Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi.

BACKGROUND: Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis. METHODS: In the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level. RESULTS: A novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410T, were compared to the results of nitrate reductase identification by biochemical test. The McNemar's Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16-6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90-0.98)]. CONCLUSIONS: The ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory.

Phylogenetic Group of Escherichia coli Isolates from Broilers in Brazilian Poultry Slaughterhouse.

The aim of the study was to determine the phylogenetic groups of E. coli strains isolated from seemingly healthy broiler and broiler condemned suspected of colibacillosis in a Brazilian slaughterhouse. Samples from respiratory tract and edible giblets (liver and heart) of broilers with and without macroscopic lesions of colibacillosis were collected at slaughter. There were 84 strains isolated from broilers condemned of which 11 were obtained from swabs of the heart, 7 from the liver, and 66 from the respiratory tract. Of the 53 E. coli strains isolated from broilers not condemned, 5 were isolated from the heart, 4 from the liver, and 44 from the respiratory tract. E coli strains were tested via PCR for phylogenetic groups A, B1, B2, C, D, E, and F. Phylogroups A and B1 were the most common phylogroups of E. coli obtained from healthy and sick-appearing broiler carcasses. The results of the study showed that phylogroups B2 and E were associated with the heart samples and phylogroup A was associated with respiratory tract samples, phylogroup B1 with not condemned carcass, and phylogroup D with liver samples.

Antimicrobial susceptibility and phylotyping profile of pathogenic Escherichia coli and Salmonella enterica isolates from calves and pigs in Minas Gerais, Brazil.

The aims of the present study were to determine (i) the profiles of phylogroup and (ii) the antimicrobial susceptibility of pathogenic Escherichia coli strains isolated from calves, and of Salmonella spp. strains isolated from calves and pigs in Minas Gerais State, Brazil. Sixty-one pathogenic E. coli strains and Salmonella spp. (n = 24) strains isolated from fecal samples of calves and Salmonella spp. (n = 39) strains previously isolated from fecal samples of growing/finishing pigs were tested. The minimum inhibitory concentration (MIC) using the agar dilution method was determined for nalidixic acid, amikacin, amoxicillin, ampicillin, cefoxitin, norfloxacin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole. All E. coli isolates were susceptible to amikacin. Tetracycline was the antimicrobial that presented the higher frequency of resistance among E. coli strains, followed by ampicillin, trimethoprim-sulfamethoxazole, amoxicillin, nalidixic acid, norfloxacin, gentamicin, and cefoxitin. E. coli (n = 61) strains isolated from calves belonged to different phylogroup namely, phylogroup A (n = 26), phylogroup B1 (n = 31), phylogroup E (n = 3), and phylogroup F (n = 1). Phylogroups B2, C, and D were not identified among the E. coli in the present study. All Salmonella spp. (n = 24) strains isolated from fecal samples of calves were susceptible to amikacin, amoxicillin, ampicillin, norfloxacin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole. Resistance to nalidixic acid and cefoxitin was detected in 16.66 and 8.33 % of the Salmonella spp. strains, respectively. Among the Salmonella spp. (n = 39) strains isolated from fecal samples of pigs, the higher frequency of resistance was observed to tetracycline, followed by amoxicillin, gentamicin, ampicillin, trimethoprim-sulfamethoxazole, nalidixic acid, cefoxitin, and norfloxacin. All strains were susceptible to amikacin. Forty-eight (78.68 %) of the E. coli strains were classified as multidrug-resistant, whereas among Salmonella spp. strains, the percentage of multidrug resistance was 57.14 %, being all multidrug-resistant strains isolated from pigs (92.30 %). The results from the present study indicate a high frequency of antimicrobial resistance among pathogenic E. coli strains isolated from calves and Salmonella spp. strains isolated from pigs and a high rate of susceptibility to most antimicrobials tested among Salmonella spp. strains isolated from calves. Our study highlights the presence of multidrug-resistant strains of E. coli and Salmonella spp. isolated from food-producing animals in Minas Gerais, Brazil.

