflected in differences in serum IgG titers between healthy and diseased sheep. This may have influenced animal body weight and might have systemic health and economic consequences. The data suggest that susceptible and non-susceptible sheep can be identified for periodontal research.

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.