Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats.
An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins.
Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.
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-γ)-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-γ-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.
A field trial to evaluate a whole cell vaccine for the prevention of caseous lymphadenitis (CLA) in sheep and goats was performed in one goat herd and one sheep flock over a period of three years. In goats, there was a nonstatistically significant trend for fewer cases of CLA in the vaccinated animals compared to the controls. In sheep, from six months to 36 months postinitial vaccination, the proportion of vaccinated sheep that developed CLA was significantly less (p less than 0.05) than in the control sheep. The antibody titers to Corynebacterium pseudotuberculosis as detected by microagglutination assay were significantly different (p less than 0.0001) at all times except at the initial vaccination. Swellings occurred at the vaccination site at an incidence level of 29.6% in goats and 34.1% in sheep. The vaccine appeared to be efficacious in reducing the proportion of sheep that developed CLA when challenged naturally in a field situation.
Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity.
Methodology and Findings
We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer.
These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.
Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable economic losses for herd owners.
We assessed the seroprevalence of infection with C. pseudotuberculosis in 805 sheep from 23 sheep farms that supply slaughterhouses in the state of Minas Gerais; we also analyzed management practices that could be associated with CLA occurrence, used on these and nearby farms that also supplied animals to the slaughterhouse (n = 60). The serum samples for assaying CLA infection were taken at the slaughterhouse. Frequency of infection with C. pseudotuberculosis was estimated at 43.7%, and farm frequency was estimated at 100%. Management practices were analyzed through a questionnaire. All farmers (60/60) had extensive/semi-extensive rearing system; 70.0% (42/60) identified sheep individually; 11.7% (7/60) had periodical technical assistance; 41.7% (25/60) disinfected the facilities; 86.7% (52/60) used barbed wire fences and did not implement adequate CLA control measures; only 11.7% (7/60) of breeders reported vaccination against C. pseudotuberculosis; 13.3% (8/60) took note of animals with clinical signs of CLA; 1.7% (1/60) opened and sanitized abscesses, and isolated the infected animals; 10.0% (6/60) knew the zoonotic potential of this disease and 1.7% (1/60) of the farmers culled animals in case of recurrence of abscesses.
It can be concluded that C. pseudotuberculosis infection is widely spread in sheep flocks in Minas Gerais state in Brazil and that there is a lack of good management measures and vaccination, allowing transmission of this infectious agent throughout the production network.
Caseous lymphadenitis; Corynebacterium pseudotuberculosis; sheep; slaughterhouse; Minas Gerais
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.
Corynebacterium pseudotuberculosis is a facultative intracellular pathogen and the causative agent of several infectious and contagious chronic diseases, including caseous lymphadenitis, ulcerative lymphangitis, mastitis, and edematous skin disease, in a broad spectrum of hosts. In addition, Corynebacterium pseudotuberculosis infections pose a rising worldwide economic problem in ruminants. The complete genome sequences of 15 C. pseudotuberculosis strains isolated from different hosts and countries were comparatively analyzed using a pan-genomic strategy. Phylogenomic, pan-genomic, core genomic, and singleton analyses revealed close relationships among pathogenic corynebacteria, the clonal-like behavior of C. pseudotuberculosis and slow increases in the sizes of pan-genomes. According to extrapolations based on the pan-genomes, core genomes and singletons, the C. pseudotuberculosis biovar ovis shows a more clonal-like behavior than the C. pseudotuberculosis biovar equi. Most of the variable genes of the biovar ovis strains were acquired in a block through horizontal gene transfer and are highly conserved, whereas the biovar equi strains contain great variability, both intra- and inter-biovar, in the 16 detected pathogenicity islands (PAIs). With respect to the gene content of the PAIs, the most interesting finding is the high similarity of the pilus genes in the biovar ovis strains compared with the great variability of these genes in the biovar equi strains. Concluding, the polymerization of complete pilus structures in biovar ovis could be responsible for a remarkable ability of these strains to spread throughout host tissues and penetrate cells to live intracellularly, in contrast with the biovar equi, which rarely attacks visceral organs. Intracellularly, the biovar ovis strains are expected to have less contact with other organisms than the biovar equi strains, thereby explaining the significant clonal-like behavior of the biovar ovis strains.
