The occurrence, prevalence and transmission of Bacteroides nodosus infection in cattle

Following reports of findings of ovine foot-rot flora in the feet of cattle, a study was undertaken to determine the prevalence of Bacteroides nodosus infection in the apparently normal cattle population. We found that 34.5 to 74.2 per cent of the animals examined on different farms had B nodosus present in one or more feet. B nodosus was not the most prevalent bacterium observed in smears from cattle. Other Gram negative species including Fusiformis necrophorus and many Gram positive cocci and coccobacilli were also present. Macroscopic lesions in the interdigital skin characterised by erosion and hyperkeratosis were usually associated with the occurrence of B nodosus. B nodosus isolated from cattle induced mild interdigital dermatitis in experimental cattle and sheep and the infection was transmitted to recipient cattle and sheep under field conditions. Virulent foot-rot of sheep was not transmitted to recipient cattle in conditions where the disease spread to susceptible sheep.     

The comparative susceptibility of five breeds of sheep to foot-rot

Five breeds of sheep, Romney Marsh, Dorset Horn, Border Leicester, Peppin Merinos and Saxon Merinos were examined for their susceptibility to foot-rot by exposure to natural transmission of infection on irrigated pasture or by the application of pure cultures of Bacteroides nodosus to each foot in a pen experiment. On pasture, the sheep encountered a moderate challenge and the British breeds were more resistant than Merinos to the development of severe foot-rot. Resistance was manifested by a rapid resolution of relatively benign lesions in the interdigital skin, rather than a reduction in the number of feet affected. However, under more severe challenges with foot-rot in pens, all breeds were equally susceptible. There was little difference between resistant and susceptible sheep in the kinetics and magnitude of their antibacterial immune responses indicating that resistance did not depend on pre-existing antibody or a more rapid induction of antibody production. In each experiment, humoral immune responses against B. nodosus were not greatly elevated until under-running lesions of the hoof developed.     

Differences between strains of Bacteroides nodosus in their effects on the severity of foot-rot, bodyweight and wool growth in Merino sheep

The effects of 3 ovine and one bovine strains of Bacteroides nodosus on the severity of foot-rot, bodyweight and wool growth were compared in Merino sheep in a field experiment. Based on the severity of the induced foot lesions, one strain was classed as virulent (causing underrunning lesions in most feet), one was benign (causing lesions of the interdigital skin only), and 2, including the bovine strain, were of intermediate virulence (causing underrunning lesions in a small proportion of feet). Differences among strains in their effect on foot-rot severity and bodyweight were significant when compared over the whole experimental period, but were not significant at any single time of measurement, because of large differences between replicates. Bodyweight loss and severity of foot-rot caused by the virulent strain were significantly greater than that caused by the benign strain. The intermediate strains lay between these 2 extremes in terms of both bodyweight and foot-rot scores but were not significantly different from either in a statistical sense. Total greasy wool weight did not differ among groups over the whole experiment, but the rate of wool growth during the period when foot lesions were most prevalent and severe was reduced appreciably by the virulent strain and to a lesser extent by the intermediate strains.     

Microbiological study of foot-rot in lambs: isolation, elastolytic activity and antimicrobial susceptibility.

A microbiological study was made of an outbreak of foot-rot in lambs. Elastolytic activity was detected in all 10 Bacteroides nodosus strains isolated from the hooves of 9 affected animals. Among 14 other isolated strictly anaerobic microorganisms belonging to different species or genera only 4 strains showed a low degree of elastolysis. The 14 antimicrobial agents studied effectively inhibited "in vitro" growth of B. nodosus.     

Comparative antigenic analysis of extracellular proteins of Bacteroides nodosus isolated from virulent and benign ovine footrot

Antigens in the extracellular protein (ECP) complexes of Bacteroides nodosus, isolated from sheep with either benign or virulent footrot, were studied by immunoelectrophoresis (IEP). Rabbit antisera against ECP from virulent and benign strains, were used in homologous and heterologous crossed IEP. Four precipitin peaks unique to the virulent strain, and five peaks unique to the benign strain were identified. In an attempt to characterize the different antigens in ECP, rabbit antisera were raised against an outer membrane protein (OMP, mol. wt. 35 000 daltons), pili and various proteases of virulent and benign strains of B. nodosus. No precipitin band was observed when ECP from both B. nodosus strains were reacted against anti-OMP and anti-pilus antisera. However, single precipitin bands unique to one protease from the benign strain and one protease from the virulent strain were identified. The results suggest that specific antigens other than proteases or pili are important in determining whether a B. nodosus isolate is virulent or benign.     

