Immunohistochemical detection of Clostridia species in paraffin-embedded tissues of experimentally inoculated guinea pigs

Abstract

Blackleg is caused by Clostridium chauvoei, whereas malignant oedema is caused by C. chauvoei, C. septicum, C. sordellii, C. perfringens type A, and/or C. novyi type A. Anti-C. chauvoei, anti-C. septicum, anti-C. sordellii and anti-C. novyi type A polyclonal antibodies were produced in rabbits and purified in a column of DEAE-cellulose. Aliquots of the antisera were conjugated with fluorescein isothiocyanate and the remaining was used for the streptavidin biotin peroxidase technique (SBPT). SBPT was standardized to detect C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs. SBPT was compared to a fluorescent antibody technique (FAT). Sections and smears of muscle from inoculation area (MIA), heart, liver, spleen and kidney, were obtained for both SBPT and FAT. Cross-reactions between the different Clostridial species were not observed. C. chauvoei and C. septicum were detected in all specimens from the animals inoculated with these microorganisms, while only sections of muscle obtained from all the animals inoculated with C. sordellii and C. novyi type A were positive. The same results observed by the SBPT, were obtained on tissue smears of these microorganisms stained by the FAT. The results indicate that SBPT is suitable for detection of C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs.

Comparative analysis of lesions caused by histotoxic clostridia in experimentally induced myonecrosis

“…The bacterins of each of five histotoxic clostridia, were obtained, as previous described for Assis et al (2005), and emulsified (Sorvall mixer Omini-17106-Sorvall, USA) in a 1:1 ratio, with complete and incomplete Freund's adjuvant (Sigma-Aldrich, USA) for the first (day 0) and other inoculations (days 28, 49, 70 and 91), respectively. To obtain hyperimmune serum, three rabbits for each bacterin were used.…”

Detection of several clostridia by a direct fluorescent antibody test in formalin-fixed, paraffin-embedded tissues

“…Immunohistochemical techniques have been used to demonstrate several clostridial organisms in formalin-fixed, paraffin-embedded tissues (Vanelli and Uzal, 1996a;Vanelli and Uzal, 1996b;Assis et al, 2005). These techniques are particularly useful when only formalin-fixed tissues are available for diagnosis.…”

“…In this study, the tissues sections were fixed in formalin by 24 hours to avoid antigenic masking due to protein link (Gimeno, 1995).…”

“…This technique will be particularly useful in areas where laboratory facilities for anaerobic cultivation are not readily available and in any situation in which fixed rather than fresh tissues are submitted to the laboratory. In a recent work, carried out by Assis et al (2005) comparing the techniques of FAT and immunohistochemistry using the streptavidinbiotin technique to detect several clostridia, showed that both techniques present the same sensibility and specificity. However, the FAT has important advantages in comparison with different immunohistochemical techniques.…”

Reação em cadeia da polimerase para detecção de Clostridium chauvoei em tecidos de Cavia porcellus

“…Assis et al (2005) também usaram blocos parafinizados, para defecção imunohistoquímica (IHQ) de C. chauvoei, C. septicum, C. sordellii e C. novyi tipo A, e verificaram que a quantidade de bacilos C. chauvoei, nos diferentes órgãos, foi decrescendo progressivamente a partir do músculo da área de inoculação. Além disso, o fato de os materiais de todos os animais com inoculação de C. chauvoei terem sido positivos pela IHQ (Tabela 1), e o fato de as secções de baço terem sido negativas pela reação em cadeia da polimerase realizada neste estudo, podem ser atribuídos à presença de substâncias inibitórias da PCR (Miller et al, 1997).…”