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.

In silico approaches for predicting natural compounds with therapeutic potential and vaccine candidates against Streptococcus equi.

Equine strangles is a prevalent disease that affects the upper respiratory in horses and is caused by the Gram-positive bacterium Streptococcus equi. In addition to strangles, other clinical conditions are caused by the two S. equi subspecies, equi and zooepidemicus, which present relevant zoonotic potential. Treatment of infections caused by S. equi has become challenging due to the worldwide spreading of infected horses and the unavailability of effective therapeutics and vaccines. Penicillin treatment is often recommended, but multidrug resistance issues arised. We explored the whole genome sequence of 18 S. equi isolates to identify candidate proteins to be targeted by natural drug-like compounds or explored as immunogens. We considered only proteins shared among the sequenced strains of subspecies equi and zooepidemicus, absent in the equine host and predicted to be essential and involved in virulence. Of these, 4 proteins with cytoplasmic subcellular location were selected for molecular docking with a library of 5008 compounds, while 6 proteins were proposed as prominent immunogens against S. equi due to their probabilities of behaving as adhesins. The molecular docking analyses revealed the best ten ligands for each of the 4 drug target candidates, and they were ranked according to their binding affinities and the number of hydrogen bonds for complex stability. Finally, the natural 5-ring compound C25H20F3N5O3 excelled in molecular dynamics simulations for the increased stability in the interaction with UDP-N-acetylenolpyruvoylglucosamine reductase (MurB). This research paves the way to developing new therapeutics to minimize the impacts caused by S. equi infections.Communicated by Ramaswamy H. Sarma.

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.

First description of Candida haemulonii infecting a snake Boa constrictor: Molecular, pathological and antifungal sensitivity characteristics.

Candida haemulonii is an emergent infectious pathogen that affects human presenting comorbidities and/or immunodepression. Little is known about other possible hosts. For the first time, this fungus was found causing a cutaneous infection in a snake, Boa constrictor, characterized by scale opacity and several ulcerative lesions. This C. haemulonii was isolated, identified using molecular techniques and a phylogenetic study, and had its growth totally inhibited by all the drugs tested; however, no fungicide effect was seen for fluconazole and itraconazole. The B. constrictor clinical signals subsided after a treatment using a biogenic silver nanoparticle-based ointment. These findings, along with the B. constrictor presence near human habitats, warn for the necessity of wildlife health monitoring for emergent and opportunistic diseases in peri-urban environments.

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.

Photodynamic inactivation of different Candida species and inhibition of biofilm formation induced by water-soluble porphyrins.

BACKGROUND: Candida spp. is the main fungal genus related to infections in humans, and its treatment has become a challenge due to the production of biofilm and its resistance/multi-resistance profile to conventional antifungals. Antimicrobial photodynamic therapy stands out as a treatment characterized by a broad spectrum of antimicrobial action, being able to induce oxidative stress in pathogens, and porphyrins are photosensitizers with high selectivity to pathogens. Thus, this work aimed to analyze the photoinactivation of different species of Candida by two cationic (4-H2TMeP+ and 3-H2TMeP+) and one anionic (4-H2TPSP‒) porphyrins. MATERIALS AND METHODS: Microdilution assays were performed to determine the MIC100, with subsequent determination of MFC100. Determination of oxidative species was done through the use of scavengers, while biofilm morphological features were investigated using the atomic force microscopy. RESULTS: Cationic porphyrins were significantly efficient in inactivating Candida albicans and non-albicans species with 100% growth inhibition and fungicidal activity (MFC100/MIC100 ≤ 4.0). The cationic porphyrins were also able to interfere in Candida spp biofilm formation. The photo-oxidative mechanism activated by 3-H2TMeP+ in Candida spp. is concurrent with the production of singlet oxygen and oxygen radical species. In the AFM analysis, 3-H2TMeP+ was able to reduce yeast adhesion to the surface. CONCLUSIONS: Cationic porphyrins can photo-inactivate different species of Candida in both planktonic and biofilm-associated forms, and reduce the adhesion of these fungi to the surface.

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.

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.

The glycoproteomics of hawk and caiman tears.