Experimental evaluation of a commercial footrot vaccine against native Canadian strains of Dichelobacter nodosus.

Two serotypes of the anaerobic bacterium Dichelobacter nodosus were used to experimentally infect young sheep resulting in infectious pododermatitis or footrot characteristic of the natural disease in sheep. The specific serotypes of D. nodosus were reisolated from the feet and identified using immunofluorescent microscopy of hoof scrapings. Prior immunization of sheep with a commercially available bacterin containing whole cell preparations of ten strains of D. nodosus resulted in serum IgG reactive to a serotype of D. nodosus common to the vaccine. Immunization also produced serum IgG reactive to a serotype of D. nodosus not incorporated in the vaccine. A less severe infection occurred in the immunized sheep than in the controls regardless of the serotype of bacteria used to infect them. Clinical lameness and lesion severity were milder in sheep infected with the serotype of D. nodosus common to the vaccine. Western blot analysis of sera from convalescent sheep showed cross-reactive antibodies to nonfimbrial cell surface proteins, as well as bacterial lipopolysaccharide. Such cross-reactivity may explain the partial protection seen in animals infected with a serotype distinctive from the ones in the vaccine. Despite the historical emphasis of fimbrial immunogens in ovine footrot this study using a new model of experimental ovine footrot suggests other surface antigens may also be important in protective immunity.     

Extensive diversity in New Zealand Dichelobacter nodosus strains from infected sheep and goats

Footrot is a contagious bacterial disease of ruminants spread by the Gram-negative, anaerobic organism, Dichelobacter nodosus. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. Using the polymerase chain reaction (PCR) to amplify fragments of the fimbrial gene (fimA), D. nodosus was detected in 14 hoof scrapings, sampled from six farming regions within New Zealand. DNA sequencing revealed 15 strains covering eight serogroups on the New Zealand farms. The predominant serogroup was B which contained six strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Eleven out of the 15 D. nodosus strains had fimbriae sequences different to those previously reported and the presence of multiple strains on a single hoof was common (86% samples). Individual sheep from the same farm, or the same paddock, were often infected by a different range of strains, which suggests a host role in mediating footrot infection.     

Purification, characterization, and serologic characteristics of Bacteroides nodosus pili and use of a purified pili vaccine in sheep

Hair-like appendages (pili) were isolated and purified from Bacteroides nodosus, the etiologic agent of foot rot disease in sheep. Microscopic and biochemical analyses indicated that pili from organisms isolated in Australia, New Zealand, and the United States are morphologically and structurally similar. Pili are filamentous assemblies of identical protein subunits. Using specific antisera raised in rabbits against pili, 7 antigenic types were identified. A geographic pattern in the distribution of the pilus serotypes was not evident. In a preliminary vaccine trial, sheep vaccinated with purified pili developed resistance to challenge exposure to B nodosus. Protection was correlated positively with the serum anti-pilus antibody titers.     

Transmission of virulent footrot between sheep and goats

OBJECTIVE: To determine the infectivity of ovine and caprine strains of Dichelobacter nodosus for both sheep and goats. DESIGN: Pen experiments in which 20 sheep and 19 goats were challenged directly with the two strains, and transmission experiments on pasture, using donors infected by experimental challenge. RESULTS: Sheep and goat strains of D nodosus infected both animal species in experimental challenges. Animals so infected transmitted footrot to both sheep and goats on pasture plots. A significantly smaller proportion of goats than sheep was infected when challenged with either strain. The interval between exposure and development of footrot in goats was longer than in sheep when recipient animals were exposed to infected donors on pasture. The disease was less invasive in goats than in sheep. CONCLUSIONS: With the strains of D nodosus used there was no evidence of host specificity. Direct transmission of footrot can occur between sheep and goats in the same environment. There is a need to include goats in ovine footrot eradication programs and vice versa.     

The prevalence of salmonella infection in sheep at slaughter

The prevalence of subclinical Salmonella infection in sheep at slaughter was monitored at two slaughter-houses in the southern part of the North Island over a 17-month period during 1975 and 1976. The crude prevalence rate was 4.7% (96/2027). Prevalence rates were greatest in the 2 and 4-tooth age group, with maximum monthly rates of 30%and 32% during 1975 and 1976 respectively. The seasonal distribution of infections was similar in each age group, prevalence rates being greatest in March, April and May, and least in October, November and December.