Recent advances in Brucella abortus vaccines.

Brucella abortus vaccines play a central role in bovine brucellosis control/eradication programs and have been successfully used worldwide for decades. Strain 19 and RB51 are the approved B. abortus vaccines strains most commonly used to protect cattle against infection and abortion. However, due to some drawbacks shown by these vaccines much effort has been undertaken for the development of new vaccines, safer and more effective, that could also be used in other susceptible species of animals. In this paper, we present a review of the main aspects of the vaccines that have been used in the brucellosis control over the years and the current research advances in the development of new B. abortus vaccines.

Nanoplexes of ZnS quantum dot-poly-l-lysine/iron oxide nanoparticle-carboxymethylcellulose for photocatalytic degradation of dyes and antibacterial activity in wastewater treatment.

The contamination and pollution of wastewater with a wide diversity of chemical, microbiological, and hazardous substances is a field of raising environmental concern. In this study, we developed, for the first time, new hybrid multifunctional nanoplexes composed of ZnS semiconductor quantum dots (ZnS QDs) chemically biofunctionalized with epsilon-poly-l-lysine (ɛPL) and coupled with magnetic iron oxide nanoparticles (MION, Fe3O4) stabilized by carboxymethylcellulose (CMC) for the photodegradation (ZnS) of organic molecules and antibacterial activity (ɛPL) with a potential of recovery by an external magnetic field (Fe3O4). These nanosystems, which were synthesized entirely through a green aqueous process, were comprehensively characterized regarding their physicochemical properties combined with spectroscopic and morphological features. The results demonstrated that supramolecular colloidal nanoplexes were formed owing to the strong cationic/anionic electrostatic interactions between the biomacromolecule capping ligands of the two nanoconjugates (i.e., polypeptide in ZnS@ɛPL and polysaccharide in Fe3O4@CMC). Moreover, these nanosystems showed photocatalytic degradation of methylene blue (MB) used as a model dye pollutant in water. Besides MB, methyl orange, congo red, and rhodamine dyes were also tested for selectivity investigation of the photodegradation by the nanoplexes. The antibacterial activity ascribed to the ɛPL biomolecule was confirmed against Gram-positive and Gram-negative bacteria, including drug-resistance field strains. Hence, it is envisioned that these novel green nanoplexes offer a new avenue of alternatives to be employed for reducing organic pollutants and inactivating pathogenic bacteria in water and wastewater treatment, benefiting from easy magnetic recovery.

Bio-functionalized nanocolloids of ZnS quantum dot/amine-rich polypeptides for bioimaging cancer cells with antibacterial activity: "seeing is believing".

Among almost 200 types of cancers, glioma is considered one of the most common forms of malignant tumors located in the central nervous system (CNS). Glioblastoma (GBM), one of the deadliest types of brain cancer, remains one of the challenges faced by oncologists. Thus, smartly designed nanomaterials biofunctionalized with polypeptides can offer disruptive strategies relying on the earliest possible diagnosis ("seeing is believing") combined with more efficient therapies for fighting cancer cells. To worsen this scenario, bacteria infections very often pose a serious challenge to cancer-immunodeficient patients under chemotherapy. Thus, in this research, we report for the first time the design and synthesis of novel nanoconjugates composed of photoluminescent ZnS quantum dots (ZnS QDs), which were directly surface biofunctionalized with epsilon-poly-l-lysine (εPL), acting as an amine-rich cell-penetrating peptide (CPP) and antimicrobial peptide agent (AMP). These nanoconjugates (named ZnS@CPP-AMP) were produced through a one-step facile, eco-friendly, and biocompatible colloidal aqueous process to be applied as a proof of concept as nanoprobes for bioimaging GBM cancer cells (U87-MG) associated with synergic antibacterial activity. They were characterized regarding their physicochemical and optical properties associated with the biological activity. The results demonstrated that chemically stable aqueous colloidal nanoconjugates were effectively formed, resembling core-shell (inorganic, ZnS, organic, εPL) nanostructures with positively surface-charged features due to the cationic nature of the amine-rich polypeptide. More importantly, they demonstrated photoluminescent activity, cytocompatibility in vitro, and no significant intracellular reactive oxygen species (ROS) generation. These ZnS@CPP-AMP nanocolloids behaved as fluorescent nanoprobes for bioimaging GBM cancer cells, where the polycationic nature of the εPL biomolecule may have enhanced the cellular uptake. Additionally, they displayed mild antibacterial growth inhibition due to electrostatic interactions with bacterial membranes. Thus, it can be envisioned that these novel photoluminescent colloidal nanoconjugates offer novel nanoplatforms that can be specifically targeted with biomolecules for bioimaging to diagnose highly lethal cancers, such as GBM, and as an adjuvant in antibacterial therapy.