BACKGROUND: Glycoproteins are important tear components that participate in the stability of the ocular surface. However, the glycopeptides that are present in the tears of wild animals have not yet been described. This work aimed to describe the glycoproteomic profile of roadside hawk (Rupornis magnirostris) and caiman (Caiman latirostris) tears. METHODS: Tears collected from 10 hawks and 70 caimans using Schirmer tear test strips were used in this study. The samples were submitted to trypsin digestion and separated using a reverse-phase column coupled to a mass spectrometer associated to a nanospray ionization source. The glycoproteins were categorized as: cellular components, biological processes and molecular function, according to the UniProt Knowledgebase. RESULTS: As shown by the liquid chromatography-mass spectrometry, all glycopeptides found were classified as N-type. Of the 51 glycoproteins that were identified in the hawk tear film, the most abundant were ovotransferrin, globulins and complement system proteins. In the caiman tear film, 29 glycoproteins were identified. The most abundant caiman glycoproteins were uncharacterized proteins, ATPases, globulins and proteasome components. Ontological characterization revealed that the glycoproteins were extracellular, and the most identified molecular function was endopeptidase activity for both species. CONCLUSION: Glycoproteins are abundant in the tear film of the bird and reptile species studied herein, and all these molecules were shown to have N-type modifications. Location at the extracellular space and an endopeptidase inhibitor activity were the main cell component and molecular function for both species, respectively. These profiles showed differences when compared to human tears, are possibly linked to adaptive processes and can be the basis for further studies on the search of disease biomarkers.

Perfil de sensibilidade a antibióticos de um isolado de Providencia rettgeri proveniente de equino / Antibiotic sensitivity profile in isolate of Providencia rettgeri from a horse

Introdução: a meningite bacteriana em equinos é uma enfermidade frequente em animais jovens. Streptococcus spp., Staphylococcus aureus e Escherichia coli são as bactérias mais comumente isoladas nesses casos. Apesar da bactéria Providencia rettgeri já ter sido isolada em casos de meningite humana e de crocodilo, não há relatos de seu isolamento em equinos. Objetivo: relatar o isolamento e a identificação da bactéria P. rettgeri de um potro com sintomas neurológicos e avaliar o perfil de sensibilidade a antibióticos deste isolado. Metodologia: o isolamento foi realizado a partir do líquido cefalorraquidiano do potro, por meio de cultivo em meio ágar chocolate. Após isolamento, as colônias formadas foram identificadas por equipamento Biotyper, baseado em espectrometria de massa. O perfil de sensibilidade foi definido por teste de difusão em discos, seguindo metodologia relatada pelo CLSI M2-A8 em 2003, sendo a bactéria classificada como resistente, padrão indeterminado ou sensível aos antibióticos, de acordo com o descrito pelo EUCAST em 2021. Resultados: este é o primeiro relato do isolamento de P. rettgeri como agente etiológico de meningite em potro. Dos 15 antibióticos testados, a bactéria foi resistente a 9, sensível a 5 e com padrão indeterminado a 1 antibiótico. Conclusão: nossos resultados indicam que P. rettgeri deve ser considerada entre possíveis agentes etiológicos de quadros neurológicos em equinos e que testes de sensibilidade a antibiótico são fundamentais, uma vez que essa bactéria já apresenta resistência a diversos antibióticos disponíveis comercialmente.

Introduction: Bacterial meningitis in horses is a frequent disease in young animals. Streptococcus spp., Staphylococcus aureus and Escherichia coli are the most commonly isolated bacteria in these cases. Although Providencia rettgeri bacterium has already been isolated in cases of human and crocodile meningitis, there are no reports of its isolation in cases of meningitis in horses. Objective: to report isolation and identification of the P. rettgeri bacteria from a foal with neurological symptoms and to assess antibiotic sensitivity profile in isolate of it. Methods: isolation was performed from the foal's cerebrospinal fluid, through cultivation in chocolate agar medium. After isolation, formed colonies were identified by Biotyper equipment, based on mass spectrometry. Sensitivity profile was verified by disk diffusion test, according to methodology that was reported by CLSI M2-A8 in 2003, which classified bacteria as resistant, indeterminate pattern or sensitive to antibiotics, as described by EUCAST in 2021. Results: this is the first report on isolation of P. rettgeri as an etiologic agent of meningitis in foals. Among 15 antibiotics that were tested, results showed bacteria resistence to 9 antibiotics, bacteria sensitivity to 5, but undetermined pattern to 1 antibiotic. Conclusion: results indicate that P. rettgeri shall be considered among potential etiologic agents of neurological conditions in horses and that antibiotic sensitivity tests are essential, since this type of bacterium is already resistant to several commercially available antibiotics.

