Validation and improvement of a multiplex PCR method to detect murine Helicobacter species in feces samples of mice.

BACKGROUND: Murine Helicobacter species have gained increasing awareness in mouse facilities over the last years. Infections with Helicobacter species may have an impact effect on the health of mice and might pose a zoonotic risk to researchers. To minimize the interference with experiments and hence contribute to the 3Rs, a reliable method of monitoring Helicobacter infections in animal facilities needs to be available. The aim of this study was to improve and validate the detection of the most common murine Helicobacter species. MATERIAL AND METHODS: A multiplex PCR assay was developed for identification of Helicobacter hepaticus, H. bilis, H. muridarum, H. rodentium, and H. typhlonius that could simultaneously detect these five strains in fecal samples. To ensure the quality of the results, the method was validated based on recommendations for in-house developed tests. RESULTS: The method established was highly sensitive and specific. All five strains were detectable with a detection limit of 102 bacteria. Eight different mouse facilities were tested with the validated assay, and the following prevalence were found: H. rodentium 57%, H. hepaticus 46%, H. typhlonius 17%, H. bilis 12%, and H. muridarum 0%. CONCLUSION: The multiplex PCR is a reliable, economic, and time-saving diagnostic tool for routine health monitoring. Further prevalence studies are needed to confirm the high prevalence and hence importance of H. rodentium, as until now this agent is not yet listed in FELASA recommendations.

Avaliação dos efeitos do subgalato de bismuto na proliferação de miofibroblastos / Assessment of the effects of bismuth subgallate on proliferation of myofibroblasts: an experimental study in rats

Resumo Contexto O subgalato de bismuto é um metal pesado e insolúvel, utilizado por suas propriedades adstringentes e hemostáticas. Objetivo Avaliar os efeitos do subgalato de bismuto na cicatrização mediante observação de miofibroblastos em pele de ratos. Métodos Foram utilizados 60 ratos da linhagem Wistar, que receberam uma ferida no dorso da pele. Os animais foram divididos em dois grupos: controle (aplicação diária de cloreto de sódio a 0,9%) e experimental (aplicação diária de 0,5 mg de subgalato de bismuto). Cada grupo foi subdividido em três subgrupos, que foram reoperados para retirada da ferida em 3, 7 e 14 dias. Foi realizada coloração de hematoxilina eosina, picrosirius e imuno-histoquímica para avaliar contagem de miofibroblastos, resposta inflamatória e síntese de colágeno. Resultados Não foi encontrada diferença entre os grupos controle e experimento com relação ao processo inflamatório – subgrupos 3 dias (p = 1), 7 dias (p = 0,474) e 14 dias (p = 303). A avaliação dos colágenos tipo I e III no grupo-controle não demonstrou benefícios de cicatrização – 3 dias (p = 0,436), 7 dias (p = 0,853) e 14 dias (p = 0,436); já no grupo experimental, houve aumento dos colágenos tipos I e III nos subgrupos 3 e 14 dias (p = 0,005). A imuno-histoquímica confirmou os resultados encontrados na coloração hematoxilina eosina, na qual a área de miofibroblastos entre os subgrupos, nos grupos experimental (p = 0,4) e controle (p = 0,336), foi indiferente. Conclusão A utilização do subgalato de bismuto em ferida de pele de ratos não evidenciou benefícios na cicatrização, ou seja, não houve diferença na fibroplasia quando comparados os grupos experimental e controle.

Abstract Background Bismuth subgallate is an insoluble heavy metal that is used for its astringent and hemostatic properties. Objective To evaluate the effects of bismuth subgallate on the healing process by observation of myofibroblasts in the skin of rats. Methods A sample of 60 Wistar rats was used. Each rat was subjected to a dorsal skin wound and allocated to one of two groups: a control group, in which 0.9% sodium chloride was administered daily, or an experimental group, in which 0.5 mg of bismuth subgallate was administered daily. Each of these groups was further subdivided into three subsets, which were reoperated after 3, 7 and 14 days respectively for excision and collection of the skin wound specimens. Samples were treated with hematoxylin eosin, picrosirius, and immunohistochemical staining to enable assessment of myofibroblast counts, inflammatory response phase, and collagen synthesis. Results No inflammatory process differences were detected between the control and experimental groups at 3 days (p = 1), 7 days (p = 0.474), or 14 days (p = 303). Evaluation of types I and III collagen in the control group did not demonstrate healing benefits at 3 days (p = 0.436), 7 days (p = 0.853), or 14 days (p = 0.436); whereas in the experimental group there were increases in types I and III collagen at 3 and 14 days (p = 0.005). Immunohistochemical analysis confirmed the results of hematoxylin eosin staining, since there were no differences between subsets in terms of area of myofibroblasts, in the experimental (p = 0.4) or the control (p = 0.336) groups. Conclusions Administration of bismuth subgallate to skin wounds in rats did not result in any evidence of benefits to healing, i.e., no difference in fibroplasia was detected when experimental and control groups were compared.

Description of 'Candidatus Helicobacter heilmannii' based on DNA sequence analysis of 16S rRNA and urease genes.

While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as 'Helicobacter heilmannii' or 'Gastrospirillum hominis'. Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis. This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes. Fifteen gastrospirilla detected in humans, primates and pigs clustered with 'Candidatus Helicobacter suis', thus expanding the host range for this organism. By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species. The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name 'Candidatus Helicobacter heilmannii' is proposed.