Brucella abortus RB51: enhancing vaccine efficacy and developing multivalent vaccines

Brucella abortus vaccine strain RB51 is an attenuated, stable rough mutant that is being used in many countries to control bovine brucellosis. Our earlier study demonstrated that the protective efficacy of strain RB51 can be significantly enhanced by overexpressing Cu–Zn superoxide dismutase (SOD), a homologous protective antigen. We have also previously demonstrated that strain RB51 can be engineered to express heterologous proteins and mice vaccinated with such recombinant RB51 strains develop a strong Th1 type of immune response to the foreign proteins. The present study is aimed at combining these two characteristics to generate new recombinant RB51 vaccines with enhanced abilities to protect against brucellosis and simultaneously able to protect against infections by Mycobacterium spp. We constructed two recombinant RB51 strains, RB51SOD/85A which overexpresses SOD with simultaneous expression of the 85A, a protective protein of Mycobacterium spp., and RB51ESAT which expresses ESAT-6, another protective protein of M. bovis, as a fusion protein with the signal sequence and few additional amino terminal amino acids of SOD. Mice vaccinated with these recombinant strains developed specific immune responses to the mycobacterial proteins and significantly enhanced protection against Brucella challenge compared to the mice vaccinated with strain RB51 alone.     

Injection site lesions of footrot vaccines in sheep

Three trials were conducted to assess the size and frequency of lesions induced by an oil-based and an alum-precipitated aqueous B. nodosus commercial footrot vaccine at two injection sites (neck and brisket) with or without prior skin swabbing with methanol. Animals used were previously unvaccinated and clean and dry at time of vaccination. Trial 1 contained 57 2-tooth Booroola Merino x Coopworth ewes, trial 2 contained 60 mixed-age Corriedale ewes, and trial 3 contained 39 rams of mixed age and sex. Firm nodules or plaques with indurated edges and with a maximum dimension of 1cm or more appeared soon after vaccination and were still very common (50-100% of sheep vaccinated) 5-6 weeks after injection. A variable proportion exhibited a liquefied central area of necrosis and later discharged necrotic material. The oil-based vaccine produced significantly larger lesions than the alum-precipitated aqueous vaccine. Lesion size at the brisket site was significantly larger than the neck site in ewes but not in rams. Skin preparation with methanol had no effect on lesion frequency or size.     

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 pathogenesis of ovine footrot

Ovine footrot is a contagious and debilitating disease that is of major economic significance to the sheep meat and wool industries. The causative bacterium is the gram negative anaerobe Dichelobacter nodosus. Research that has used a classical molecular genetics approach has led to major advances in our understanding of the role of the key virulence factors of D. nodosus in the disease process. D. nodosus strains produce polar type IV fimbriae and extracellular serine proteases. Mutagenesis of the fimbrial subunit gene fimA and the pilT gene, which is required for fimbrial retraction, and subsequent testing of these mutants in sheep virulence trials has shown that type IV fimbriae-mediated twitching motility is essential for virulence. The extracellular protease genes aprV2, aprV5 and bprV have also been mutated. Analysis of these mutants has shown that ArpV5 is the major extracellular protease and that AprV2 is the thermostable protease that is responsible for the extracellular elastase activity. Structural analysis of AprV2 has revealed that it contains several novel loops, one of which appears to act as an exosite that may modulate substrate accessibility. Finally, virulence experiments in sheep have shown that the AprV2 protease is required for virulence.     

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.     

The effect of dissociation of Bacteroides nodosus pili on their efficacy as a protective antigen against ovine footrot.

Previous studies have shown that pili from homologous Bacteroides nodosus provide protective immunity in sheep against footrot, whereas denatured pilin subunits are ineffective. The aim of the present study was to examine whether pili that were dissociated into pilin subunits under less vigorous, non-denaturing treatment conditions, would provide an effective level of protective immunity. Using the techniques of gel permeation chromatography, light scattering and susceptibility to proteolysis as measures of disruption, it was shown that pili were dissociated either by the neutral detergents n-octyl-beta-D-glucopyranoside (NOG) and Tween 80 or by lowering the pH with 1 M phosphoric acid to pH 2.2. Circular dichroic spectra indicated, however that the samples were not denatured by these treatments. Electron microscopic monitoring of detergent dissociated material following exhaustive dialysis showed the presence of protein-detergent micelles and "in-line" aggregates which gave the appearance of short fibres. Within these monitored preparations, there was no evidence of native undissociated pili. Pili dissociated by NOG or acid were tested in protection trials and shown to provide protective immunity, although agglutination titres of serum taken from the vaccinated sheep were significantly lower than those of animals inoculated with intact pili.     

