A novel phage-displayed MilA ELISA for detection of antibodies against Myc. bovis in bovine milk.

AIMS: The aim of this study was to assess a phage-displayed MilA protein of Myc. bovis in an indirect ELISA for the detection of Myc. bovis antibodies in milk samples. METHODS AND RESULTS: The desired sequence of milA gene was synthesized and cloned into pCANTAB-F12 phagemid vector. The expression of the MilA on the phage surface was confirmed by Western blotting. The recombinant phage was used in the development of an indirect ELISA to detect Myc. bovis antibodies in milk samples. There was a significant agreement between the results of phage-based ELISA and recombinant GST-MilA ELISA for the detection of Myc. bovis antibodies in milk samples. CONCLUSIONS: The inexpensive and convenient phage-based ELISA can be used instead of recombinant protein/peptide ELISA as an initial screening of Myc. bovis-associated mastitis. SIGNIFICANCE AND IMPACT OF STUDY: Mastitis associated with Myc. bovis is a continuous and serious problem in the dairy industry. Sero-monitoring of Myc. bovis infection cases are one of the key factors for surveillance of the infections in dairy farms. Despite the existence of some commercially serological assays for Myc. bovis antibodies, they have some limitations regarding their sensitivity and availability. The development of accurate diagnosis tools could contribute to control programmes of Myc. bovis-associated mastitis in the dairy herds.

Cytotoxicity, apoptosis, and IL-8 gene expression induced by some foodborne pathogens in presence of Bacillus coagulans in HT-29 cells.

Food poisoning caused by bacteria is one of the most important concerns in food hygiene. The use of probiotics in prevention, control, and treatment of these infections has been considerably increased in recent years. This study evaluated the effect of B. coagulans cell free supernatant (CFS) on growth of Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, non-pathogenic Escherichia coli, and Escherichia coli 0157:H7 by the broth dilution method. The cytotoxicity, and apoptosis induced by pathogens alone and in co-culture with B. coagulans or its CFS were measured by trypan blue, and fluorescence staining methods. The expression level of interleukin-8 (IL-8) cytokine-encoding genes was also investigated by a qRT-PCR assay in all pathogens and co-cultured groups in HT-29 cells. Our results showed that 4% B. coagulans CFS reduced pathogen growth. The highest rate of growth inhibition was observed in L. monocytogenes. We also found that B. coagulans, and its 4% CFS reduced the cytotoxic effects of pathogens, with the exception of S. aureus. Non-pathogenic E. coli also had no significant cytotoxic effect on the cells. Examination of the treated cells with acridine orange/ethidium bromide staining showed reductions in the rate of cell damage (including early apoptosis, late apoptosis, and necrosis) in pathogen-probiotic co-cultures. Furthermore, we showed that co-culture of pathogens with B. coagulans significantly down-regulated IL-8 gene expression (P < 0.05). The greatest down-regulation compared with pathogen alone was observed in S. aureus. Hence, B. coagulans can be considered as an appropriate probiotic to diminish cytotoxicity, and inflammatory response of enteropathogenic bacteria.

Genetic variants within Ninjurin 2 gene are associated with risk of ischemic stroke in Iranian population.

Previous genetic and epidemiological studies have shown the contribution of genetic factors in conferring the risk of ischemic stroke. Among the acknowledged risk factors of stroke are the single nucleotide polymorphisms (SNPs) near Ninjurin 2 (NINJ2) gene which encodes a surface adhesion protein. In the current study, we investigated the role of two SNPs near this gene in ischemic stroke in Iranian population. The frequency of the A allele of the rs11833579 was significantly lower in cases compared with controls (OR (95% CI) = 0.68 (0.54-0.86), adjusted P value = 0.002). The rs11833579 was significantly associated with risk of stroke in co-dominant (AA vs. GG: OR (95% CI) = 0.39 (0.23-0.66), adjusted P value = 0.003) and recessive (OR (95% CI) = 0.44 (0.27-0.72), adjusted P value = 0.001) models. The rs3809263 was associated with risk of stroke in dominant model (OR (95% CI) = 1.5 (1.09-2.06), adjusted P value = 0.02). The A C haplotype (rs11833579 and rs3809263) decreased the risk of stroke (OR (95% CI) = 0.72 (0.57-0.91), adjusted P value = 0.03), while the G T haplotype conferred susceptibility to stroke (OR (95% CI) = 1.42 (1.11-1.82), adjusted P value = 0.02). Consequently, the present case-control study supports the role of NINJ2 as a risk locus for ischemic stroke in Iranian population.