Comparison of Brucella abortus population structure based on genotyping methods with different levels of resolution.

Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission dynamics of brucellosis worldwide. We aimed to compare the population structure of the genomes of 53 Brazilian Brucella abortus isolates using eight different genotyping methods: multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphism (SNP) detection (parSNP and NASP) from whole genomes. The strains were isolated from six different Brazilian states between 1977 and 2008 and had previously been analyzed using MLVA8, MLVA11, and MLVA16. Their whole genomes were sequenced, assembled, and subjected to MLST9 MLST21, cgMLST, and SNP analyses. All the genotypes were compared by hierarchical grouping method based on the average distances between the correlation matrices of each technique. MLST9 and MLST21 had the lowest level of resolution, both revealing only four genotypes. MLVA8, MLVA11, and MLVA16 had progressively increasing levels of resolution as more loci were analyzed, identifying 6, 16, and 44 genotypes, respectively. cgMLST showed the highest level of resolution, identifying 45 genotypes, followed by the SNP-based methods, both of which had 44 genotypes. In the assessed population, MLVA was more discriminatory than MLST and was easier and cheaper to perform. SNP techniques and cgMLST provided the highest levels of resolution and the results from the two methods were in close agreement. In conclusion, the choice of genotyping technique can strongly affect one's ability to make meaningful epidemiological conclusions but is dependent on available resources: while the VNTR based techniques are more indicated to high prevalence scenarios, the WGS methods are the ones with the best discriminative power and therefore recommended for outbreaks investigation.

Nanosilver-Functionalized Hybrid Hydrogels of Carboxymethyl Cellulose/Poly(Vinyl Alcohol) with Antibacterial Activity for Prevention and Therapy of Infections of Diabetic Chronic Wounds.

Diabetic foot ulcers (DFUs) are considered one of the most severe chronic complications of diabetes and can lead to amputation in severe cases. In addition, bacterial infections in diabetic chronic wounds aggravate this scenario by threatening human health. Wound dressings made of polymer matrices with embedded metal nanoparticles can inhibit microorganism growth and promote wound healing, although the current clinical treatments for diabetic chronic wounds remain unsatisfactory. In this view, this research reports the synthesis and characterization of innovative hybrid hydrogels made of carboxymethyl cellulose (CMC) and poly(vinyl alcohol) (PVA) chemically crosslinked by citric acid (CA) functionalized with silver nanoparticles (AgNPs) generated in situ using an eco-friendly aqueous process. The results assessed through comprehensive in vitro and in vivo assays demonstrated that these hybrid polymer hydrogels functionalized with AgNPs possess physicochemical properties, cytocompatibility, hemocompatibility, bioadhesion, antibacterial activity, and biocompatibility suitable for wound dressings to support chronic wound healing process as well as preventing and treating bacterial infections. Hence, it can be envisioned that, with further research and development, these polymer-based hybrid nanoplatforms hold great potential as an important tool for creating a new generation of smart dressings for treating chronic diabetic wounds and opportunistic bacterial infections.

Identification of virulence factors by multiplex PCR in Escherichia coli isolated from calves in Minas Gerais, Brazil.

