Activity of biogenic silver nanoparticles in planktonic and biofilm-associated Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a gram-positive bacterium and is the etiologic agent of caseous lymphadenitis (CL) in small ruminants. This disease is characterized by the development of encapsulated granulomas in visceral and superficial lymph nodes, and its clinical treatment is refractory to antibiotic therapy. An important virulence factor of the Corynebacterium genus is the ability to produce biofilm; however, little is known about the characteristics of the biofilm produced by C. pseudotuberculosis and its resistance to antimicrobials. Silver nanoparticles (AgNPs) are considered as promising antimicrobial agents, and are known to have several advantages, such as a broad-spectrum activity, low resistance induction potential, and antibiofilm activity. Therefore, we evaluate herein the activity of AgNPs in C. pseudotuberculosis, through the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antibiofilm activity, and visualization of AgNP-treated and AgNP-untreated biofilm through scanning electron microscopy. The AgNPs were able to completely inhibit bacterial growth and inactivate C. pseudotuberculosis at concentrations ranging from 0.08 to 0.312 mg/mL. The AgNPs reduced the formation of biofilm in reference strains and clinical isolates of C. pseudotuberculosis, with interference values greater than 80% at a concentration of 4 mg/mL, controlling the change between the planktonic and biofilm-associated forms, and preventing fixation and colonization. Scanning electron microscopy images showed a significant disruptive activity of AgNP on the consolidated biofilms. The results of this study demonstrate the potential of AgNPs as an effective therapeutic agent against CL.

First Report of Caseous Lymphadenitis by Corynebacterium pseudotubercolosis and Pulmonary Verminosis in a Roe Deer (Capreolus capreolus Linnaeus, 1758) in Italy.

Caseous lymphadenitis is a chronic debilitating disease typical of small ruminants, but it is also noted in several other domestic and wild species. In this report, we present the first documented case in Italy of pseudotuberculosis in a roe deer (Capreolus capreolus, Linnaeus 1758) found dead in the mountains of Forlì-Cesena province, Emilia Romagna region. The carcass underwent necropsy according to standard protocols, revealing generalized lymphadenopathy and severe apostematous pneumonia with multifocal and encapsulated abscesses. Corynebacterium pseudotuberculosis was isolated from the lung parenchyma, lymph nodes and abscesses. Additionally, severe parasitic bronchopneumonia of the caudal lobes and gastrointestinal strongyle infestation were detected. To our knowledge, this is the first documented case of CLA referable to C. pseudotubercolosis in a roe deer in Italy.

Pilot study on the distribution of caseous lymphadenitis in Korean native goats and the relationship between sex and age in disease occurrence.

Caseous lymphadenitis (CLA) caused by Corynebacterium pseudotuberculosis is a chronic disease that affects goats. The Korean native goat (KNG) is the most popular goat breed raised in Korea. In this study, the distribution of CLA in the KNG population and the relevance of sex and age in disease development were determined. From March 2020 to February 2021, 1,177 KNGs from 110 farms were assessed. The distribution of CLA in animals was determined by a veterinary inspector who performed gross examinations of goat carcasses and confirmed diagnoses via polymerase chain reaction and bacteriological examination. The CLA detection rate in the KNG population was 19.80%, and more than half of the farms were affected by the disease (56.36%). A statistically significant difference was observed among the sex groups, with castrated males (13.98%) having the lowest detection rate, followed by intact males (22.48%) and females (24.09%), suggesting that castration has a positive effect on reducing the risk of CLA (p < 0.05). In terms of age groups, an increased detection rate of 28.16% was observed in the late adult (>2 years) group. Phylogenetic analysis indicated that the RNA polymerase beta subunit-encoding gene can effectively differentiate biovar ovis from biovar equi and can be used for further epidemiological studies of C. pseudotuberculosis. This is the first nationwide surveillance study of CLA distribution to confirm the continuous occurrence of CLA in Korean goat farms. Future studies should include risk factor analyses of CLA based on herd levels to prevent and control this disease in Korea.

