The laboratory culture of Dichelobacter nodosus in a footrot eradication program

SUMMARY As part of a program to eradicate virulent footrot from Western Australia, 2745 isolates of Dichelobacter nodosus were isolated from 5263 specimens from 1883 submissions. The virulence of each isolate was assessed using protease thermostability and isoenzyme zymogram. We describe changes to the materials and methods required to handle these specimens and to reduce the interval between submission and report to 8 days.     

Molecular analysis of Dichelobacter nodosus isolated from footrot in sheep in Malaysia

Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5′GGGCCC3′), SfiI (5′GGCCNNNNNGGCC3′)and SmaI (′5CCCGGG3′) enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.     

A recently introduced Dichelobacter nodosus strain caused an outbreak of footrot in Norway

Background

In 2008, an outbreak of ovine footrot occurred in Norway. Dichelobacter nodosus isolates collected between 2008 and 2011 have been characterised. Isolates defined as virulent by the gelatin gel test (GG-test) were only found in sheep in Rogaland County, where the severe cases of footrot were registered. The majority (96%) of the virulent isolates belonged to serogroup A. It is suspected that they represent a newly introduced strain, and the aim of the present study was to investigate whether they are genetically similar. Sixty-one virulent isolates from sheep and 116 benign isolates from sheep, cattle and goats were included. Four GG-test virulent isolates from Danish sheep were also included. All isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and by PCR for pgr variant determination.

Results

The Norwegian virulent isolates were assigned to 8 pulsotypes (PTs), while the benign isolates were assigned to 66 PTs. Thirty-seven (68.5%) of the 54, virulent, serogroup A isolates belonged to the same PT, and included isolates from 2008 through 2011. Isolates belonging to this PT were defined as the outbreak strain. The remaining virulent serogroup A isolates belonged to 4 PTs differing by ≤3 bands from the outbreak strain. Two virulent, Danish, serogroup A isolates differed by 2 bands from the Norwegian outbreak strain. All but 3 (95%) of the virulent isolates had the pgrA variant while 85% of the benign isolates had the pgrB variant.

Conclusion

This study provides evidence that the footrot outbreak in Norway in 2008 most likely was caused by new introduction and local spread of one virulent D. nodosus strain.     

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.     

Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India

Footrot is a highly contagious and economically important disease of sheep and goats, caused by Dichelobacter nodosus, a slow growing anaerobic Gram-negative rod. The current Australian antigenic classification system, based on variation in the fimbriae, classifies D. nodosus into at least 10 serogroups (A-I and M) and 18 serotypes. This investigation was intended to determine the serological diversity of D. nodosus in this region of Kashmir, India. Exudates of footrot lesions were collected from 24 naturally infected sheep and 42 goats located in the Kashmir valley. Of these 66 samples, 24 yielded evidence of D. nodosus by PCR using 16SrDNA specific primers. Multiplex PCR using serogroup specific primers revealed the presence of serogroup B in all the samples except two, which showed the presence of serogroup E D. nodosus. This study also documents the isolation of D. nodosus and detection of serogroup E for the first time in India.     

The detection and characterisation of Dichelobacter nodosus from cases of ovine footrot in England and Wales

Footrot, caused by the strictly anaerobic bacterium Dichelobacter nodosus, is the most common cause of lameness in sheep in Great Britain but problems exist in association with its diagnosis and control. The fastidious nature of D. nodosus means that complex media and several weeks are required for characterisation. An alternative method to simplify and enhance the detection of D. nodosus in clinical samples is therefore highly desirable. In terms of control, anecdotal evidence from the farming community suggests that the commercially available vaccine, based on Australian isolates of D. nodosus, is not widely employed in this country due to its perceived inefficacy. Seven hundred and six isolates, collected from outbreaks in England and Wales, were therefore used to investigate these issues. A 16S rRNA PCR was adapted to detect D. nodosus in clinical material within 1 day of sampling; a 15% increase in detection compared with culture and less than 1% false negatives were achieved. This represents a major advance in the rapid diagnosis of footrot and will be of great value to practitioners and diagnostic laboratories. Bacterial virulence was tested using protease thermostability and zymogram assays, whilst serogrouping was performed by slide agglutination. All isolates demonstrated virulence patterns previously recorded in Australia and all nine serogroups of D. nodosus (A-I) were represented. Serogroup H was predominant. There was, therefore, no evidence for the presence of novel strains of D. nodosus compared with Australia suggesting the need for further investigation into farmers' views on the use of the commercial vaccine in Great Britain.     

