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.

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.

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.

Activity of Ethanolic and Supercritical Propolis Extracts in Corynebacterium pseudotuberculosis and Its Associated Biofilm.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis in small ruminants, a chronic disease characterized by the development of granulomas in superficial and visceral lymph nodes as well as in several organs. An important characteristic of the infection with this bacterium is the formation of a biofilm and the absence of effective antibiotic therapy against the disease. From this scenario, the objective of this study was to evaluate the susceptibility of C. pseudotuberculosis to conventional antibiotics and to red, green, and brown propolis extracts obtained by the supercritical and ethanolic extraction methods as well as its activity in the bacterial biofilm. The results of the sensitivity test using antibiotics indicated a sensitivity of C. pseudotuberculosis strains to the antimicrobial agents. The ethanolic extract of green propolis and the supercritical red propolis extract showed the best antibacterial activities against planktonic C. pseudotuberculosis. A lower antimicrobial activity of the brown propolis extract was identified. Propolis extracts were effective in interfering with the formation of the C. pseudotuberculosis biofilm but had little activity on the consolidated biofilm. In conclusion, propolis extracts are more effective against C. pseudotuberculosis in the planktonic stage, being able to interfere with the formation of bacterial biofilm. However, the action of propolis extracts in a sessile and structured microbial biofilm is reduced.

Brazilian Green Propolis as a Therapeutic Agent for the Post-surgical Treatment of Caseous Lymphadenitis in Sheep.

As antibiotics are ineffective when used to treat caseous lymphadenitis, the surgical excision of lesions is often required. Iodine solution (10%) is currently the choice for the post-surgical treatment; however, it may cause histotoxicity. Propolis are resinous substances composed by a mixture of different plants parts and molecules secreted by bees. As green propolis has already proven to possess anti-bacterial and wound healing properties, this study aimed to evaluate the use of a green propolis-based ointment as a therapeutic agent for the post-surgical treatment of caseous lymphadenitis. The caseous lesions of 28 sheep were surgically excised before dividing animals into two groups: (1) iodine-treated animals and (2) sheep treated with an ointment made with a previously characterized green propolis extract. Clinical data of animals, size of the scar area, the presence of moisture and secretion in the surgical wound, the humoral immune response against the bacterium and the susceptibility of C. pseudotuberculosis clinical isolates to the green propolis extract were analyzed. The green propolis-treated group presented complete healing of the surgical wound 1 week before the iodine-treated group. Additionally, animals treated with the green propolis ointment had fewer cases of wound secretion, but it was not statistically different from the iodine-treated group. No clinical signs indicating green propolis toxicity or other side effects were found, associated with a faster and more organized hair recovery by propolis use. The green propolis extract was able to inhibit the growth of 23 from the 27 C. pseudotuberculosis clinical isolates, with minimum inhibitory and minimum bactericide concentrations ranging from 01 to 08 mg/mL, and did not interfere with the humoral immune response against the bacterium. In addition, green propolis was able to inhibit biofilm formation by four of the C. pseudotuberculosis clinical isolates. We concluded that green propolis is a promising therapeutic agent to be used in the post-surgical treatment of caseous lymphadenitis in small ruminants due to its effects on surgical wound healing, hair recovery, inhibition of wound contamination and bacterial growth.