Efficacy of an oral live vaccine for veterinary use against pseudotuberculosis

Pseudotuberculosis, an infection caused by the ubiquitous enteropathogenic bacterium Yersinia pseudotuberculosis, is a recurrent veterinary problem in livestock and zoo animals. The only vaccine currently available in zoos is Pseudovac (a mixture of killed strains of various serotypes), but its efficacy is not well established. We show here that Pseudovac does not protect guinea pigs against a severe Y. pseudotuberculosis infection. We thus evaluated the possibility of using a live attenuated Y. pseudotuberculosis strain (IP32680) as an oral vaccine against animal pseudotuberculosis. We report that IP32680 is avirulent for guinea pigs and induces a strong IgG response against various serotypes of Y. pseudotuberculosis. One and two oral inoculations of IP32680 provided 50% and 83% protection, respectively against a severe infection with a highly pathogenic strain. The avirulent Y. pseudotuberculosis IP32680 is therefore much more protective than Pseudovac and may represent a valuable oral vaccine against pseudotuberculosis in zoo animals.     

Single-dose oral pharmacokinetics of pergolide mesylate in healthy adult mares

This pilot study evaluated protection of an equine autogenous bacterin-toxoid vaccine against Corynebacterium pseudotuberculosis infection. Twenty-four BALB/c mice were inoculated with two doses of bacterin-toxoid vaccine or two injections of a placebo. Clinical, microbiologic, and pathologic outcomes were assessed after intradermal infection with one of two equine-origin C. pseudotuberculosis strains. Mice receiving bacterin-toxoid from fast-growing C. pseudotuberculosis showed significant protection from challenge infection, as evidenced by a higher survival rate, fewer gross and histopathologic lesions, and lower bacterial levels on culture. Successful protection via a vaccine against equine internal abscesses might provide supplementary management options against an important, potentially fatal disease.     

Efficacy of DNA vaccination by different routes of immunisation in sheep

DNA vaccination, delivered through various routes, has been used extensively in laboratory animals. Few studies have focused on veterinary species and while results obtained in laboratory animals can often be extrapolated to veterinary species this is not always the case. In this study we have compared the effect of the route of immunisation with DNA on the induction of immune responses and protection of sheep to challenge with live Corynebacterium pseudotuberculosis. Intramuscular injection of plasmid DNA encoding an inactivated form of the phospholipase D (PLD) antigen linked to CTLA4-Ig resulted in the induction of a strong memory response and sterile immunity following challenge in 45% of the animals. In contrast, gene gun delivery or subcutaneous (SC) injection of the DNA vaccine induced comparatively poor responses and insignificant levels of protection. Thus, DNA vaccine efficacy in sheep is strongly influenced by the route of vaccination. Amongst intramuscular vaccinates, protected sheep had significantly elevated IgG2 responses compared to unprotected animals, while both subgroups had equivalent IgG1 levels. This suggests that the presence of IgG2 antibodies and hence a Th1-like response, induced by the DNA vaccine gave rise to protective immunity against C. pseudotuberculosis.     

The presence of anti-Yersinia pseudotuberculosis immunoglobulins in equine serum

The research was conducted on clinically healthy mares (n = 40) and foals (n = 78) during Y. pseudotuberculosis associated enzootics. The animals were divided into groups: I to IV--mares, IA to IVA--their offsprings, IB to IVB--foals which mothers were not treated with any medicaments. The animals in group I, IA and IB were injected with PBS; in group II, IIA and IIB--with Y. pseudotuberculosis strain-based vaccine, in group III, IIIA and IIIB--with P. acnes strain-based immunostimulator; in group IV, IVA and IVB--with P. acnes strain-based immunostimulator and (5 days after the immunostimulator injection) Y. pseudotuberculosis strain-based vaccine. The presence of antibodies was determined by means of ELISA. The study revealed anti-Yersinia pseudotuberculosis IgG only in 19 mares before, and in 25 mares and 26 foals 3 weeks after vaccination. The mean extinction 3 weeks after vaccination amounted to: II-0.489, IV-2.578, iiA-0.572, IVA-0.974, IIB-0.312, iVB-0.418. The cut-off extinction value was 0.154. The presence of anti-Yersinia pseudotuberculosis IgG before vaccination in the sera of clinically healthy mares may suggest that Y. pseudotuberculosis infection occurs definitely more often than is expected. Vaccination preceded by immunostimulation appeared to be the most efficient method of treatment against yersiniosis.     

