Protection of sheep against experimental footrot by vaccination with pili purified from Bacteroides nodosus

Merino sheep vaccinated with either whole Bacteroides nodosus organisms, a crude surface antigen preparation or highly purified pili (>99% homogeneity) in oil adjuvant, developed significant resistance to artificial footrot infection when compared with unvaccinated control sheep inoculated with saline-in-oil emulsion (Freund;s incomplete adjuvant) alone. The pili-vaccinated sheep generally had higher K-agglutinating antibody titres than sheep vaccinated with whole B. nodosus. These results confirmed the role of B. nodosus pilus protein both as a protective antigen and the K-agglutinogen. Vaccines prepared with Freund;s incomplete adjuvant containing either purified pili, crude pili or B. nodosus whole cells did not produce significantly different injection-site reactions.     

Protection of sheep against experimental footrot by vaccination with pili purified from Bacteroides nodosus

Merino sheep vaccinated with either whole Bucteroides nodosus organisms, a crude surface antigen preparation or highly purified pili (>99% homogeneity) in oil adjuvant, developed significant resistance to artificial footrot infection when compared with unvaccinated control sheep inoculated with saline-in-oil emulsion (Freund's incomplete adjuvant) alone. The pili-vaccinated sheep generally had higher K-agglutinating antibody titres than sheep vaccinated with whole B. nodosus. These results confirmed the role of B. nodosus pilus protein both as a protective antigen and the K-agglutinogen.

Vaccines prepared with Freund's incomplete adjuvant containing either purified pili, crude pili or B. nodosus whole cells did not produce significantly different injection-site reactions.     

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.     

Duration of resistance to experimental footrot infection in Romney and Merino sheep vaccinated with Bacteroides nodosus oil adjuvant vaccine

Groups of 10-12 Romney and Merino wethers were challenged simultaneously with homologous experimental footrot infection after having received the second of 2 doses of Bacteroides nodosus (strain 198) vaccine 0, 4, 8, 12, 16 and 20 weeks previously. Inoculations were carried out 28 days apart and unvaccinated sheep of both breeds were challenged as controls. Most Romneys that had been vaccinated up to 16 weeks prior to challenge were resistant to footrot whereas 8 of 10 controls were susceptible. This resistance was lost by about 20 weeks after vaccination. By contrast, protection against challenge in vaccinated Merinos lasted only about 4-5 weeks, although residual benefits of vaccination were apparent after longer intervals from the reduced number and severity of foot lesions among vaccinates compared with controls. Agglutinin titres, which did not differ markedly after similar intervals between Romneys and Merinos, reached maximum levels between 4 and 8 weeks after the second vaccine dose and subsequently declined. Although peak titres were generally recorded at the time of maximum protection in Merinos, the relationship between agglutinin levels and resistance in the Romneys was ill-defined.     

Field trials with a killed, nine-strain, oil adjuvanted Bacteroides nodosus footrot vaccine in sheep

Field trials were set up on five properties to determine the cure and protection rates of a killed, nine-strain, oil adjuvanted Bacteriodes nodosus footrot vaccine, under natural conditions. Foot infections were scored at the time of the first vaccination and at approximately four weeks and ten weeks later. Cure rates were 34-47% for vaccinated sheep, and 18-47% for untreated sheep. Using the Australian Licensing Authorities calculation, the cure rates for the vaccine ranged from -40% to 44%. A lack of suitable conditions for spread of footrot meant that protection rates could not be determined.     

Effect of pilus dose and type of Freund's adjuvant on the antibody and protective responses of vaccinated sheep to Bacteroides nodosus

Groups of sheep were immunised twice with one or other of six vaccines consisting of purified pili from Bacteroides nodosus at three dose levels (10, 38 and 154 micrograms) and emulsified with either complete (CFA) or incomplete Freund's adjuvant (IFA). Beginning one month after vaccination the sheep were homologously challenged on irrigated pasture, with naturally transmitted foot rot for a period of 26 weeks. Statistical analyses of the number of feet per sheep with severe foot rot demonstrated that there was a significant effect of vaccinal dose but neither an adjuvant effect nor an interaction between dose and adjuvant. Similar conclusions were reached when the titres of antipilus agglutinins in the serum were analysed. By both criteria the responses to doses of 154 and 38 micrograms of pili were significantly better than to 10 micrograms, but not significantly different from each other. The IFA vaccines caused less reaction at the sites of injection than the CFA vaccines and within the former the vaccines containing 10 and 38 micrograms pilus produced less reaction than those containing 154 micrograms. Hence a vaccine containing 38 micrograms of purified pili in IFA is nearly optimal for homologous protection against severe foot rot and is acceptable in terms of the reaction at the injection site.     

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.     

