Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice

Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.     

Vaccination against Brucella ovis infection in sheep

Extract

Dairy cows in New Zealand subsist chiefly on pasture or pasture products such as hay and silage, whilst concentrates are fed only in exceptional cases. Town supply herds are concerned with the production of winter milk and payment by the dairy factory is based on their milk production assessment during the months of June, July, and August. It is therefore necessary to have maximum milk production during these winter months when pastures are at their minimum rate of growth. As a food supplement, therefore, many town supply herds augment their feeding with crops and wet brewers' grains. The grains are fed from hoppers in the bail and are also spread out in the paddocks or placed in large wooden troughs. Most grains are purchased on a contract basis from the brewers, more being available during the summer months, when more beer is consumed. Friday's load of grains is usually stored and the consumption spread out over the week-end until the first fresh load is brought in on Monday morning.     

Enhanced efficacy of recombinant Brucella abortus RB51 vaccines against B. melitensis infection in mice

Brucella abortus strain RB51 is an attenuated rough strain, currently being used as the official live vaccine for bovine brucellosis in the USA and several other countries. In strain RB51, the wboA gene, encoding a glycosyltransferase required for the O-side chain synthesis, is disrupted by an IS711 element. Recently, we have demonstrated that strain RB51WboA, RB51 complemented with a functional wboA gene, remains rough but expresses low quantities of O-side chain in the cytoplasm. Mice vaccinated with strain RB51WboA develop greatly enhanced resistance against challenge with B. abortus virulent strain 2308. We have also demonstrated that overexpression of Cu/Zn superoxide dismutase (SOD) in strain RB51 (RB51SOD) significantly increases its vaccine efficacy against strain 2308 challenge. In this study, we constructed a new recombinant strain, RB51SOD/WboA, that over expresses SOD with simultaneous expression of O-side chain in the cytoplasm. We tested the vaccine potential of strains RB51SOD, RB51WboA, RB51SOD/WboA against challenge with virulent Brucella melitensis 16M and B. abortus 2308 in mice. In comparison with strain RB51, strain RB51SOD induced better protection against strain 2308, but not strain 16M, challenge. Similar to strain RB51WboA, vaccination with strain RB51SOD/WboA resulted in complete protection of the mice from infection with strain 2308. When challenged with strain 16M, mice vaccinated with either strain RB51WboA or strain RB51SOD/WboA were significantly better protected than those vaccinated with strain RB51 or RB51SOD. These results suggest that strains RB51WboA and RB51SOD/WboA are good vaccine candidates for inducing enhanced protection against B. melitensis infection.     

Production of immunity in rams against Brucella ovis infection

Extract

During earlier investigations in New Zealand into the bacterial and mycotic flora of hedgehogs (Erinaceus europaeus), strains of Salmonella typhimurium were obtained from the gut of a number of animals. It was therefore decided to examine these animals more thoroughly for the presence of salmonellae. This paper embodies the results of a small survey subsequently carried out in the Hamilton suburban area during February, 1964.     

Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis

To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.     

Immunization of mice and calves against Salmonella dublin with attenuated live and inactivated vaccines.

Previous findings, viz. that mice can be successfully immunized against infection with Salmonella dublin with either live or inactivated vaccine, were confirmed. Immunity lasted for at least 12 weeks in mice which had been immunized with inactivated alum-precipitated vaccine. The immunogenicity of inactivated vaccine gradually decreased on storage at 4 degrees C, but this was only detectable if a single injection was used for immunization: 2 injections virtually eliminated this phenomenon. The immunogenicity of live vaccine in mice was not enhanced by levamizole or the simultaneous injection of inactivated organisms. Both live and inactivated vaccines provided immunity in calves. A single injection of lyophilized vaccine, prepared from live rough Salmonella dublin strain (HB 1/17),protected 3 out of 6 calves, while 2 injections of a formalin-inactivated, alum-precipated vaccine, containing 1% packed cells of S. dublin strain 2652 V, protected 5 out of 6 calves against intraduodenal challenge with 2 x 10(9), S. dublin strain 2652 V. Two calves which had been immunized with an inactivated oil adjuvant vaccine were also solidly immune to this challenge. Serum antibody response in calves was poor when measured by the tube agglutination and the haemagglutination tests. Similarly, the sera had only marginal protective values when tested by means of a passive protection test in mice. Antibody titres alone are not a valid measure therefore, for the immune status of immunized animals.     

