Prototheca zopfii genotype II induces mitochondrial apoptosis in models of bovine mastitis.

Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of P. zopfii (genotype I and II (GT-I and -II)), P. zopfii GT-II is the genotype associated with acute mastitis and decreased milk production, although its pathogenesis is not well known. The objective was to determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance (ΔΨm) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model, P. zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-α, IL-1ß and Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis.

Virulence-Related Genes and Coenteropathogens Associated with Clinical Outcomes of Enteropathogenic Escherichia coli Infections in Children from the Brazilian Semiarid Region: a Case-Control Study of Diarrhea.

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in children from developing countries and presents high genetic variability. We aimed to characterize the EPEC virulence-related gene (VRG) distribution and copathogens associated with diarrhea and nutrition-related outcomes in children from the low-income Brazilian semiarid region. A cross-sectional case-control study of diarrhea was conducted in 1,191 children aged 2 to 36 months from the northeast region of Brazil. Stool samples were collected and clinical, epidemiological, and anthropometric data were identified from each child. A broad molecular evaluation of enteropathogens was performed, and EPEC-positive samples were further investigated for 18 VRGs using five multiplex PCRs. EPEC was detected in 28.2% of the study population, with similar proportions among cases and controls. Typical EPEC (tEPEC) infections were more often associated with diarrhea than atypical EPEC (aEPEC) infections, while aEPEC infections presented a higher prevalence. The VRG ler, a negative regulator of the locus of enterocyte effacement, was associated with the absence of diarrhea in aEPEC-positive children; espB, a major component of the type 3 secretion system, was associated with diarrhea in tEPEC-positive children; the presence of procolonization VRGs-the combination of cesT positivity, espP negativity, and the presence of the map gene-was associated with undernutrition; and Campylobacter spp., norovirus, and enteroaggregative E. coli (EAEC) coinfections were associated with increased clinical severity in EPEC-infected children. These data identified tEPEC strains associated with diarrhea and specific VRGs of EPEC (ler, espB, cesT, and map genes) and Campylobacter spp., norovirus, and EAEC to be major contributors to diarrhea and undernutrition in children from a low-income Brazilian region.

Alanyl-glutamine Protects Against Damage Induced by Enteroaggregative Escherichia coli Strains in Intestinal Cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an important pathogen causing enteric infections worldwide. This pathotype is linked to malnutrition in children from developing countries. Alanyl-glutamine (Ala-Gln) is an immune modulator nutrient that acts during intestinal damage and/or inflammation. This study investigated the effect of EAEC infection and Ala-Gln on cell viability, cell death, and inflammation of intestinal epithelium cells (IEC-6). METHODS: Cells were infected with an EAEC prototype 042 strain, an EAEC wild-type strain isolated from a Brazilian malnourished child, and a commensal E coli HS. Gene transcription and protein levels of caspases-3, -8, and -9 and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL1) were evaluated using RT-qPCR, western blot analysis, and ELISA. RESULTS: Infections with both EAEC strains decreased cell viability and induced apoptosis and necrosis after 24 hours. Ala-Gln supplementation increased cell proliferation and reduced cell death in infected cells. Likewise, EAEC strain 042 significantly increased the transcript levels of caspases-3, -8, and -9 when compared to the control group, and Ala-Gln treatment reversed this effect. Furthermore, EAEC induced CXCL1 protein levels, which were also reduced by Ala-Gln supplementation. CONCLUSION: These findings suggest that EAEC infection promotes apoptosis, necrosis, and intestinal inflammation with involvement of caspases. Supplementation of Ala-Gln inhibits cell death, increases cell proliferation, attenuates mediators associated with cell death, and inflammatory pathways in infected cells.

Determinant Variables, Enteric Pathogen Burden, Gut Function and Immune-related Inflammatory Biomarkers Associated With Childhood Malnutrition: A Prospective Case-Control Study in Northeastern Brazil.

