Emergence of livestock-associated Mammaliicoccus sciuri ST71 co-harbouring mecA and mecC genes in Brazil.

The discovery and tracking of antimicrobial resistance genes are essential for understanding the evolution of bacterial resistance and restraining its dispersion. Mammaliicoccus sciuri (formerly Staphylococcus sciuri) is the most probable evolutionary repository of the mecA gene, that later disseminated to S. aureus. In this study, we describe the first double mecA/mecC homologue-positive non-aureus staphylococci and mammaliicocci (NASM) from the American continent, also representing the first report of mecC-positive NASM in Brazil. Two clonally related methicillin-resistant M. sciuri strains co-carrying mecA and mecC genes were isolated from the teat skin swab and milk sample collected from an ewe's left udder half. Both M. sciuri strains belonged to the sequence type (ST) 71. Besides mecA and mecC genes, the M. sciuri strains carried broad resistomes for clinically important antimicrobial agents, including ß-lactams, tetracyclines, lincosamide, streptogramin, streptomycin, and aminoglycosides. Virulome analysis showed the presence of the clumping factor B (clfB), ATP-dependent protease ClpP (ClpP) and serine-aspartate repeat proteins (sdrC and sdrE) virulence-associated genes. Phylogenomic analysis revealed that these M. sciuri strains are part of a globally disseminated branch, associated with farm and companion animals and even with food. Our findings suggest that M. sciuri is likely to emerge as a pathogen of global interest, carrying a broad repertoire of antimicrobial resistance genes with a remarkable co-presence of mecA and mecC genes. Finally, we strongly encourage to monitor M. sciuri under the One Health umbrella since this bacterial species is spreading at the human-animal-environment interface.

Bovine-associated staphylococci and mammaliicocci trigger T-lymphocyte proliferative response and cytokine production differently.

We examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B- and T-lymphocyte proliferation and interleukin (IL)-17A and interferon (IFN)-γ production by peripheral blood mononuclear cells in nulliparous, primiparous, and multiparous dairy cows. Flow cytometry was used to measure lymphocyte proliferation with the Ki67 antibody, and specific monoclonal antibodies were used to identify CD3, CD4, and CD8 T lymphocyte and CD21 B lymphocyte populations. The supernatant of the peripheral blood mononuclear cell culture was used to measure IL-17A and IFN-γ production. Two distinct, inactivated strains of bovine-associated Staphylococcus aureus [one causing a persistent intramammary infection (IMI) and the other from the nose], 2 inactivated Staphylococcus chromogenes strains [one causing an IMI and the other from a teat apex), as well as an inactivated Mammaliicoccus fleurettii strain originating from sawdust from a dairy farm, and the mitogens concanavalin A and phytohemagglutinin M-form (both specifically to measure lymphocyte proliferation) were studied. In contrast to the "commensal" Staph. aureus strain originating from the nose, the Staph. aureus strain causing a persistent IMI triggered proliferation of CD4+ and CD8+ subpopulations of T lymphocytes. The M. fleurettii strain and the 2 Staph. chromogenes strains had no effect on T- or B-cell proliferation. Furthermore, both Staph. aureus and Staph. chromogenes strains causing persistent IMI significantly increased IL-17A and IFN-γ production by peripheral blood mononuclear cells. Overall, multiparous cows tended to have a higher B-lymphocyte and a lower T-lymphocyte proliferative response than primiparous and nulliparous cows. Peripheral blood mononuclear cells of multiparous cows also produced significantly more IL-17A and IFN-γ. In contrast to concanavalin A, phytohemagglutinin M-form selectively stimulated T-cell proliferation.

Comparative analysis of fasting effects on the cecum microbiome in three guinea pig breeds: Andina, Inti, and Peru.