Lameness in rams following vaccination against Brucella ovis infection

Extract

Since the introduction in 1957 of a vaccination procedure to control Brucella ovis infection in sheep, many thousands of rams have been vaccinated annually throughout New Zealand almost without complications.     

Immunization with Brucella melitensis Rev 1 against Brucella ovis infection of rams

The efficacy of Brucella Melitensis Rev 1 vaccine (Rev 1) for the prophylaxis of Brucella ovis ram epididymitis was evaluated. Twenty-nine 3-month-old rams were vaccinated with 2 X 10(9) Rev 1 and 14 were revaccinated with 5 X 10(8) at 14 months of age. Six rams remained unvaccinated as a control group. All rams were challenged with 5 X 10(8) B. ovis at 21 months of age. Before being slaughtered 8 weeks later, only one vaccinated ram developed epididymitis while four of the six control rams developed testicular alterations. Genital and selected extragenital organs and lymph nodes were removed at slaughter and inoculated on selective media. B. ovis was isolated from 26.6% of the vaccinated rams, 21.4% of the revaccinated rams and 100% of control rams. Portions of epididymis, testes and vesicular glands were also used for pathological studies. More severe lesions were observed in control rams than in vaccinated ones. In conclusion, these results show that vaccination of young lambs, followed or not by revaccination, is a suitable method for the prophylaxis of B. ovis infection of rams.     

Newly attenuated Mycobacterium bovis mutants as vaccines against bovine tuberculosis, particularly for possums

Cattle vaccinated with a single dose of subunit vaccine containing the capsid and 3C proteinase coding regions of foot-and-mouth disease virus (FMDV) Asia I/HNK/CHA/05 strain were protected when challenged 28 days later with a homologous virus. Here, the 50% bovine protective dose (PD(50)) test was performed to assess the potency of the subunit vaccine. When challenged with two Chinese isolates, the subunit vaccine could achieve 6.5 PD(50) (challenged with Asia I/HNK/CHA/05 strain) and 5.2 PD(50) (challenged with Asia I/JSL/05 strain) per dose.     

Experimental Brucella ovis infection in pregnant ewes.

Forty yearling Brucella-free ewes were inoculated with Brucella ovis by the conjunctival route in mid or late first pregnancy. Only a few ewes excreted B ovis during pregnancy and gave birth to stillborn lambs, but most of them excreted the organism at lambing or during lactation. One of the 11 lambs which were born alive but died before they were weaned was found to be infected postmortem. In contrast, none of the 46 surviving lambs which were reared in isolation until adulthood, was found to be infected. At weaning, the 40 ewes were mated again with five Brucella-free rams. Although many of the ewes excreted B ovis, none of the rams was found to be infected when necropsied after mating. Most of the ewes that became pregnant, all having excreted B ovis during their first pregnancy, cleared the infection during the second pregnancy. However, three remained persistently infected and excreted B ovis in their milk throughout the second lactation. None of the lambs born to these three ewes was found to be infected when necropsied at weaning.     

Galactose epimeraseless mutants of Salmonella typhimurium as live vaccines for calves.

The purpose of the study was to evaluate the safety and efficacy of a galactose epimeraseless mutant of Salmonella typhimurium administered as an oral vaccine to one week old calves and to investigate properties of galactose epimeraseless mutants which affect their virulence and immunogenicity. The galactose epimeraseless mutant S. typhimurium strain G30D caused diarrhea and fever in three calves to which it was administered orally at a dose of 10(10) organisms; all three calves died following challenge with virulent S. typhimurium ten days postvaccination. Mild illness developed in four calves vaccinated with a dose of 9 X 10(6) organisms and one of these calves survived challenge. Three unvaccinated calves died following challenge. The vaccine organism persisted in tissues and was shed for a prolonged period by calves which received 10(10) organisms. Studies of characteristics of galactose epimeraseless mutants of S. typhimurium showed that, in the presence of galactose, there is selection for secondary mutants which are galactose resistant. The studies indicate that galactose epimeraseless mutants of S. typhimurium are not good candidate live vaccine organisms for use in calves.     