In Alberta, caseous lymphadenitis (CLA) is one of the leading causes of lamb and mutton carcass condemnation. In this study, serologic results confirmed a high (50-94%) incidence of exposure to Corynebacterium pseudotuberculosis, the causative agent of CLA, in mature, unvaccinated sheep in southern Alberta. To assess the efficacy and impact of vaccination with 2 commercial (Glanvac-6 and Case-Vac) and 1 experimental (WC+ MDP-GDP) CLA vaccines, a series of 3 field trials in 3249 ewes and lambs was conducted in affected flocks from 1992-1996. Efficacy was assessed from the serological response to vaccination, prevalence and size of injection site reactions by treatment, and the incidence of CLA abscesses. Overall, agglutinating antibody titres to C. pseudotuberculosis in lambs vaccinated with WC+MDP-GDP and Case-Vac remained significantly elevated above nonvaccinated control lambs for the 12 mo period after the initial vaccination. Lambs vaccinated with the WC/MDP-GDP maintained higher titres (P < 0.06) than those vaccinated with Case-Vac for the period from 6 to 12 mo after vaccination. Agglutinating antibody titres for lambs vaccinated with Glanvac did not differ from those of controls at any point during the 12 mo period after vaccination. The number of injection site reactions was elevated in lambs vaccinated with Glanvac as compared to those vaccinated with WC+MDP-GDP but the size of injection site reactions did not significantly differ. Sheep vaccinated with WC+ MDP-GDP also had a reduced incidence of putative CLA abscesses, although confirmation of the presence of C. pseudotuberculosis was only successful in a small number of instances.
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.
Caseous lymphadenitis is a chronic suppurative disease caused by Corynebacterium pseudotuberculosis and is responsible for serious economic losses to the sheep and goat industry. Caseous lymphadenitis was first reported for goats in the United Kingdom in 1990 and for sheep in 1991. Recent evidence suggests that the prevalence of the disease within the national flock is increasing. Fifty isolates of C. pseudotuberculosis from the United Kingdom comprising sheep and horse isolates, the original goat outbreak strain, and the type strain were characterized by biotyping, antimicrobial susceptibility, production of phospholipase D, and genotyping by pulsed-field gel electrophoresis using SfiI and SmaI. All of the isolates were confirmed as C. pseudotuberculosis, and all produced phospholipase D but none reduced nitrate. Restriction with SfiI generated 16 to 18 bands between 48.5 and 290 kb and differentiated six pulsotypes. We conclude that 80% of the strains tested were epidemiologically related to the outbreak strain and that the equine profile was distinct both phenotypically and genotypically.
A large-scale DNA vaccination trial was performed with sheep to
investigate whether an antigen targeted by CTLA-4 enhanced and
accelerated the humoral immune response. Vaccination with genetically
detoxified phospholipase D (ΔPLD) has been shown to be effective, at
least partially, against Corynebacterium
pseudotuberculosis, the causal agent of caseous lymphadenitis in
sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used
to direct the fusion antigens to sites of immune induction. Here we
demonstrated that targeting ΔPLD as a CTLA-4 fusion protein
significantly enhanced the speed, magnitude, and longevity of the
antibody response compared to that obtained with DNA encoding ΔPLD.
While all groups of sheep vaccinated with DNA encoding ΔPLD were
afforded better protection against an experimental challenge with
C. pseudotuberculosis than those immunized with an
irrelevant plasmid or those left unimmunized, the best protection was
provided by the targeted DNA vaccine. We propose that targeting
antigens to antigen-presenting cells offers a generic strategy for
enhancing the efficacy of DNA vaccines.
Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. We identified and characterized the hsp60-hsp10 bicistronic operon of the animal pathogen Corynebacterium pseudotuberculosis, a Gram-positive bacterium of the class Actinobacteria, which causes caseous lymphadenitis (CLA) in small ruminants.
To construct the DNA vaccine, the hsp60 gene of C. pseudotuberculosis was cloned in a mammalian expression vector. BALB/c mice were immunized by intramuscular injection with the recombinant plasmid (pVAX1/hsp60).
This vaccination induced significant anti-hsp60 IgG, IgG1 and IgG2a isotype production. However, immunization with this DNA vaccine did not confer protective immunity.
Caseous lymphadenitis (CLA) is a disease of small ruminants caused by Corynebacterium pseudotuberculosis. The pathogenesis of CLA is a slow process, and produces a chronic rather than an acute disease state. Acute phase proteins (APP) such as haptoglobin (Hp) serum amyloid A (SAA) and α1 acid glycoprotein (AGP) are produced by the liver and released into the circulation in response to pro-inflammatory cytokines. The concentration of Hp in serum increases in experimental CLA but it is not known if SAA and AGP respond in parallel or have differing response profiles.