Guinea pigs have historically been used as a food source and are also an important model for studying the human intestines. Fasting is the act of temporarily stopping the intake of food. This process can alter the microbiota of various animals. This study is the first to investigate the impact of fasting on the cecum microbiome of three guinea pig breeds. We investigated the impact of fasting on the microbiome population structure in the cecum of three guinea pig breeds. This was done by sequencing and analyzing the V4 hypervariable region of the 16S rRNA gene in bacterial communities found in cecum mucosa samples. To achieve this, we established two treatment groups (fasting and fed), for each of the three guinea pig breeds: Andina, Inti, and Peru. The study involved twenty-eight guinea pigs, which were divided into the following groups: Andina-fed (five), Andina-fasting (five), Inti-fed (four), Inti-fasting (five), Peru-fed (five), and Peru-fasting (four). The results indicated a significant difference in beta diversity between the treatment groups for the Peru breed (P-value = 0.049), but not for the treatment groups of the Andina and Inti breeds. The dominant phyla across all groups were Firmicutes and Bacteroidetes. We observed variations in the abundance of different taxa in the cecum microbiota when comparing the treatment groups for each breed. Additionally, there was a higher number of unique taxa observed in the fasting groups compared to the fed groups. We discovered that the genus Victivallis was the only one present in all fasting groups across all breeds. Despite the findings, the resilience of the gut microbiome was not challenged in all three breeds, which can lead to disruptive changes that may affect the overall maintenance of the cecum microbiome. Based on the observed differences in the treatment groups of the Peru breed, it can be suggested that fasting has a greater impact on this particular breed.

Staphylococcus aureus-Cure-Associated Antigens Elicit Type 3 Immune Memory T Cells.

BACKGROUND: Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work. METHODS: Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus. RESULTS: Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A+ cells among CD8+ T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment. CONCLUSION: The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.

Memory CD4+ and CD8+ T lymphocyte proliferation in vaccinated dairy cows with different histories of Staphylococcus aureus mastitis.

Staphylococcus aureus mastitis constitutes a serious threat to dairy cows. The reasons why available vaccines are not fully effective remain poorly understood; thus, in the present study, we investigated CD4+ and CD8+ T lymphocyte proliferation in dairy cows vaccinated with a polyvalent mastitis vaccine that had distinct precedent Staphylococcus aureus mastitis. We studied 17 S. aureus-infected dairy cows (11 vaccinated and six unvaccinated) and eight vaccinated healthy dairy cows with no previous S. aureus mastitis infections. Flow cytometry was used to assess lymphocyte proliferation using an anti-Ki67 antibody, and monoclonal antibodies were used to identify T cell subsets. S. aureus-infected cows exhibited reduced overall lymphocyte proliferation, including CD4+ T lymphocyte proliferation, and memory lymphocyte proliferation in response to S. aureus isolate stimulus. Immunization did not influence the expansion of blood lymphocyte populations. Furthermore, CD8+ T cells, memory CD8+ T lymphocytes, and effector memory CD8+ T lymphocytes displayed reduced proliferation 21 days after the third vaccine dose compared with before vaccination at time zero. The present data demonstrates an overall negative regulation of the T-cell response suggesting its detrimental impact leading to the persistence of S. aureus intramammary infections. Furthermore, the lack of vaccination effect on T-cell mediated immunity (e.g., proliferation) may be related to poor vaccine efficacy.

Staphylococcus aureus Protection-Related Type 3 Cell-Mediated Immune Response Elicited by Recombinant Proteins and GM-CSF DNA Vaccine.