In this study, multiplex PCR was employed to investigate the virulence factors of Escherichia coli strains isolated from 60-day-old calves. Faecal samples were collected from 54 calves at 12 dairy farms in the state of Minas Gerais, Brazil. A total of 156 isolates were obtained after culture and microbiological isolation and were tested by multiplex PCR for the presence of genes encoding toxins (Stx1, Stx2 and STa) and adherence factors (intimin, F41 and F5). Seventy of 156 isolates were positive for at least one virulence factor: ten (14.3 %) from diarrhoeic animals and 60 (85.7 %) from healthy calves. The virulence markers identified were: Stx1 (82.8 %), eae (24.3 %), F41 (11.4 %), F5 (10 %), STa (4.28 %) and Stx2 (4 %). In diarrhoeic animals, Stx1 (70 %) and F41 (30 %) were identified, while Stx1 (83.3 %), eae (28.3 %), F41 (8.3 %), F5 (11.6 %), STa (5 %) and Stx2 (1.6 %) were detected in isolates from healthy calves. Mixed infections with pathotypes Shiga toxin-producing E. coli (STEC)/enteropathogenic E. coli, STEC/enterohaemorrhagic E. coli and STEC/other (eae/F5, Stx1/STa) were detected in five healthy calves. Pathogenic E. coli were identified in 59.26 % of all calves and on 75 % of the dairy farms studied, not only in diarrhoeic (five of six) but also in healthy calves (27 of 48), which demonstrates the importance of this agent in the aetiology of diarrhoea in calves in the state of Minas Gerais.

Exploring the MALDI Biotyper for the Identification of Corynebacterium pseudotuberculosis biovar Ovis and Equi.

Biochemical, serological, and molecular methods have been developed for the laboratory diagnosis of diseases caused by C. pseudotuberculosis (CP), but the identification of the pathogen and biovars differentiation may be time-consuming, expensive, and confusing compared with other bacteria. This study aimed to evaluate MALDI Biotyper and Overall Genome Relatedness Index (OGRI) analysis to optimize the identification and differentiation of biovars of C. pseudotuberculosis. Out of 230 strains isolated from several hosts and countries, 202 (87.8%) were precisely classified using MALDI Biotyper and the BioNumerics platform. The classification accuracies for the Ovis and Equi biovars were 80 (88.75%) and 82 (92.68%), respectively. When analyzing a sampling of these strains by Average Nucleotide Identity based on BLAST and TETRA analyses using genomic sequence data, it was possible to differentiate 100% of the strains in Equi and Ovis. Our data show that MALDI Biotyper and OGRI analysis help identify C. pseudotuberculosis at the species and biovar levels.

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil.

BACKGROUND: Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable economic losses for herd owners. RESULTS: We assessed the seroprevalence of infection with C. pseudotuberculosis in 805 sheep from 23 sheep farms that supply slaughterhouses in the state of Minas Gerais; we also analyzed management practices that could be associated with CLA occurrence, used on these and nearby farms that also supplied animals to the slaughterhouse (n = 60). The serum samples for assaying CLA infection were taken at the slaughterhouse. Frequency of infection with C. pseudotuberculosis was estimated at 43.7%, and farm frequency was estimated at 100%. Management practices were analyzed through a questionnaire. All farmers (60/60) had extensive/semi-extensive rearing system; 70.0% (42/60) identified sheep individually; 11.7% (7/60) had periodical technical assistance; 41.7% (25/60) disinfected the facilities; 86.7% (52/60) used barbed wire fences and did not implement adequate CLA control measures; only 11.7% (7/60) of breeders reported vaccination against C. pseudotuberculosis; 13.3% (8/60) took note of animals with clinical signs of CLA; 1.7% (1/60) opened and sanitized abscesses, and isolated the infected animals; 10.0% (6/60) knew the zoonotic potential of this disease and 1.7% (1/60) of the farmers culled animals in case of recurrence of abscesses. CONCLUSIONS: It can be concluded that C. pseudotuberculosis infection is widely spread in sheep flocks in Minas Gerais state in Brazil and that there is a lack of good management measures and vaccination, allowing transmission of this infectious agent throughout the production network.