Evaluation of Salmonella enterica serovar Typhimurium and Choleraesuis slyA mutant strains for use in live attenuated oral vaccines

SlyA protein plays a key role in virulence in Salmonella enterica. In this study, we evaluated the ability of the slyA mutant strain of S. enterica serovar Choleraesuis (S. choleraesuis) to protect against swine salmonellosis. Using a murine model infected with S. enterica serovar Typhimurium (S. typhimurium), we showed that the Salmonella strain with a deletion of slyA could be used as a highly immunogenic, effective and safe vaccine in mice. Based on these data, a slyA mutant of S. enterica serovar Choleraesuis strain RF-1 was constructed, and the ability of this mutant to protect immunized pigs from S. choleraesuis infection was examined. As with the S. typhimurium slyA mutant, immunization of pigs with the S. choleraesuis slyA mutant strain provided significant protection against subsequent challenge by the wild-type RF-1. These results demonstrate that SlyA is a potential target in the development of a novel live attenuated vaccine against S. enterica.     

Detection of antibodies to Brucella ovis in sheep milk using B. ovis and B. canis antigen

The diagnostic techniques most widely used for detecting brucellosis caused by Brucella ovis are serological tests such as complement fixation (CFT), agar gel immunodiffusion (AGID), and ELISAs. However, to our knowledge, milk tests, with the advantage that samples may be taken in a non invasive manner, have not been investigated as diagnostic tools. We studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using Brucella canis and B. ovis antigens. A group of 75 ewes in an uninfected flock were serologically negative to rapid slide agglutination test (RSAT), indirect ELISA (IELISA)-B. canis, AGID and IELISA-B. ovis. The milk of these ewes had an IELISA-B. canis mean (%P) value of 16.18 (S.D. 5.63), while the IELISA-B. ovis had a mean (%P) value of 12.52 (S.D. 4.94). A cut-off value of (%P) 27.44 (+2 S.D.) or (%P) 33 (+3 S.D.) was determined by milk-ELISA-B. canis and (%P) 22.4 (+2 S.D.) and (%P) 27.34 (+3 S.D.) by milk-IELISA-B. ovis. These cut-off values were adjusted by receiver-operator characteristics (ROC) analysis using 23 positive samples from infected ewes, which indicated a milk-IELISA-B. canis cut-off value of (%P) 33 and milk-IELISA-B. ovis of (%P) 26 with 100% sensitivity and specificity. Based on our results, we propose that, following a study of a larger number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis brucellosis in lactating ewes.     

Evaluation of electrophoretic immunoblotting for Brucella ovis infection in deer using ram and deer serum

AIMS: Recently the first case of natural infection of deer with Brucella ovis was discovered. The aim of this study was to develop and evaluate an electrophoretic immunoblotting method for testing deer serum for specific B. ovis antibodies. METHODS: An existing immunoblotting method for sheep serum was altered by using a recombinant protein G-alkaline phosphatase conjugate and Tris-buffered saline containing 3% non-fat dry milk powder for the blocking step and the serum and conjugate dilutions. The method was evaluated using 106 sheep sera from B. ovis - negative, accredited flocks, 69 sera from chronically infected rams shedding B. ovis in their semen, 110 sera from a B. ovis-infected flock, 18 sera from stags from which B. ovis was isolated, and 48 sera from deer flocks free from B. ovis infections. The immunoblotting method was applied to another 85 deer sera. RESULTS: The sensitivity of the new immunoblotting method was 98.6% for sheep and 94.4% for deer, and the specificity 99.1% for sheep and 100% for deer. Sixty-nine out of 97 deer sera, originating from the property from which the first B. ovis deer case had been reported, tested positive or suspicious in the complement fixation test. Of these, 53 sera exhibited staining patterns in blots typical for B. ovis infections and also one serum which was negative in the CFT. Only six out of 1498 deer sera. from throughout New Zealand had positive or suspicious reactions in the B. ovis complement fixation test. Of these, one exhibited a staining pattern in the blot suggestive of a B. ovis infection, while four showed patterns of suspicious reactions. CONCLUSION: The new immunoblotting technique is useful as a confirmatory serological test method for B. ovis infections in deer.     