Feline Leishmaniasis Caused by Leishmania infantum: Parasite Sequencing, Seropositivity, and Clinical Characterization in an Endemic Area From Brazil.

Zoonotic leishmaniasis caused by Leishmania infantum is a disease of One Health concern since human and animal cases and environmental damage are interconnected. L. infantum has a complex epidemiological cycle with multiple hosts, including mammals-humans, domestic, and wild animals-and arthropod vectors. Knowledge on mammal infections in endemic areas is crucial for developing control strategies. This work aimed to detect and characterize L. infantum infection in domestic cats from areas where human and canine leishmaniasis cases occur. No cases of feline leishmaniasis (FeL) had been previously reported in those areas. Five municipalities from Bahia state were chosen, comprising 2,480.8 km2 with 1,103,866 inhabitants. Ninety domiciliated and/or sheltered cats underwent clinical examination and serology by a rapid reference test recommended by the Brazilian government. Cytology, PCR, and parasite DNA sequencing were performed in bone marrow samples. Rapid tests detected antibodies in 5.6% (5/90) of the cats. Leishmania infantum infection was confirmed in 7.8% (7/90) of the cats by PCR, sequencing, and parasite isolation. Three out of the five municipalities (60%) had infected cats, and PCR positivity varied from 6.9 to 29%. One cat was categorized as harboring active L. infantum infection with amastigote forms in bone marrow smears. No clinical signs were detected at the first clinical exam, but 1 month later the cat developed severe FeL. The cat isolate was grown in culture, typed and its DNA sequence was homologous to the L. infantum reference strain (PP75). In conclusion, cats are potential hosts and may acquire L. infantum in endemic areas where canine and human cases occur. For cats, the need for surveillance, differential diagnosis and clinical care is highly recommended since a fast clinical progression of FeL developed in a subclinical animal. An accurate standardized immunodiagnostic assay for FeL is warranted.

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.

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.

Leishmania infantum-Derived Glycoinositolphospholipids in the Immunodiagnosis of Subclinically Infected Dogs.

Lipophosphoglycan (LPG), when used as an ELISA target, confers high specificity and sensitivity to the detection of Leishmania infantum antibodies in dogs. Glycoconjugates are economically viable because the yield is very high after extraction/purification. In addition, they are very stable, which allows their use in point-of-care testing without special storage conditions. During the glycoconjugate extraction, a glycoinositolphospholipid (GIPL)-enriched fraction is obtained in similar quantities as LPG. Since GIPLs can be extracted from the same parasite pellet as LPGs, this work aimed to evaluate the immune recognition of GIPLs by Leishmania infantum-infected dogs and its use for canine leishmaniasis (CanL) immunodiagnosis. Like LPG, GIPLs were recognized by sera from L. infantum-infected dogs, but with less sensitivity (83.8%). However, 80% (16/20) of subclinically infected dogs were detected as positive in the assay. Different from LPG, the GIPL-based assay achieved a lower specificity (73.7%) and cross-reactions occurred with T. cruzi and L. braziliensis-infected dogs. Although GIPLs exhibited a similar performance to LPG for subclinically L. infantum-infected dogs, the occurrence of cross-reactivities with other protozoa and a lower sensitivity hinders its use for an immunodiagnostic test. In places where those diseases do not co-exist such as in the Mediterranean region, its use for subclinically dogs could be an alternative.

Hyperresistinemia in Obese Female Dogs With Mammary Carcinoma in Benign-Mixed Tumors and Its Correlation With Tumor Aggressiveness and Survival.

