RESUMEN
OBJECTIVE: To compare the efficacy of three different tissue stains, namely haematoxylin and eosin (H&E), periodic-acid Schiff (PAS) and immunohistochemical (IHC) stains for detection of Entamoeba histolytica (E. histolytica) trophozoites in abscessed liver tissues of hamster. METHODS: Amoebic liver abscess was experimentally induced in a hamster by injecting 1 × 10(6) of axenically cultured virulent E. histolytica trophozoites (HM1-IMSS strain) into the portal vein. After a week post-inoculation, the hamster was sacrificed and the liver tissue sections were stained with H&E, PAS and IHC stains to detect the amoebic trophozoite. RESULTS: The three stains revealed tissue necrosis and amoebic trophozoites, but with varying clarity. H&E and PAS stained the trophozoites pink and magenta, respectively, however it was difficult to differentiate the stained trophozoites from the macrophages because of their similarity in size and morphology. On the other hand, IHC stain revealed distinct brown appearance of the trophozoites in the infected liver tissues. CONCLUSIONS: It can be concluded that out of the three stains, IHC is the best for identification of E. histolytica trophozoites in tissue sections.
Asunto(s)
Entamoeba histolytica/aislamiento & purificación , Absceso Hepático Amebiano/diagnóstico , Absceso Hepático Amebiano/patología , Parasitología/métodos , Coloración y Etiquetado/métodos , Animales , Modelos Animales de Enfermedad , Entamoeba histolytica/citología , Histocitoquímica/métodos , Inmunohistoquímica/métodos , Masculino , Mesocricetus , Microscopía , Trofozoítos/citologíaRESUMEN
Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/rtxA mutated vaccine candidate against O139 V. cholerae.
Asunto(s)
Aciltransferasas/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Vacunas contra el Cólera , Vibrio cholerae O139/genética , Animales , Toxinas Bacterianas/toxicidad , Línea Celular Tumoral , Eliminación de Gen , Genes Bacterianos , Humanos , Intestinos/microbiología , Ratones , Mutagénesis Insercional , Conejos , Serotipificación , Vibrio cholerae O139/crecimiento & desarrollo , Vibrio cholerae O139/metabolismo , Vibrio cholerae O139/patogenicidad , Factores de Virulencia/genéticaRESUMEN
Vibrio cholerae is the causative agent of the infectious disease, cholera. The bacteria adhere to the mucosal membrane and release cholera toxin, leading to watery diarrhea. There are >100 serovars of V. cholerae, but the O1 and O139 serovars are the main causative agents of cholera. The present study aimed to compare the severity of intestinal mucosal infection caused by O1 El Tor and O139 V. cholerae in a rabbit ileal loop model. The results showed that although the fluid accumulation was similar in the loops inoculated with O1 and O139 V. cholerae, the presence of blood was detected only in the loops inoculated with the O139 serovar. Serosal hemorrhage was confirmed by histopathological examination and the loops inoculated with O139 showed massive destruction of villi and loss of intestinal glands. The submucosa and muscularis mucosa of the ileum showed the presence of edema with congested blood vessels, while severe hemorrhage was seen in the muscularis propria layer. The loops inoculated with O1 El Tor showed only minimal damage, with intact intestinal villi and glands. Diffuse colonies of the O139 serovar were seen to have infiltrated deep into the submucosal layer of the intestine. Although the infection caused by the O1 serovar was focal and invasive, it was more superficial than that due to O139, and involved only the villi. These observations were confirmed by immunostaining with O1 and O139 V. cholerae-specific monoclonal antibodies. The peroxidase reaction demonstrated involvement of tissues down to the submucosal layer in O139 V. cholerae infection, while in O1 El Tor infection, the reaction was confined mainly to the villi, and was greatly reduced in the submucosal region. This is the first reported study to clearly demonstrate the histopathological differences between infections caused by the O139 Bengal and O1 El Tor pathogenic serovars of V. cholerae.