Eosinophilic Inflammation and Equine Herpesvirus-1 Associated With Haemorrhagic Cystitis in a Horse. Case Report.

Equine idiopathic haemorrhagic cystitis (EIHC) is a recently described form of aseptic cystitis in horses in which there is no discernible underlying cause. This case report describes a 9-year-old Thoroughbred gelding that presented with stranguria, pollakiuria, and haematuria. Cystoscopy revealed ulceration and haemorrhage of the bladder mucosa, diffuse mural hyperaemia and marked urine sedimentation. Histopathological evaluation of the bladder revealed chronic active ulcerative neutrophilic, lymphoplasmacytic, and eosinophilic cystitis. There was no bacterial or fungal growth upon culture but polymerase chain reaction (PCR) testing and sequencing for equine herpesvirus-1 (EHV-1) on bladder mucosa was positive. Conservative therapy with broad spectrum antimicrobials and non-steroidal anti-inflammatory therapy yielded complete resolution of clinical signs with significant improvement of macroscopic lesions in 14 days. Although a positive EHV-1 PCR suggests a viral cause, the horse's clinical signs, histology and recovery rate are more consistent with equine idiopathic haemorrhagic cystitis (EIHC). Neutrophilic and lymphoplasmacytic inflammation is a known feature of EIHC but eosinophilic infiltrates have not been previously described. The significance of the eosinophilic involvement is not certain; however, their presence has been associated with fungal, viral, parasitic, and immune-mediated aetiologies in other body systems. This is the first report of a horse with possible EIHC in Australia, as well as the first case with eosinophilic infiltrates and testing positive for EHV-1.

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.

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.

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.