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.     

Occurrence of different strains of Dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections

OBJECTIVE: To investigate the occurrence of S1, U1 and T strains of Dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections. DESIGN: Seventy-seven donor sheep were grazed with 84 recipients for 33 weeks. The donor sheep were Merinos with a history of clinically virulent footrot associated with protease type S1, U1 and T strains of D nodosus that hybridised with gene sequences pJIR314B, pJIR318 and/or pB645-335. The recipient sheep were Merinos with no history of footrot. PROCEDURE: Each fortnight, all feet were examined, their lesion scores were recorded and samples of lesion material were taken for laboratory tests. RESULTS: Eighty-nine percent (299 of 336) of feet of recipient sheep developed new clinical lesions. S1, U1 and T strains of D nodosus were recovered from 58%, 22% and 18%, respectively, of these lesions at a ratio that remained constant during two apparent peaks in footrot transmission. Gene sequences homologous to pJIR314B and pB645-335 were detected in 56% (93 of 166) and 29% (48 of 166), respectively, of S1 strains of D nodosus at a ratio that was not constant during the experiment. CONCLUSIONS: S1 was the dominant protease type of D nodosus in new clinical lesions. The occurrence of S1 strains did not increase relative to U1 and T strains of D nodosus during the experiment. S1, U1 and T strains of D nodosus remained in equilibrium despite changes in environment, genetic types in the population of S1 strains, and host resistance to footrot.     

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.     

Update on ovine footrot in New Zealand: isolation, identification, and characterization of Dichelobacter nodosus strains [corrected

Dichelobacter nodosus, a Gram-negative strict anaerobe, is the essential causative agent of ovine footrot. Despite its worldwide presence, the disease has significant economic impact in those sheep-farming countries with a temperate climate and moderate to high rainfall, such as New Zealand (NZ) and Australia. In this study, we aimed to isolate, identify, and characterize as many D. nodosus strains as possible from NZ farms by using polymerase chain reaction (PCR)-based technology. Understanding the virulence of this bacterium and showing extensive genomic variation in the fimbrial subunit gene (fimA) in different D. nodosus strains was very important to produce serogroup specific and effective vaccine for NZ. More than 100 footrot samples were collected from four different farming regions in NZ. Thousands of primary plates were cultured anaerobically and examined with Gram-staining in order to detect single colonies of D. nodosus. Approximately 500 plates that had potential D. nodosus colonies were subcultured several times to eliminate contaminating colonies until single colonies were obtained. Variable and a part of the conserved regions of the fimbrial subunit gene (fimA) were amplified directly from bacterial DNA extracted from footrot lesions and also from cultured NZ D. nodosus isolates, using the polymerase chain reaction. Different fimA amplimers were analyzed by DNA sequencing. On the basis of DNA sequence analysis, 16 new D. nodosus isolates belonging to eight different serogroups were identified from NZ. These new D. nodosus fimA sequences from NZ were different to previously reported strains and strains used in a commercial vaccine.     

