Iridaea cordata lipid extract associated with the rCP01850 protein of C. pseudotuberculosis elicited a Th1 immune response in immunized sheep.

Sheep farming contributes to the socioeconomic development of small and medium-scale livestock farmers. However, several factors can hinder successful animal production, as is the case for infectious diseases, such as the one caused by Corynebacterium pseudotuberculosis, known as caseous lymphadenitis (CLA). CLA has >90% prevalence in Brazilian herds and antibiotic treatment is not effective, consequently causing significant economic losses to farmers. Given the above, effective vaccines need to be developed to prevent this disease. This study aimed to evaluate the adjuvant activity of the lipid extract from the macroalgae Iridaea cordata as a candidate for developing an effective vaccine formulation. For such, four groups of six sheep each were inoculated with sterile 0.9% saline solution (G1), rCP01850 (G2), rCP01850 + I. cordata (G3), and rCP01850 + saponin (G4). Each sheep received two vaccine doses 30 days apart. Total IgG production levels significantly increased in experimental groups G3 and G4 on days 30, 60, and 90. On day 90, G3 showed higher total IgG production (p < 0.05) when compared to G4. When analyzing cytokine production, G3 was the only experimental group with significantly increased IFN-γ, IL-12, TNF-α, and IL-10 mRNA expression levels. Our results show the vaccine formulation containing rCP01850 adjuvanted with the I. cordata lipid extract elicited a Th1 immune response in sheep, indicating I. cordata lipid extract may be a promising adjuvant for developing an effective vaccine against infection caused by C. pseudotuberculosis.

Lactobacillus acidophilus protects against Corynebacterium pseudotuberculosis infection by regulating the autophagy of macrophages and maintaining gut microbiota homeostasis in C57BL/6 mice.

Corynebacterium pseudotuberculosis (C. p), a facultative intracellular bacterium, is an important zoonotic pathogen that causes abscesses and pyogenic granulomas. The relationship between gut microbiota and host health or diseases has received increasing attention. However, the role of gut microbiota in the process of C. p infection is still unclear. In this study, we established a C. p infection model in C57BL/6 mice and examined the impact of preemptive oral administration Lactobacillus acidophilus (L. acidophilus) on infection. Our findings revealed that C. p infection led to pronounced pathological alterations in the liver and kidneys, characterized by abscess formation, intense inflammatory responses, and bacterial overload. Remarkably, these deleterious effects were greatly relieved by oral administration of L. acidophilus before infection with C. p. Additionally, we further found that during C. p infection, peritoneal macrophages (PMs) of mice orally administered with L. acidophilus accumulated more rapidly at sites of infection. Furthermore, our results showed that PMs from mice with oral L. acidophilus administration showed a stronger C. p clearance effect, and this was mediated by high expression of LC3-II protein. Meanwhile, oral administration of L. acidophilus protected the gut microbiota disorder in C57BL/6 mice caused by C. p infection. In summary, our study demonstrates that oral administration of L. acidophilus confers effective protection against C. p infection in C57BL/6 mice by modulating macrophage autophagy, thereby augmenting bacterial clearance and preserving gut microbiota and function stability. These findings position L. acidophilus as a viable probiotic candidate for the clinical prevention of C. p infection. IMPORTANCE: Corynebacterium pseudotuberculosis (C. p) is known to induce a range of chronic diseases in both animals and humans. Currently, clinical treatment for C. p infection mainly relies on antibiotic therapy or surgical intervention. However, excessive use of antibiotics may increase the risk of drug-resistant strains, and the effectiveness of treatment remains unsatisfactory. Furthermore, surgical procedures do not completely eradicate pathogens and can easily cause environmental pollution. Probiotic interventions are receiving increasing attention for improving the body's immune system and maintaining health. In this study, we established a C. p infection model in C57BL/6 mice to explore the impact of Lactobacillus acidophilus during C. p infection. Our results showed that L. acidophilus effectively protected against C. p infection by regulating the autophagy of macrophages and maintaining intestinal microbiota homeostasis. This study may provide a new strategy for the prevention of C. p infection.

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.

Effect of Antimicrobial Prophylaxis on Corynebacterium bovis Infection and the Skin Microbiome of Immunodeficient Mice.

