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.

EFFECTS OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS ON SHEEP KIDNEYS

Light and electron microscopic examinations were performed on kidneys of sheep infected with caseous lymphadenitis ( CLA ) caused by Corynebacterium pseudotuberculosis Membranproliferative glomerulonephritis was demonstrated. There were irregular thickenings of the glomeruelar basement membrane and glomerular cell proliferation, in contrast, no such lesions were found in the control lambs, indicating that CLA play a role in inducing glomerulorephritis

In Silico Screening of Putative Corynebacterium pseudotuberculosis Antigens and Serological Diagnosis for Caseous Lymphadenitis in Sheep by Enzyme-Linked Immunosorbent Assay

Corynebacterium pseudotuberculosis is the etiologic agent of Caseous Lymphadenitis (CLA), a disease leading to severe damage in sheep and goats farming due to the lack of serological diagnosis, treatment, and effective prophylaxis. In this context, several strategies in an attempt to discover new antigens to compose diagnosis assays or vaccines are fundamental. Therefore, this study aimed to use bioinformatics software to evaluate the critical chemical characteristics of unknown proteins of C. pseudotuberculosis by selecting them for heterologous expression in Escherichia coli. For this purpose, six protein sequences of ascorbate transporter subunit, UPF protein, MMPL family transporter, Ribonuclease, Iron ABC transporter domain-containing permease, and fimbrial subunit were obtained. In silico analyses were performed using amino acid sequences to access immunodominant epitopes and their antigenic and allergenic potential and physicochemical characterization. The expressed proteins were used as an antigen for serological diagnosis by ELISA. All proteins showed distinct immunodominant epitopes and potential antigenic characteristics. The only proteins expressed were PTS and Ribonuclease. In parallel, we expressed CP40 and all were used with ELISA antigen in 49 CLA positive sera and 26 CLA negative sera. The proteins alone showed 100% sensitivity and 96.2%, 92.3%, and 88.5% specificity for rPTS, rRibonuclease, and rCP40, respectively. When proteins were combined, they showed 100% sensitivity and 84.6%, 92.3%, 88.5%, and 92.3% specificity for rPTS/rCp40, rRibonuclease/rCP40, rPTS/rRibonuclease, and rPTS/rRibonuclease/rCP40, respectively. The results of this study show an excellent correlation of sensitivity and specificity with all proteins. None of the specificity values preclude the potential of rPTS, rRibonuclease, or rCP40 for use in ELISA diagnostic assays since the results of this work are superior to those of other studies on CLA diagnosis described in the literature.

Molecular Characterization of Corynebacterium pseudotuberculosis Isolated over a 15-Year Period in Switzerland

Corynebacteriumpseudotuberculosis biovar Ovis is the etiological agent of the contagious and chronic disease caseous lymphadenitis (CLA) in sheep and goats. The economic impact of CLA in Switzerland remains largely unknown, and the transmission modalities, as well as the genetic diversity of circulating strains, are poorly understood. This work presents further characterization data for 215 C. pseudotuberculosis isolates from sheep, goats and a dromedary originating from Switzerland and the Principality of Liechtenstein, collected over a 15-year period. The isolates were classified into the two biovars Ovis and Equi, analyzed for the presence of the diphtheria-like toxin gene and characterized using MLSA. All sheep and goat isolates were classified as C. pseudotuberculosis biovar Ovis. The isolate from a dromedary was classified as biovar Equi. No isolates harboring the diphtheria-like toxin gene were detected. Phylogenetic analysis of the concatenated sequences of four genes revealed the existence of 24 clusters. There was no correlation between MLSA sequence types, year of isolation and the geographical origin of the isolates. These findings confirm the presence of several MLSA sequence types in the study area and over a 15-year period. Moreover, no sheep- and goat-specific MLSA sequence types were found.

Pathology, bacteriology and molecular studies on caseous lymphadenitis in Camelus dromedarius in the Emirate of Abu Dhabi, UAE, 2015-2020