Impact of footrot vaccination and antibiotic therapy on footrot and contagious ovine digital dermatitis

Footrot and contagious ovine digital dermatitis (CODD) are common causes of foot disease of sheep in the UK. The study reported here is a split flock randomised treatment trial undertaken on a group of 748 fattening lambs on a UK sheep farm affected by CODD and footrot. The sheep were randomly assigned to one of two treatment protocols. In protocol A, all sheep were given two doses of footrot vaccine (Footvax, MSD), plus targeted antibiotic therapy (long-acting amoxicillin, Betamox LA, Norbrook Pharmaceuticals) to sheep with foot lesions likely to be associated with a bacterial infection. In protocol B, the sheep only received targeted antibiotic therapy. Sheep were re-examined and foot lesions recorded five and nine weeks later. New infection rates in the footrot vaccinated group were lower compared with the vaccinated group for both CODD (18.2 per cent compared with 26.4 per cent, P=0.014) and footrot (12.55 per cent compared with 27.5 per cent, P<0.001). Recovery rates were unaffected for CODD (80.46 per cent compared with 70.97 per cent, P=0.14) but higher for footrot (92.09 per cent compared with 81.54 per cent, P=0.005) in sheep which received the vaccine. On this farm, a footrot vaccine efficacy of 62 per cent was identified against footrot and 32 per cent against CODD infection. An association between a sheep having footrot at visit 1 and subsequently acquiring CODD was identified (odds ratio [OR] 3.83, 95 per cent CI 2.61 to 5.62, P<0.001). These results suggest a role for infection with Dichelobacter nodosus in the aetiopathogenesis of CODD on this farm.     

Equine influenza vaccine efficacy: the significance of antigenic variation

To investigate the level of cross-protection induced by equine influenza H3N8 vaccines derived from different lineages, two studies have been carried out with ponies vaccinated with 'American-like' and 'European-like' vaccines and experimentally challenged with a European-like strain. The results demonstrated that equine influenza vaccines clearly protect against challenge with homologous virus if serum antibody titres are sufficiently high. On the other hand, protection is incomplete even when animals vaccinated with heterologous strains have comparative antibody levels. Nevertheless, the protection afforded by heterologous viruses can be improved by stimulating high levels of antibody. It would be advisable to update equine influenza vaccine strains regularly so that they contain similar strains to variants that are circulating in the field.     

Influence of formulation on the efficacy of experimental oil-emulsion Newcastle disease vaccines

Twenty-one experimental oil-emulsion vaccines with different emulsifier contents, aqueous-to-oil ratios, and antigen concentrations were compared by immunization of 4-week-old chickens. Vaccines that contained oil-phase (Arlacel 80) and aqueous-phase (Tween 80) emulsifiers induced 2-to-4-fold higher hemagglutination-inhibition titers than vaccines with only the oil-phase emulsifier. The emulsion vaccines containing both emulsifiers were also more stable at 37 C and less viscous than those containing only the oil-phase emulsifier. Vaccines that had different aqueous-to-oil ratios and contained different quantities of allantoic-fluid antigen (1.2% to 50% of the vaccine volume) induced similar protection against challenge, but hemagglutination-inhibition titers were proportional to the amount of antigen added. Vaccines that had different aqueous-to-oil ratios but contained equal amounts of antigen induced similar hemagglutination-inhibition titers and similar protection against challenge.     

The preparation and efficacy of manually emulsified Newcastle disease oil-emulsion vaccines

Experimental Newcastle disease oil-emulsion vaccines were prepared by manually shaking small quantities of antigen, surfactant, and mineral oil and compared with oil-emulsion vaccines made by the more conventional mechanical methods. Emulsion stability (prolonged emulsification of aqueous antigen) of the manually prepared vaccines was achieved by four different processes and tested for efficacy in broilers. White rock broilers were vaccinated at 3-5 weeks of age and bled at 1- to 2 week intervals thereafter for 8 weeks. Cumulative 8-week hemagglutination-inhibition (HI) mean titers (reciprocals) ranged from 15 to 250 for manually emulsified vaccines and from 18 to 240 for mechanically emulsified vaccines. Eight-week cumulative mean HI responses induced by manually emulsified vaccines were never significantly (P less than 0.05) lower than their specific mechanically emulsified comparison vaccines and were occasionally significantly higher. Highest HI titers were induced when hydrophile-lipophile balances of 7, 9, and 6 were used for the preparation of manually emulsified vaccines. In general, most of the vaccine emulsions were stable for more than 30 days. Vaccine efficacy, however, was not always diminished by poor emulsion stability. These results indicate that manual emulsification can be used for the production of oil-emulsion vaccines of high efficacy that can provide benefits over existing mechanical methods.     