A serum NMR metabolomic analysis of the Corynebacterium pseudotuberculosis infection in goats.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants, is highly prevalent worldwide. Economic losses have already been associated with the disease, and little is known about the host-pathogen relationship associated with the disease. The present study aimed to perform a metabolomic study of the C. pseudotuberculosis infection in goats. Serum samples were collected from a herd of 173 goats. The animals were classified as controls (not infected), asymptomatic (seropositives but without detectable CLA clinical signs), and symptomatic (seropositive animals presenting CLA lesions), according to microbiological isolation and immunodiagnosis. The serum samples were analyzed using nuclear magnetic resonance (1H-NMR), nuclear Overhauser effect spectroscopy (NOESY), and Carr-Purcell-Meiboom-Gill (CPMG) sequences. The NMR data were analyzed using chemometrics, and principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were performed to discover specific biomarkers responsible for discrimination between the groups. A high dissemination of the infection by C. pseudotuberculosis was observed, being 74.57% asymptomatic and 11.56% symptomatic. In the evaluation of 62 serum samples by NMR, the techniques were satisfactory in the discrimination of the groups, being also complementary and mutually confirming, demonstrating possible biomarkers for the infection by the bacterium. Twenty metabolites of interest were identified by NOESY and 29 by CPMG, such as tryptophan, polyunsaturated fatty acids, formic acid, NAD+, and 3-hydroxybutyrate, opening promising possibilities for the use of these results in new therapeutic, immunodiagnosis, and immunoprophylactic tools, as well as for studies of the immune response against C. pseudotuberculosis. KEY POINTS: • Sixty-two samples from healthy, CLA asymptomatic, and symptomatic goats were screened • Twenty metabolites of interest were identified by NOESY and 29 by CPMG • 1H-NMR NOESY and CPMG were complementary and mutually confirming.

Evaluation of the Association of Recombinant Proteins NanH and PknG from Corynebacterium pseudotuberculosis Using Different Adjuvants as a Recombinant Vaccine in Mice.

Caseous lymphadenitis is a chronic contagious disease that causes economic losses worldwide. Treatments are ineffective, thus demonstrating the importance of vaccination. In this study, rNanH and rPknG proteins from Corynebacterium pseudotuberculosis were associated with saponin or aluminum hydroxide adjuvants. Three experimental groups (10 animals each) were immunized with sterile 0.9% saline solution (G1), rNanH + rPknG + Saponin (G2), rNanH + rPknG + Al(OH)3 (G3). The mice received two vaccine doses 21 days apart. Animals were challenged 21 days after the last immunization and evaluated for 50 days, with endpoint criteria applied when needed. The total IgG production levels of the experimental groups increased significantly on day 42 when compared to the control (p < 0.05). When tested against rNanH, G2 had a better rate of anti-rNanH antibodies compared to G3. In the anti-rPknG ELISA, the levels of total IgG, IgG1, and IgG2a antibodies were higher in G2. The vaccines generated partial protection, with 40% of the animals surviving the challenge. The association of recombinant NanH and PknG proteins led to promising protection rates in mice, and although using different adjuvants did not interfere with the survival rate, it influenced the immune response generated by the vaccine formulations.

Isolation and Molecular Characterization of Corynebacterium pseudotuberculosis: Association with Proinflammatory Cytokines in Caseous Lymphadenitis Pyogranulomas.

Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a causative agent of numerous chronic diseases, including caseous lymphadenitis (CLA) in sheep and goats, which has a zoonotic potential in humans in addition to a poor therapeutic response. In this study, out of 120 collected samples, only 12 (10%) were positive for C. pseudotuberculosis by PCR and by intraperitoneal injection of male Guinea pigs and then characterized for antimicrobial susceptibility and its genetic-relatedness by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), which showed 2-4 bands ranging from 100 to 3000 bp that can be clustered into four clusters (C1-C4). Despite the serotype biovar 1 only infecting sheep and goats, ERIC-PCR reveals intra-subtyping variation. Examination of affected LNs and organs revealed marked enlargement with either thick creamy green pus or multiple abscesses of variable sizes with a central caseated core surrounded by dense fibrous capsule. A histopathological examination revealed a central necrotic core surrounded by a peripheral mantle of mononuclear cells and a fibrous capsule. Positive immune expression of nuclear factor kappa B (NF-κB/p65) and interleukin-1ß (IL-1ß) and negative expression of tumor necrosis factor (TNF) in CLA is the first report to our knowledge. Conclusion: In CLA pyogranulomas, IL1ß is a more crucial proinflammatory cytokine than TNF in the regulation of C. pseudotuberculosis infection, which is accompanied by marked NF-κB immunoexpression. Therefore, the NF-κB/p65 signaling pathway is involved in the activation of IL1ß, and additional immunohistochemical studies are required to determine the various roles of NF-κB/p65 in the inflammatory response within CLA pyogranulomas to control this pathogen.

