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.     

Comparison of alum-absorbed or non-alum-absorbed oil emulsion vaccines containing either pilate or non-pilate Bacteroides nodosus cells in inducing and maintaining resistance of sheep to experimental foot rot

Highly pilate (P) or non-pilate (NP) cells of Bacteroides nodosus were compounded into oil emulsion (O) either with or without prior absorption onto alum (A). The abilities of these four preparations (referred to as PAO, NPAO, PO and NPO vaccines) to stimulate antibody production and to protect sheep from foot rot were compared. Two injections of PAO vaccine protected sheep against homologous challenge 12 weeks after the second dose by PO, NPO and NPAO vaccines were less effective. Sheep were protected against homologous challenge for 14 weeks after a single dose of PAO vaccine and for 22 weeks after three doses; an ameliorative effect was still evident 40 weeks after the third dose. Protection against challenge with two heterologous strains was demonstrated at six weeks after three doses of vaccine. A numerical system of scoring the lesions also confirmed that foot rot in vaccinated sheep challenged outside the 'protective' period of the vaccine was somewhat less severe than in controls. PAO vaccine induced much higher and more persistent titres of agglutinins than the other vaccines tested. There was a relationship between agglutinin titres and resistance to homologous challenge.     

坏死杆菌白细胞毒素作为腐蹄病亚单位疫苗候选抗原的研究前景

腐蹄病（foot rot）是侵害反刍动物趾间皮肤及深层软组织为主的,严重影响奶牛生产性能和产奶质量的一种常见疾病。由于传统的灭活菌苗具有免疫效果差、副反应严重及大量生产困难等缺点,使腐蹄病基因工程疫苗的研究成为热点。笔者对坏死杆菌白细胞毒素作为腐蹄病亚单位疫苗候选抗原研究的最新进展进行综述,希望为腐蹄病亚单位疫苗的研究提供参考。     

Cross-protection from Bacteroides nodosus vaccines and the interaction of pili and adjuvants

The effects of vaccination of Merino sheep with the purified pili or the whole cells of Bacteroides nodosus strain 198, either in oil or alum-oil adjuvant, on the severity of foot-rot induced with the homologous strain (198) and a heterologous strain (217) were determined in a field experiment, on flood irrigated pasture. The efficacy of the whole cell vaccines was comparable to that of purified pili vaccines, against homologous challenge, when both had a similar content of pilus antigen although the purified pili vaccines induced significantly greater homologous pilus agglutinating antibody titres than the whole cell vaccines. However, against heterologous challenge, the whole cell vaccines in oil (CO) or alum-oil (CAO) provided significantly greater protection than a purified pili-in-oil (PPO) vaccine, the number of severely affected feet in sheep vaccinated with PPO being similar to that of the unvaccinated group. The group vaccinated with purified pili in alum-oil (PPAO) was intermediate between these two extremes. The superior performance of the PPAO in comparison to the PPO vaccine, against heterologous challenge, was associated with significantly higher mean ELISA titres to the outer membrane complex. Western blot analyses implicated a role in cross-protection for outer membrane proteins, in particular a protein Mr 78,000. The PPO vaccine produced fewer, smaller and less persistent vaccination reactions at the inoculation sites than did the other vaccines. Bodyweight gains in the period prior to challenge were much lower for the groups vaccinated with CO and CAO than for the controls and those vaccinated with purified pili, due presumably to the larger vaccination reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusobacterium necrophorum and Bacteroides melaninogenicus as etiologic agents of foot rot in cattle.

Fusobacterium necrophorum (Sphaerophorus necrophorus) and Bacteroides melaninogenicus were the predominant bacteria isolated from biopsy specimens of lesions in cattle affected with foot rot. Mixed inoculums of the 2 bacteria, applied to the scarified interdigital skin or inoculated intradermally into the interdigital skin of test cattle, induced typical lesions of foot rot. Both bacteria were reisolated in large numbers from the induced lesions.     

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.     