The efficacy of three commercial Mycoplasma gallisepticum vaccines in laying hens

The efficacy of three commercial Mycoplasma gallisepticum (MG) immunizing agents-a bacterin, a recombinant fowlpox-MG vaccine, and a live F-strain vaccine-was compared in specific-pathogen-free hens in egg production. Three groups of 25 chickens were vaccinated with one of the vaccines at 10 wk of age and 25 birds were not vaccinated. At 25 wk of age (and approximately 50% egg production), 20 birds from each of the three vaccinated groups and 15 nonvaccinated controls were challenged with virulent R-strain via aerosol; the birds were necropsied and evaluated at 10 days post-challenge. The MG bacterin and live F-strain vaccinations were both protective and resulted in significant differences in air sac lesions, tracheal lesions, and ovarian regression compared to the nonvaccinated controls and the recombinant fowlpox-MG vaccine (P < or = 0.05). The evaluation of ovarian regression is a useful method of testing the efficacy of MG vaccines in laying hens.     

不同氟源对鸡新城疫疫苗免疫效果的影响

微量元素氟在动物体内具有多种生理作用 ,但它又是一种具有中等毒性的元素。摄入过量会引起中毒。在养殖业中 ,氟中毒过去多见于牛羊等大家畜 ,但是近年来随着养禽业的迅速发展 ,因高氟饲料引起的家禽氟中毒的报道屡见不鲜。众多研究表明 ,畜禽氟中毒是一种多器官受损害性疾病 ,高氟对动物的肾脏、肝脏、神经系统、内分泌系统、免疫系统、软组织等都有损害 ,而并非仅限于骨骼和牙齿[1,2 ] 。本试验采用不同氟化物人工诱发雏鸡氟中毒 ,以研究高氟对鸡新成疫 (ND)疫苗免疫效果的影响。为探讨氟中毒对家禽免疫机制的影响提供科学依据。1　材料…     

Effect of thimerosal concentration on the efficacy of inactivated Newcastle disease oil-emulsion vaccines

Different quantities of the preservative thimerosal in inactivated Newcastle disease oil-emulsion vaccines were tested to determine the influence on the hemagglutination-inhibition (HI) response of broilers. The effect of thimerosal was measured in vaccines that had been stored for 1, 21, and 52 weeks; HI serology was conducted at 2, 4, and 6 weeks after vaccination. Mean HI titers 4 weeks after vaccination decreased at a significant rate (P less than or equal to 0.001) with increasing concentrations of thimerosal. HI titers 4 weeks after vaccination with 1-week-old vaccine were significantly (P less than or equal to 0.05) higher than those after vaccination with 52-week-old vaccine at all thimerosal concentrations tested. Titers were also significantly higher (P less than or equal to 0.05) after vaccination with 1-week-old vaccine than after vaccination with 21-week-old vaccine at all thimerosal concentrations below about 8.25 mg/ml of antigen. Thimerosal at the levels recommended in commercial vaccines does not significantly decrease vaccine efficacy.     

Effect of climatic region on the clinical expression of footrot of lesser clinical severity (intermediate footrot) in sheep

OBJECTIVE: To determine if the clinical classification of intermediate footrot (IFR) is changed to virulent footrot (VFR) by a transfer of the infected flock to a region where climatic conditions are more favourable for the transmission of the disease. DESIGN: Clinical examination of two groups of Merino wethers infected with IFR; one group of 309 in a region considered less favourable for footrot and another group of 343 at a second site considered more favourable. PROCEDURES: After characterising the form of footrot at the first site, infection was established at the second site by mixing 142 wethers from the first site with 201 unrelated wethers considered to be free of IFR and VFR. Observations of clinical characteristics were made over a 16 month period during which an outbreak of footrot occurred. Clinical assessments were made by inspecting every foot of every sheep at regular intervals and allocating a footscore. Evidence that the same clonal lines of D. nodosus were responsible for the footrot at both sites was provided by serotyping of isolates and using omp gene RFLP as a molecular epidemiological tool. RESULTS: The disease at the first site was classified as IFR because 7% of the sheep developed a maximum footscore (MFS) of 4, the most severe category, despite relatively low rates of transmission. When the outbreak occurred at the second site, which was more suitable for footrot transmission, the maximum proportion of the flock that developed a MFS of 4 was 3.6%, confirming the initial classification of IFR. CONCLUSIONS: When a flock infected with IFR was moved to a region where climatic conditions were more favourable for footrot transmission, the clinical classification of the disease remained the same in both the original flock and in sheep exposed to the infection for the first time.