Corynebacterium bovis is an opportunistic pathogen of the skin of immunodeficient mice and is sensitive to oral antibiotics that reach therapeutic blood concentrations. However, prophylactic antibiotics are considered to be ineffective at preventing C. bovis infection. In addition, the effect of C. bovis on the skin microbiome (SM) of common immunodeficient mouse strains has yet to be characterized. Consequently, we evaluated whether oral prophylactic antibiotics prevent C. bovis infection after inoculation. An infectious dose of C. bovis was applied to the skin of Hsd:Athymic Nude (nude) and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were then housed individually and assigned randomly to receive either untreated drinking water (Cb+Abx-group) or prophylactic amoxicillin-clavulanic acid in the drinking water (0.375 mg/mL) for 14 d (Cb+Abx+group). A third treatment group of each mouse strain was uninoculated and untreated (Cb-Abx-group). Mice from all groups were serially sampled by using dermal swabs to monitor C. bovis infection via quantitative real-time PCR and the SM via 16S rRNA sequence analysis. Fourteen days of prophylactic antibiotics prevented the perpetuation of C. bovis skin infection in both strains. Only the combination of C. bovis inoculation and oral antibiotics (Cb+Abx+) significantly affected the SM of NSG mice at day 14; this effect resolved by the end of the study (day 70). In mice that did not receive antibiotics, C. bovis significantly altered the SM of nude mice but not NSG mice at days 14 and 70. These findings demonstrate the potential benefit of prophylactic antibiotics for prevention of C. bovis infection. However, indirect effect of antibiotics on commensal bacteria and potential effects on xenograft models must be considered.

Peptide vaccines designed with the aid of immunoinformatic against Caseous Lymphadenitis promotes humoral and cellular response induction in mice.

Caseous Lymphadenitis (CLA) is a chronic disease that affects also small ruminants. CLA is caused by Corynebacterium pseudotuberculosis and is responsible for high economic losses due to the formation of superficial and visceral granulomas, the latter is considered as asymptomatic CLA causing high levels of dissemination. Several vaccination strategies, in which the use of synthetic peptides stands out. Thus, this work aimed to evaluate the protective potential of peptide vaccines designed to determine the immunodominant epitopes of CP40 against CLA in mice. The animals were divided into eight groups separated in controls (G1-PBS, G2-Saponin and G9-rCP40) and experimental (G3-pep1, G4- pep2, G5-pep3, G6-pep4, G7-pep5 and G8-pep6), these were vaccinated on days 0 and 15 by a subcutaneous route. 60 days after the first immunization, all animals were challenged with C. pseudotuberculosis. On days 0, 15, 60, and 120 after the first immunization, blood samples were taken to measure immunoglobulins. On the same day of the challenge, the splenocytes were isolated and assayed for the production of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17, and IL-10. After vaccinations, the animals were challenged and all of them were affected by the disease which led to their death. The G6 and G8 groups provided 10% protection and the G7 provided 20%. The G3 and G4 groups provided 30% and 40% protection respectively. The peptides showed the production of Total IgG antibodies and cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α), indicating a possible activation of the Th1 type response. However, groups G3, G5, G6, and G8 showed production of IL-17. None of the study groups showed IL-10 production. The immunogenicity of the peptides was not enough to protect these animals and it is believed that the use of adjuvants based on PAMPs may improve the immune response offered by these peptides.

Proteins from the core genome of Corynebacterium ulcerans respond for pathogenicity and reveal promising vaccine targets for diphtheria.

Corynebacterium ulcerans is an emerging pathogen able to transmit the acute infection diphtheria to humans. Although there is a well-established vaccine based on the toxin produced by Corynebacterium diphtheriae, another species of this genus known to cause the disease, there is still no vaccine formulations described for C. ulcerans; this fact contributes to the increase in cases of infection that has been observed. In this study, we want to provide information at the genomic level of this bacterium in order to suggest proteins as possible vaccine targets. We carried out an in silico prospection of vaccine candidates through reverse vaccinology for targets that exhibit antigenic potential against diphtheria. We found important virulence factors, such as adhesion-related ones, that are responsible for pathogen-host interaction after infection, but we did not find the diphtheria toxin, which is the main component of the currently available vaccine. This study provides detailed information about the exoproteome and hypothetical proteins from the core genome of C. ulcerans, suggesting vaccine targets to be further tested in vitro for the development of a new vaccine against diphtheria.

Saponin-adjuvanted recombinant vaccines containing rCP00660, rCP09720 or rCP01850 proteins against Corynebacterium pseudotuberculosis infection in mice.

