Use of a toxoid vaccine to protect goats against intradermal challenge exposure to Corynebacterium pseudotuberculosis

Two groups of male, 9-week-old goats (5 goats/group) were vaccinated subcutaneously with formalized exotoxin of Corynebacterium pseudotuberculosis, with Freund's incomplete adjuvant. Each goat was given 2 vaccinations, 2 weeks apart. At each vaccination, each group 1 goat was given 0.5 ml of toxoid, and each group 2 goat was given 1 ml of toxoid. Twenty days after the 2nd vaccination, vaccinated goats and 5 nonvaccinated 12-week-old goats (controls) were inoculated intradermally (challenge exposed) with live C pseudotuberculosis, monitored for 13 weeks, and euthanatized. At necropsy, 5 of the 10 vaccinated goats did not have C pseudotuberculosis lesions, 3 had abscesses limited to the inoculation site and draining lymph node, and 2 had disseminated bacterial lesions. Of the 5 nonvaccinated controls, 4 had disseminated abscesses and 1 had a single abscess in an internal node. Serologically, 9 of the 10 vaccinated goats developed positive (greater than or equal to 1:8) antibody titers against the exotoxin within 1 week after inoculation; the 10th goat seroconverted 2 weeks after inoculation, whereas control goats required 3 weeks to develop a positive antibody response. Therefore, early during an infection with C pseudotuberculosis, antibodies against the exotoxin may protect a goat against spread of the organism. All goats were injected intradermally before challenge exposure, 10 days after challenge exposure, and at 4, 8, and 12 weeks after challenge exposure with a skin-test reagent composed of fragmented bacterial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved Corynebacterium pseudotuberculosis vaccine for sheep

Extensive experiments in mice confirmed that the immunogenicity of a Corynebacterium pseudotuberculosis vaccine could not be significantly improved with the use of various adjuvants. Immunity against C. pseudotuberculosis likewise could not be enhanced by incorporating various immunostimulants into the vaccine or by the use of live vaccines. However, a combination of aluminium hydroxide gel and saponin as adjuvant did have a beneficial effect. This vaccine was tolerated better, and a smaller dose apparently protected sheep more effectively against intralymph node challenge than the currently available alum-precipitated vaccine.     

Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10⁷ C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.     

An improved protocol for electrotransformation of Corynebacterium pseudotuberculosis

We developed an improved protocol for the electrotransformation of Corynebacterium pseudotuberculosis, testing variations of parameters in the procedures that are routinely used for the preparation of electrocompetent cells of this species, including (i) culture conditions, (ii) cell growth phase, (iii) electroporation solutions and (iv) quantity of plasmid DNA. We obtained the greatest efficiency of transformation when the cells were grown until the stationary phase and then washed with 10% glycerol electroporation solution. The transformation efficiency was inversely proportional to the quantity of plasmid DNA. The transformation efficiency reached 10(5) colony-forming units (cfu)/mug plasmid DNA. This protocol would be useful for genetic studies of C. pseudotuberculosis.     

Protection in alpacas against Corynebacterium pseudotuberculosis using different bacterial components

Corynebacterium pseudotuberculosis is a Gram positive bacterium that produces caseous lymphadenitis in sheep and goats, and a granulomatous lymphadenitis in llamas and alpacas. To evaluate the immune potential of different doses of cell wall and toxin components of C. pseudotuberculosis from alpaca origin, 12 adult alpacas were allotted at random to four groups, and SC inoculated in the left flank with vaccines composed of low and high doses of bacterial crude antigens, cell wall: 250 and 500 microg/ml and toxin: 133 and 265 microg/ml, respectively. The vaccines were supplemented with 20 microg/ml of muramyl dipeptide as adjuvant. Three alpacas were sham inoculated with adjuvant as a control. After 3 weeks, immunized and naive alpacas were challenged intradermally in the right flank with 1 x 10(6) colony forming units (CFU) of C. pseudotuberculosis. The alpacas were sacrificed at days 28, 58 and 112 after inoculation, and the degree of protection induced by vaccines was demonstrated by the absence of abscesses and/or bacteria. The alpacas vaccinated with high dose of toxin, did not show abscesses. In contrast, the alpacas vaccinated with a low dose of toxin showed abscesses at the inoculation site, regional, and renal lymph nodes. The cell wall vaccinated alpacas showed a lesser degree of protection than the other groups with superficial and internal abscesses. The control alpacas had persistent fever and abscesses at the inoculation site, regional, and internal lymph nodes. In addition, a robust and early humoral response was observed in all vaccinated alpacas after challenge, lasting at least 3 months. The results suggest that the toxin of C. pseudotuberculosis is a very important antigen, inducing a dose dependant protective immunity against this bacterium in alpacas.     

Effect of levamisole on immunity to Corynebacterium pseudotuberculosis in mice and sheep

The stimulating effect of levamisole on immunity to Corynebacterium pseudotuberculosis in mice was marginal, while no enhancement of immunity could be detected in sheep. The conclusion was reached that, as levamisole has no potentiating effect on immunity to C. pseudotuberculosis in normal sheep, it is of no practical value as an immunostimulant in this instance.     