Efficacy of a multivalent Bacteroides nodosus vaccine against foot rot in sheep in Britain

A multivalent Bacteroides nodosus foot rot vaccine was tested by comparing the prevalence of foot rot in 317 vaccinated ewes and lambs with the prevalence in 422 unvaccinated control sheep. The vaccine conferred statistically significant protection against foot rot. The vaccine also demonstrated a therapeutic effect in animals with foot rot, after one injection.     

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.     

The effect of time between doses on serological response to a recombinant multivalent pilus vaccine against footrot in sheep

SUMMARY The response of sheep to a recombinant multivalent footrot vaccine containing pilus antigens was examined after the administration of two doses of vaccine at Intervals ranging from 2 to 52 weeks. Agglutinating antibody titres were measured 3 weeks after the second vaccination and showed that lengthening of the interdose interval results in higher agglutinin titres. The capability of sheep to mount an increasingly strong immune response as the interval between doses is increased provides an opportunity to maximise the usefulness of vaccination by administering the first dose well before an expected footrot transmission period. This advantage of increasing the interdose interval has not been reported for traditional, whole-cell footrot vaccines, and use of the new pilus vaccine in this manner may improve prospects for disease control. Furthermore, sheep given a third dose either 6 or 12 months after their initial two-dose vaccination program achieved significantly higher titres than those elicited after the second dose, suggesting the likelihood of further improvement in disease control in successive seasons.     

Generation, maintenance and reactivity of ovine T-lymphocyte clones derived from sheep immunized with pili from Bacteroides nodosus

Peripheral blood leucocytes (PBL) from sheep immunized with pilus protein purified from Bacteroides nodosus serogroup A were cultivated in vitro and cloned in the presence of the specific antigen and autologous antigen-presenting cells (APC). The efficiency of cloning was enhanced by high proliferative responses to pili during the initial week of cultivation, and the provision of recombinant human interleukin-2 (rec-IL-2). After three passages at weekly intervals, bulk cultures of PBL and cloned T-lymphocytes were greater than 99% CD4+, CD8-, sIg-, i.e. the characteristic phenotype of helper T-lymphocytes. Cloned T-lymphocytes were devoid of allo-reactivity, and were restricted by class II antigens of the major histocompatibility complex (MHC). Both bulk PBL and cloned T-lymphocytes exhibited similar patterns of reactivity against pili from different serogroups of B. nodosus and the T-lymphocytes reacted to three of six peptides synthesized from the amino-acid sequence of pilus from serogroup A. Although clones of T-lymphocytes could retain antigen specificity for up to 2 months of cultivation, several attempts to recover clones with specific reactivity after storage in liquid nitrogen were unsuccessful.     

Comparative in vitro susceptibility of Bacteroides and Fusobacterium isolated from footrot in sheep to 28 antimicrobial agents

The agar dilution method was used to determine the bacteriostatic activity of 28 antimicrobial agents against 141 strains to the genus Bacteroides and 29 strains from the genus Fusobacterium. All organisms were isolated from clinical cases of ovine footrot. The strains were isolated from 125 Merino sheep, over a period of 2 years, from January 1987 to December 1988. The three ureidopenicillins studied (azlocillin, mezlocillin and piperacillin) proved to be the most effective antimicrobial agents. Chloramphenicol, metronidazole and tinidazole effectively inhibited the growth of Bacteroides spp., while phosphomycin was active against Fusobacterium spp.     

The role of elastase in the differentiation of Bacteroides nodosus infections in sheep and cattle

Eighty-seven Bacteroides nodosus isolates were examined for elastase production by clearing of elastin particles in TAS agar medium. These included 54 ovine virulent isolates, 28 ovine benign isolates and five bovine isolates. In addition 22 ovine virulent, 16 ovine benign and two bovine isolates were examined for decline in proteolytic activity over a 13-day period in the degrading proteinase test using hide power-azure as substrate. There was a remarkable correlation between elastase production, relative stability of proteolytic activity in the hide powder-azure test and virulence of B nodosus. Ovine virulent isolates invariably produced elastase whereas ovine benign isolates and bovine isolates were elastase negative. Bovine isolates produced only mild lesions in the feet of challenged sheep.     

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.     

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.     

羊场环境中绵羊腐蹄病病原的检测

以近年来国内外最新研究资料为基础,采用PCR方法对采集于绵羊腐蹄病发病地区水样和土样环境中坏死杆菌和节瘤拟杆菌的存在情况进行检测。结果显示:34份样品中有1份水样为坏死杆菌阳性,其余为阴性,节瘤拟杆菌检测全为阴性。将扩增所得基因KY130428通过BLAST序列比对,发现该基因片段与FJ230831序列同源性为99%,表明样品中存在坏死杆菌。从羊圈周围动物饮水中检测到坏死杆菌的存在,国内尚未见相关报道,本研究旨在摸清羊场环境中腐蹄病病原的分布情况,为该病的防制提供参考依据。