The effects of probiotic Bacillus coagulans on the cytotoxicity and expression of alpha toxin gene of Clostridium perfringens type A.

Around the world, Clostridium perfringens type A is known to be a common foodborne pathogen. Therefore, the control and treatment of food poisoning caused by this pathogen are important. This study investigated, in vitro, the effects of Bacillus coagulans and its culture extracts on alpha toxin gene expression, growth inhibition, cytotoxicity, and apoptosis induced by C. perfringens spore, germinated spore and its enterotoxin. Flow cytometry was used to evaluate the apoptosis rate, and MTT test was used to evaluate cytotoxicity. Minimum inhibitory concentration was also used to measure the percentage of inhibition in the broth medium. Finally, RT-qPCR was used to evaluate alpha toxin gene expression. The results showed that the B. coagulans culture extract was able to inhibit the growth of the germinated spore of C. perfringens. Moreover, treating the extract with pepsin can reduce growth in the broth medium. MTT and flow cytometry showed that both B. coagulans and its extract can significantly reduce the cytotoxicity and apoptosis rate induced by C. perfringens type A. In addition, it was shown that the co-culture of B. coagulans and C. perfringens decreases alpha toxin gene expression. The findings of this study indicate that B. coagulans, with growth inhibition and reduced expression of alpha toxin in C. perfringens, can reduce the cytotoxicity and apoptosis rate induced on HT-29 cells.

The effect of Bacillus coagulans on cytotoxicity and apoptosis induced by Salmonella Typhimurium in HT-29 cell culture.

BACKGROUND AND OBJECTIVES: Human epithelial cells have been widely used to study the interaction between intestinal cells and pathogens, in vitro. In this study, the effect of probiotic bacteria Bacillus coagulans and its supernatant on the growth inhibition, cytotoxicity and induction of apoptosis caused by Salmonella Typhimurium and its adhesion to HT-29 cells were investigated. MATERIALS AND METHODS: B. coagulans supernatant was used to obtain the minimum inhibitory concentration. To evaluate the cytotoxicity and percent of apoptotic cells, B. coagulans and its supernatant (2, 4, 6 and 8% concentrations) with S. Typhimurium was added to HT-29 cells. The MTT assay was used in order to evaluate the cytotoxicity. Percent of apoptotic cells was reported using a fluorescence staining method. Additionally, the adhesion of S. Typhimurium to HT-29 cells was investigated. The effect of B. coagulans on the level of adhesion was also studied. RESULTS: The most inhibitory effect was shown at the concentration of 80000 µg/ml supernatant of B. coagulans (54.77% ± 1.43). The simultaneous culture of S. Typhimurium with B. coagulans had the lowest amount of cytotoxicity and induced apoptosis among the all co-culture groups of S. Typhimurium with B. coagulans or its supernatant. The determined cytotoxicity and induced apoptosis were 26.06 % ± 3.79 and 17.63 % ± 2.14 respectively. In the adhesion test, it was observed that B. coagulans can significantly prevent adhesion of S. Typhimurium to HT-29 cell. CONCLUSION: B. coagulans can reduce the adhesion, cytotoxicity and induction of apoptosis caused by S. Typhimurium in HT-29 cells in vitro.