Staphylococcus aureus mastitis remains a major challenge for dairy farming. Here, 24 mice were immunized and divided into four groups: G1: control; G2: Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) DNA vaccine; G3: F0F1 ATP synthase subunit α (SAS), succinyl-diaminopimelate (SDD), and cysteinyl-tRNA synthetase (CTS) recombinant proteins; and G4: SAS+SDD+CTS plus GM-CSF DNA vaccine. The lymphocyte subpopulations, and the intracellular interleukin-17A (IL-17A) and interferon-γ production in the draining lymph node cells were immunophenotyped by flow cytometry. The immunophenotyping and lymphocyte proliferation was determined in spleen cells cultured with and without S. aureus stimulus. Immunization with S. aureus recombinant proteins generated memory cells in draining lymph nodes. Immunization with the three recombinant proteins plus GM-CSF DNA led to an increase in the percentage of IL-17A+ cells among overall CD44+ (memory), T CD4+, CD4+ T CD44+ CD27-, γδ TCR, γδ TCR+ CD44+ CD27+, and TCRVγ4+ cells. Vaccination with S. aureus recombinant proteins associated with GM-CSF DNA vaccine downregulated TH2 immunity. Immunization with the three recombinant proteins plus the GM-CSF DNA led to a proliferation of overall memory T, CD4+, and CD4+ TEM cells upon S. aureus stimulus. This approach fostered type 3 immunity, suggesting the development of a protective immune response against S. aureus.

Staphylococcus aureus protection-related type 3 cell-mediated immune response elicited by recombinant proteins and GM-CSF DNA vaccine

Staphylococcus aureus mastitis remains a major challenge for dairy farming. Here, 24 mice were immunized and divided into four groups: G1: control; G2: Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) DNA vaccine; G3: F0F1 ATP synthase subunit α (SAS), succinyl-diaminopimelate (SDD), and cysteinyl-tRNA synthetase (CTS) recombinant proteins; and G4: SAS+SDD+CTS plus GM-CSF DNA vaccine. The lymphocyte subpopulations, and the intracellular interleukin-17A (IL-17A) and interferon-γ production in the draining lymph node cells were immunophenotyped by flow cytometry. The immunophenotyping and lymphocyte proliferation was determined in spleen cells cultured with and without S. aureus stimulus. Immunization with S. aureus recombinant proteins generated memory cells in draining lymph nodes. Immunization with the three recombinant proteins plus GM-CSF DNA led to an increase in the percentage of IL-17A+ cells among overall CD44+ (memory), T CD4+, CD4+ T CD44+ CD27−, γδ TCR, γδ TCR+ CD44+ CD27+, and TCRVγ4+ cells. Vaccination with S. aureus recombinant proteins associated with GM-CSF DNA vaccine downregulated TH2 immunity. Immunization with the three recombinant proteins plus the GM-CSF DNA led to a proliferation of overall memory T, CD4+, and CD4+ TEM cells upon S. aureus stimulus. This approach fostered type 3 immunity, suggesting the development of a protective immune response against S. aureus.

Orange-Emitting ZnSe: Mn2+ quantum dots as nanoprobes for macrophages

The biocompatibility, bionanointeraction, uptake efficiency, and entry pathway of luminescent nanomaterials are the key factors to understand development of an efficient bionanoprobe. The foremost objective of this work is to explore the potential of 3-mercaptopropionic acid (3-MPA) capped ZnSe:xMn2+ (x = 5, 10, and 15 mol %) quantum dots (QDs) for the development of bionanoprobe used in future biological and clinical applications. For this purpose, highly intense orange-emitting activator Mn2+ ion doped ZnSe QDs were synthesized via a high-temperature organometallic method and rendered water-soluble by a ligand exchange approach. The morphological and physicochemical characterizations displayed the ultrasmall zinc-blend cubic crystal structure of QDs with an elliptical shape nanocrystals and average diameter of 4 nm. The luminescent nanomaterials exhibited orange emission centered at 584 nm under excitation at 385 nm. The biocompatibility, time-dependent cellular uptake, and the uptake mechanism of QDs were studied in RAW 264.7 macrophages, accomplished by various cytotoxicity assays, CytoViva hyperspectral enhanced dark-field and dual-mode fluorescence (DMF) microscopy, and transmission electron microscopy (TEM) images. The cytotoxicity study did not confirm any noticeable deleterious effect of QDs within incubation for 6 h. The fluorescence images of cells incubated with QDs showed efficient emission, which is a manifestation that QDs are photochemically stable in the intracellular environment. The cellular uptake findings demonstrated that the QDs were predominantly internalized via clathrin- and caveolae-mediated pathways. After the uptake, QDs aggregates appeared inside the vesicles in the cytoplasm, and their number and size gradually increased as a function of time. Nevertheless, the fluorescent QDs presented remarkable colloidal stability in various media, biocompatibility within the designated time, efficient time-dependent uptake, and distinct entry pathway in RAW macrophages, suggesting promising candidates to explore for the development of future bionanoprobes.

