Immune response of BALB/c mice infected with two strains of Corynebacterium pseudotuberculosis.

This work evaluated aspects of the immune response of BALB/c mice infected with Corynebacterium pseudotuberculosis (T1 and C57). The fifteen BALB/c mice were euthanized after 70 days of infection and morphologically evaluated, also analyzing the innate and adaptive immune responses. The C57 strain induced more pronounced morphological changes than the T1 strain. There was an increase in CD4+ and CD8+ T cells identified during infection with the C57 strain. Cytokines of the inflammatory profile IL-1α and IL-6 and regulatory IL-13 and IL-10 presented significant differences. Cytokines IL-2, IL-4, INF-γ, IL-22, IL-21, and IL-27 did not differ significantly between groups. The obtained results contribute to a better understanding of the type of response and the immunological mechanisms involved during infection with different strains of C. pseudotuberculosis.

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.

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.

Trypanosoma sp. infection in Boa constrictor snakes: morphological, hematological, clinical biochemistry, molecular, and phylogenetic characteristics.

There are few reports of Trypanosoma in snakes, as well as little information about its pathogenicity in these animals. Thus, the present study aimed to characterize Trypanosoma found in Boa constrictor snakes, to verify the influence of the parasitism on hematological and clinical biochemistry parameters, and to perform a phylogenetic study of the isolates. Blood samples from sixty-one boas were analyzed for the presence of trypanosomatids and by hematological and clinical biochemistry assays. The flagellates that were found in this analysis were used for cell culture, morphometry, and molecular analysis. Later, molecular typing phylogenetic studies were performed. Nine positive animals (14.75%) were identified by microscopy analysis. The hematological results showed that parasitized animals presented significantly lower levels of packed cell volume, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin. In the leukogram, eosinophils and heterophils counts were higher in parasitized animals. Considering the molecular analyses, the isolates presented a higher identity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the 18S small subunit ribosomal RNA (SSU rRNA) gene fragments with Trypanosoma serpentis. The phylogenetic tree, using the GAPDH, clustered all isolates with T. serpentis and Trypanosoma cascavelli. This is the first description of T. serpentis parasitizing boas and of the clinical changes caused by trypanosomatid infection in snakes.

AFFINITY OF BRAZILIAN WILD MAMMAL IMMUNOGLOBULINS TO BACTERIAL PROTEINS A AND G.

Staphylococcal A and streptococcal G proteins are widely used in immunoassays when specific immunological reagents are unavailable, such as for wild animals. The affinity of bacterial proteins A and G to the immunoglobulins of seven Brazilian mammals were tested, including black-tufted marmoset (Callithrix penicillata, n = 5), golden-bellied capuchin (Sapajus xanthosternos, n = 13), woolly mouse opossum (Micoureus demerarae, n = 6), long-nosed armadillo (Dasypus novemcinctus, n = 5), collared anteater (Tamandua tetradactyla, n = 5), ocelot (Leopardus pardalis, n = 6), and vampire bat (Desmodus rotundus, n = 5). Blood samples were collected from animals that were rescued in peri-urban rainforest fragments. Sera pools of each species were tested by ELISA to determine the intensity of each bacterial protein affinity to the immunoglobulins. When comparing the affinity to both proteins, immunoglobulins from D. rotundus, S. xanthosternos, and T. tetradactyla presented a higher affinity to protein G, whereas a higher affinity to protein A was found for immunoglobulins of C. penicillata and L. pardalis. The only species that presented a very low affinity to both bacterial proteins was M. demerarae. This study can be used as a reference for further studies on the development of sensitive and specific immunodiagnostic assays to be used for the monitoring of the health of these wild mammals.

Mutational spectrum of breast cancer susceptibility genes among women ascertained in a cancer risk clinic in Northeast Brazil.

