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At least 19 recordsLinked to original sources
PubMed1981

Phospholipase D activity of Corynebacterium pseudotuberculosis (Corynebacterium ovis) and Corynebacterium ulcerans, a distinctive marker within the genus Corynebacterium.

A search has been made for corynebacterial phospholipase D, "ovis toxin," a sphingomyelinase (phosphatidylcholine phosphohydrolase, EC 3.1.4.4), among a wide variety of corynebacteria. Phospholipase D activity has been found in strains exhibiting the biochemical properties characteristic of Corynebacterium pseudotuberculosis or of Corynebacterium ulcerans and in no other species of Corynebacterium. Methods for the assay of phospholipase D as a sphingomyelinase and methods for screening for phospholipase D in the presence of Corynebacterium equi on washed sheep blood agar are discussed.

Corynebacterium↗
PubMed1997

Attenuation and vaccine potential of aroQ mutants of Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis, a gram-positive intracellular bacterial pathogen, is the etiological agent of the disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel live veterinary vaccine vectors. We have cloned and sequenced the aroB and aroQ genes from C. pseudotuberculosis C231. By allelic exchange, aroQ mutants of both C231, designated CS100, and a pld mutant strain TB521, designated CS200, were constructed. Infection of BALB/c mice indicated that introduction of the aroQ mutation into C231 and TB521 attenuated both strains. In sublethally infected BALB/c mice, both CS100 and CS200 were cleared from spleens and livers by day 8 postinfection. The in vivo persistence of these strains was increased when the intact aroQ gene was supplied on a plasmid in trans. Mice infected with TB521 harbored bacteria in organs at least till day 8 postinfection without ill effect. When used as a vaccine, only the maximum tolerated dose of CS100 had the capacity to protect mice from homologous challenge. Vaccination with TB521 also elicited protective immunity, and this was associated with gamma interferon (IFN-gamma) production from splenocytes stimulated 7 days postvaccination. The role of IFN-gamma in controlling primary infections with C. pseudotuberculosis was examined in mice deficient for the IFN-gamma receptor (IFN-gammaR(-/-) mice). IFN-gammaR(-/-) mice cleared an infection with CS100 but were significantly more susceptible than control littermates to infection with C231 or TB521. These studies support an important role for IFN-gamma in control of primary C. pseudotuberculosis infections and indicate that aroQ mutants remain attenuated even in immunocompromised animals. This is the first report of an aroQ mutant of a bacterial pathogen, and the results may have implications for the construction of aromatic mutants of Mycobacterium tuberculosis for use as vaccines.

Amino Acid Sequence↗
PubMed1990

Alterations in the phospholipid composition and morphology of ovine erythrocytes after intravenous inoculation of Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis produces a sphingomyelin-specific phospholipase D exotoxin that is a major determinant in the pathogenesis of caseous lymphadenitis. The effect of this exotoxin on erythrocytes was assessed during experimentally induced infection of sheep. Blood was drawn at timed intervals, and the phospholipid composition of erythrocytes was determined by use of high-performance liquid chromatographic analysis of membrane extracts. Erythrocyte morphology was determined by use of transmission electron microscopy. Significant (P less than or equal to 0.05) decreases in erythrocyte membrane sphingomyelin content and significant (P less than or equal to 0.05) increases in phosphatidylglycerol content were observed 30 minutes after IV inoculation of C pseudotuberculosis. The concentration of other phospholipids remained unchanged. Initially, spherostomatocytes were formed that later became pitted at the cell surface. These pits or invaginations appeared as numerous vacuoles at the periphery of thin-sectioned cells. Pitting became progressively worse, leading to an extensive scalloped cell surface. Alterations in the phospholipid composition and morphology of ovine erythrocytes may contribute to pathophysiologic findings in sheep with acute infection induced by C pseudotuberculosis.

Animals↗
PubMed2005

In vitro IFN-gamma production by goat blood cells after stimulation with somatic and secreted Corynebacterium pseudotuberculosis antigens.

