Differences between breeds of sheep in their responses to Bacteroides nodosus vaccines

In a field experiment on irrigated pasture, sheep of several breeds were vaccinated twice, subcutaneously, in the upper neck, with Bacteroides nodosus vaccine containing either depiliated cells (DC vaccine), or whole, piliated cells (WC vaccine) and the responses were measured over the following 14 weeks. DC vaccine was as effective as WC vaccine in protecting against the development of foot-rot under conditions of moderate challenge, although the WC vaccine induced significantly higher pilus agglutinating antibody titres. Foot-rot developed in significantly more vaccinated Merinos (Peppin and Saxon strains) than in Romney Marsh, Dorset Horn or Border Leicester breeds. Agglutinating antibody titres after WC vaccination were significantly lower in the Peppin Merino than in the other sheep for the first 6 weeks, while after DC vaccination the titres remained elevated longer in the Border Leicester and Saxon Merino and were significantly higher from 6 weeks onwards. Reactions at the inoculation sites were generally larger in the British breeds than in the Merinos and among the former the reactions were largest, most numerous and most frequently discharged their contents in the Dorset Horn. Bodyweight gains in all vaccinated sheep were initially reduced, compared with controls, but the differences were no longer significant after the eighth week.     

Colostral transfer of Bacteroides nodosus antibodies in sheep.

Ovine contagious foot rot may cause lameness in sheep, resulting in decreased wool growth and low weight gain. Affected neonatal lambs are difficult to treat, and treatment is labor intensive; thus, a method of prevention is warranted. Vaccination of ewes with a multivalent vaccine in an oil adjuvant induced development of antibody to the somatic O antigen of Bacteroides nodosus, and this antibody was detected in serum of newborn lambs after consumption of colostrum from the vaccinated ewes. Antibody titers were determined in 48 unvaccinated ewe/lamb pairs, and in 50 once-vaccinated and 78 twice-vaccinated pairs. Serum and colostrum O-agglutinin titers to B nodosus were determined by a microtitration agglutination test. Lambs from vaccinated ewes had significantly (P less than 0.05) higher O-agglutinin titers than those from unvaccinated ewes, and double vaccination of ewes resulted in the highest potentially protective titers (greater than 1:2,400) in ewes and lambs.     

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.     

Effect of adjuvants on antibody responses of sheep immunised with recombinant pili from Dichelobacter nodosus

Objective To compare the effects of two oil emulsion adjuvants (incomplete Freunds adjuvant and a proprietary oil adjuvant), DEAE-dextran, L-tyrosine particles and Quil A on the humoral immune responses of sheep immunised with recombinant pili of Dichelobacter nodosus (strain A).
Procedure Antibody titres were studied for up to 32 weeks and were measured by bacterial agglutination and ELISA. The relative avidity of antibodies for pili was determined and the incidence and severity of adverse reactions at the site of injection of vaccines were recorded.
Results The oil emulsion adjuvants and Quil A were more effective than either DEAE-dextran or L-tyrosine at stimulating antibodies in sheep. The incidence and severity of adverse reactions was lower in sheep which received vaccines containing either Quil A or DEAE-dextran than in sheep which received vaccines containing oil emulsion adjuvants. L-tyrosine had no adverse effects.
Conclusion Quil A was as effective as oil adjuvants at stimulating high levels of antibodies against recombinant pili in sheep and had the significant advantage of being less irritant after subcutaneous injection.     

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.     

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.     

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.     

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.     

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.     

Failure of oral zinc therapy to alleviate Bacteroides nodosus infections in cattle and sheep

Cattle and sheep with Bacteroides nodosus infection were treated orally with both high (65 mg Zn/kg and 82 mg Zn/kg) and low (1 mg Zn/kg and 8.6 mg Zn/kg) doses of zinc sulphate respectively. The lower dose rates administered weekly for one month, in the case of cattle, or daily for 2 weeks, in the case of sheep, had no effect either on serum zinc levels or the prevalence or severity of infection in treated animals. High dose rates of zinc (approximately 2.5 g Zn per head per day) were required to elevate serum levels above those normally present in both cattle and sheep. Even these dose rates continued daily for about 2 weeks had no beneficial effect on B. nodosus infection in either species.     

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 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.     

Quantitation by ELISA of pili and sheep antibodies to the pili of Bacteroides nodosus

ELISA systems have been developed to quantitate the isotypic antibody response of sheep naturally infected with B. nodosus isolate 198 or injected with pili from isolate 198 in oil emulsion vaccines. The predominant humoral antibody detected following vaccination was IgG1, with substantially lower amounts of IgG2 and IgM. The antibody response was relatively specific for the pilus antigen from isolate 198. Although weak cross reactivities were detected with antiserums to some other isolates, ELISA IgG antibody titres in excess of 200 offer a tentative identification of the isolates of B. nodosus involved in natural outbreaks of footrot. A related ELISA was also developed to quantitate the amount of pili in cell suspensions and crude preparations of pili used in vaccines.     

Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection

Humoral immune responses of sheep to natural bluetongue virus (BTV) infection were studied on a temporal basis. The temporal development of viral protein-specific IgG was determined by western immunoblotting; virus neutralization and agar gel immunodiffusion (AGID) were conducted for comparative purposes. Prior to the emergence of the arthropod vector and the associated transmission of BTV, virus-neutralizing antibody was absent from all sentinel sheep; 3 sheep had pre-existing AGID antibody and all sheep had IgG, specific for 4 viral proteins, as determined by immunoblotting. Following emergence of the BTV vector, 9 of 11 sheep became infected, as determined by virus isolation, with BTV. All sheep developed virus-neutralizing and AGID antibody. However, only those sheep with a demonstrable viremia experienced an increase in viral protein-specific antibody. Development of viral protein-specific IgG varied with the individual animal and no obvious correlation between a specific response and protective immunity or viral clearance was noted. From a diagnostic viewpoint, the immunoblotting procedure was superior in identifying past exposure to BTV, as compared with neutralization and AGID. In addition, the application of immunoblotting to paired serum samples appeared to be a sensitive indicator of viremia.     

Motility in relation to virulence of Bacteroides nodosus

Fourteen Bacteroides nodosus isolates from footrot lesions of sheep were examined microscopically and all were found to have twitching motility. The mean percentage of cells showing motility was 40% and 9% for virulent and benign strains, respectively. This corresponded with mean agar colony diameters of 17 mm and 7 mm, respectively, for these strains. Two strains of intermediate virulence had values of motility and colony diameter similar to the benign strains. However, the intermediate and the virulent strains produced relatively stable protease compared to the benign strains. All virulent, benign and intermediate strains produced abundant pili. Included for comparison in this study was an avirulent variant strain which was highly motile, formed large colonies and produced stable protease, but showed no pili on electron microscopy. It was concluded that the properties of motility and protease stability may be used to distinguish, in the laboratory, wild-type virulent, benign and intermediate strains of B. nodosus.     

Serum and skin surface antibody responses in merino sheep given three successive inoculations with Dermatophilus congolensis

Three antigens prepared from different phases of the life cycle of Dermatophilus congolensis were used in an enzyme-linked immunosorbent assay to measure serum and skin surface antibody responses in sheep after a first, second and third inoculation with D. congolensis. After the first inoculation, a strong antibody response to the flagella, filament and soluble antigens was detected after 7-21 days in the sera from sheep that were regularly biopsied; the antibody response at the skin surface was detected 28-42 days after inoculation, when the lesions were resolving. Strong anamnestic responses were detected in the serum of sheep that were biopsied and some of the nonbiopsied sheep after the second and third inoculations, but the skin surface antibody response at these times was variable.     

Importance of pilus-associated antigen in Bacteroides nodosus vaccines

The performances of two cellular vaccines, one sparsely piliated and the other well piliated, were compared on irrigated pasture with those of vaccines containing their respective, purified pili. There were statistically significant differences among the four vaccines in the mean numbers of feet per sheep which developed severe foot rot during 27 weeks of exposure. The incidence of severe foot rot was significantly lower in the two pili-vaccinated groups than in the group vaccinated with well-piliated cells, which were in turn substantially more effective than the sparsely piliated. However, the two pili vaccines did not prevent infection of the interdigital skin so that the differences in vaccine performance were much less marked when interdigital skin lesions were included. The number of feet affected by blowfly strike was closely associated with the presence of the most severe lesions and consequently there were far fewer struck feet in the two pili vaccine groups than in the two cellular vaccine groups. Analysis of serum agglutinin titres led to the same assessment of vaccinal efficacy as that derived from the analysis of severe foot rot lesions. Individually the agglutinin response to vaccination was not universally associated with resistance or susceptibility to severe foot rot but mean titres were significantly higher in the two pili vaccine groups than in the highly piliated-cell vaccine group, which were all in turn significantly higher than in the poorly piliated-cell vaccine group.     

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.     

Method of production of and antibody response to an oil adjuvant Bacteroides nodosus bacterin.

A procedure is described for the production of a highly concentrated Bacteroides nodosus bacterin. An oil adjuvant bacterin which elicited the best antibody response in rabbits produced a response in sheep which lasted for at least 6 months.