Caseous lymphadenitis (CLA) or pseudotuberculosis is a chronic zoonotic bacterial disease caused by Corynebacterium pseudotuberculosis, which affects livestock and humans. This study aimed to describe the pathology, bacteriology and confirm the identity of the pathogen by 16S rRNA gene sequencing in Camelus dromedarius. A total of 12 camels with suspected CLA in three regions of Abu Dhabi Emirate (Abu Dhabi, Al Ain and Al Dhafra), United Arab Emirate (UAE) were subjected to clinical and postmortem examinations from January 2015 to December 2020. Clinically, camels were emaciated and showed the presence of external caseous abscesses suggestive of CLA. Postmortem examination showed multiple abscesses of variable sizes with caseous material encapsulated by fibrous tissue in the liver, lungs, muscle, and lymph nodes. Following clinical and postmortem examination, blood, pus and different tissue samples were collected for subsequent analysis. Histopathological examination of all organs stained with Hematoxylin and Eosin (H&E) indicated a central caseo-necrotic core that was admixed with bacterial colonies and infiltration of chronic inflammatory cells, surrounded by a pyogenic membrane, and an outer fibrous connective tissue capsule. Bacterial culture identified the isolates of Corynebacterium pseudotuberculosis biotype ovis strain, and these isolates were shown to be sensitive to all antibiotics tested (penicillin, ampicillin, Co-trimoxazole, enrofloxacin and tetracycline). Moreover, the identity of the isolates was confirmed by partial sequencing of the 16S rRNA gene which showed a 100% identity to Corynebacterium pseudotuberculosis. Phylogenetic analysis based on 16S rRNA gene sequence clearly differentiates Corynebacterium pseudotuberculosis from other species of Corynebacterium. Briefly, this study provided the basic information for infection of Corynebacterium pseudotuberculosis in Camels and will help in controlling of this pathogen in the region.

Intestinal Caseous Lymphadenitis in Sheep

Background: Caseous Lymphadenitis (CL) is a chronic infectious disease caused by the bacterium Corynebacterium pseudotuberculosis, which is considered the main agent responsible for abscess lesions. In the visceral form it can affect the internal organs of sheep and goats, which could negatively affect animal health and cause large economic losses for producers.Case: This study aims to report a case of intestinal CL in sheep, with suspected diagnosis during physical examination and identification during the performance of the oophorectomy procedure, adopted as a management approach. It is a mixed breed sheep, aged over 5 years; weight 28 kg; emaciated on physical examination; with pale pink and moist eyelid mucosa; heart and respiratory rate: 81 beats/min and 22 movements/min, respectively; body temperature 39.2°C; ruminal movements at 1 movement/min; without identification of lymphadenomegaly on palpation, however, it was observed that the right submandibular lymph node presented tissue retraction compatible with the healing process. For the surgical procedure, an 18-h fast was used and pre-anesthetic medication with 2% xylazine (0.1 mg/kg), 10% ketamine (5 mg/kg) and 50 mg/mL tramadol (2 mg/kg) administrated intramuscularly. The animal was placed in the left lateral decubitus position, then was performed trichotomy and epidural administration of 2% lidocaine (4 mg/kg) and maintenance with propofol 10 mg/mL intravenous dose-effect and oxygen mask 3 liters/min, antibiotic prophylaxis was performed with 10% enrofloxacin (2.5 mg/kg). Flank oophorectomy was performed according to the classic technique and during abdominal inspection, abscess lesions were found in the mesentery and intestinal loops. Incisional biopsy was performed to collect samples in the jejunal segment. At the end of the procedure, 50 mg/mL (2.2 mg/kg) of flunixin meglumine was administered intravenously. A 0.6 cm x 0.2 cm sample, was submitted to histological analysis, which showed the presence of central necrosis areas formed by concentric lamellae, with the presence of large bacterial colonies and foci of mineralization, surrounded by a strip of inflammatory infiltrate with epithelial macrophages and few neutrophils. In the adjacent layer, lymphocytes and plasmocytes were found and the entire lesion was delimited by fibrous conjunctive tissue, compatible with lesions caused by Corynebacterium pseudotuberculosis, the causative agent of CL. During the whole postoperative period the animal was kept isolated from the herd. Due to the unfavorable prognosis and histological confirmation of visceral CL, euthanasia was indicated. The animal was sent for necropsy and no lesions compatible with CL were found in other organs or tissues besides the anatomical structures where the lesions were previously described (intestine and mesentery).Discussion: As it is an infectious disease, isolation and euthanasia should be indicated in cases of CL, in order to not compromise the health of the herd. Thus, justifying the orientation of euthanasia after histological confirmation. The case did not have other possibilities of diagnostic aid, however, histological lesions of CL are characteristic but not pathognomonic, as it could be confused with lesions caused by other pyogenic pathogens. In this way, clinical considerations and complementary exams are relevant to support the diagnostic. To conclude, the observation and physical examination of the herd were fundamental tools for raising the diagnostic hypothesis ante mortem. Complementary tests allowed the confirmation of the disease and prevented the spread in the herd, which could lead to large economic losses for producers and negatively affect animal health.