PURPOSE: rCP01850, rCP09729 and rCP00660 proteins from Corynebacterium pseudotuberculosis, predicted as the three best targets to be used in vaccines against Caseous Lymphadenitis in mature epitope density (MED) analysis were tested as vaccinal targets in association to saponin as adjuvant. METHODOLOGY: rCP00660, rCP09720 and rCP01850 were expressed in E. coli and purified for immunization assay. Balb/c mice were divided into five groups of sixteen animals each. G1 was injected with saline solution (0.9% NaCl), G2 with saponin, G3, G4 and G5 with, respectively, rCP00660, rCP09720 and rCP01850 added by saponin. Two doses were administered within a 21-days interval, and blood samples were collected for IgG quantification. Twenty-one days after the last immunization, ten mice in each group were challenged with virulent C. pseudotuberculosis MIC-6 strain, and mortality was recorded for 40 days. Meanwhile six mice in each group were used for cytokine quantification by qPCR. RESULTS: G2, G3, G4 and G5 presented protection rates of 10, 30, 40 and 60%, respectively. In spite of levels of total IgG were higher in G4 and G5, production of IgG2a was higher than IgG1 for G5. G3, G4 and G5 presented significant high IFN-γ levels, however, only G5 showed high TNF-α while G3 and G4 showed high IL-17. CONCLUSION: rCP01850 added by saponin was able to protect efficiently mice against C. pseudotuberculosis challenge, and to induce high IgG, IFN-γ and TNF-α levels. In spite of rCP00660 and rCP09720 had not same adequate protection levels, significant IgG, IFN-γ, and IL-17 levels and further studies aiming to improve protection rates should be conducted.

Vaccines for caseous lymphadenitis: up-to-date and forward-looking strategies.

Caseous lymphadenitis (CLA) is an infectious chronic disease responsible for economic losses in sheep and goat breeding worldwide. CLA has no effective treatment, evidencing the vaccination schedule as the best control strategy. Although some commercial vaccines have been available, none of them provides total protection, which is sometimes insufficient and does not reach the same efficiency when compared in sheep and goats. They also have questionable safety levels and side effects. In light of this, several experimental vaccines are in development in order to improve safety, reproducibility, and protective immune response against the etiologic agent of CLA, Corynebacterium pseudotuberculosis. In this review, we discussed aspects as antigen, adjuvant, routes of administration, protection level, and animal models used in CLA vaccine development, as well the challenges and future perspectives. KEY POINTS: Caseous lymphadenitis (CLA) does not have an appropriate commercial vaccine. Different experimental vaccines are in development aiming to protect against Corynebacterium pseudotuberculosis. An ideal vaccine for CLA is necessary for the disease control.

A novel approach for an immunogen against Corynebacterium pseudotuberculosis infection: An Escherichia coli bacterin expressing phospholipase D.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA) in small ruminants. There is still needed an immunoprophylaxis model, which induces a protective and sustained immune response against the bacteria. In this study, we evaluated a recombinant Escherichia coli bacterin expressing the recombinant phospholipase D (rPLD) protein, the most relevant virulence factor of C. pseudotuberculosis, as a potential vaccine formulation. E. coli BL21 (DE3) Star strain was used for rPLD protein expression and was then inactivated by formaldehyde. Four groups with 10 Balb/c mice each were immunized twice within a 21 days interval: G1-control - 0.9% saline solution; G2- E. coli bacterin/pAE (naked plasmid); G3- E. coli bacterin/pAE/pld; G4-purified recombinant rPLD. Subsequently, the animals were challenged with a C. pseudotuberculosis virulent strain and evaluated for 40 days. The highest survival rate was observed for G3 with 40% protection, followed by 30% in the purified rPLD group (G4). These two groups also showed considerable IgG production when compared with the control group (G1). Also, a higher significant expression of interferon-γ was observed for the experimental groups G2, G3, and G4 when compared with a control group (G1) (p < 0.05). These results represent that a recombinant bacterin can be seen as a promising approach for vaccinal antigens against CLA, being possible to be used in association of different vaccine strategies.

NanH and PknG putative virulence factors as a recombinant subunit immunogen against Corynebacterium pseudotuberculosis infection in mice.