Salmonella typhimurium was found in 78% of the infected sheep, and phage-type 1 caused 72% of these infections. Other serotypes isolated were S. bovis-morbificans, S. derby, S. newingion, S. saintpaul, S. anatutn and S. enieritidis. The organisms were all fully sensitive to nine antibiotics. On the grounds that 87% of the infected sheep had salmonellae in their intestinal lymph nodes, it was deemed likely that most of the infected sheep harboured their infections while still on the farms of origin.     

Severity and persistence of footrot in Merino sheep experimentally infected with a protease thermostable strain of Dichelobacter nodosus at five sites

Objective To test the hypothesis that ovine footrot associated with a thermostable protease strain of Dichelobacter nodosus undergoes self cure or is sustained as an annually recurring disease, depending on the environment.
Design and procedure Forty Merino sheep from a single blood line were infected with a protease thermostable strain of D nodosus a t each of five sites in Western Australia. Footrot lesions and microscopic evidence of D nodosus were recorded every fortnight for 2.5 years, supplemented by laboratory culture. Rainfall, soil and air temperature, pasture quantity and composition and soil types were also recorded. Flocks that apparently self cured were relocated to a more favourable site for footrot in the final spring season.
Results The maximum prevalence of feet with clinical footrot lesions was 80.6, 1.3, 14.4, 3.8 and 88.1% at the five sites. Severe footrot occurred for three consecutive spring seasons at one site that had clay loam soil and at least 3500 kg/ha total pasture dry matter annually. However, the infection was asymptomatic for up to 10 weeks between outbreaks. D nodosus was isolated from flocks for 2.5 years at only two sites, although there was microscopic evidence of the organism at other sites in the final year. A thermolabile variant (strain U6) of D nodosus was isolated from the two sites where footrot persisted.
Conclusion Depending on time and location, ovine footrot induced by a protease thermostable strain of D nodosus either self cured or persisted as annual outbreaks interspersed with periods of asymptomatic infection.     

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 Dichelobacter 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.     

Effect of Quil A,a saponin derivative,on the response of sheep to alum precipitatedBacteroides nodosus vaccines

Alum precipitatedBacteroides nodosus vaccines prepared from two antigens with and without the saponin derivative, Quil A, did not induce adverse tissue reactions in sheep injected subcutaneously. Vaccinated, non-infected Merino sheep had higher agglutinin antibody titres when the vaccines included Quil A. Moreover, the recovery rates in vaccinated sheep affected with foot-rot were higher when the vaccines included Quil A.     

Treatment of ovine foot-rot by vaccination with the specific aetiological agent Bacteroides nodosus

Accelerated healing of foot-rot cases after vaccination with Bacteroides nodosus confirms the aetiological role of this organism in the disease. There is a variation in the degree of response. It may range from an increased body weight of affected vaccinated sheep to complete resolution of clinical signs and lesions.The degree of response following vaccination is influenced by the adjuvant incorporated in vaccines. Oil emulsion vaccines were superior to aqueous alum-precipitated preparations. A saponin derivative, Quil A, enhanced the effect of alum-precipitated vaccines in a therapeutic experiment.High agglutinin titres to vaccine strains develop in immunised sheep. Recovery from infection has been demonstrated in animals with high agglutinin titres to the immunising B. nodosus but with low titres to the strain of B. nodosus with which sheep were infected. Antibodies other than those directed against the pili of B. nodosus may be involved in the mediation of the demonstrated therapeutic response.     

Serum antibody responses in sheep after natural infection with Bacteroides nodosus

Soluble outer membrane protein of Bacteroides nodosus extracted with potassium thiocyanate (KSCN) was employed as antigen in an enzyme linked immunosorbent assay (ELISA) to detect serum antibody in sheep naturally infected with a heterologous serogroup. Serum antibody responses in 55 sheep were monitored for 2 years and maximum levels were directly related to the severity of clinical foot lesions. Serum antibody levels rose 2 weeks after foot lesions developed and declined within several months of resolution of lesions. After the first footrot transmission period, antibody levels persisted significantly (P less than 0.001) longer in sheep that did not become affected in the next transmission period compared with sheep in which footrot recurred. Antibody response did not appear to result in resolution of foot lesions. ELISA using KSCN antigen gave similar results to whole cell ELISA where cells prepared from an homologous serogroup were used as antigen. Both these assays were more sensitive than ELISA in which heterologous whole cell antigen was used. Proteins extracted from the outer membrane of B. nodosus, which are known to be immunogenic in natural infection and common to different serogroups of B. nodosus, appear to be useful antigens for serological investigations of ovine footrot.     

Serological classification and virulence determination of Dichelobacter nodosus isolated from Alberta and British Columbia sheep.