Transformation of Corynebacterium pseudotuberculosis by electroporation

Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistance plasmid from C diphtheriae. Corynebacterium pseudotuberculosis cultivated in brain-heart infusion broth was washed 3 times with water, and resuspended to a final concentration of about 5 x 10(13) colony-forming units/ml. An electroporator constructed in our laboratory incorporated an electrode with 0.8-mm interelectrode gap, using disposable spectrophotometer cuvettes as containers for electroporation. The pNG2 was prepared in Escherichia coli and 4 to 16 micrograms of pNG2 DNA was mixed with 400-microliters amounts of cell suspension in prechilled cuvettes. After incubation on ice for 5 to 10 minutes, the mixture was electroporated at field strengths of up to 18 kV/cm, mixed with 1.5 ml of brain-heart infusion broth, and incubated at 37 C for 2 hours with agitation. Aliquots were then plated on brain-heart infusion blood agar with 15 micrograms of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of approximately 4 x 10(4) transformants/micrograms of pNG2 DNA. Most total transformants and most transformants per microgram of pNG2 were generated at a field strength of 18 kV/cm. When the concentration of pNG2 DNA was varied, the average total number of transformants increased through a concentration of 30 micrograms/ml, but the efficiency of transformation was highest at the lowest DNA concentration. Transformants contained unmodified pNG2.     

Corynebacterium pseudotuberculosis infection in goats. VIII. The effect of vaccination against experimental infection

The effect of an inactivated vaccine against C. pseudotuberculosis infection was tested on castrated male kids from a herd free from caseous lymphadenitis. The animals were divided into 3 groups with 8 animals in each. Group 1 was immunized with crude filtrated C. pseudotuberculosis toxoid and whole killed organisms, while Group 2 in addition was given levamisole. The kids were vaccinated twice at an interval of 4 weeks. Group 3 consisted of unvaccinated animals. All groups were challenged subcutaneously with live bacteria 4 weeks after the last vaccination. Unvaccinated animals showed the most severe course of illness after challenge. Development of abscesses in the regional lymph nodes (Inn. subiliaci) was significantly more common in unvaccinated than in vaccinated kids at necropsy 2 months after challenge. There was, however, no such difference between the vaccinated groups, and there was no difference between any of the groups as regards abscess formation at the inoculation site. In each of the 2 vaccinated groups, there was a titre rise following vaccination in the hemolysis inhibition test, whereas no such rise was seen in the bacterial agglutination test. The titre values in both tests increased significantly after challenge in all the groups, the increase being most rapid in the vaccinated animals. The present investigation indicates that development of caseous lesions in lymph nodes in goats, following subcutaneous inoculation with C. pseudotuberculosis, can be reduced by an inactivated vaccine containing whole organisms and crude toxin.     

Corynebacterium pseudotuberculosis infection in goats. IX. The effect of vaccination against natural infection

A vaccination trial was carried out in 10 infected herds. The trial included 247 female kids, the number of animals in each herd varying from 15 to 38. About half of the animals in each herd were vaccinated twice at 3 to 4 week intervals, the first vaccination being carried out before the age of 4 months. A combination of a crude filtrate of C. pseudotuberculosis toxoid with whole organisms, was used. Overall, the prevalence of animals with superficial swellings was higher in the unvaccinated than in the vaccinated group during the first 1-2 years following immunization. However, in some herds superficial swellings were as common in vaccinated as in unvaccinated animals. An antibody response following vaccination was demonstrated in the hemolysis inhibition test, but not in the bacterial agglutination test. Superficial swellings were more common in vaccinated animals which were negative than in animals which were positive in the hemolysis inhibition test at 1 1/2 months after vaccination. The vaccine used in the present study, was not sufficiently efficacious to be recommended as the only protective measure against caseous lymphadenitis in Norwegian goat herds.     

Effect of muramyl dipeptide on immunogenicity of Corynebacterium pseudotuberculosis whole-cell vaccines in mice and lambs

Colostrum-deprived lambs and CF1 mice were vaccinated with water-in-oil emulsion vaccines containing nonviable whole cells (WC) of Corynebacterium pseudotuberculosis with and without muramyl dipeptide (MDP). Efficacy of vaccines was determined from the survival of mice and lesions in lambs after IV injection of 10(4) colony-forming units of C pseudotuberculosis. In mice, protection was related to the concentration of WC in the vaccine. At 50, 100, or 150 micrograms of WC, protection was good (78.8%). At 10 or 25 micrograms of WC, protection was considerably less (54.7%). At high WC concentrations, protection could only be moderately increased to 82.3% with high (50 and 100 micrograms) concentrations of MDP or increased to 90% protection with low (5 and 10 micrograms) concentrations of MDP. At low WC concentrations, protection significantly decreased to 32% (P less than 0.025) with high concentrations of MDP, but significantly increased to 72.5% (P less than 0.025) with low concentrations of MDP. Therefore, the amount of protection with lower concentrations of WC and MDP was comparable with the amount of protection with higher concentrations of WC without MDP. In lambs, high prechallenge antibody titers (geometric mean titers from 5.1 to 5.4 by day 35) were observed after vaccination with WC. Protection and vaccination site abscesses in lambs were related to the concentration of WC and MDP. Pulmonary or vaccination site abscesses were not observed in 4 of 4 lambs vaccinated with 1 mg of WC + 50 micrograms of MDP.     