Despite the economic and zoonotic relevance of caseous lymphadenitis, a competent immunoprophylaxis tool is still necessary. Here, we evaluated two putative virulence factors of Corynebacterium pseudotuberculosis, rNanH, and rPknG, as recombinant subunit vaccines in a murine model against the infection by C. pseudotuberculosis. Three groups of ten Balb/c mice each were inoculated with a sterile 0.9% saline solution (G1), rNanH (G2), or rPknG (G3) in formulations containing saponin as an adjuvant. The mice received two vaccine doses intercalated by a 21-day interval and were challenged with 2 × 104 CFU/mL of the C. pseudotuberculosis MIC-6 strain 21 days after the last immunization. The total IgG, IgG1, and IgG2a production levels increased significantly in the experimental groups (G2 and G3) on day 42. The highest levels of IgG2a antibodies in G2 and G3 were observed compared to IgG1 levels. G3 showed a significant (p < 0.05) humoral response through higher production of total IgG at day 42 when compared to G2. A significant increase of mRNA expression levels of interleukin (IL)-17, tumor necrosis factor, and interferon-γ was observed only in G2, while IL-4 was significantly produced only by G3. The levels of IL-10 and IL-12 obtained were not significant in any group. The survival rates after the challenge were 20% for G3 and 60% for G2 (p < 0.05). Our findings suggest that the formulation containing rNanH and saponin (G2) resulted in the best protection against the challenge and was able to elicit a Th1 immune response in mice, and can be considered as a promising antigen in the development of an effective vaccine against caseous lymphadenitis.

Histopathological changes of reproductive organs of goats immunized with Corynebacterium pseudotuberculosis killed vaccine.

Caseous lymphadenitis (CLA) caused by Corynebacterium pseudotuberculosis is characterized by the development of abscesses, mainly in superficial and internal lymph nodes, visceral and reproductive organs in small ruminants. This study aims to examine the histopathological changes in reproductive organs of goats immunized with killed vaccine of C. pseudotuberculosis. In this study, twenty four (24) clinically healthy bucks and does were divided into four groups A, B, C and D. Animals in groups A and B were immunized with 0.5 and 1% formalin killed vaccine, respectively; followed by a booster dose. After the booster dose of immunization, groups A, B and C were challenged with C. pseudotuberculosis at 106 cfu/ml. Goats in group D were immunize and unchallenged and left as control group. All C. pseudotuberculosis infected animals were euthanized humanely 12 weeks post-challenged. Tissue samples such as testes, epididymis, spermatic cord, penis, pituitary gland, mammary gland, vulva, vagina, cervix, uterus, fallopian tube and ovaries were collected for histopathology study. Microscopic examination of all tissues (testes, seminiferous tubules, spermatic cord, penile tissues and the pituitary gland) in the male reproductive organs of the bucks that were inoculated with 2 ml of 0.5% and 1.0% of C. pseudotuberculosis killed vaccine showed normal (animals inoculated with 1.0%) to mild (animals inoculated with 0.5%) histopathological changes when compared with those from group C which showed varying degrees of histopathological changes (p < 0.01) in their various tissues. For the female does, similar histopathological changes were observed for the various tissues examined (ovaries, fallopian tubes, uterine horns, uterine tissues, cervix, vaginal, vulva, mammary glands and the pituitary glands) in which the vaccinated groups A &B showed a significantly (p < 0.001) less histopathological changes when compared with those in group C that showed varying degrees of histopathological changes in the reproductive organs investigated. This study showed the efficacy of C. pseudotuberculosis killed vaccine protecting against reproductive tissue damages cause by the active infection with the live bacteria in both bucks and does in the study area.

Metaphylactic Antibiotic Treatment to Prevent the Transmission of Corynebacterium bovis to Immunocompromised Mouse Offspring.

Current methods for eradicating Corynebacterium bovis, such as depopulation, embryo transfer, and cesarean rederivation followed by cross fostering, are expensive, complex, and time-consuming. We investigated a novel method to produce immunocompromised offspring free of C. bovis from infected NOD. Cg-PrkdcscidIl2rgtm1Wgl/SzJ (NSG) breeding pairs. Adult NSG mice were infected with C. bovis, paired, and randomly assigned to either a no-antibiotic control group (NAB, n = 8) or a group that received amoxicillin-clavulanic acid (0.375 mg/mL) in their drinking water for a mean duration of 7 wk (AB group, n = 7), spanning the time from pairing of breeders to weaning of litters. The AB group also underwent weekly cage changes for 3 wk after pairing to decrease intracage C. bovis contamination, whereas the NAB mice received bi-weekly cage changes. Antibiotics were withdrawn at the time of weaning. All litters (n = 7) in the AB group were culture- and qPCR-negative for C. bovis and remained negative for the duration of the study, whereas all litters in the NAB group (n = 6) remained C. bovis positive. A single adult from each breeding pair was sampled at weaning and at 5 and 10 wk after weaning to confirm the maintenance of (NAB) or to diagnose the reemergence (AB) of C. bovis infection. By the end of the study, C. bovis infection had returned in 3 of the 7 (43%) tested AB adults. Our data suggest that metaphylactic antibiotic use can decrease viable C. bovis organisms from adult breeder mice and protect offspring from infection. However, using antibiotics with frequent cage changing negatively affected breeding performance. Nevertheless, this technique can be used to produce C. bovis-free NSG offspring from infected adults and may be an option for salvaging infected immunocompromised strains of mice that are not easily replaced.