Efficacy of amoxicillin trihydrate for the treatment of experimentally induced foot rot in cattle

Twenty holstein heifers were intradermally inoculated in the interdigital skin with a suspension containing Fusobacterium necrophorum and Bacteroides melaninogenicus to induce acute foot rot. Lesions, lameness, and swelling were evaluated during the study, using a subjective scoring system. Rectal temperature, species and number of bacteria isolated, and change in body weight were monitored throughout the study. Ten heifers (treated) were given amoxicillin trihydrate (10 mg/kg of body weight, IM) for 5 days, beginning at the onset of lameness. The remaining 10 heifers (controls) were given physiologic saline solution IM. Treated heifers had less severe lesions and greater weight gain than did control heifers. Rectal temperatures of treated heifers did not differ significantly from those of control heifers. It was concluded that administration of amoxicillin trihydrate early in the course of acute foot rot may reduce the severity of lesions associated with foot rot in cattle.     

The protective efficacy of pili from different strains of Moraxella bovis within the same serogroup against infectious bovine keratoconjunctivitis.

Three groups of ten calves were each immunised with a total of 400 micrograms pili prepared from three separate strains of Moraxella bovis in Alhydrogel-oil adjuvant as two divided, equal doses 21 days apart. Groups 1 and 2 each received a monovalent vaccine made from strain 4L and S276R respectively, which belonged to pili serogroup A. Group 3 received vaccine made from pili of strain Maff1, belonging to serogroup F. A further group of ten calves served as non-vaccinated controls. Calves in groups 1 and 2 had developed serogroup A-specific antibody and those in group 3 developed serogroup F-specific antibody, and some evidence of cross-reacting antibody was also detected when measured by an agglutination test using formalin-killed piliated cells of serogroup A strain 4L. Although antibody titres measured against purified pili by ELISA were highest with homologous serogroup antigens, cross-reactive titres to shared epitopes of M. bovis pili were also detected by this method. Ocular challenge of the 40 calves with virulent M. bovis of serogroup A strain S276R was carried out 14 days after the second vaccine dose. All non-vaccinated calves developed infectious bovine keratoconjunctivitis (IBK). The percentage protection in groups 1 (strain 4L) and 2 (strain S276R) was 60% and 80% respectively (P less than 0.05), with mean lesion scores of 0.7 and 0.3 out of a possible 6.0. The percentage protection of calves in group 3 (strain Maff1) was only 30%, with a mean lesion score of 1.4 compared with 2.2 for non-vaccinated controls. The present findings, together with other evidence indicating that immunity to IBK is serogroup-specific, suggest that inclusion of pili from one representative strain from each of the seven Australian and British serogroups in a polyvalent, subunit vaccine should effectively protect the majority of cattle against IBK caused by most field strains of M. bovis encountered in Australia and the United Kingdom.     

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.     

Pathogenicity and immunogenicity of piliated and nonpiliated phases of Moraxella bovis in calves

Pathogenicity evaluations of Moraxella bovis strain EPP 63 grown in the piliated and nonpiliated phase indicated that the organism grown in the piliated phase induced clinical keratoconjunctivitis, whereas the organism grown in the nonpiliated phase did not induce disease under identical challenge conditions. Calves inoculated with culture grown in the piliated phase developed significantly (P less than 0.01) higher lesion scores than did calves inoculated with culture grown in the nonpiliated phase. Vaccination/challenge exposure evaluations indicated that calves immunized with vaccine prepared from piliated culture had significantly lower lesion scores than did control calves (P less than 0.001) or calves immunized with vaccine prepared from nonpiliated culture (P less than 0.05). Similarly, calves immunized with piliated vaccine developed a significantly higher antibody titer against purified pili. Nonvaccinated controls and calves immunized with nonpiliated vaccine did not develop a significant increase in antibody titer. The results indicate that M bovis pili constitute an important factor in the pathogenicity of keratoconjunctivitis and may be used as a vaccine in the control of keratoconjunctivitis in cattle.     

Vaccination against infectious bovine keratoconjunctivitis: protective efficacy and antibody response induced by pili of homologous and heterologous strains of Moraxella bovis

