Ozone Therapy in the Integrated Treatment of Female Dogs with Mammary Cancer: Oxidative Profile and Quality of Life.

Considering the high frequency of malignant breast tumors, there is a growing search for new therapeutic strategies that control neoplastic growth and dissemination, combined with fewer adverse reactions. Therefore, this study evaluated the effects of ozone therapy in female dogs with mammary cancer undergoing chemotherapy treatment. Twenty-five canines diagnosed with malignant mammary neoplasia were divided into two groups: one treated with carboplatin alone (n = 11) and the other with carboplatin associated with ozone therapy (n = 14). Clinical and laboratory evaluations, mastectomy, analysis of the oxidative profile based on total antioxidant capacity (TAC) and serum concentrations of malondialdehyde (MDA), survival rate, and quality of life were performed. Animals in the ozone therapy group had higher concentrations of red blood cells and platelets, significantly improving the survival rate and quality of life. Furthermore, adverse reactions were less intense and frequent in this group, which was associated with an increase in TAC and a reduction in MDA. These results indicate that the combination of carboplatin and ozone therapy represents a promising complementary treatment for female dogs with mammary cancer, as it was associated with fewer adverse reactions and a better oxidative profile.

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.

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.

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.

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.

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.

Biogenic Silver Nanoparticles as a Post-surgical Treatment for Corynebacterium pseudotuberculosis Infection in Small Ruminants.

Caseous lymphadenitis (CL) is an infectious and zoonotic disease characterized by the development of granulomas in the lymph nodes and internal organs of small ruminants. The etiological agent of this disease is Corynebacterium pseudotuberculosis, a Gram-positive and facultative intracellular bacterium. The conventional treatment for CL consists of drainage and chemical cauterization of the lesions using a 10% iodine solution. However, this type of treatment is not effective, due to iodine's cytotoxic profile and low antibacterial activity. Currently, silver nanoparticles (AgNPs) can be seen as an alternative treatment for CL due to their antimicrobial activity and wound healing effects. Therefore, the present study aimed to evaluate AgNPs as a post-surgical treatment for CL. Twenty-nine goats and sheep with clinical signs of CL were selected. Surgical intervention was performed to excise the caseous lesions. To treat the lesions, an ointment formulation based on AgNP mixed with natural waxes and oils was used in the experimental group, and the conventional treatment with 10% iodine was used in the control group. Bacteria were isolated from the excised caseous material. The animals were observed for 8 weeks after the surgical treatment, and blood samples were taken weekly. The surgical wounds of sheep treated with AgNP healed faster, and the surgical wound area was smaller during the observation period; the latter effect was also observed in goats. AgNP-treated animals also had less purulent discharge and less moisture in the surgical wounds. The AgNP-treated animals had lower leukocyte counts and lower titers of anti-C. pseudotuberculosis antibodies. There was no statistically significant difference between the groups with regard to the hemogram results. The results of the susceptibility testing of C. pseudotuberculosis strains (T1, 1002, FRC41, and VD57 strains) and clinical isolates to AgNPs showed growth inhibition, even at low concentrations. It can be concluded that post-surgical treatment of CL using the AgNP-based ointment may be a promising tool in the control of CL, through faster healing, decreased wound contamination, and no apparent toxic effects.

Sensibilidade in vitro a antimicrobianos de Corynebacterium pseudotuberculosis isolados de pequenos ruminantes da região Centro- Norte do Estado da Bahia / In vitro antimicrobial susceptibility of Corynebacterium pseudotuberculosis isolated from small ruminants from the North-Central region of the State of Bahia

Introdução: corynebacterium pseudotuberculosis é uma bactéria com importância em medicina veterinária por ser o agente etiológico da Linfadenite Caseosa (LC) em pequenos ruminantes. A doença leva a perdas econômicas devido a condenação de carcaças, diminuição de produtividade e redução do valor comercial do couro dos animais acometidos. O tratamento da doença é pouco eficaz, pois os agentes antimicrobianos não conseguem atravessar a barreira dos granulomas, mas tem sido proposta a inoculação intracapsular de antibióticos como tratamento e como medida de prevenção de contaminação ambiental. Objetivo: devido a esse fato, o presente estudo teve como objetivo avaliar a sensibilidade in vitro de isolados clínicos de Corynebacterium pseudotuberculosis isolados de animais da região Centro-Norte do estado da Bahia. Metodologia: foram retirados material de granulomas de 11 animais de uma fazenda na região acima mencionada, e as bactérias foram isoladas a partir desse material. Esses isolados então foram submetidos a metodologia de difusão em disco de ágar utilizando-se discos impregnados com doze antibióticos diferentes. Resultados: 90,9% (10/11) dos isolados foram resistentes a oxacilina. Com relação à sensibilidade, 100% dos isolados foram sensíveis a ampicilina, amicacina, amoxicilina, cefalotina, ceftriaxona, ciprofloxacina, enrofloxacina, neomicina e penicilina, enquanto 90,9% destes apresentaram sensibilidade a bactericina e doxiciclina. Conclusão: pode-se concluir que os isolados de C. pseudotuberculosis da região estudada apresentam sensibilidade a diversos antibióticos, o que pode facilitar na escolha de um antimicrobiano com menor toxicidade para fins de tratamento com inoculação intracapsular do medicamento.