R-Phycoerythrin-labeled Mannheimia haemolytica for the simultaneous measurement of phagocytosis and intracellular reactive oxygen species production in bovine blood and bronchoalveolar lavage cells.

The present study aimed to validate the use of R-phycoerythrin (R-PE)-labeled Mannheimia haemolytica to simultaneously stimulate phagocytosis and intracellular production of reactive oxygen species (ROS) by blood phagocytes in bronchoalveolar lavage (BAL) fluid. Initially, R-PE-labeled M. haemolytica was inactivated using a water bath at 60 °C for 60 min. Afterwards, R-PE labelling of bacteria was confirmed by flow cytometry. The geometric mean fluorescence intensity of R-PE-labeled bacteria (FL2 detector, 585 ±â€¯42 nm) was analyzed by flow cytometry and was 41.5-fold higher than the respective unlabeled controls, confirming the success of bacterial conjugation to R-PE. Phagocytosis and intracellular production of ROS by blood neutrophils and monocytes, and by BAL CD14+ macrophages, in 12 healthy 6-month-old male calves were then performed using R-PE-labeled bacteria and 2',7'-dichlorofluorescein diacetate (DCFH-DA) as probes. Confocal microscopy was used to confirm phagocytosis of R-PE-labeled M. haemolytica by phagocytes and to exclude erroneous measurements of bacteria adhering to the leukocyte membrane. The present study showed that there is no difference in the ROS production without stimulus and in the presence of M. haemolytica by peripheral blood neutrophils and monocytes, in contrast to the increased ROS production by local alveolar macrophages upon stimulation by M. haemolytica. This emphasizes the importance of alveolar macrophages in the maintenance of homeostasis and health of the respiratory system, which can be supported during the inflammatory process by the rapid recruitment of neutrophils with high microbicidal and phagocytic capacity. The method described here provides an easy and feasible tool to measure phagocytosis and intracellular ROS production by phagocytes, especially when commonly used probes for intracellular ROS production were used, such as DCFH-DA and dihydrorhodamine 123.

Asymptomatic infections in blood donors harbouring Plasmodium: an invisible risk detected by molecular and serological tools.

BACKGROUND: Transfusion-transmitted malaria due to asymptomatic Plasmodium infections is a challenge for blood banks. There is a lack of data on the prevalence of asymptomatic infected blood donors and the incidence of transfusion-transmitted malaria in low endemicity areas worldwide. We estimated the frequency of blood donors harbouring Plasmodium in an area in which asymptomatic infections have been reported. MATERIAL AND METHODS: To estimate the frequency of blood donors harbouring Plasmodium we used microscopy and molecular tools. Serological tests were applied to measure the exposure of candidates to Plasmodium antigens. Venous blood was collected from 91 candidates attending the "Pró-Sangue" Blood Centre Foundation in São Paulo, who lived in the municipality of Juquitiba, São Paulo, Brazil, where sporadic autochthonous cases of malaria have been described. Blood samples were used for parasitological, molecular and serological studies. RESULTS: Among the 91 samples examined, rare Plasmodium forms were observed in two donors. Genus real-time polymerase chain reaction analysis demonstrated Plasmodium amplification in three candidates and species-specific nested polymerase chain reaction identified P. malariae in two. ELISA-IgG was reactive in 42.9% of samples for P. vivax (Pv-MSP119) and in 6.6% for P. falciparum (Pf-Zw). ELISA-IgM was reactive in 2.2% of samples for P. vivax and in 4.4% for P. falciparum. An indirect immunofluorescence assay was reactive for P. malariae in 15.4% of cases. DISCUSSION: Reservoirs of Plasmodium represent a challenge for blood banks, since studies have shown that high levels of submicroscopic infections can occur in low transmission areas. The risk of transfusion-transmitted malaria presented here points to the need to conduct molecular investigations of candidate donors with any positive malarial antibody test.

Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions.

The last step of Leishmania intracellular life cycle is the egress of amastigotes from the host cell and their uptake by adjacent cells. Using multidimensional live imaging of long-term-infected macrophage cultures we observed that Leishmania amazonensis amastigotes were transferred from cell to cell when the donor host macrophage delivers warning signs of imminent apoptosis. They were extruded from the macrophage within zeiotic structures (membrane blebs, an apoptotic feature) rich in phagolysosomal membrane components. The extrusions containing amastigotes were selectively internalized by vicinal macrophages and the rescued amastigotes remain viable in recipient macrophages. Host cell apoptosis induced by micro-irradiation of infected macrophage nuclei promoted amastigotes extrusion, which were rescued by non-irradiated vicinal macrophages. Using amastigotes isolated from LAMP1/LAMP2 knockout fibroblasts, we observed that the presence of these lysosomal components on amastigotes increases interleukin 10 production. Enclosed within host cell membranes, amastigotes can be transferred from cell to cell without full exposure to the extracellular milieu, what represents an important strategy developed by the parasite to evade host immune system.

Luminescent material based on the [Eu(TTA)3(H2O)2] complex incorporated into modified silica particles for biological applications.

Amino-functionalized luminescent silica particles were investigated for use in immunoassays. The particles were prepared by the Stöber method where the [Eu(TTA)3(H2O)2] complex (TTA: 3-thenoyltrifluoroacetonate) was incorporated into silica particles during the hydrolysis and condensation of TEOS: tetraethylorthosilicate. Then, the amino groups were introduced in the particle surface using APTS: 3-aminopropyltriethoxisilane. The resulting particles were characterized by scanning electron microscopy (SEM), X ray diffraction (XRD) and photoluminescence spectroscopy. In order to demonstrate the viability of the use of luminescent particles as optical markers, an enzyme-substrate reaction was performed using HRP: horseradish peroxidase. It was possible to verify the binding of HRP-oxidized LDL (low density lipoprotein) and anti-oxLDL antibody-luminescent silica particles through the evaluation of the presence of HRP. The bioassay data open a broad field for the development of protein-tagged luminescent particles for use in biomedical sciences.

Divergence between hepatic insulin-like growth factor (IGF)-I mRNA expression and IGF-I serum levels in Leishmania (Leishmania) infantum chagasi-infected dogs.

Visceral leishmaniasis (VL) caused in the New World by Leishmania (Leishmania) infantum chagasi has dog as important peridomestic reservoir in its transmission cycle. Since VL in infected animals is similar to human VL, its study is interesting for human pathology. During Leishmania infection, insulin-like growth factor-I (IGF-I) plays a role in the host-parasite interaction, favoring parasite growth, particularly acting directly on Leishmania. We evaluated IGF-I mRNA expression in different organs/tissues, which was differently modulated in dogs naturally infected by L. (L.) infantum chagasi. We also evaluated the hepatic IGF-I mRNA and serum IGF-I levels in infected dogs. Hepatic mRNA IGF-I expression was higher in the infected dogs than in control animals. However, the serum levels of IGF-I, which are related to the production of this factor in the liver, were reduced in the infected dogs compared with the non-infected controls. Thus, we suggest interference in post-transcriptional processing in IGF-I production in active visceral leishmaniasis.