PURPOSE: There is a paucity of data on the spectrum and prevalence of pathogenic variants among women of African ancestry in the Northeast region of Brazil. METHODS: We performed BROCA panel sequencing to identify inherited loss-of-function variants in breast cancer susceptibility genes among 292 Brazilian women referred to a single institution cancer risk assessment program. RESULTS: The study included a convenient cohort of 173 women with invasive breast cancer (cases) and 119 women who were cancer-free at the time of ascertainment. The majority of the women self-reported as African-descended (67% for cases and 90.8% for unaffected volunteers). Thirty-seven pathogenic variants were found in 36 (20.8%) patients. While the spectrum of pathogenic variants was heterogeneous, the majority (70.3%) of the pathogenic variants were detected in high-risk genes BRCA1, BRCA2, PALB2, and TP53. Pathogenic variants were also found in the ATM, BARD1, BRIP1, FAM175A, FANCM, NBN, and SLX4 genes in 6.4% of the affected women. Four recurrent pathogenic variants were detected in 11 patients of African ancestry. Only one unaffected woman had a pathogenic variant in the RAD51C gene. Different risk assessment models examined performed well in predicting risk of carrying germline loss-of-function variants in BRCA1 and/or BRCA2 in breast cancer cases. CONCLUSION: The high prevalence and heterogenous spectrum of pathogenic variants identified among self-reported African descendants in Northeast Brazil is consistent with studies in other African ancestry populations with a high burden of aggressive young onset breast cancer. It underscores the need to integrate comprehensive cancer risk assessment and genomic testing in the management of newly diagnosed Black women with breast cancer across the African Diaspora, enabling improved cancer control in admixed underserved and understudied populations.

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.

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.

In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli.

Recombinant proteins are generally fused with solubility enhancer tags to improve the folding and solubility of the target protein of interest. However, the fusion protein strategy usually requires expensive proteases to perform in vitro proteolysis and additional chromatographic steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, simplify the recombinant protein purification process, and promote the screening of molecules that fail to remain soluble after tag removal. High yields of soluble target proteins have already been achieved using these protease co-expression systems. Herein, we review approaches for controlled intracellular processing systems tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular processing of recombinant proteins regarding system design features, advantages, and limitations of the various strategies.

CD4+ T Cell Profile and Activation Response in Sickle Cell Disease Patients with Osteonecrosis.

Recent evidence suggests that abnormalities involving CD4+T lymphocytes are associated with the pathophysiology of osteonecrosis (ON); however, few studies have addressed the CD4+T cells in ON related to sickle cell disease (SCD/ON). In addition, T cells producing multiple cytokines simultaneously are often present in the inflammatory milieu and may be implicated in the immune response observed in SCD/ON. In the present study, we aimed to characterize the functional status of CD4+T cells in SCD by simultaneously determining the frequency of IFN-γ +, IL-4+, and IL-17+ CD4+T in cell cultures under exogenous stimuli. Peripheral blood mononuclear cells (PB-MNCs) from 9 steady-state SCD patients, 15 SCD/ON patients, and 19 healthy controls had functional status of CD4+T cells analyzed. Bone marrow mononuclear cells (BM-MNCs) from 24 SCD/ON patients (SCD BM) and 18 patients with ON not related to SCD (non-SCD BM) were also analyzed. We found that PB-MNC of SCD patients with or without ON presented significantly reduced TCD4+, TCD8+, and TCD4+ naïve cell frequencies and increased frequency of circulating CD4+T cells able to simultaneously produce IFN-γ +/IL4+ and IL-17+/IL4+ compared to healthy controls. Conversely, the polyclonal stimulation of BM-MNC induced an increased frequency of CD4+IFN-γ + and CD4+IL-17+ in SCD BM compared to non-SCD BM. The increased proportion of CD4+ T cells able to produce a broad spectrum of proinflammatory cytokines after a strong stimulus indicates that the immune system in SCD/ON patients presents an expressive pool of partially differentiated cells ready to take on effector function. It is possible that this increased subpopulation may extend to inflammatory sites of target organs and may contribute to the maintenance of inflammation and the pathophysiology of osteonecrosis in sickle cell disease.