一株奶山羊伪结核棒状杆菌的分离与鉴定

为获得陕西某羊场病羊脓包中的病原菌,以无菌采集的病羊颈部脓汁为材料,进行细菌分离培养,鉴定分离菌株的形态特征、培养特性、生化特性,挑取分离菌株进行协同溶血试验(cAMP)和16SrRNA基因的PCR扩增,并对序列进行分析,NJ法构建系统发育树;用分离菌株分别接种Balb/c小鼠和奶山羊;药敏试验鉴定分离株的耐药性。结果显示,分离菌株的形态特征、培养特性、生化特性与伯杰细菌鉴定手册所述伪结核棒状杆菌特性一致;测序显示分离菌株与GenBank中已收录伪结核棒状杆菌相似性达97%以上;Balb/c小鼠致病性试验和奶山羊动物回归试验表明分离株有强致病性;药敏试验表明分离菌株对红霉素、环丙沙星、克拉霉素、强力霉素、四环素、头孢唑啉、新霉素、氧氟沙星等多种抗生素敏感,对复方新诺明耐药。     

Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR

Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.     

Efficacy of Corynebacterium pseudotuberculosis bacterin for the immunologic protection of sheep against development of caseous lymphadenitis

The efficacy of a Corynebacterium pseudotuberculosis bacterin to protect sheep immunologically against development of caseous lymphadenitis was evaluated in controlled challenge-exposure experiments. Sixty-three mixed-breed, white-faced lambs were used. The lambs were 10 to 12 weeks old and were randomly assigned to 3 groups (21 lambs/group). Group 1 was vaccinated once, using 2 ml of a C pseudotuberculosis bacterin (given subcutaneously) in the right axillary region at the beginning of the study. Group 2 was vaccinated twice; the 1st vaccination was given at the same time that lambs in group 1 were vaccinated and the 2nd vaccination was given 4 weeks later. Group 3 (nonvaccinated controls) was given physiologic saline solution (2 ml, subcutaneously). Each lamb was challenge exposed (ie, given 2 ml of live Corynebacterium pseudotuberculosis inoculum [6 X 10(6) colony-forming units/ml], subcutaneously at 4 different sites) during the 20th week of the study. All lambs were killed and necropsied during week 33. The mean number of abscesses per lamb was 7 for group 1, 4 for group 2, and 32 for group 3. Significant differences in the size of the abscesses were not found between the groups. Results of the study indicated that the vaccine provided immunologic protection of lambs against challenge exposure to Corynebacterium pseudotuberculosis.     

Immunisation against ovine caseous lymphadenitis: comparison of Corynebacterium pseudotuberculosis vaccines with and without bacterial cells

Sheep were immunised with Corynebacterium pseudotuberculosis vaccines prepared from cell-free toxoid or from toxoid with formalin-killed cells of C pseudotuberculosis added. Resistance of sheep to infection was tested 6 months after immunisation by inoculation with caseous lymphadenitis pus. The outcome was assessed 3 months later by slaughter and inspection of the sheep for lesions of caseous lymphadenitis. immunised sheep were adequately protected against infection as shown by a significant reduction in the number of sheep exhibiting lesions compared with control sheep, and by fewer abscesses in affected vaccinated sheep than in affected control sheep. The protective potency of the vaccines was not improved by the inclusion of cells of C pseudotuberculosis.     

Comparative molecular characterization of Corynebacterium pseudotuberculosis of different origin

Ribotyping and susceptibility to 17 antimicrobial agents were used to compare 37 isolates of Corynebacterium pseudotuberculosis (28 from horses, 1 from cattle, 3 from sheep and 5 from goats) derived from various types of lesions, and different geographic locations. According to the presence of nitrate reductase, all but one isolate from horses reduced nitrate (nitrate-positive), whereas all isolates from sheep and goats were unable to reduce nitrate (nitrate-negative). The ribotype of the nitrate-negative isolate from a horse with ulcerative lymphangitis was identical to all the other isolates from horses, and different than the ribotype of nitrate-negative isolates from sheep and goats. Ribotyping with one of the restriction endonucleases, Apa I, revealed differences between, but not within, the two biotypes. However, ribotyping with Pst I endonuclease revealed one variant within the equine biotype and one variant within the ovine biotype. The minimum inhibitory concentration (MIC; μg/ml) of antimicrobial agents against isolates from nitrate-negative and nitrate-positive groups was very similar, with the exception of isolates from sheep and goats which had a higher MIC for amikacin than isolates from horses and cattle.