Corynebacterium pseudotuberculosis infection in goats. V. Relationship between the infection and lesions resulting from vaccination against paratuberculosis

In 2 goat herds, one infected with Gorynebacterium pseudo-tuberculosis and one free from the infection,, goats were examined for superficial swellings on the shoulder and chest. All animals in this study had been vaccinated against paratuberculosis before the age of 4 weeks. The vaccine had been applied subcutaneously behind the shoulder. Twenty-two of 40 (55 %) and 31 of 45 (69 %) goats had such lesions in the infected and non-infected herds, respectively. The difference between the herds was not significant, P > 0.05.Swellings found behind the shoulder in 19 goat carcasses derived from 4 herds in which G. pseudotuberculosis infection occurred were examined bacteriologically. No bacteria could be isolated from such lesions in 15 animals, while G. pseudotuberculosis in pure culture was isolated from 3 carcasses, and a mixed bacterial flora from the re-maining carcass. Bacteria could not be isolated from lesions situated behind the shoulder in 7 carcasses from 3 herds free from G. pseudo-tuberculosis infection.It is concluded that most swellings on the shoulder and chest in goats were granulomas resulting from vaccination against paratuber-culosis.     

Efficacy of a flock-specific pseudotuberculosis vaccine in goats

In a flock with 33 adult goats repeated abscesses due to Pseudotuberculosis occurred. For the owners refused a sanitation program based on testing and removing, the goats were vaccinated with a flock specific, whole-cells lysate vaccine of Corynebacterium pseudotuberculosis. Partly simultaneously or 14 days later vaccinations against Clostridiosis (Covexin8) and against Erysipelas (Erysorb plus) were performed at different application sites. After a twofold basal vaccination in a 4 week interval the goats were revaccinated in half year intervals. The local (swelling at the injection site) and systemic reactions (temperature elevation, antibody activity) after vaccination and the clinical status of the flock (core body temperature, adspection, abscesses, lymph nodes) were investigated over a period of two years. Due to the vaccination program the rate of new cases of clinical pseudotuberculosis were reduced significantly. In the last 3 months of the investigation no clinical cases occurred. The vaccination with the C. pseudotuberculosis-vaccine induced a significant increase in antibody activities detected by ELISA. Due to interaction of maternal antibodies with the antigen of the vaccine lambs should be vaccinated at the earliest at an age of 9 weeks. Vaccinating the C. pseudotuberculosis-vaccine separately in time to Covexin8 and Erysorb plus induced with increasing numbers of revaccinations higher antibody activities against C. pseudotuberculosis than simultaneous vaccinations with all the 3 vaccines. But this difference does not justify the additional expense of separate vaccinations in time. Despite considerable local and systemic reaction the C. pseudotuberculosis-vaccine is recommended to support sanitation programs.     

陕南白山羊伪结核棒状杆菌分离鉴定及灭活铝胶疫苗研制

羊干酪性淋巴结炎又称羊伪结核病,是由伪结核棒状杆菌感染引起的一种人畜共患慢性接触性传染病,对山羊危害最为严重,目前尚无有效治疗措施。陕西省石泉县陕南白山羊饲养量较大,羊群也存在伪结核棒状杆菌的感染,造成较大的经济损失。为筛选对当地伪结核棒状杆菌敏感的药物和研制灭活疫苗,本研究从疑似伪结核病患羊采取多份新鲜的脓液,进行细菌分离鉴定;对获得的1株分离菌进行药物敏感性试验并用其研制灭活疫苗。结果显示,分离菌在固体培养基上形成白色、干燥、扁平、不透明、边缘整齐的中等大小菌落,革兰染色阳性,经16SrRNA检测和序列分析,证实分离到的菌株为羊伪结核棒状杆菌。药物敏感性试验结果显示,该分离菌对环丙沙星、头孢曲松、红霉素、卡那霉素、头孢噻肟、妥布霉素、强力霉素、庆大霉素、氯霉素高度敏感;对四环素、利福平、链霉素中度敏感。以该分离菌为种子菌制备的灭活铝胶疫苗无菌检验合格,试验接种山羊无不良反应,目前已经在采样羊场进行临床应用,进一步评价免疫效果。     