Corynebacterium pseudotuberculosis is the causal agent of caseous lymphadenitis, a chronic illness that attacks goats and sheep characterized by pyogranulomas formation in lymph nodes and organs. Regarding the current knowledge of the pathogenesis of the caseous lymphadenitis, there is evidence that besides the humoral response the induction of a durable cellular response is fundamental for its control. In this sense, research on antigens of C. pseudotuberculosis that are capable to inducing cellular immunity is an important step for the development of diagnosis tests and more efficient vaccines. In the present study, the interferon-gamma production in cultures of whole blood from infected goats stimulated with secreted bacterial antigen or somatic antigen were used to evaluate the cellular response. The results demonstrated a significant difference in the ability of the two antigens to induce a cellular response. That is, IFN-gamma production was high with cells from infected animals in response to the secreted antigen while IFN-gamma production was low when somatic antigen was used. The concomitant use of these antigens with PWM also showed differences. That is, the secreted antigen increased the IFN-gamma production induced by PWM, while the somatic antigen seems not to have altered the response to PWM.

Animals↗
PubMed1991

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.

Animals↗
PubMed1995

[Corynebacterium pseudotuberculosis adenitis in a shepherd].

Corynebacterium pseudotuberculosis causes suppurative lymphadenitis in sheep and other domestic animals. Human infection has been reported in few instances. We report on a case of Corynebacterium pseudotuberculosis lymphadenitis in a human being. A 34-years old previously healthy shepherd was attended for presenting a painful lymph node in right groin with one year of evolution. Cultures of an aspirate and ganglionar tissue yielded growth of Corynebacterium pseudotuberculosis in pure culture. Histological examination of the excised lymph node showed a suppurative granulomatous lymphadenitis with areas of necrosis in which there were clusters of bacillary organisms. After surgical excision and administration of erythromycin clinical signs disappeared without complications. We have carried out a review of the literature and we have found no case reported in Spain. Corynebacterium pseudotuberculosis lymphadenitis in human beings is a rare entity that principally affects persons in contact with animals, principally sheep. Most cases have been reported in Australia. In accordance with the reviewed literature this is the first time this disease is reported in Spain. Corynebacterium pseudotuberculosis infection should be considered in the differential diagnosis of localized granulomatous lymphadenitis.

Adult↗
PubMed1980

Diagnostic utilization of hemolytically active exosubstances of certain Gram-positive bacteria. II. Detection of staphylococcal hemolysins by means of a pre-purified hemolytically active exosubstance of Corynebacterium pseudotuberculosis.

The use of Corynebacterium pseudotuberculosis or of its hemolytically active exosubstance for the detection of staphylococcal hemolysins represents a suitable alterative to the use of the beta-toxin producing strain of Staphylococcus aureus, or of isolated beta-toxin. An increased capture of beta-toxin production was recorded when the CAMP-factor of Streptococcus agalactiae was used. A total of 446 Staphylococcus aureus strains and 48 coagulase-negative staphylococal strains were tested against the prepurified form of hemolytically active exosubstance of Corynebacterium pseudotuberculosis and against the CAMP-factor. The results obtained are in agreement with the testing by means of staphylococcal beta-toxin and of the CAMP-factor. The two modifications of the method for the detection of staphylococcal hemolysins using one plate as proposed by the authors give good results. Their ability of assessment, however, is higher by one degree when the exocubstance of Corynebacterium pseudotuberculosis is employed.

Bacterial Toxins↗
PubMed1998

Vaccine potential of attenuated mutants of Corynebacterium pseudotuberculosis in sheep.

Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C. pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable specific gamma interferon (IFN-gamma)-secreting lymphocytes and induced low levels of antibodies to C. pseudotuberculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-gamma-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines or as vaccine vectors.

Animals↗
PubMed1984

Corynebacterium ulcerans and Corynebacterium pseudotuberculosis responses to DNA probes derived from corynephage beta and Corynebacterium diphtheriae.