3D sponges of chemically functionalized chitosan for potential environmental pollution remediation: biosorbents for anionic dye adsorption and 'antibiotic-free' antibacterial activity.

In this work, it was developed three-dimensional (3D) porous hydrogel sponges produced by the freeze-dried process using chitosan polymer functionalized by 11-mercaptoundecanoic acid (MUA). These chitosan-based sponges were used as cationic adsorbents for the removal of anionic methyl orange (MO) dye, simulating a model organic pollutant in aqueous medium. Moreover, these porous 3D constructs were also evaluated as 'antibiotic-free' antibacterial materials against gram-negative and gram-positive bacteria, Pseudomonas aeruginosa and Staphylococcus aureus, respectively, which were used as model pathogens possibly found in contaminated hospital discharges. These 3D hydrogels were comprehensively characterized through morphological methods such as scanning electron microscopy and X-ray micro-computed tomography techniques, combined with FTIR, Raman, and UV-visible spectroscopy analyses. Additionally, the surface area, the degree of swelling, and the adsorption profiles and kinetics of these scaffolds were systematically investigated. The chemically thiolated chitosan (CHI-MUA) hydrogels were successfully produced with a supramolecular polymeric network based on hydrogen bonds, disulfide bonds, and hydrophobic interactions that resulted in higher stability in aqueous medium than hydrogels of pristine chitosan. CHI-MUA exhibited sponge-like three-dimensional structures, with highly interconnected and hierarchical pore size distribution with high porosity and surface area. These architectural aspects of the 3D sponges favoured the high adsorption capacity for MO dye (∼388 mg.g-1) in water with removal efficiency greater than 90% for MO solutions (from 20 mg.L-1-1200 mg.L-1). The adsorption data followed a pseudo-second-order kinetic model and adsorption isotherm analysis and spectroscopy studies suggested a multilayer behaviour with coexistence of adsorbent-adsorbate and adsorbate-adsorbate interactions. Additionally, the in vitro evaluation of toxicity (MTT and LIVE-DEAD® assays) of 3D-sponges revealed a non-toxic response and preliminary suitability for bio-related applications. Importantly, the 3D-sponges composed of chitosan-thiolated derivative proved high antibacterial activity, specificity against P. aeruginosa (model hazardous pathogen), equivalent to conventional antibiotic drugs, while no lethality against S. aureus (reference commensal bacteria) was observed.

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.

Effects of vaccination against brucellosis and clostridia on the intake, performance, feeding behavior, blood parameters, and immune responses of dairy heifers calves.

The aim of this study was to identify possible effects of different vaccination strategies (concomitantly or not) against brucellosis and clostridia on intake, performance, feeding behavior, blood parameters, and immune responses of dairy heifers calves. Fifty heifers calves were enrolled [38 Gyr (Zebu, Bos taurus indicus) and 12 5/8 Holstein × Gyr]. At 120 d of age, animals were randomly distributed among 3 groups: B (n = 18), vaccinated against brucellosis; C (n = 14), vaccinated against clostridia and CB (n = 18), vaccinated concomitantly for both. Rectal and thermographic temperatures were evaluated on days -1, 0, 1, 2, 3, 5, 7,10, 14, and 28 relatives to the vaccination day. Feed and water intake, body weight (BW), and feeding behavior were monitored daily by an electronic feeding system. Blood was sampled on days 0, 3, 7, 14, and 28, relative to the vaccination day for determination of glucose and ß -hydroxybutyrate (BHBA) concentrations. Blood sampled on day 0 (prevaccination) and on days 28 and 42 were used to evaluate the immune response against Brucella abortus and clostridia. There was an increase in rectal temperature between the first and the third day postvaccination in the 3 groups. The thermography revealed an increase of local temperature for 7 d on groups B and CB. Group C had increased local temperature for a longer period, lasting for up to 14 d. Dry mater intake was reduced for groups B and CB, but no alteration was observed for group C. No alterations regarding initial BW, final BW, average daily weight gain, and feed efficiency were observed. No differences were observed for the 3 vaccination groups for blood parameters throughout the evaluation period. The concomitant vaccination against brucellosis and clostridia led to lower neutralizing antibody titers against epsilon toxin of Clostridium perfringens and botulinum toxin type C of C. botulinum (C > CB > B). When cellular proliferation assay and serological tests to B. abortus were evaluated, no differences were observed between groups B and CB. The present results indicate that the concomitant vaccination against brucellosis and clostridia has no relevant impact on the intake, performance, and feeding behavior of dairy calves. However, the concomitant vaccination of vaccines against these 2 pathogens impacts animal immunity against clostridial infections.