The protective effect of 2 Moraxella bovis pili vaccines against infectious bovine keratoconjunctivitis (IBK) experimentally induced by homologous or heterologous strain challenge with virulent, haemolytic M. bovis strain, Dal 2d, was measured in trials using weaned calves aged 3 to 7 months. Purified pili vaccines were prepared from haemolytic strain Dal 2d, (pilus serogroup IV), and haemolytic strain Epp 63, (pilus serogroup III). Calves were challenged by conjunctival instillation of 1 x 10(9) colony forming units of virulent M. bovis strain Dal 2d 14 days after the second of 2 subcutaneous doses of vaccine. Each consisted of 200 micrograms of pili in alum-oil adjuvant administered at an interval of 21 days. In trial 1 the level of protection against challenge with the homologous strain was 46.7% (p less than 0.01). Small, rapidly resolving lesions of IBK occurred in some vaccinates compared with a larger proportion of severe lesions that required treatment in non-vaccinated calves (p less than 0.025). In trial 2, the level of protection against IBK after exposure of vaccinates to the homologous Dal 2d strain was 72.7%, but no significant level of protection or reduction in the size and duration of lesions was apparent in similarly challenged calves vaccinated with Epp 63 pili when contrasted with susceptible, non-vaccinated controls. No marked reduction in the duration of infection with M. bovis Dal 2d following challenge resulted from vaccination with pili of either of the serogroups III or IV. Rising homologous serum IgG antibody titres to serogroups III and IV pili were recorded in response to vaccination with each antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lameness on milk yield in dairy cows

OBJECTIVE: To examine the relationship between lameness and milk yield in dairy cows. DESIGN: Cohort study. ANIMALS: 531 dairy cows. PROCEDURE: Cows affected with lameness were classified into 1 of 3 groups on the basis of type of diseases or lesions observed, including interdigital phlegmon (foot rot), papillomatous digital dermatitis (foot warts), or claw lesions. Cows not affected with lameness were classified as healthy. From Dairy Herd Improvement Association records, 305-day mature equivalent milk yield data were collected at the end of lactation or when the cow left the herd. Milk yield was compared between cows affected with lameness and healthy cows. RESULTS: 167 (31%) cows were affected with lameness during lactation. Lame cows had claw lesions (60%), papillomatous digital dermatitis (31%), or interdigital phlegmon (9%). Milk yield in lame cows with interdigital phlegmon (mean, 17,122 lb) was significantly less, compared with healthy cows (19,007 lb). CONCLUSIONS AND CLINICAL RELEVANCE: In this herd, interdigital phlegmon was associated with a 10% decrease in milk production. Lame cows with claw lesions or papillomatous digital dermatitis produced less milk than healthy cows, but the difference was not significant.     

Some relationships between spirochaete infections and digital dermatitis in four UK dairy herds

Digital dermatitis, presenting as a focal necrotic dermatitis on the skin of cows' feet adjacent to the interdigital space, has been associated with dermal infection by spirochaetes. Lesions cause severe, transient lameness. An enzyme-linked immunoassay (ELISA) was used to measure IgG(2) antibody titres against three different spirochaetes: USA bovine Treponema isolates 1-9185MED and 2-1498, and a UK strain of Borrelia burgdorferi B31. Logistic models were used to describe lesion prevalence in terms of the antibody titres, herd and milking status, year, lactation number, and yield. The antibody titres and milking status (P<0.001) dominated the logistic fits, with the other variables adding little. Generalised linear models were used to describe the antibody titres in terms of the presence or absence of lesions, herd, year, lactation number, and yield. The presence or absence of lesions had the most significant effect for all three antibodies (P<0.001). Neither the herd effects nor the effects due to lactation number or yield showed consistent patterns.     

A field trial to assess the therapeutic and prophylactic effect of a foot rot vaccine in sheep

An 18-week field trial was conducted on a sheep ranch to evaluate the therapeutic and prophylactic effect of a commercial foot rot vaccine. Two hundred sheep were included in the study, 100 with detectable foot rot lesions and 100 without. Approximately 50 sheep from each group were selected randomly and vaccinated twice against foot rot at a 6-week interval in the late spring 1984; the remaining sheep acted as nonvaccinated controls. Therapeutic effect of the vaccine was demonstrated by a cure rate of 53% in vaccinated, foot rot-affected sheep vs a cure rate of 19% in nonvaccinated, foot rot-affected sheep. Prophylactic effect of the vaccine was demonstrated by a foot rot prevalence at the end of the 18-week period of 9% for vaccinated sheep vs 53% for nonvaccinated sheep. Associations of foot rot lesions and vaccination with body condition were found to be significant, as was the association between foot rot lesions and mortality.     