Strains of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (Corynebacterium ovis) were examined for the production of diphtheria toxin. A majority of C. ulcerans strains (25 of 37) and 1 C. pseudotuberculosis strain (1 of 14) gave a positive Elek test for diphtheria toxin, and for all strains but 1, production of diphtheria toxin was inhibited at the same level of Fe2+ as was the Corynebacterium diphtheriae control. All Elek-positive cultures as well as two Elek-negative isolates of C. ulcerans gave a positive signal when hybridized with a DNA probe unambiguous for the diphtheria toxin gene (tox) under conditions of high stringency. The majority of probe-positive C. ulcerans strains contained three or more DNA restriction fragments that hybridized with converting corynephage beta, suggesting that in C. ulcerans as in C. diphtheriae there may be a relationship between toxinogeny and carriage of beta-related phage. Selected strains of C. diphtheriae, C. ulcerans, and C. pseudotuberculosis were examined for DNA homology by a semiquantitative technique. There was very little homology between C. diphtheriae and members of the other two species. Strains of C. ulcerans and C. pseudotuberculosis, although more closely related, appeared to belong to distinct species as well.

Bacteriophages↗
PubMed1990

Changes in ovine erythrocyte morphology due to sphingomyelin degradation by Corynebacterium pseudotuberculosis phospholipase D.

Corynebacterium pseudotuberculosis produces a sphingomyelin-specific phospholipase D exotoxin that is a major determinant in the pathogenesis of caseous lymphadenitis. We assessed the changes induced in the morphology and sphingomyelin concentration of ovine erythrocytes after incubation with broth culture filtrates or purified exotoxin. Sphingomyelin was hydrolysed nearly in parallel with shape change. Morphologically, spherostomatocytes were seen initially, and later these contained numerous vacuoles at the periphery. Vacuoles seen in thin sections with transmission electron microscopy appeared as pits with scanning electron microscopy. Pitting became progressively worse with time, leading to extensive scalloping of the membrane surface. Chemically, significant decreases (P less than or equal to 0.05) in erythrocyte membrane sphingomyelin content and significant increases (P less than or equal to 0.05) in the content of glycerophospholipids (i.e. phosphatidylglycerol) were observed in erythrocytes incubated with broth culture filtrates or purified exotoxin. These alterations may contribute to the pathophysiology of acute infections induced by C. pseudotuberculosis.

Animals↗
PubMed1997

Cytokine gene expression in sheep following experimental infection with various strains of Corynebacterium pseudotuberculosis differing in virulence.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis in sheep and goats. This disease is characterized by the development of pyogranulomas in the lymph nodes and lung tissue. To measure the cytokine gene expression in C pseudotuberculosis lesions, sheep were inoculated with two attenuated strains (Tox- and PLD-t) and a wild-type (WT) strain of C pseudotuberculosis and were necropsied at 7 or 28 days post-inoculation. The Tox- strain showed a strong reduction in virulence as assessed by the absence of disseminating lesions in the lymph nodes draining the inoculation site in contrast with the WT strain. The PLD-t strain showed an intermediate reduction in virulence. The two attenuated strains, however, induced the same amount of antibodies and IFN-gamma production as the WT strain. Using a semi-quantitative RT-PCR technique, the expression of inflammatory cytokines was found to be higher in the inoculation site, whereas expression of T-cell associated cytokines was more intense in the draining lymph node. On the whole, the infected sheep produced high levels of cytokines in at least one organ on days 7 or 28 post-inoculation. No significant differences in cytokine gene expression were shown between sheep infected with strains differing in virulence. Higher cytokine expression was measured in sheep with pyogranulomas in the draining lymph nodes as compared to those without, especially for interleukin-1 beta and interleukin-8. Overall, these results taken together confirmed the attenuation of virulence in Tox- and PLD-t strains of C pseudotuberculosis and showed the important role of PLD in disseminating the bacteria from the inoculation site to the draining lymph nodes. The pathogenesis of ovine caseious lymphadenitis was shown to be associated with production of cytokines at the pyogranuloma level, but the local cytokine patterns associated with different courses of infection were not distinguished.

Animals↗
PubMed2005

Identification of macrophage induced genes of Corynebacterium pseudotuberculosis by differential fluorescence induction.