Background: corynebacterium pseudotuberculosisis important bacteria in veterinary medicine because it is the Etiologic Agent of Caseous Lymphadenitis (CL) in small ruminants. The disease leads to economic losses due to condemnation of carcasses, productivity decrease and leather commercial value reduction of affected animals affected. The treatment of the disease is not very effective, because antimicrobial agents cannot cross the barrier of granulomas, but it has been proposed the intracapsular inoculation of antibiotics as treatment and as a measure of prevention of environmental contamination. Objective: due to this fact, the present study aimed to evaluate the in vitro susceptibility of clinical isolates of Corynebacterium pseudotuberculosis isolated from animals in the North Central region of the State of Bahia. Methodology: granuloma material were removed from 11 animals from a farm in the above mentioned area, and the bacteria were isolated from this material. These isolated bacteria were then subjected to disk diffusion method of agar using disks impregnated with twelve different antibiotics. Results: 90,9% (10/11) the isolated bacteria were resistant to oxacillin.With respect to sensitivity, 100% of the isolates bacteria were susceptible to amikacin, ampicillin, amoxicillin, cephalotin, ceftriaxone, ciprofloxacin, enrofloxacin, neomycin and penicillin, while 90.9% of these showed sensitivity to bacitracin and doxycycline. Conclusion: it can be concluded that the isolated bacteria of C. pseudotuberculosis from the region studied present sensitivity to different antibiotics, which can facilitate the choice of a less toxic antimicrobial for treating with intracapsular inoculation of the medicine.

The genome anatomy of Corynebacterium pseudotuberculosis VD57 a highly virulent strain causing Caseous lymphadenitis.

Corynebacterium pseudotuberculosis strain VD57 (Cp_VD57), a highly virulent, nonmotile, non-sporulating, and a mesophilic bacterium, was isolated from a goat's granulomatous lesion in the municipality of Juazeiro, Bahia State, Brazil. Here, we describe a set of features of the strain, together with the details of its complete genome sequence and annotation. The genome comprises of a 2.5 Mbp long, single circular genome with 2,101 protein-coding genes, 12 rRNA, 49 tRNA and 47 pseudogenes and a G + C content of 52.85 %. Genetic variation was detected in Cp_VD57 using C. pseudotuberculosis strain 1002 as reference, wherein small genomic insertions and deletions were identified. The comparative analysis of the genome sequence provides means to better understand the host pathogen interactions of this strain and can also help us to understand the molecular and genetic basis of virulence of this bacterium.

An iron-acquisition-deficient mutant of Corynebacterium pseudotuberculosis efficiently protects mice against challenge.

Caseous lymphadenitis (CLA) is a chronic disease that affects sheep and goats worldwide, and its etiological agent is Corynebacterium pseudotuberculosis. Despite the economic losses caused by CLA, there is little information about the molecular mechanisms of bacterial pathogenesis, and current immune prophylaxis against infection has been unable to reduce the incidence of CLA in goats. Recently, 21 different mutant strains of C. pseudotuberculosis were identified by random mutagenesis. In this study, these previously generated mutants were used in mice vaccination trials to develop new immunogens against CLA. Based on this analysis, CZ171053, an iron-acquisition-deficient mutant strain, was selected. After challenge with a virulent strain, 80% of the animals that were immunized with the CZ171053 strain survived. Furthermore, this vaccination elicited both humoral and cellular responses. Intracellular survival of the bacterium was determined using murine J774 cells; in this assay, the CZ171053 had reduced intracellular viability. Because iron acquisition in intracellular bacteria is considered one of their most important virulence factors during infection, these results demonstrate the immunogenic potential of this mutant against CLA.

Acaricides efficiency on Rhipicephalus (Boophilus) microplus from Bahia state north-central region / Eficiência de acaricidas sobre Rhipicephalus (Boophilus) microplus na região centro-norte, Bahia

The cattle tick Rhipicephalus (Boophilus) microplus is responsible for major losses in the Brazilian livestock, mainly due to reduction in cattle productive performance. Resistance development to major classes of acaricide widely used nowadays has been extensively reported, as well as the occurrence of residues from these compounds in animal products and the environment. This study aimed to evaluate the efficiency of acaricides on R. (B.) microplus collected from rural properties in the North-Central region of Bahia State. Ticks were collected in several cattle farms in the cited region, and an in vitro acaricide efficiency assay was performed based on the immersion of ticks in acaricide solutions made according to manufacturers' recommendations. The results obtained in the experiments indicated varying degrees of efficiency of the several bases studied, with the products benzofenilurea, macrocyclic lactone and fipronil showing the highest levels of efficiency, 100%, 100% and 97.34%, respectively. It was possible to conclude that, for the region studied, there are different degrees of commercial acaricides efficiency, and many present less than 95% effectiveness, value determined as acceptable by the Brazilian legislation.

O carrapato Rhipicephalus (Boophilus) microplus é responsável por grandes perdas na pecuária brasileira, principalmente devido à redução no desempenho produtivo dos animais. A resistência do parasita a diferentes classes de acaricidas tem sido amplamente relatada, bem como os problemas de ocorrência de resíduos desses compostos em produtos de origem animal e no ambiente. Esse estudo teve como objetivo avaliar a eficiência de diversos acaricidas sobre populações de R. (B.) microplus provenientes de propriedades rurais da Região Centro-Norte do Estado da Bahia, Brasil. Teleóginas ingurgitadas foram colhidas em diferentes propriedades da região e submetidas a ensaio in vitro de eficiência de acaricidas, baseado na imersão dos carrapatos em soluções de acaricidas feitas seguindo as recomendações dos fabricantes. Os resultados obtidos indicam níveis variados de eficiência das bases estudadas, com os acaricidas benzofenilureia, lactona macrocíclica e fipronil apresentando os índices mais altos de eficiência, 100%, 100% e 97.34%, respectivamente. Pode-se concluir que, para a região estudada, os acaricidas apresentam níveis diversos de eficiência, e muitos deles possuem eficácia de menos de 95%, como recomendado pela legislação brasileira.