Antigen specificity and activity of ovine antibodies induced by immunization with Corynebacterium pseudotuberculosis culture filtrate

Sheep were immunized three times with a vaccine composed of filtrate from a 36 h culture of Corynebacterium pseudotuberculosis and a block polymer adjuvant. Immunization resulted in the development of exotoxin-neutralizing antibody. This corresponded to the recognition of a 31.6 kDa protein on sequential immunoblots of ammonium sulfate-precipitated filtrate. In addition sera from vaccinated sheep recognized at least eight bacterial cellular antigens on immunoblots of ether-extracted C. pseudotuberculosis, including bands of 12, 25.1, 31.6, 36.3, 39.8, 63.1, 70, 75 or 79.4 kDa. Sera from these sheep altered the colony growth characteristics of C. pseudotuberculosis in vitro. These results indicate that immunization with soluble C. pseudotuberculosis in vitro. These results indicate that immunization with soluble C. pseudotuberculosis antigen preparations that have been used in toxoid vaccines induces antibody responses to numerous cellular antigens in addition to exotoxin and suggest that serologically mediated antibacterial effects could be an important component in the protection from disease that has been reported following immunization with C. pseudotuberculosis toxoids.     

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.     

An interferon-gamma assay for diagnosis of Corynebacterium pseudotuberculosis infection in adult sheep from a research flock

The optimal method of control of caseous lymphadenitis of sheep caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals. Current serological approaches to identification of infected sheep are generally hampered by low sensitivity and specificity of available tests. The objective of this study was to develop a whole blood assay for detection of C. pseudotuberculosis-infected sheep, based on detection of IFN-gamma response to whole cell C. pseudotuberculosis antigens, and to determine the reliability of the assay. A commercially available bovine interferon-gamma assay enzyme-linked immunosorbent assay was used and the test optimised using experimentally infected sheep. The assay was also tested on known CLA-negative sheep. Setting a IFN-gamma optical density cut-off at 0.100 as positive under the conditions used, the test detected C. pseudotuberculosis experimentally infected sheep over a 450-day period with a reliability of 95.7%. It identified known non-infected sheep with a reliability of 95.5%. Repeated vaccination of three uninfected sheep with a commercially available bacterin-toxoid vaccine did not interfere with the assay. The IFN-gamma response of sheep whole blood to C. pseudotuberculosis antigens offers promise for use in a test-and-removal approach to eradication of CLA in sheep.     

Acetone-treated agglutinin against Yersinia pseudotuberculosis in free-living rats

Retrospective investigation was done on Yersinia pseudotuberculosis infection in barn rat populations living in an Yersinia endemic area and in zoo rat populations living in a nonendemic area. The OH agglutinins against Y pseudotuberculosis were normally distributed within both populations. Most of the OH agglutinin titers and many O agglutinin titers were reduced by treatment of sera with acetone. Sera with O and OH agglutinin titers were assigned to 2 groups, one with a titer less than 1:8 and the other with a titer of greater than or equal to 1:8. Rats harboring the organism did not possess O or OH agglutinin titers greater than 1:8 in acetone-treated sera. In most nontreated sera, OH agglutinin titers were higher than were the O agglutinin titers. However, in most acetone-treated sera, O agglutinin titers were equivalent to OH agglutinin titers. Differences by region and age were observed in acetone-treated O agglutinin titers of greater than or equal to 1:8 against Y pseudotuberculosis. Therefore, it is recommended that to detect antibody against Y pseudotuberculosis in rats, sera should be treated with acetone before the test and O antigen should be used.     

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.     