Potential in vitro action of an adenosine analog and synergism with penicillin against Corynebacterium pseudotuberculosis.

Caseous lymphadenitis is a well-known disease caused by Corynebacterium pseudotuberculosis affecting small ruminants with small significance to human health because of its minor zoonotic potential. In both cases, few treatment options are available and conventional antimicrobial therapy is commonly refractory due to development of pyogranulomatous reactions, bringing great interest in discovering novel therapeutics for more suitable approaches. Dideoxynucleotides presented antibacterial action against various bacteria but were never described for C. pseudotuberculosis. Hypothesizing the antimicrobial action of 2',3'-dideoxiadenosine (ddATP) against C. pseudotuberculosis, we performed for the first time an investigation of its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in the ATCC® 19,410 strain and a well-characterized clinical isolate of C. pseudotuberculosis. We also assessed potential synergism with penicillin. ddATP showed a growth delay effect for C. pseudotuberculosis at 2 µmol/mL and a MIC and MBC of 4 µmol/mL against the ATCC® 19,410 strain, but not for the clinical strain. An antimicrobial effect was observed when using concentrations lower than the MIC of ddATP associated with penicillin for both strains tested. Our data suggest the potential of nucleotide analogs, especially adenosine, and its combination with penicillin, as a possible novel treatment for C. pseudotuberculosis-induced infections, and contributes with knowledge regarding alternative drugs to treat C. pseudotuberculosis infections.

Bioinformatic Approach of B and T Cell Epitopes of PLD and CP40 Proteins of Corynebacterium pseudotuberculosis ovis Mexican Isolate 2J-L towards a Peptide-Based Vaccine.

Mapping B and T cell epitopes constitutes an important action for peptide vaccine design. PLD and CP40 virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a causal agent of Caseous Lymphadenitis, have been evaluated in a murine model as good candidates for vaccine development. Therefore, the goal of this work was to in silico analyze B and T cell epitopes of the PLD and CP40 proteins of a Mexican isolate of Corynebacterium pseudotuberculosis ovis. The Immune Epitope Data Base and Resource website was employed to predict the linear and conformational B-cell, T CD4+, and T CD8+ epitopes of PLD and CP40 proteins of Corynebacterium pseudotuberculosis ovis Mexican strain 2J-L. Fifty B cell epitopes for PLD 2J-L and forty-seven for CP40 2J-L were estimated. In addition, T CD4+ and CD8+ cell epitopes were predicted for PLD 2J-L (MHC I:16 epitopes, MHC II:10 epitopes) and CP40 2J-L (MHC I: 15 epitopes, MHC II: 13 epitopes). This study provides epitopes, paying particular attention to sequences selected by different predictor programs and overlap sequences as B and T cell epitopes. PLD 2J-L and CP40 2J-L protein epitopes may aid in the design of a promising peptide-based vaccine against Caseous Lymphadenitis in Mexico.

Proteomics of Toxigenic Corynebacteria.

Within the genus Corynebacterium, six species are potential carriers of the tox gene, which encodes the highly potent diphtheria exotoxin: Corynebacterium diphtheriae, Corynebacterium belfantii, Corynebacterium rouxii, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis and Corynebacterium silvaticum. Based on their potential to infect different host species and cause either human infections, zoonotic diseases or infections of economically important animals, these bacteria are of high scientific and economic interest and different research groups have carried out proteome analyses. These showed that especially the combination of MS-based proteomics with bioinformatic tools helped significantly to elucidate the functional aspects of corynebacterial genomes and to handle the genome and proteome complexity. The combination of proteomic and bioinformatic approaches was also used to discover new vaccine and drug targets. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been established as a fast and precise tool for the identification of these bacteria.