The detection of Brucella ovis infection in rams

Abstract

Extract

Each year in New Zealand, many rams are palpated for lesions of the epididymes and testes. Although regarded initially as a method of detecting infertile rams (Crawford and James, 1950 Biberstein, E. L. and McGowan, B. 1958. Cornell Vet., 48: 31–31.  [Google Scholar]), the technique is now regarded primarily as a method of detecting infection with the organism for which Buddie (1956 Buddle, M. B. 1956. J.Hyg., 54: 351–351.  [Google Scholar]) proposed the name Brucella ovis.     

Evaluation of an enzyme linked-immunosorbent assay for the diagnosis of Brucella ovis infection in rams

A simple enzyme linked immunosorbent assay (ELISA) was developed for the serological diagnosis of Brucella ovis infections in rams. Serums from brucellosis accredited-free flocks and flocks known to be infected with B. ovis were tested and the results correlated with warm complement fixation (CF) test and bacteriological examination of semen. Both the ELISA and the CF test detected 0.5% false positive reactions in rams from clinically negative flocks. However the ELISA detected significantly more positive reactors in infected flocks and the CF test failed to detect some rams excreting B. ovis. The ELISA proved to be a valuable test in eradicating brucellosis from infected flocks.     

Use of Brucella canis antigen for detection of ovine serum antibodies against Brucella ovis

Brucella ovis causes a genital disease of sheep manifested by epididymitis in rams and placentitis in ewes producing reduced fertility in the flock. Clinical diagnosis is not sensitive enough and bacteriological testing is not feasible for detection of the disease in large numbers of animals. Indirect methods of serological testing are preferred for routine diagnosis, of which agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA tests are recommended as the most efficient. Since B. ovis shares antigenic components with Brucella canis, it would seem that either strain could be used as antigen with the same results; however, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination tests. We present data on AGID and IELISA tests using B. ovis antigen and rapid screening agglutination test (RSAT), 2-mercapto-ethanol RSAT (2ME-RSAT) and IELISA using B. canis antigen. We tested 225 animals. The cut-off values were adjusted by ROC analysis using 51 negative and 32 positive sera; the IELISA-B. canis cut-off value was 39 (%P) and IELISA-B. ovis, 51 (%P), with 100% sensitivity and specificity. Of the 32 positive sera from the infected flock RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Of the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, IELISA-B. canis and IELISA-B. ovis, 20 positive only with RSAT and 2 positive only by both IELISAs. RSAT is a very sensitive screening test that, because of its simplicity and easy interpretation, following a study in larger sample, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis. The IELISA-B. canis or IELISA-B. ovis could be used as confirmatory tests, since they show equal specificity and sensitivity.     

The serological response of rams to four methods of vaccination against Brucella ovis infection

A comparative study was made of the serological response of rams to an inactivated Brucella ovis saline in-oil vaccine administered either once or twice by either the subcutaneous or the intraperitoneal route. The serological response of rams to two spaced doses of vaccine was more consistent and more persistent than when a single dose of vaccine was used. The rise in titre was more rapid and the final titre of greater magnitude when the subcutaneous route of administration was used in comparison with the intraperitoneal route. On a serological basis, the most satisfactory vaccination technique investigated consisted of two subcutaneous injections of vaccine administered ten weeks apart. The least satisfactory technique was a single intraperitoneal injection.