Ovine footrot is a contagious disease of sheep that occurs in temperature climates. It is caused by the strict anaerobe, Dichelobacter nodosus. Benign and virulent organisms are differentiated according to serotype and protease production. This study was conducted to identify the presence of virulent serotypes of D. nodosus in sheep flocks in Alberta and British Columbia. Dichelobacter nodosus was detected in lame sheep from 11 of 15 (73%) flocks in Alberta and in 4 of 5 (80%) British Columbia flocks. It was recovered from 57 of 107 (53%) lame sheep. In Alberta, 4 distinct serotypes were isolated from the 11 positive flocks while in British Columbia a total of 6 different serotypes were isolated. One British Columbia isolate could not be classified into existing serotypes. Of the 19 field strains tested, all but 3 were defined as virulent based upon the rapid rise in protease activity in vitro which was maintained between 3 and 5 d. The knowledge of the serotype and virulence of the D. nodosus isolated from affected animals can assist in the control and prevention of ovine footrot.     

Fusobacterium necrophorum,and not Dichelobacter nodosus,is associated with equine hoof thrush

The aim of this study was to determine which of the two species, Fusobacterium necrophorum or Dichelobacter nodosus, are associated with hoof thrush in horses. Fourteen hoof samples, collected from eight horses with thrush and 14 samples collected from eight horses with healthy hooves, were examined for the presence of F. necrophorum, Fusobacterium equinum and D. nodosus. Only isolates with phenotypic characteristics representing Fusobacterium could be cultured. Total DNA extracted from the 28 hoof samples was amplified by using DNA primers designed from gene lktA, present in F. necrophorum subsp. necrophorum, F. necrophorum subsp. funduliforme and F. equinum, and gene fimA, present in D. nodosus. The lktA gene was amplified from five of the 14 infected hoof samples and from one hoof sample without thrush. The DNA sequence of the amplified ltkA gene was identical to the lktA gene of the type strain of F. necrophorum (GenBank accession number AF312861). The isolates were phenotypically differentiated from F. equinum. No DNA was amplified using the fimA primer set, suggesting that F. necrophorum, and not D. nodosus, is associated with equine hoof thrush. Hoof thrush in horses is thus caused by F. necrophorum in the absence D. nodosus. This is different from footrot in sheep, goats, cattle and pigs, which is caused by the synergistic action of F. necrophorum and D. nodosus.     

Novel fimbrial subunit genes of Dichelobacter nodosus: recombination in vivo or in vitro?

Polymerase chain reaction (PCR) was used to amplify the variable region of the fimbrial subunit encoding gene (fimA) of Dichelobacter nodosus from sheep and goats infected with footrot. Two amplimers (designated X and Y) generated single-strand conformation polymorphism (SSCP) patterns different to those of previously identified serogroups and serotypes. DNA sequencing revealed that these two fragments were novel. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1, whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype F1 and the upstream primer specific for serotype M or E1, generated PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artifacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It, therefore, seems more likely that fragments X and Y reflect genuine fimA genes of D. nodosus which have resulted from in vivo DNA recombination rather than from a PCR recombination artifact.     

Prevalence of footrot in Swedish slaughter lambs

Background

Footrot is a world-wide contagious disease in sheep and goats. It is an infection of the epidermis of the interdigital skin, and the germinal layers of the horn tissue of the feet. The first case of footrot in Swedish sheep was diagnosed in 2004. Due to difficulties in distinguishing benign footrot from early cases of virulent footrot and because there is no possibility for virulence testing of strains of Dichelobacter nodosus in Sweden, the diagnosis is based of the presence or absence of clinical signs of footrot in sheep flocks. Ever since the first diagnosed case the Swedish Animal Health Service has worked intensively to stop the spread of infection and control the disease at flock level. However, to continue this work effectively it is important to have knowledge about the distribution of the disease both nationally and regionally. Therefore, the aims of this study were to estimate the prevalence of footrot in Swedish lambs at abattoirs and to assess the geographical distribution of the disease.

Methods

A prevalence study on footrot in Swedish lambs was performed by visual examination of 2000 feet from 500 lambs submitted from six slaughter houses. Each foot was scored according to a 0 to 5 scoring system, where feet with score ≥2 were defined as having footrot. Moreover, samples from feet with footrot were examined for Dichelobacter nodosus by culture and PCR.

Results

The prevalence of footrot at the individual sheep level was 5.8%, and Dichelobacter nodosus was found by culture and PCR in 83% and 97% of the samples from feet with footrot, respectively. Some minor differences in geographical distribution of footrot were found in this study.

Conclusions

In a national context, the findings indicate that footrot is fairly common in Swedish slaughter lambs, and should be regarded seriously.