Hematological and clinical biochemistry profiles in Canindé goats infected by Corynebacterium pseudotuberculosis and bred in a tropical semi-arid region.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis in goats and sheep, is highly prevalent worldwide and is characterized by economic losses in small ruminant production. Currently available techniques for clinical and laboratory diagnosis of the disease lack market availability and/or sensitivity, and therefore, infected animals can remain in the herd, serving as a source of infection for other animals. The present study aimed to verify hematological and clinical biochemistry changes in goats naturally infected by C. pseudotuberculosis. One hundred seventy-three Canindé goats were included in this study, from which blood samples and caseous lesions were collected. The animals were classified as uninfected, asymptomatic, and symptomatic according to microbiological isolation and serological assays. A high dissemination of the infection was observed in the herd, with 86.13% of positive animals, being 74.57% asymptomatic and 11.56% symptomatic. In the hemogram and clinical biochemistry analyses, the only statistical difference found was a higher level of serum urea in asymptomatic individuals than in non-infected animals. In addition, this study points to the possibility of chronic CLA being potentially reflected in hepatic and renal biochemical markers.

Multi-Omics of Corynebacterium Pseudotuberculosis 12CS0282 and an In Silico Reverse Vaccinology Approach Reveal Novel Vaccine and Drug Targets.

Corynebacterium pseudotuberculosis is an important animal pathogen, which is also able to infect humans. An optimal treatment of infections with this pathogen is not available today and consequently, more research is necessary to understand the infection process. Here, we present a combined -omics and bioinformatics approach to characterize C. pseudotuberculosis 12CS0282. The genome sequence of strain 12CS0282 was determined, analyzed in comparison with the available 130 C. pseudotuberculosis sequences and used as a basis for proteome analyses. In a reverse vaccinology approach, putative vaccine and drug targets for 12CS0208 were identified. Mass spectrometry analyses revealed the presence of multiple virulence factors even without host contact. In macrophage interaction studies, C. pseudotuberculosis 12CS0282 was highly resistant against human phagocytes and even multiplied within human THP-1 cells. Taken together, the data indicate a high pathogenic potential of the strain.

A Seroepidemiological Survey of Corynebacterium pseudotuberculosis Infection in South Tyrol, Italy.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, a bacterial infection that can affect livestock. This infection can cause low growth rates and milk yields and reproductive failure, along with the infection of humans, especially those in close contact with the animals. The aim of this study was to evaluate the local CLA prevalence, highlighting the parameters for the potential predisposition to infection in goats, and to implement a monitoring program based on the newly acquired scientific evidence. Of a total of 2365 goat farms in South Tyrol, 384 farms were selected for the present study. A statistically significant number of animals were subjected to serologic analysis for the detection of C. pseutotubercolosis antibodies. A total of 2948 goats belonging to the selected farms were tested for CLA, 713 of which showed a positive result. The data analysis led to an estimated CLA seroprevalence of 21.85%. The results achieved can enable the evaluation and possible implementation of a voluntary program that permits us to test a larger number of animals using serological techniques. This program would be of great importance, especially for those farms dedicated to the production of milk and dairy products, as some manufacturing practices may increase the risk of transmission of zoonotic pathogens such as C. pseudotuberculosis to humans.

Immunoprophylactic properties of the Corynebacterium pseudotuberculosis-derived MBP:PLD:CP40 fusion protein.