Malnutrition results in serious consequences for growth and cognitive development in children. We studied select child and maternal biologic factors, socioeconomic factors, enteric pathogenic burden and gut function biomarkers in 402 children 6-24 months of age in Northeastern Brazil. In this prospective case-control study, not being fed colostrum [odds ratio (OR): 3.29, 95% confidence interval (CI): 1.73-6.26], maternal age ≥18 years (OR: 1.88, 95% CI: 1.10-3.22) and no electric fan (OR: 2.46, 95% CI: 1.22-4.96) or bicycle (OR: 1.80, 95% CI: 1.10-2.95) in the household were positively associated, and higher birth weight (OR: 0.27, 95% CI: 0.19-0.38), larger head circumference (OR: 0.74, 95% CI: 0.66-0.82) and shortness of breath in the last 2 weeks (OR: 0.49, 95% CI: 0.27-0.90) were negatively associated with malnutrition. Subclinical enteric pathogen infections were common, and enteroaggregative Escherichia coli infections were more prevalent in malnourished children (P = 0.045). Biomarkers such as the lactulose-mannitol test, myeloperoxidase, neopterin and calprotectin were highly elevated in both malnourished and nourished children. Nourished children had a better systemic immune response than the malnourished children, as detected by elevated serum amyloid A-1 and soluble cluster of differentiation protein 14 biomarkers (P < 0.001). Serum amyloid A-1 and soluble cluster of differentiation protein 14 were also associated with better nutritional Z scores. Neonatal, maternal and socioeconomic factors were associated with malnutrition in children. There was a substantial subclinical enteric pathogen burden, particularly with enteroaggregative E. coli, in malnourished children.

Biomarkers of Environmental Enteropathy, Inflammation, Stunting, and Impaired Growth in Children in Northeast Brazil.

Critical to the design and assessment of interventions for enteropathy and its developmental consequences in children living in impoverished conditions are non-invasive biomarkers that can detect intestinal damage and predict its effects on growth and development. We therefore assessed fecal, urinary and systemic biomarkers of enteropathy and growth predictors in 375 6-26 month-old children with varying degrees of malnutrition (stunting or wasting) in Northeast Brazil. 301 of these children returned for followup anthropometry after 2-6m. Biomarkers that correlated with stunting included plasma IgA anti-LPS and anti-FliC, zonulin (if >12m old), and intestinal FABP (I-FABP, suggesting prior barrier disruption); and with citrulline, tryptophan and with lower serum amyloid A (SAA) (suggesting impaired defenses). In contrast, subsequent growth was predicted in those with higher fecal MPO or A1AT and also by higher L/M, plasma LPS, I-FABP and SAA (showing intestinal barrier disruption and inflammation). Better growth was predicted in girls with higher plasma citrulline and in boys with higher plasma tryptophan. Interactions were also seen with fecal MPO and neopterin in predicting subsequent growth impairment. Biomarkers clustered into markers of 1) functional intestinal barrier disruption and translocation, 2) structural intestinal barrier disruption and inflammation and 3) systemic inflammation. Principle components pathway analyses also showed that L/M with %L, I-FABP and MPO associate with impaired growth, while also (like MPO) associating with a systemic inflammation cluster of kynurenine, LBP, sCD14, SAA and K/T. Systemic evidence of LPS translocation associated with stunting, while markers of barrier disruption or repair (A1AT and Reg1 with low zonulin) associated with fecal MPO and neopterin. We conclude that key noninvasive biomarkers of intestinal barrier disruption, LPS translocation and of intestinal and systemic inflammation can help elucidate how we recognize, understand, and assess effective interventions for enteropathy and its growth and developmental consequences in children in impoverished settings.

Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. METHODS: Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 µg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). RESULTS: Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. CONCLUSIONS: These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

Campylobacter jejuni infection and virulence-associated genes in children with moderate to severe diarrhoea admitted to emergency rooms in northeastern Brazil.

Campylobacter is an important cause of foodborne gastroenteritis. We determined the occurrence of Campylobacter jejuni and Campylobacter coli, using culture-based methods and PCRs targeting virulence-associated genes (VAGs) among children aged ≤14 years who were treated for diarrhoea at emergency rooms in northeastern Brazil. Genomic DNA was extracted directly from stool samples collected from 366 children. A questionnaire was also applied to qualify the clinical conditions presented by each child at the time of admission. C. jejuni and C. coli were detected in 16.4 % (60/366) and 1.4 % (5/366) of the diarrhoeal samples, respectively, by PCR, a much higher proportion than that detected by conventional methods. C. jejuni VAGs were detected in the following proportions of hipO-positive samples: ciaB, 95 % (57/60); dnaJ, 86.7 % (52/60); racR, 98.3 % (59/60); flaA, 80 % (48/60); pldA, 45 % (27/60); cdtABC, 95 % (57/60); and pVir 0 % (0/60). Particular symptoms, such as blood in faeces, vomiting, fever, and/or abdominal pain, were not associated with detection of C. jejuni nor were they associated with any particular VAG or combination of VAGs (P>0.05). C. jejuni and its VAGs were detected in a substantial proportion of the children admitted. Further efforts shall be directed towards elucidating whether these genetic factors or their expressed proteins play a role in Campylobacter pathogenesis.