Resistin is associated with metabolic, inflammatory, and neoplastic disorders, and is also considered a prognostic marker in human oncology. Canine mammary tumors have epidemiological, clinical, biological, and genetic characteristics similar to those of women and are proposed as a comparative study model. Here, we evaluate the serum levels of resistin in female dogs with or without mammary carcinoma in mixed tumors (CBMT) and its correlation with the proliferative potential of the tumor, obesity, and survival. Eighty dogs grouped according to the presence (50) or absence (30) of CBMT, reproductive status and body condition were assessed for weight, fat percentage, and canine body mass index. The characteristic of the proliferative potential of the tumor (Ki-67) was evaluated. Ki-67 levels (p = 0.024), staging (p = 0.004), and grade (p = 0.016) influenced the survival of the female dogs. Through a multifactorial analysis, it could be seen that the parameters proliferation index (Ki-67) (p = 0.044) and staging (p = 0.036) influenced the survival of the animals. Neutered and overweight dogs from the control and CBMT groups showed hyperresistinemia. Ki-67 expression and resistin levels in dogs with CBMT were higher in overweight dogs than in dogs with normal weight (p = 0.0001). The survival rate of dogs with CBMT, obese and with high levels of resistin (8,400 µg L-1) was lower when compared to those with lower levels of resistin. These results showed an important relationship between hyperresistinemia, tumor proliferative potential and excessive body fat, suggesting that resistin levels may act as an interesting prognostic marker in patients with CBMT.

Cell wall glycolipids from Corynebacterium pseudotuberculosis strains with different virulences differ in terms of composition and immune recognition.

Caseous lymphadenitis (CLA) is an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants and is characterized by the development of granulomas in the lymph nodes, spleen, liver, and lungs. Although little is known about the host-pathogen relationship of this bacterium, it was previously reported that the pathogen's lipids are important for its taxonomic classification and survival inside macrophages. However, there are no studies regarding the composition of these molecules. In this study, cell wall glycolipids from two C. pseudotuberculosis strains presenting different virulence profiles were purified and its composition was characterized. A difference was observed between the electrophoretic and chromatogram profiles for cell wall components from the two strains, mainly among molecules with low molecular weights. IgM from sheep with acute CLA recognized antigens with an estimated molecular weight of 11 kDa of the low-pathogenicity strain, while low-molecular weight antigens from the high-pathogenicity strain presented a lower recognition by these antibodies. Mass spectrometry analysis showed that the cell wall of the high-pathogenicity strain contained glycolipids with high amounts of unsaturated fatty acids and glycerophosphoinositols, which may contribute to the capacity of this strain to cause severe disease. In conclusion, it is indicated that cell wall non-protein antigens can play a key role in C. pseudotuberculosis virulence.

The combination of Brazilian red propolis and recombinant protein rCP01850 in the immunoprophylaxis of Corynebacterium pseudotuberculosis infection in mice.

The immunomodulatory properties of Brazilian red propolis (BRP) have been already described. Also, propolis have been proved to have antibacterial activity on Corynebacterium pseudotuberculosis. An adjuvant effect of red propolis oil was able to induce a significant anti-C. pseudotuberculosis humoral immune response. Here, we demonstrate for the first time the immunostimulant property of BRP hydroalcoholic extract (BRPHE) in a recombinant vaccine against caseous lymphadenitis. Mice BALB/c were allocated in three groups inoculated with: sterile saline solution (G1); BRPHE (G2); or BRPHE combined with the C. pseudotuberculosis rCP01850 recombinant protein (G3) in two doses within a 21-days-interval. Blood samples were collected for the total IgG, IgG1 and IgG2a measurement. Mice were challenged with a virulent C. pseudotuberculosis strain, and other 6 mice were used for IFN-γ and IL-10 levels determination after splenocyte stimulation with the recombinant antigen. G3 showed higher significant levels of antibodies on the 42nd experimental day, with a high IgG2a/IgG1 proportion. G2 and G3 presented significant production of IFN-γ and IL-10, while G3 presented the higher levels of IFN-γ (p < 0.05). After challenge, G2 showed a survival rate of 20%, while 70% of mice from G3 survived the experimental challenge. In conclusion, BRPHE used alone has immunostimulant properties specially on cellular immune response, and when used in combination with the recombinant protein rCP01850 induces cellular and humoral immune responses as well as a significant survival of inoculated mice.