Caseous lymphadenitis (CLA) is a disease that affects small ruminants, and the best way to prevent its spread on a herd is through immunoprophylaxis. Thus, we aimed to evaluate the MBP:PLD:CP40 fusion protein as a new CLA immunogen. The fusion protein was constructed by combining Corynebacterium pseudotuberculosis PLD and CP40 proteins with maltose-binding protein (MBP) as an intrinsic adjuvant. The antigenicity, allergenic potential, prediction of B epitopes, binding to MHC receptors, and docking on the Toll-Like 2 receptor were evaluated in silico. MBP:PLD:CP40 was expressed and purified. 40 BALB/c were divided into four groups (G1 - control, G2 - Saponin, G3 - MBP:PLD:CP40, and G4 - rPLD + rCP40). Total IgG, IgG1, and IgG2a were quantified, and the expressions of cytokines after splenocyte in vitro stimulation were assessed. Mice were challenged 42 days after the first immunization. The in silico analysis showed that MBP:PLD:CP40 has immunogenic potential, does not have allergic properties, and can dock on the TRL2 receptor. MBP:PLD:CP40 stimulated the production of IgG1 antibodies in a fivefold proportion to IgG2a, and TNF and IL-17 were significantly expressed in response to the antigenic stimuli. When rPLD and rCP40 were used together for immunization, they could induce IFN-γ and IL-12, but with no detectable antibody production. The G3 and G4 groups presented a survival of 57.14% and 42.86%, respectively, while the G1 and G2 mice were all dead 15 days after the challenge. MBP:PLD:CP40 partially protected the mice against C. pseudotuberculosis infection and can be considered a potential new CLA immunogen. KEY POINTS: • The fusion protein induced more IgG1 than IgG2a antibodies; • The fusion protein also induced the expression of the TNF and IL-17 cytokines; • Mice inoculated with MBP:PLD:CP40 presented a 57.14% survival.

In silico approach to identify microsatellite candidate biomarkers to differentiate the biovar of Corynebacterium pseudotuberculosis genomes.

Corynebacterium pseudotuberculosis is the causative bacterial agent of the zoonotic disease known as caseous lymphadenitis, and it presents several mechanisms of response to host defenses, including the presence of virulence factors (VFs). The genomes of these bacteria have several polymorphic markers known as microsatellites, or simple sequence repeats (SSRs), that can be used to characterize the genome, to study possible polymorphisms existing among strains, and to verify the effects of such polymorphic markers in coding regions and regions associated with VFs. In this study, several SSRs were identified within coding regions throughout the 54 genomes of this species, revealing possible polymorphisms associated with coding regions that could be used as strain-specific or serotype-specific identifiers of C. pseudotuberculosis. The similarities associated with SSRs amongst the different serum variants of C. pseudotuberculosis, biovars equi and ovis, were also evaluated, and it was possible to identify SSRs located in coding regions responsible for a VF enrolled in pathogenesis known to mediate bacterial adherence (SpaH-type pili virulence factor). Phylogenetic analyses revealed that strains sharing SSR patterns, including the possible polymorphisms identified in the same position of gene-coding regions, were displayed by strains with a common ancestor, corroborating with the Genome Tree Report of the NCBI. Statistical analysis showed that the microsatellite groups belonging to equi and ovis biovars have a significance of 0.006 (p-value) in similarity, thus indicating them as good biomarker candidates for C. pseudotuberculosis.

P22 protein complex in the serodiagnosis of animal tuberculosis: Antigenic stability and cross-reactivity with Corynebacterium pseudotuberculosis infection.

The P22 ELISA was recently developed for the serodiagnosis of animal tuberculosis. Herein, the stability of the P22 antigen in different presentations and storage conditions, and the cross-reactivity with Corynebacterium pseudotuberculosis infection in small ruminants were evaluated. For the stability assay, serum samples from cows, sheep, goats, alpacas, badgers, and wild boar were used in the P22 ELISA. The cross-reactivity analysis used sera from sheep and goats with caseous lymphadenitis (CLA). Differences in the immune recognition of P22 were found when the antigen was stored at 40 °C, but without altering the negative or positive status of each sample. P22 ELISA presented 5.71 % cross-reactivity when CLA-positive sheep were evaluated, but no cross-reaction was observed among CLA-positive goat serum samples. This study showed that the P22 protein complex is stable under different formulations and temperatures, and that the assay presents a low cross-reactivity with CLA.

The Association of Bacterin and Recombinant Proteins Induces a Humoral Response in Sheep against Caseous Lymphadenitis.