The differential diagnosis of Salmonella abortus ovis and Yersinia pseudotuberculosis in sheep abortions

55 strains of Salmonella abortus ovis and 3 strains of Yersinia pseudotuberculosis (two serotypes I, one serotype II), isolated out of aborted ovine fetuses from northern Bavaria, were investigated and compared culturally, biochemically and serologically. The identification of S. abortus ovis (group B) could not be achieved by use of Api 20 E. The code-number 4004500/47 identified S. typhi (group D) only. Y. pseudotuberculosis serotype II but not serotype I agglutinated with the polyvalent and the specific anti-0: 4-Salmonella serum. No agglutination was observed with the flagella anti-H-sera.     

Corynebacterium pseudotuberculosis infection in adult ewes by inoculation in the external ear

Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, was studied in ewes to devise a reliable method of induced infection that would help to understand mechanisms by which abscesses develop in lymph nodes or internal organs, especially the lungs. Fifteen adult ewes, from a flock where the occurrence of caseous lymphadenitis was unknown, were inoculated SC in the auricle with various doses of a virulent strain of C pseudotuberculosis. Ewes inoculated with at least 10(8) viable corynebacteria develop a strong local reaction that was apparent 4 hours after challenge exposure and lasted 7 to 14 days. Of these ewes, all developed at least 1 abscess and 3 developed multiple abscesses in lymph nodes draining the inoculated auricle and in the lungs. All infected ewes developed a strong antibody response against C pseudotuberculosis exotoxin, which persisted for 3 months. Of 9 ewes infected with 1.2 X 10(8) corynebacteria, 3 developed lesions because of field strains of C pseudotuberculosis and had an increased capacity to limit the dissemination of challenge-inoculum bacteria by developing only one abscess in the draining lymph node (parotid or prescapular lymph node). Seemingly, inoculation in the external ear was a suitable method for obtaining a reproducible infection and indicated that a primary infection might protect against reinfection by limiting dissemination of C pseudotuberculosis.     

Seroepidemiological survey of Corynebacterium pseudotuberculosis infection in sheep in Japan using enzyme-linked immunosorbent assay and immunodiffusion

Sera from 1186 apparently healthy sheep in Hokkaido were examined by enzyme-linked immunosorbent assay (ELISA) and immunodiffusion (ID) for the presence of antibodies against Corynebacterium pseudotuberculosis. ELISA-positives were 466 (39.3%) while ID-positives were 330 (27.8%). Spread of caseous lymphadenitis in sheep in Hokkaido was thus clarified. Although ID was less sensitive than ELISA in detecting the antibodies against C. pseudotuberculosis it did not give any non-specific reaction. From the results and in view of the simplicity of the test procedure, ID was found to be of practical diagnostic value. Distribution by age group of anti-C. pseudotuberculosis antibodies in 758 sheep in a herd detected by both tests showed that the ratio of positives was low in sheep aged less than 1 year, and the ratio increased significantly in those aged 1 year and continued to increase with age until it reached a plateau at the age of 4-5 years.     

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.     

Experimental infections with wild and mutant Yersinia pseudotuberculosis strains in rabbits

Experimental oral infections of rabbits with a wild-type Yersinia pseudotuberculosis strain (pIB102), and two null-mutants (yopK and ypkA) were carried out with the aim to explore the possibility to use mutant strains of Y. pseudotuberculosis as live carrier vaccine strains. The infectious process of the three strains proceed with passing hyperthermia, leucocytosis with granulocytosis, moderate monocytosis and a transient lymphopenia, better demonstrated at mutant strain infections. Short-term bacterial dissemination into the brain and viscera was observed at yopK infection. An augmented resistance to bactericidal activity of leucocytes at the initial phase of infection was followed by an increased sensitivity discovered earlier in case of yopK strain accompanied by at least 70- and 20-fold, respectively, for ypkA lower virulence for mice. The level of attenuation of yopK was accompanied by significant Yersinia specific IgG and IgM antibody response. Inflammatory foci were found by morphological examination in brain, lung and small intestines after infection with the wild-type strain, while such foci were only observed in brain and mesenterial lymph nodes after infection with the yopK mutant. After infection with the ypkA mutant foci were found in brain and spleen of the infected animals. Morphological changes in the lymphatic tissue of rabbits infected with mutant strains were consistent with induction of immunogenesis. The data suggest that genetically constructed yopK null-mutant exhibits characteristics that makes the strain suitable to be used as a live carrier vaccine to deliver heterologous antigens.     