Ticks and Associated Pathogens From Rescued Wild Animals in Rainforest Fragments of Northeastern Brazil.

The Ixodidae family comprises ticks that are hematophagous ectoparasites and are considered vectors of several hemoparasites from the Anaplasmataceae family and the genus Hepatozoon, Babesia, and Rickettsia. These ectoparasites parasitize domestic and wild animals belonging to several vertebrate groups. Ticks are highly adapted to different biomes and thus possess a wide geographical distribution. In the Brazilian state of Bahia, localized in the Northeast region, there are large rainforest fragments. Studies have rarely been carried out on ticks, and their hemoparasites, that parasitize wild animals in this region. Thus, this study aimed to identify the tick species parasitizing wild animals rescued in rainforest fragments of Bahia and investigate the presence of hemoparasites in tick tissues. During a 2-year period, 238 ticks were collected from 41 wild mammalians, reptiles, and amphibians. These ectoparasites were taxonomically classified according to their morphological characteristics. The ticks identified belonged to five different species from the Ixodidae family: Amblyomma varium, Amblyomma rotundatum, Amblyomma nodosum, Ixodes loricatus, and Rhipicephalus sanguineus. For the first time, an A. rotundatum parasitizing the Mesoclemmys tuberculata turtle was described. PCR assays using DNA extracted from salivary glands or midgut of the ticks were performed to detect specific DNA fragments of hemoparasites from the genus Rickettsia, Ehrlichia, Babesia, Hepatozoon, and from the Anaplasmataceae family. The results showed positive detection of the Rickettsia genus (7.9%), Anaplasmataceae family (15.8%), and Hepatozoon genus (15.8%). Specific DNA from the Ehrlichia and Babesia genera were not detected in these samples. Specific DNA from members of the Anaplasmataceae family was detected in A. varium for the first time. The present work showed that amphibians, reptiles, and mammals from Bahia's Atlantic Forest areparasitized by different tick species, and that these ectoparasites present pathogens in their tissues that impact both humans and animals due to their zoonotic potential.

Ticks and associated pathogens from rescued wild animals in rainforest fragments of northeastern Brazil

The Ixodidae family comprises ticks that are hematophagous ectoparasites and are considered vectors of several hemoparasites from the Anaplasmataceae family and the genus Hepatozoon, Babesia, and Rickettsia. These ectoparasites parasitize domestic and wild animals belonging to several vertebrate groups. Ticks are highly adapted to different biomes and thus possess a wide geographical distribution. In the Brazilian state of Bahia, localized in the Northeast region, there are large rainforest fragments. Studies have rarely been carried out on ticks, and their hemoparasites, that parasitize wild animals in this region. Thus, this study aimed to identify the tick species parasitizing wild animals rescued in rainforest fragments of Bahia and investigate the presence of hemoparasites in tick tissues. During a 2-year period, 238 ticks were collected from 41 wild mammalians, reptiles, and amphibians. These ectoparasites were taxonomically classified according to their morphological characteristics. The ticks identified belonged to five different species from the Ixodidae family: Amblyomma varium, Amblyomma rotundatum, Amblyomma nodosum, Ixodes loricatus, and Rhipicephalus sanguineus. For the first time, an A. rotundatum parasitizing the Mesoclemmys tuberculata turtle was described. PCR assays using DNA extracted from salivary glands or midgut of the ticks were performed to detect specific DNA fragments of hemoparasites from the genus Rickettsia, Ehrlichia, Babesia, Hepatozoon, and from the Anaplasmataceae family. The results showed positive detection of the Rickettsia genus (7.9%), Anaplasmataceae family (15.8%), and Hepatozoon genus (15.8%). Specific DNA from the Ehrlichia and Babesia genera were not detected in these samples. Specific DNA from members of the Anaplasmataceae family was detected in A. varium for the first time. The present work showed that amphibians, reptiles, and mammals from Bahia's Atlantic Forest areparasitized by different tick species, and that these ectoparasites present pathogens in their tissues that impact both humans and animals due to their zoonotic potential.