Persistent Corynebacterium bovis Infectious Hyperkeratotic Dermatitis in Immunocompetent Epidermal-Mutant dep/dep Mice.

During a previously reported program-wide Corynebacterium bovis outbreak, both immunocompetent depilated (dep/dep) mutant mice and transgenic mice that express the papillomavirus E6 oncoprotein became persistently infected with C. bovis. An orthokeratotic, hyperkeratotic, acanthotic dermatitis developed in the C. bovis-infected dep/dep mice, which remained C. bovis PCR-positive for >45 days prior to euthanasia as part of the program-wide C. bovis eradication effort. Since both affected strains of mice have altered skin homeostasis, immune status or the presence of hair may not alone be sufficient to explain strain susceptibility to C. bovis-related cutaneous disease. In order to avoid invalidation of preclinical studies due to C. bovis infection, it may be necessary to isolate immunodeficient mouse strains, implement facililty-wide surveillance for C. bovis, and sterilize equipment with vaporized hydrogen peroxide.

Oxytetracycline concentrations in interstitial fluid from tissue chambers inoculated with Corynebacterium pseudotuberculosis after intramuscular or intrachamber administration in sheep.

OBJECTIVE: To determine oxytetracycline concentrations in plasma and in fluid from Corynebacterium pseudotuberculosis (CPT)-inoculated tissue chambers (used as experimental abscess models) and uninoculated (control) tissue chambers in sheep after IM or local administration of the drug and to investigate whether CPT growth was reduced or eliminated by these treatments. ANIMALS: 10 clinically normal female sheep. PROCEDURES: Sterile tissue chambers were surgically implanted in both paralumbar fossae of each sheep; ≥ 2 weeks later (day -6), 1 randomly selected chamber was inoculated with CPT, and the opposite chamber was injected with sterile growth medium. Sheep received oxytetracycline IM (n = 5) or by percutaneous injection into CPT-inoculated (4) or uninoculated (1) chambers on day 0. Tissue fluid from each chamber and venous blood samples for plasma collection were obtained at predetermined times over 6 days for bacterial counts (tissue chambers) and analysis of oxytetracycline concentrations (tissue chambers and plasma). Sheep were euthanized on day 6. Regional lymph nodes were collected bilaterally from each sheep for culture. RESULTS: Measurable concentrations of oxytetracycline were present in each chamber throughout the study, regardless of administration route or presence of CPT. No CPT growth was detected after the 48-hour time point in inoculated chambers injected with oxytetracycline; however, CPT was isolated from all inoculated chambers throughout the study after IM drug administration. One regional lymph node (ipsilateral to a CPT-inoculated, oxytetracycline-injected chamber with no CPT growth after 48 hours) was culture positive for CPT. CONCLUSIONS AND CLINICAL RELEVANCE: Intralesional administration of oxytetracycline may eliminate growth of CPT locally, but complete elimination of the organism remains difficult.

Establishment of an objective endpoint in mice model for caseous lymphadenitis vaccine trials.

For a long time, the scientific community has described the need for a continued update in practices that ensure the welfare of animals undergoing experimentation. In addition to approaches on principles of care and use of animals, there is a more current emerging concern: defining an appropriate end point in experiments that use animals for research, teaching and testing. The term "endpoint" is defined as the point at which an experimental animal's pain and/or distress is terminated, minimized, or reduced humanely. In the present study, we established an endpoint in Balb/C mice for caseous lymphadenitis vaccine trials, which can be considered as a highly important parameter since several studies are being developed to control the disease efficiently. Mice were monitored daily until the 30th day after infection with pathogenic strain of C. pseudotuberculosis MIC-6 using the most relevant parameters for the appearance of clinical signs of caseous lymphadenitis (CLA), such as abscesses, lethargy, and loss of weight and hair. The endpoint was found to be a weight loss of 0.2167 g after five days or 10% weight loss in less than five days. In conclusion, the findings reported here will help improve animal's well-being during vaccine trials for CLA and consequently represent significant contribution to animal's welfare.