Virulence factors and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis in Brazil.

This study aimed to evaluate virulence factors and genetic markers of antimicrobial resistance in 400 Staphylococcus aureus strains isolated from bovine mastitis in four Brazilian states, as well as to assess the association between these characteristics and field information. Virulence factors and drug resistance genes were identified by PCR screening. Biofilm-forming and hemolytic phenotype were detected using Congo red Tryptic Soy Broth and defibrinated sheep blood agar, respectively. Of all isolates, 83.5% were biofilm-forming and 98.5% strains exhibited biofilm gene icaAD, and a significant association between phenotype and genotype for biofilm was observed (P = 0.0005). Hemolysin genes were observed in 82.85% (hla+hlb+), 16.5% (hla+) and 0.75% (hlb+) isolates, whereas the hemolytic phenotype exhibited was complete and incomplete hemolysis in 64.25%, complete in 28.25%, incomplete in 4.75%, and negative in 2.75% of the strains. Virulence factors genes luk, seb, sec, sed, and tst were observed in 3.5%, 0.5%, 1%, 0.25%, and 0.74% isolates, respectively. The gene blaZ was detected in 82.03% of penicillin-resistant isolates, whereas tetK and aac(6')-Ie-aph(2')-Ia were observed in 33.87% and 45.15% of the tetracycline and aminoglycosides-resistant isolates, respectively. Fluoroquinolone resistance gene mepA was detected for the first time in S. aureus from bovine mastitis. Resistance genes tetM (3.22%), tetL (1.61%), ermA (14.29%), ermB (14.29%), ermC (33.3%), ermT (9.52%), ermY (4.76%), msrA (9.52%), and mphC (9.52%) were also detected among resistant isolates. No association between virulence factors or antimicrobial-resistant genes and year of isolation, geographic origin, or antimicrobial resistance profile was observed. Our results showed that S. aureus strains isolated from bovine mastitis in the four Brazilian states sampled are mainly biofilm-forming and hemolytic, whereas virulence genes associated with enterotoxins, luk and tst, were less frequently observed. Moreover, a wide variety of resistance genes that confer resistance to almost all classes of antimicrobial agents approved for use in animals and humans were found. Overall, the data point to a great pathogenic potential of S. aureus associated with bovine mastitis and to the non-negligible risks to public health of staphylococcal infections from animal origin.

Genetic diversity and antimicrobial resistance in Staphylococcus aureus and coagulase-negative Staphylococcus isolates from bovine mastitis in Minas Gerais, Brazil.

