In vitro potency test for evaluation of Clostridium perfringens type D epsilon toxoid Avaliação in vitro da potência de toxoide épsilon de Clostridium perfringens tipo D

The control of enterotoxemia caused by the epsilon toxin, produced by Clostridium perfringens type D, is based on vaccination with epsilon toxoid. The potency test for this immunogen is conducted using seroneutralization in mice. Here, an in vitro test for detection of neutralizing antibodies with Madin-Darby Canine Kidney (MDCK) cells was standardized in order to study alternative methodologies for the potency test. Titers observed in the in vivo and in vitro seroneutralization tests had a correlation of 99.73%.


RESUMO:
O controle da enterotoxemia causada pela toxina épsilon produzida por Clostridium perfringens tipo D é baseado na vacinação com toxoide.O teste de potência desse imunógeno é realizado utilizando-se a técnica de soroneutralização em camundongos.Objetivando-se estudar metodologias alternativas a essa técnica, padronizou-se um teste in vitro para detecção de anticorpos neutralizantes utilizando-se a linhagem celular Madin-Darby Canine Kidney (MDCK).Os títulos observados nas soroneutralizações in vivo e in vitro apresentaram correlação de 99,73%.In vitro potency test for evaluation of Clostridium perfringens type D epsilon toxoid Enterotoxemia is a widespread disease of domestic ruminants caused by epsilon toxin of Clostridium perfringens type D that results in significant economical losses for the meat and dairy industry (Lobato et al., 2010).The control and prophylaxis are based on good management practices and systematic vaccination.The standard technique proposed by Brazilian legislation for potency tests of clostridial vaccines is seroneutralization (SN) in mice (MAPA, 1997).Despite the known sensitivity and reliability of this potency test in mice, seroneutralization is time-consuming, relatively expensive, and considered ethically debatable by humanitarian groups and researchers aiming to protect animal welfare (Metz et al., 2002).

PALAVRAS-CHAVE:
These bioethical issues have inspired further studies regarding the development of in vitro assays that yield quick, reliable results with good sensitivity and specificity at a decreased cost.Therefore, cell culture methods have been regarded as possible alternatives to SN in mice (Borrmann et al., 2006).This work aimed to standardize an in vitro SN test to evaluate the potency of epsilon toxoid and to compare this cell culturebased method to standard animal bioassays.
The production of epsilon toxin was performed as previously described by Azevedo et al. (1998).A standard serum (National Institute for Biological Standards and Control -NIBSC, UK) with 10 IU/mL of anti-epsilon toxin antibodies was used for epsilon-toxin standardization.Test sera used in both the in vitro and in vivo SN were obtained from pools of rabbits immunized with clostridial vaccines containing epsilon toxoid and standard serum with different concentrations.Negative control rabbits were inoculated with 0.85% saline solution under the same conditions.These test sera were titrated in mice according to Lobato et al. (2000) at the L+/10 level (British Pharmacopeia, 1998).The use of animals in this study was approved by the Ethics Committee for Animal Experimentation (CETEA), Federal University of Minas Gerais, nº 40/2008.The cell line used was Madin-Darby Canine Kidney (MDCK -ATCC/CCL-34) cultivated in Eagle's minimum essential medium (EMEM -Gibco Laboratories, USA; Souza Júnior et al., 2010).
The epsilon toxin was standardized in vitro to the test level L+/50.Titration of the test sera was performed in 96 wells plates.Four repetitions of two-fold serial dilutions from 1:2.5 to 1:320 were prepared to a final volume of 50 µL/well.Another 50 µL was then added to each well containing standardized toxin at the test level L+/50.The plates were homogenized, re-incubated, and cellular suspensions (4.8 x 10 4 cells/50 µL) were added.Positive control wells contained 50 µL of standardized toxin, 50 µL of EMEM, and 50 µL of cells.Negative control wells had 100 µL of EMEM and 50 µL of cells.Serum control wells had 50 µL of the test serum, 50 µL of EMEM, and 50 µL of cells.The plate was incubated at 37°C in a humid chamber (Sheldon Manufacturing Inc -Oregon, USA) with a controlled atmosphere (5% CO 2 and 95% O 2 ) for 48 hours.The supernatant was removed after this period.Staining was done with 100 μL of crystal violet solution (0.1% in alcohol) to observe the cytopathic effect (CPE) (Souza Júnior et al., 2010).Titers of the test sera were calculated as the highest dilution, resulting in 70% destruction of the cell monolayer (Knight et al., 1990).Retrotitration with standard antitoxin was used to check the standardization of the toxin (Salvarani et al., 2010).Results of in vivo and in vitro SN were analyzed and compared with a parametrical correlation using Pearson's correlation (Sampaio, 1998).
The results of in vivo and in vitro SN are shown in Table 1.Titers obtained by in vitro SN were within intervals of the in vivo SN titers.The correlation between the results was 99.73% (p < 0.05), which was superior to the 91% previously demonstrated by Knight et al. (1990).In vitro repeatability was 99.97%.Similar results were also previously reported by Salvarani et al. (2010) and Lima et al. (2011), which reported a correlation greater than 98% for alpha toxin of Clostridium septicum and for alpha toxin of Clostridium novyi type B, respectively.The viability of substituting the animal bioassay with in vitro models is thus proved.
Cell culture has a number of advantages over the animal bioassays.These include a significant decrease in the number of animals utilized, greater sensitivity and lower variation among individual responses.Additionally, an in vitro model that indicates cytotoxicity allows for the observation and study of the biological activity of the epsilon toxin, which is not attainable through other in vitro techniques such as ELISA and agar gel immunodiffusion.
This study demonstrates that detection of anti-epsilontoxin by in vitro SN in MDCK cells is a viable option to replace the animal bioassays that are typically utilized in potency tests for clostridial vaccines containing epsilon toxoid.
This study was conducted according to the ethical principles of animal experimentation established by the Ethics Committee for Animal Experimentation (CETEA/UFMG), protocol n. 40/2008, as determined by the declaration emitted by the Coordinator of the Committee on June 3, 2008.

Table 1 .
Titers (IU/mL) of anti-epsilon-toxin in serum pool of vaccinated rabbits and standard serum obtained by seroneutralization in the mouse and MDCK cells.