Humoral responses to a multivalent vaccine in age-matched lambs of different bodyweight and nutrition

K-agglutination, pilus-enzyme-linked immunosorbent assay (ELISA) and outer membrane protein-ELISAs were used to assess humoral responses after vaccination with a commercial, multivalent, ovine foot rot vaccine (Dichelobacter nodosus whole cells) in three groups of nine-month-old lambs of markedly different bodyweight, nutritional history and dietary protein supply. Mean bodyweights of lambs in low (L), medium (M) and high (H) bodyweight/nutrition groups were 22, 32 and 48 kg, respectively, at the time of vaccination. Few significant differences in humoral responses to vaccine antigens were found between groups. However, lambs in group H tended to have lower levels of antibody to a greater number of component antigens than did lambs in the other groups. These results suggest that low bodyweight due to poor nutrition is unlikely to affect the response of sheep to multivalent foot rot vaccines.     

Cultural characteristics and virulence of strains of Fusobacterium necrophorum isolated from the feet of cattle and sheep

Sixty-one isolates of Fusobacterium necrophorum were recovered for study. Thirty-one were obtained from lesions of foot abscess in cattle (25) and sheep (6), 28 were from interdigital lesions in cattle and 2 were from the normal interdigital skin of cattle. The majority of isolates from lesions of foot abscess were virulent, belonged to biotype AB (Fievez 1963), produced flat, irregular shaped, greyish colonies and haemolysis on blood agar, and grew as turbid filamentous suspensions in liquid media. They produced a soluble exotoxin, a leucocidin, and were pathogenic for cattle and mice. Virulent isolates also produced a haemolysin which most readily lysed bovine, equine and chicken erythrocytes; those from sheep were less susceptible while those of rabbit and pig were the most resistant. Isolates recovered from lesions of the feet not classified as foot abscess and from clinically normal feet were predominantly of the B biotype and caused few experimental lesions, produced convex, round, yellow colonies, flocculated and sedimented while growing in liquid medium and produced little or no haemolysin or leucocidin. Routine differentiation between virulent and non-virulent bovine isolates of F. necrophorum could be achieved by assessing the colour, morphology, and degree of haemolytic activity of colonies grown on blood agar.     

Genetic-associated resistance to foot rot in selected Targhee sheep

Three Targhee rams obtained from the Ohio Agricultural and Experimental Station had been identified as foot rot resistant on the basis of results of challenge exposure. In the first breeding trial, when rams were bred to 20 foot rot-susceptible ewes, the percentages of foot rot-resistant offspring from the 3 foot rot-resistant rams were 68, 82, and 100, compared with 55 and 60 for foot rot-resistant offspring from 2 known foot rot-susceptible rams. In the second year, the foot rot-resistant rams were mated with ewes of unknown foot rot status. The foot rot-resistant status of their lambs was compared with that of range-raised lambs whose parents' foot rot status was unknown. During the first year, challenge exposure to the disease consisted of confinement of the lambs in moist or wet pens with sheep affected with the naturally acquired disease. This protocol was repeated for lambs born during the second-year breeding trial. In addition, the right front foot of each lamb was inoculated with a broth culture of Bacteroides nodosus. During the second year, when data that included infected feet from all lambs were analyzed, 41% of the progeny of the foot rot-resistant rams and 17% of the offspring of parents of unknown foot rot status were unaffected by the disease. When the B nodosus-inoculated foot was not included in the analysis, however, 61% of the progeny of the foot rot-resistant rams and 29% of the others were unaffected. The resistance to foot rot undoubtedly is hereditary. The mechanism of resistance may be in the interdigital skin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Whole-bacterial cell enzyme-linked immunosorbent assay for cell-bound Moraxella bovis pili

Infectious bovine keratoconjunctivitis (IBK), caused by Moraxella bovis, is a disease of major importance in cattle industry. M. bovis has several virulence factors among which pili are crucial antigen for the protective capacity of vaccines against this disease. The production of vaccines against IBK therefore requires a reliable technique for cellular piliation level assessment on cells to be included as vaccine components. In this study we describe a specific whole-bacterial cell enzyme-linked immunosorbent assay (bact-ELISA) capable of detecting pili antigen on M. bovis cell surface. A sequential competitive bact-ELISA was developed using highly piliated M. bovis cells as antigen. Samples to be analyzed were allowed to react with anti-pilus serum prior to incubation in wells coated with piliated cells of M. bovis. This assay proved useful for the rapid, sensitive and reproducible evaluation of piliation on M. bovis cells, and represents an important tool for cellular piliation monitoring daburing M. bovis cells production in stirred bioreactors.     

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.     

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.