In this study, we investigated the capacity of the recombinant proteins SpaC, NanH, SodC, and PLD of C. pseudotuberculosis to trigger protective humoral and cellular immune responses against experimentally induced C. pseudotuberculosis infection in sheep. The antigens were produced in a heterologous system and were purified by affinity chromatography. Nine sheep were randomly divided into three groups, which were immunized as follows: Group 1 (control)-a mix of adjuvants composed of the inactivated T1 strain of C. pseudotuberculosis and commercial Montanide™ISA 61 VG (T1M); Group 2-rSpaC, rSodC, rPLD, and T1M; Group 3-rNanH, rSodC, rPLD, and T1M. All groups were immunized twice (on days 0 and 30) and challenged on day 90 of the experiment. Humoral and cellular immune responses were evaluated by Enzyme-Linked Immunosorbent Assay (ELISA) to quantify the IgG antibodies and interferon-gamma (IFN-y). Both vaccine formulations with recombinant proteins (groups 2 and 3) could induce a significant humoral IgG immune response in sheep. The proteins rSodC, rPLD, and rNanH were more immunogenic, inducing significant levels of IgG antibodies after the first dose of the vaccine or after the challenge, maintaining constant levels until the end of the experiment. However, it was not possible to differentiate between the cellular responses induced by the vaccines. This lack of effectiveness points toward the need for further studies to improve the efficacy of this subunit-based vaccine approach.

Corynebacterium pseudotuberculosis Infections in Alpacas (Vicugna pacos).

Alpacas are the major camelid species in Europe held for hobbies, animal-aided therapy, and commercial reasons. As a result, health-related issues associated with alpacas are of growing significance. This especially holds true for one of the most serious infectious diseases, caseous lymphadenitis, which is caused by the bacterial pathogen Corynebacterium (C.) pseudotuberculosis. Our study focuses on post-mortem examinations, the laboratory diagnostic tool ELISA, and the immunoblot technique for the detection of specific antibodies against C. pseudotuberculosis and detection of the causative pathogen in alpaca herds. We examined a total of 232 alpacas living in three herds. Four of these alpacas were submitted for post-mortem examination, revealing abscesses, apostematous and fibrinous inflammation in inner organs, pleura, and peritoneum. Serological investigation using a commercial ELISA based on phospholipase D (PLD) as antigen and an in-lab ELISA based on whole cell antigens (WCA) revealed an overall seroprevalence of 56% and 61.2%, respectively. A total of 247 alpaca sera originating from 232 animals were tested comparatively using the in-lab and the commercial ELISA and showed a substantial degree of agreement, of 89.5% (Cohen's kappa coefficient of 0.784), for both tests. Further comparative serological studies using the two ELISAs and the immunoblot technique were carried out on selected sera originating from 12 breeding stallions and six breeding mares for which epidemiological data and partial C. pseudotuberculosis isolates were available. The results showed the immunoblot to have a sensitivity that was superior to both ELISAs. In this context, it should be emphasized that evaluation of these investigations and the epidemiological data suggest an incubation period of one to two months. Antibiotic susceptibility testing of 13 C. pseudotuberculosis isolates based on the determination of minimal inhibitory concentrations using the broth microdilution method revealed uniform susceptibility to aminopenicillins, cephalosporines, macrolides, enrofloxacin, florfenicol, tetracycline, sulfonamid/trimethoprime, tiamulin, gentamicin, neomycin, spectinomycin, and vancomycin, but resistance to colistin, nitrofurantoin, and oxacillin.

Cytopathological and bacteriological studies on caseous lymphadenitis in cattle slaughtered at Bishoftu municipal abattoir, Ethiopia.