The concentration in serum of Hp, SAA and AGP in 6 sheep challenged with 2 × 105 cells of C. pseudotuberculosis showed significant increases (P < 0.05) compared to 3 unchallenged control sheep. By day 7 post infection. (p.i.) the Hp and SAA concentrations reached mean (± SEM) values of 1.65 ± 0.21 g/L and 18.1 ± 5.2 mg/L respectively. Thereafter, their concentrations fell with no significant difference to those of the control sheep by day 18 p.i.. In contrast, the serum AGP concentration in infected sheep continued to rise to a peak of 0.38 ± 0.05 g/L on day 13 p.i., after which a slow decline occurred, although the mean concentration remained significantly higher (P < 0.05) than the control group up to 29 days p.i.. Specific IgG to phospholidase D of C. pseudotuberculosis became detectable at 11 days p.i. and continued to rise throughout the experiment.
The serum concentrations of Hp, SAA and AGP were raised in sheep in an experimental model of CLA. An extended response was found for AGP which occurred at a point when the infection was likely to have been transforming from an acute to a chronic phase. The results suggest that AGP could have a role as a marker for chronic conditions in sheep.
A double antibody sandwich ELISA developed by ID-DLO, Lelystad to detect Corynebacterium pseudotuberculosis infection was used on 329 sheep from four pedigree Suffolk flocks in which clinical cases of caseous lymphadenitis (CLA) had occurred. At subsequent necropsy, typical CLA lesions were seen in 133 sheep, and the diagnosis was confirmed on culture. Lesions were most commonly seen in lungs (n = 46), parotid lymph nodes (n = 44), prescapular lymph nodes (n = 38) and mediastinal lymph nodes (n = 31). The sensitivity of the ELISA test for detecting culture-positive sheep was 0.88, while the specificity of the test was 0.55. The antibody ELISA detected 87.5 per cent of sheep that had CLA lesions restricted to internal organs only. It was concluded that the ELISA test has a valuable role in detecting sheep with both clinical and subclinical CLA.
Sheep; Caseous lymphadenitis; Serology; Corynebacterium pseudotuberculosis
A survey of caseous lymphadenitis was conducted at a goat and sheep slaughterhouse in Northeastern Brazil One hundred and fifty-eight goats and 43 sheep were examined for the presence of abscesses, with bacterial culturing of purulent material to define the etiological agent. Blood was collected simultaneously for determination of serological titer via the synergistic hemolysis-inhibition test which measures antibodies to an exotoxin of Corynebacterium pseudotuberculosis. Thirteen and nine-tenths percent of the goats had abscesses, with a high proportion having mediastinal or pulmonary lesions (9.5%). Two sheep had abscesses, both with internal organ involvement. Corynebacterium pseudotuberculosis was the most frequently isolated organism. Of 22 goats with abscesses, 20 were positive via the synergistic hemolysis-inhibition test. Both of the sheep with abscesses had positive synergistic hemolysis-inhibition titers. The proportion of serological reactors was greater than the proportion of animals with abscesses. The synergistic hemolysis-inhibition test may be detecting subclinically infected animals.
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).
From January to February 2008, 468 sheep carcasses (335 male and 133 female) in a Khosroshahr (suburb of Tabriz, East Azerbaijan province, Iran) abattoir were randomly selected for inspection. The aim of the study was to estimate the frequency of caseous lymphadenitis (CLA) in sheep and to compare the results of bacterial cultures and histopathology of suspected cases. The mean age of the population was 2.5 years. One hundred ninety-seven cases containing 153 (77.7%) males and 44 (22.3%) females had prominent enlargement of one of the lymph nodes (i.e., prescapular, prefemoral, inguinal, supramammary, or midiastinal); these were removed with the surrounding tissue for further evaluation. For confirmed diagnosis of CLA, samples were sent for microbiology and pathology analysis. Standard bacteriological culture methods for isolation of Corynebacterium pseudotuberculosis and tissue preparations for histopathological sections were performed. To evaluate the effect of age on the frequency of CLA, animals were categorized in four groups: under 1, 1–2, 2–3, and over 3 years of age. Based on the results, in 59 (12.60%) carcasses C. pseudotuberculosis was isolated, and in 94 (20.08%) of the cases histopathological studies revealed pathognomonic signs (lamellated exudates or onion ring) of CLA. The frequency of CLA based on bacteriological culture was 12.60% and on histopathological study 20.08%. In 37 (18.8%) of the carcasses, both bacteriological and histopathological studies confirmed CLA. The frequency of CLA following microscopic examination (20.08%) presented a more precise diagnosis compared to bacteriological culture (12.60%) and macroscopic evaluation of the lymph nodes (P < 0.05). Furthermore, there was a positive correlation rate between the bacteriological culture and histopathological study (r = 0.196, P = 0.006). The prescapular lymph node had the highest infection rate with 54 (1.70 ± 0.97) and supramammary lymph node had the lowest with two (0.07 ± 0.41) (P < 0.05). There was an increase in CLA detection with increasing age (P < 0.05), the mean age of animals with a positive CLA test were 2.92 years and in the oldest age group 31 (47%) cases had the highest infection.