The aims of this study were to determine the antimicrobial susceptibility profile and genetic diversity of Staphylococcus spp. isolated from dairy cows in Minas Gerais, Brazil, and to assess the relationship among the isolates' susceptibility profiles and pulsed-field gel electrophoresis (PFGE) genotypes. Seventy-nine isolates were used, including S. aureus (n = 71) and coagulase-negative staphylococci (CoNS) (n = 8). Susceptibility to 12 antimicrobial agents was performed. All Staphylococcus spp. were subjected to PFGE. Staphylococcus aureus and CoNS isolates exhibited full susceptibility only to cephalothin. The greatest percentages of resistance among Staphylococcus spp. were observed to penicillins, folate pathway inhibitors, and tetracyclines. Twelve S. aureus and four CoNS were classified as multidrug resistance strains. Percentage of MRSA was also higher among CoNS (75%), compared to S. aureus isolates (2.81%). Adopting 100% of similarity, 34 different genotypes were identified. Association of minimum-spanning tree (MST) analysis with data from municipalities, herds, methicillin-resistant S. aureus (MRSA), and resistance patterns for all isolates did not show any clustering. However, a clustering pattern of bacterial species was observed. Results from this study indicate a high frequency of antimicrobial resistance, especially among CoNS, and a high genetic diversity among Staphylococcus spp. isolated from dairy cows with mastitis in Minas Gerais, Brazil.

Modulation of the bovine trophoblastic innate immune response by Brucella abortus.

Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between Brucella abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella-induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B. abortus. Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real-time reverse transcription-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression in placentomes from experimentally infected cows was evaluated. Expression of proinflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant upregulation of CXC chemokines, namely, CXCL6 (GCP-2) and CXCL8 (interleukin 8), was observed at 12 but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing the expression of proinflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of proinflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus-induced placentitis.

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.

Viability of Campylobacter spp. in frozen and chilled broiler carcasses according to real-time PCR with propidium monoazide pretreatment.

The aim of this study was to evaluate the viability of Campylobacter spp. in frozen and chilled broiler carcasses using real-time PCR with propidium monoazide (PMA) pretreatment. Sixty broiler carcasses were collected: 30 frozen and 30 chilled. Each carcass was submitted to 2 real-time PCR protocols to detect and quantify Campylobacter spp.: one using pretreatment with PMA, which blocks the amplification of DNA from dead bacteria, and the other without PMA. The results showed that PMA-pretreated carcasses, either frozen or chilled, had a lower positivity rate compared to untreated samples (P < 0.001). Regarding storage temperatures, PMA-pretreated frozen carcasses that tested positive were in a lesser number than chilled carcasses (P < 0.05). However, the quantification of total and live bacteria in PMA-pretreated frozen carcasses that tested positive showed no significant difference compared to chilled carcasses. It was concluded that the real-time PCR with PMA pretreatment was a sensitive method for evaluating the viability of Campylobacter spp. in broiler carcasses. Chilled broiler carcasses would represent greater hazard to public health concerning Campylobacter transmission.

Molecular epidemiology of Corynebacterium pseudotuberculosis isolated from horses in California.

Corynebacterium pseudotuberculosis biovar Equi is an important pathogen of horses. It is increasing in frequency in the United States, and is responsible for various clinical forms of infection, including external abscesses, internal abscesses of the abdominal or thoracic cavities, and ulcerative lymphangitis. The host/pathogen factors dictating the form or severity of infection are currently unknown. Our recent investigations have shown that genotyping C. pseudotuberculosis isolates using enterobacterial repetitive intergenic consensus (ERIC)-PCR is useful for understanding the evolutionary genetics of the species as well for molecular epidemiology studies. The aims of the present study were to assess (i) the genetic diversity of C. pseudotuberculosis strains isolated from horses in California, United States and (ii) the epidemiologic relationships among isolates. One hundred and seven C. pseudotuberculosis biovar Equi isolates from ninety-five horses, and two C. pseudotuberculosis biovar Ovis strains, C. pseudotuberculosis ATCC 19410T type strain and C. pseudotuberculosis 1002 vaccine strain, were fingerprinted using the ERIC 1+2-PCR. C. pseudotuberculosis isolated from horses showed a high genetic diversity, clustering in twenty-seven genotypes with a diversity index of 0.91. Minimal spanning tree showed four major clonal complexes with a pattern of temporal clustering. Strains isolated from the same horse showed identical ERIC 1+2-PCR genotype, with the exception of two strains isolated from the same animal that showed distinct genotypes, suggesting a co-infection. We found no strong genetic signals related to clinical form (including internal versus external infections). However, temporal clustering of genotypes was observed.