Leader gene of Corynebacterium pseudotuberculosis may be useful in vaccines against caseous lymphadenitis of goats: a bioinformatics approach.

We conducted an in silico analysis to search for important genes in the pathogenesis of Caseous Lymphadenitis (CL), with prospects for use in formulating effective vaccines against this disease. For this, we performed a survey of proteins expressed by Corynebacterium pseudotuberculosis, using protein sequences collected from the NCBI GenPept database and the keywords "caseous lymphadenitis" and "Corynebacterium pseudotuberculosis" and "goats". A network was developed using the STRING 10 database, with a confidence score of 0.900. For every gene interaction identified, we summed the interaction score of each gene, generating a combined association score to obtain a single score named weighted number of links (WNL). Genes with the highest WNL were named "leader genes". Ontological analysis was extracted from the STRING database through Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A search in the GenPept database revealed 2,124 proteins. By using and plotting with STRING 10, we then developed an in silico network model comprised of 1,243 genes/proteins interconnecting through 3,330 interactions. The highest WNL values were identified in the rplB gene, which was named the leader gene. Our ontological analysis shows that this protein acts effectively mainly on Metabolic pathways and Biosynthesis of secondary metabolites. In conclusion, the in silico analyses showed that rplB has good potential for vaccine development. However, functional assays are needed to make sure that this protein can potentially induce both humoral and cellular immune responses against C. pseudotuberculosis in goats.

Recombinant M. bovis BCG expressing the PLD protein promotes survival in mice challenged with a C. pseudotuberculosis virulent strain.

The aim of this study was to evaluate the survival of mice inoculated with M. bovis BCG Pasteur recombinant expressing the PLD protein and challenged with a C. pseudotuberculosis virulent strain. Four groups were immunized with a sterile 0.9% saline solution (G1), 106 CFU of M. bovis BCG Pasteur (G2), 106 CFU of M. bovis BCG/pld (G3) or 106 CFU of M. bovis BCG/pld with a booster with rPLD (G4) and challenged with 104 CFU of C. pseudotuberculosis MIC-6 strain. The highest survival rate of 88% was observed in G4, followed by 77% in G3 and 66% in G2. A significant statistical difference was observed in the levels of cytokines IFN-γ and IL-10 in vaccinated groups (G3 and G4) when compared with the control group (G1) (p < 0.05). The results seem promising as the recombinant vaccine elicited a cellular immune response and provided significant survival after a high virulent challenge.

Association of Corynebacterium pseudotuberculosis recombinant proteins rCP09720 or rCP01850 with rPLD as immunogens in caseous lymphadenitis immunoprophylaxis.

Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLD + rCP09720 (G3), and rPLD + rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21 days after the last immunization. The animals were evaluated daily for 40 days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (p < .05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (p < .05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLD + rCP09720), and 50% (rPLD + rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.

Identification of new Corynebacterium pseudotuberculosis antigens by immunoscreening of gene expression library.

BACKGROUND: Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. RESULTS: A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family. CONCLUSION: Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.

Recombinant esterase from Corynebacterium pseudotuberculosis in DNA and subunit recombinant vaccines partially protects mice against challenge.

PURPOSE: We tested the efficacy of the esterase encoded by cp1002_RS09720 from Corynebacteriumpseudotuberculosis in recombinant subunit and DNA caseous lymphadenitis (CLA) vaccines. This target was predicted as one of the best CLA vaccine candidates by mature epitope density analysis. METHODOLOGY: Gene cp1002_RS09720 was cloned into two different vectors (pAE for subunit vaccine and pTARGET for DNA vaccine). Four groups of 15 mice each were immunized with the recombinant esterase rCP09720 associated with aluminium hydroxide adjuvant (G1), pTARGET/cp09720 DNA vaccine (G2), a naked pTARGET (G3) or PBS as a negative control (G4). Immunization occurred in two doses intercalated by a 21 day interval. Twenty-one days after the last dose administration, animals were challenged with a virulent C. pseudotuberculosis MIC-6 strain. RESULTS: G1 showed high levels of IgG1 and IgG2a on days 21 and 42 post-immunization and a significant level of IFN-γ (P<0.05), suggesting a Th1 response. The protection levels obtained were 58.3 and 16.6 % for G1 and G2, respectively. CONCLUSION: The subunit vaccine composed of the recombinant esterase rCP09720 and Al(OH)3 is a promising antigenic formulation for use against CLA.