Caseous lymphadenitis; Sheep; Frequency; Tabriz; Iran
The phospholipase D (PLD) gene (pld) has been deleted from the Corynebacterium pseudotuberculosis chromosome by using site-specific mutagenesis. Sheep infection trials indicate that the PLD-negative C. pseudotuberculosis strain (Toxminus) is incapable of inducing caseous lymphadentis (cheesy gland) even at doses two logs higher than that at which the wild-type strain produces the disease. This clearly establishes PLD as a major C. pseudotuberculosis virulence factor. Vaccination of sheep with live Toxminus C. pseudotuberculosis elicits strong humoral and cell-mediated immune responses and protects the animals from wild-type challenge.
Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated.
Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence.
The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.
In this work we report the genome of Corynebacterium pseudotuberculosis strain 267, isolated from a llama. This pathogen is of great veterinary and economic importance, as it is the cause of caseous lymphadenitis in several livestock species around the world and causes significant losses due to the high cost of treatment.
The dual concepts of pan and core genomes have been widely adopted as means to assess the distribution of gene families within microbial species and genera. The core genome is the set of genes shared by a group of organisms; the pan genome is the set of all genes seen in any of these organisms. A variety of methods have provided drastically different estimates of the sizes of pan and core genomes from sequenced representatives of the same groups of bacteria.
We use a combination of mathematical, statistical and computational methods to show that current predictions of pan and core genome sizes may have no correspondence to true values. Pan and core genome size estimates are problematic because they depend on the estimation of the occurrence of rare genes and genomes, respectively, which are difficult to estimate precisely because they are rare. Instead, we introduce and evaluate a robust metric - genomic fluidity - to categorize the gene-level similarity among groups of sequenced isolates. Genomic fluidity is a measure of the dissimilarity of genomes evaluated at the gene level.
The genomic fluidity of a population can be estimated accurately given a small number of sequenced genomes. Further, the genomic fluidity of groups of organisms can be compared robustly despite variation in algorithms used to identify genes and their homologs. As such, we recommend that genomic fluidity be used in place of pan and core genome size estimates when assessing gene diversity within genomes of a species or a group of closely related organisms.
Caseous lymphadenits (CL) is a chronic and subclinical disease that affects goats and sheep and, consequently, causes economic losses, especially to small producers. The purpose of this study, through use of Polymerase Chain Reaction (PCR), was to verify the presence of virulence genes of phospholipase D (PLD), integral membrane protein (FagA), iron enterobactin transporter (FagB), ATP binding cytoplasmic membrane protein (FagC) and iron siderophore binding protein (FagD) in 168 isolates of C. pseudotuberculosis obtained from cases of caseous lymphadenitis in goats and sheep. FagA, FagB and PLD genes were detected in all 145 strains isolated from abscesses in superficial lymph nodes and in 23 strains isolated from viscera. The FagC gene was positive in 167 (99.40%) isolates. The FagD gene was detected in 160 (95.23%) isolates. All virulence factors analyzed were found more frequently among isolates collected in the viscera of animals with CL, indicating a multifactorial nature, as well as variations, in the invasive potential of C. pseudotuberculosis strains.
virulence genes; C. pseudotuberculosis; caseous lymphadenits
The bacterium Corynebacterium pseudotuberculosis is of major veterinary importance because it affects livestock, particularly sheep, goats, and horses, in several countries, including Australia, Brazil, the United States, and Canada, resulting in significant economic losses. In the present study, we describe the complete genome of the Corynebacterium pseudotuberculosis Cp316 strain, biovar equi, isolated from the abscess of a North American horse.
Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N2 fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.
Twenty-five strains of Corynebacterium pseudotuberculosis isolated from lesions of caseous lymphadenitis in goats were examined for their biochemical characteristics, antimicrobial susceptibility, and phospholipase D activity. The strains were uniform in biochemical reactions, cultural characteristics, and susceptibility to antimicrobial agents. Presence of urease and phospholipase D and absence of pyrazinamidase were valuable criteria in the identification of C. pseudotuberculosis.