Failure to eradicate ovine footrot associated with Dichelobacter nodosus strain A198 by repeated daily footbathing in zinc sulphate with surfactant

OBJECTIVE: To investigate the effect of repeated daily footbathing in zinc sulphate on virulent ovine footrot associated with S1, U1 and U5 zymogram types of Dichelobacter nodosus, including the highly virulent S1 strain A198. DESIGN: A field trial with experimentally infected sheep. PROCEDURE: At week 0, 50 sheep were infected with D. nodosus strains A198 (S1), C305 (U1), BC3993 (U5) and BC3995 (U5). At weeks 1 and 47, respectively, 169 and 235 uninfected sheep were added. At week 60, sheep were allocated to control and treatment groups each containing 220 sheep. Every 2 or 4 weeks to week 113, feet were inspected, and lesions were scored and sampled. Treatment sheep were footbathed in 15 to 18% (w/v) zinc sulphate with surfactant for 5 consecutive days (10 min per day) during week 61. During week 110, the footbathing protocol was applied again, this time to all surviving treatment and control sheep. RESULTS: BC3993 and A198 were isolated from 57% (162 of 285) and 20% (58 of 285), respectively, of new lesions sampled between weeks 3 and 13, and 21% (57 of 271) and 50% (136 of 271) between weeks 49 and 59. Percentages of new lesions associated with C305 and BC3995 remained constant. During the initial 17 weeks after footbathing at week 61, 90% (75 of 83) and 19% (95 of 490) of lesions in treatment and control sheep, respectively, were score 4 or 5, and 94% (47 of 50) and 38% (33 of 87) of those were associated with A198. CONCLUSIONS: Repeated daily footbathing did not eradicate virulent ovine footrot because strain A198 produced deep, covert lesions that facilitated the survival of D. nodosus.     

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.     

Minimum inhibitory concentrations for 25 selected antimicrobial agents against Dichelobacter nodosus and Fusobacterium strains isolated from footrot in sheep of Portugal and Spain

The agar dilution method was used to determine the inhibitory activity of 25 antimicrobial agents against 69 strains of Dichelobacter nodosus and 108 strains of the genus Fusobacterium, all of which were isolated from 90 clinical cases of ovine footrot between October 1998 and November 2000. In the case of the micro-organisms belonging to the genus Fusobacterium, the six beta-lactams studied (benzyl penicillin, ampicillin, cloxacillin, cefadroxil, cefuroxime and cephalexine) proved to be, in general, the most effective antimicrobial agents. Chloramphenicol, clindamycin and doxycycline were also quite active against Fusobacterium spp. With regard to the 69 strains of D. nodosus tested, the levels of resistance remain low.     

An evaluation of the ability of Dichelobacter nodosus to survive in soil

Background

Dichelobacter nodosus is the causative agent of footrot in sheep. The survival of the bacterium in soil is of importance for the epidemiology of the disease. The investigation evaluates the survival of D. nodosus in soil with and without added hoof powder stored under different temperatures.

Results

An experimental setup was used with bacteriological culture and real-time polymerase chain reaction (PCR), and the results indicate that the bacteria can survive in soil for longer time than previously expected. The survival time was found to be dependent on temperature and the addition of hoof powder to the soil, with the longest survival time estimated to be 24 days in soil samples with hoof powder stored at 5°C.

Conclusion

Our findings indicate that the survival time of D. nodosus and its ability to infect susceptible sheep on pasture under different climatic conditions should be studied further.     

Dichelobacter nodosus serotype M fimbrial subunit gene: implications for serological classification

Dichelobacter nodosus fimbrial subunit gene (fimA) from a serotype M strain (M-SPAHL) was investigated in this study. A primer set targeting the relatively conserved fimA regions and based on the published sequence from Nepalese serogroup M isolates (Nepalese M), failed to amplify the fimA of M-SPAHL. However, when the downstream primer was substituted with a primer that is specific for other serogroups of D. nodosus, the fimA was successfully amplified. Cloning followed by DNA sequencing, revealed that the M-SPAHL fimA was different to the Nepalese M fimA. The predicted amino acid sequence of the M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not Nepalese M. The consequences of serological relatedness and sequence dissimilarity are discussed.