The 16MΔvjbR as an ideal live attenuated vaccine candidate for differentiation between Brucella vaccination and infection

Brucellosis brings great economic burdens for developing countries. Live attenuated vaccines are the most efficient means for prevention and control of animal Brucellosis. However, the difficulties of differentiating of infection from vaccine immunization, which is essential for eradication programs, limit their applications. Therefore, the development of a vaccine that could differentiate infection from immunization will overcome the limitations and get extensive application. VjbR is a quorum sensing regulator involving in Brucella's intracellular survival. The vjbR∷Tn5 mutants have been proven effective against wild type strain challenge, implying its possibility of use in vaccine candidate development. To further evaluate this candidate gene, in the present study, the antigenicity of purified recombinant VjbR protein was analyzed. Antibodies to Brucella melitensis VjbR could be detected in sera from patients and animals with brucellosis but not in control ones, implying the potential use of this protein as a diagnostic antigen. Then a vjbR mutant of B. melitensis 16M was constructed by replacing the vjbR with kanamycin gene. The mutant showed reduced survival in macrophage and mice. Vaccination of BALB/c mice with 16MΔvjbR conferred significant protective immunity against B. melitensis strain 16M challenges, being equivalent to which induced by the license vaccine Rev.1. The vjbR deletion mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon and interleukin-10. The most importance is that, the use of vjbR mutants as vaccines in association with diagnostic tests based on the VjbR antigen would allow the serological differentiation between infected and vaccinated animals. These results suggest that 16MΔvjbR is an ideal live attenuated vaccine candidate against B. melitensis and deserves further evaluation for vaccine development.     

Effect of the interval between shearing and dipping on the spread of Corynebacterium pseudotuberculosis infection in sheep

OBJECTIVE: To determine whether the spread of Corynebacterium pseudotuberculosis infection to sheep in dips could be controlled by increasing the time between shearing and dipping. DESIGN: A controlled treatment trial where only the time between shearing and dipping was varied. ANIMALS AND PROCEDURE: One hundred and ninety-five sheep were found to be negative for C. pseudotuberculosis exposure by assay of CLA toxin antibody, were divided into four treatment groups. Each was shorn at either 0, 2, 4 or 8 weeks before dipping in a solution containing C. pseudotuberculosis. Blood samples were taken 6 weeks after dipping and sheep were slaughtered 12 weeks after dipping. A fifth smaller group of 14 sheep shorn 26 weeks before dipping, was also exposed to C. pseudotuberculosis and was slaughtered with the other sheep. RESULTS: The occurrence of caseous lymphadenitis abscesses did not differ between groups or with sheep shorn 26 weeks before dipping. The proportion of sheep that seroconverted to the C. pseudotuberculosis toxin and cell wall ELISA was larger in sheep dipped immediately after shearing than in sheep in the other groups. CONCLUSIONS: Delaying dipping until 8 weeks after shearing did not decrease the C. pseudotuberculosis infection rate due to dipping. Sheep dipped immediately after shearing developed higher concentrations of antibody to C. pseudotuberculosis than sheep when dipping occurred between 2 and 8 weeks and later after shearing.     

Immunisation against ovine caseous lymphadenitis: efficacy of monocomponent Corynebacterium pseudotuberculosis toxoid vaccine and combined clostridial-corynebacterial vaccines

Sheep were immunised with Corynebacterium pseudotuberculosis toxoid formulated as a monocomponent vaccine with aluminium adjuvant or in combination with 5 clostridial antigens, and also in the combined form with sodium selenate. Immunised and control sheep were experimentally infected 16 days after vaccination and slaughtered and inspected after a further 3 months to determine their resistance to infection. All 3 vaccines afforded an equal and high level of protection; 91% of vaccinated sheep exhibiting no lesions of caseous lymphadenitis compared with 51.5% affected sheep in the control group. Average lesion counts were 1.2 per affected vaccinated sheep and 4.5 per affected control sheep. Antitoxin responses to the clostridial toxoids incorporated in the combined vaccines were not affected by inclusion of the C pseudotuberculosis toxoid or the sodium selenate.