Corynebacterium pseudotuberculosis is the etiological agent of the sheep disease caseous lymphadenitis. We have developed a promoter reporter system for this organism based on expression of the green fluorescent protein (gfp) gene from Aequorea victoria. A promoterless vector, pSM20, containing the gfp gene was constructed, and promoters were inserted upstream of the gfp gene. Upon transformation into C. pseudotuberculosis, fluorescence could be visualised by fluorescence microscopy, and relative promoter strength measured by flow cytometry. The usefulness of this system for measuring changes in gene expression was demonstrated by measuring fluorescence levels of heat shocked C. pseudotuberculosis carrying a dnaK promoter construct. Replication of C. pseudotuberculosis within J774 macrophages could be monitored by fluorescence microscopy. The establishment of the system allowed the use of differential fluorescence to identify genes that showed up-regulation following macrophage infection. Genes coding for a non-ribosomal peptide synthetase and the beta chain of a propionyl CoA carboxylase were identified as possessing promoters that demonstrated enhanced activity following macrophage infection by C. pseudotuberculosis.

Corynebacterium pseudotuberculosis↗
PubMed2006

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.

Animals↗
PubMed2006

Clinical, humoral, and pathologic findings in adult alpacas with experimentally induced Corynebacterium pseudotuberculosis infection.

OBJECTIVE: To experimentally infect adult alpacas by ID inoculation of Corynebacterium pseudotuberculosis, follow the clinical and pathologic course of disease, and study the humoral response to infection. ANIMALS: 13 adult alpacas. PROCEDURES: 9 alpacas were inoculated with 1.1 X 10(6) CFUs of C pseudotuberculosis from llama (n = 4) or alpaca (5) origin, and 4 alpacas were sham inoculated as controls. Alpacas were clinically observed after inoculation and euthanatized on days 16, 58, 93, or 128 after inoculation; necropsy examination and histologic evaluation were performed. An indirect ELISA, which made use of the C pseudotuberculosis cell wall as the antigen, was used to measure antibody titers in serum samples. RESULTS: Alpacas had a persistent febrile response, a local severe inflammatory response, and leucocytosis (> 30 X 10(3) WBCs/microL). Internal abscesses that localized mainly in the renal lymph node were observed. Corynebacterium pseudotuberculosis was recovered from the inoculation site 1 week after inoculation and from internal abscesses at 58 days after inoculation. Initial lesions were typical pyogranulomas with central caseous necrosis, whereas later lesions consisted of connective tissue, mononuclear cells, abundant neutrophils, and liquefactive necrosis. Infected alpacas had detectable serum antibody titers starting on day 16 that persisted until day 93 after inoculation. Shaminoculated alpacas did not develop serum antibody titers, clinical signs of infection, or lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Alpacas inoculated with C pseudotuberculosis developed abscesses at the inoculation site and internally in the renal lymph nodes, without lung lesions. Corynebacterium pseudotuberculosis isolates from llama and alpaca origin were found to be pathogenically indistinct.

Abscess↗
PubMed1994

Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis.

An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector.

Administration, Oral↗
PubMed1995

Molecular and biochemical characterization of a protective 40-kilodalton antigen from Corynebacterium pseudotuberculosis.

A 40-kDa protein from Corynebacterium pseudotuberculosis has been previously identified as a protective antigen against ovine caseous lymphadenitis. From genomic DNA libraries of C. pseudotuberculosis, we have cloned and sequenced the 40-kDa protein gene, which was found to contain an open reading frame of 1,137 bp encoding a protein of 379 amino acids. No significant homology with previously published DNA or amino acid sequence data was found in databases, suggesting that this is a novel protein. Recombinant 40-kDa protein was overexpressed as a fusion protein to 15% of total cell proteins in Escherichia coli. Biochemical analysis of native and recombinant 40-kDa proteins has revealed associated proteolytic activity, which was shown to be of the serine protease type through the use of specific inhibitors. We suggest that this novel protective antigen be termed corynebacterial protease 40 (CP40).

Amino Acid Sequence↗