BACKGROUND: Caseous lymphadenitis (CLA) is a chronic bacterial infectious disease that affects cattle, shoats, and other domestic and wild ruminants. METHODS: A purposive cross-sectional study was conducted on 30 cattle with enlarged lymph nodes to investigate CLA using cytopathological and bacteriological techniques from cattle slaughtered at Bishoftu municipal abattoir. RESULTS: From a total of 30 cattle subjected to clinical and post-mortem examinations, only one bull was found to be infected with a rare case of CLA in Bishoftu municipal abattoir, Ethiopia. Enlargement of the pre-scapular lymph node was the only clinical finding during ante-mortem inspection of the bull. The gross pathological lesion showed a pre-scapular lymph node with a caseo-necrotic dystrophic calcification that was accompanied by a rough texture and white to grayish hue. Histopathologically, the lymph node was characterized by central liquefactive necrosis that was surrounded by coagulative necrosis containing multiple foci of mineralization, infiltration of polymorphonuclear neutrophils and mononuclear immature fibrosis containing inflammatory cells and also with some sort of a thick layer of mature fibrosis that defines the magnitude of the lesion. Cytologically, multi-lobulated (intact and degenerated) neutrophils, a few reactive lymphocytes, macrophages and some crenated histocytes have been recognized. The bacterial culture of the sample revealed small, white cream, dry, waxy colonies with a narrow area of ß-haemolysis. The isolate of the sample was a Gram-positive cocci-bacilli that was arranged in a Chinese pattern on Gram staining, and catalase and urease were positive in the biochemical analysis of this organism, which was able to ferment glucose and maltose but not trehalose and xylose. CONCLUSIONS: The present investigation indicated that CLA was prevalent as sporadic cases among cattle slaughtered in Bishoftu municipal abattoir. Thus, effective preventive and control measures, such as good sanitation and hygiene, should be followed during meat inspection.

Chemokine production induced by Corynebacterium pseudotuberculosis in a murine model.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis. The main clinical sign of this disease is the development of granulomas, especially in small ruminants; however, the pathways that are involved in the formation and maintenance of these granulomas are unknown. Cytokines and chemokines are responsible for the migration of immune cells to specific sites and tissues; therefore, it is possible that chemokines participate in abscess formation. This study aimed to evaluate the induction of chemokine production by two C. pseudotuberculosis strains in a murine model. A highly pathogenic (VD57) and an attenuated (T1) strain of C. pseudotuberculosis, as well as somatic and secreted antigens derived from these strains, was used to stimulate murine splenocytes. Then, the concentrations of the chemokines CCL-2, CCL-3, CCL-4, and CCL-5 and the cytokines IL-1 and TNF were measured in the culture supernatants. The VD57 strain had a higher ability to stimulate the production of chemokines when compared to T1 strain, especially in the early stages of stimulation, which can have an impact on granuloma formation. The T1 lysate antigen was able to stimulate most of the chemokines studied herein when compared to the other antigenic fractions of both strains. These results indicate that C. pseudotuberculosis is a chemokine production inducer, and the bacterial strains differ in their induction pattern, a situation that can be related to the specific behavior of each strain.

Epidemiological, bacteriological and molecular studies on caseous lymphadenitis in sheep of Dakhlia, Egypt.

Caseous lymphadenitis (CLA) is a chronic and insidious disease that mainly affects small ruminants and caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The aims of this research were to identify C. pseudotuberculosis by PCR from pyogenic lesions, to study the phylogenetic analysis of C. pseudotuberculosis and to detect the prevalence based on the detected superficial lesions of CLA in Dakahlia governorate, Egypt. Out of 3471 clinically examined animals, 129 (3.71%) animals were affected with CLA. The isolation rate of C. pseudotuberculosis in abscess of sheep was 45.74% (59/129). Out of 129 samples examined by PCR assay, 63 (48.83%) were positive phospholipase D (PLD) indicated at fragment size 203 bp. This is the first phylogenetic analysis study of C. pseudotuberculosis isolate in Egypt which was isolated from infected sheep. Nucleotide sequence identity data demonstrated that C. pseudotuberculosis PLD gene (MW187942) Dakahlia share homology 99.01%, 98.83 and 98.48% with Zagazig, Egypt (MN867024), Tamil nadu, India (MG720636) and Sudan (MG692441), respectively. In conclusion, this study provided information on the molecular detection and phylogeny of C. pseudotuberculosis in Egypt. Findings of this study can be conducted in other CLA endemic countries with similar animal breeding practices in the Middle East and developing countries.