Large scale purification of Clostridium perfringens toxins: a review

Purificação de toxinas produzidas por Clostridium perfringens: uma revisão

Abstracts

Clostridium perfringens, a Gram-positive anaerobic bacterium, is widespread in the environment and commonly found in the intestines of animals, including humans. C. perfringens strains are classified into five toxinotypes (A, B, C, D and E) based on the production of four major toxins (±, ², µ, ¹). However the toxins (theta, delta, lambda and enterotoxin) are also synthesized by C. perfringens strain. Many attempts to purify the toxins produced by C. perfringens have been proposed. In this review we discuss the purification methods used to isolate toxins from C. perfringens reported in last four decades.

Clostridium perfringens; Purification; Toxin; Downstream

Clostridium perfringensé uma bactéria anaeróbia Gram-positiva, amplamente distribuída no meio ambiente e comumente encontrada no intestino de animais, incluindo o homem. As espécies de C. perfringensestão classificadas em cinco tipos toxigênicos (A, B, C, D, E) em função da produção de quatro toxinas (±, ², µ, ¹). Entretanto, as toxinas teta, delta, lambda e enterotoxina são também sintetizadas por outras espécies dessa bactéria. Muitas metodologias para purificação das toxinas produzidas por C. perfringens têm sido propostas e, portanto, nesta revisão foram apresentados e discutidos os métodos e resultados de purificação dessas toxinas relatados nas últimas quatro décadas.

Clostridium perfringens; Purificação; Toxina; Processos

REVIEW

Large scale purification of Clostridium perfringens toxins: a review

Purificação de toxinas produzidas por Clostridium perfringens: uma revisão

Maria Taciana Holanda Cavalcanti^{I, II}; Tatiana Porto^{I, II}; Ana Lúcia Figueiredo Porto^III; Igor Viana Brandi^IV; José Luiz de Lima Filho^I; Adalberto Pessoa Junior^II,*

^ILaboratório de Imunopatologia Keizo-Asami (LIKA), Universidade Federal de Pernambuco

IIDepartamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo

^IIIDepartamento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco

^IVInstituto de Ciências Biomédicas, Universidade de São Paulo

ABSTRACT

Clostridium perfringens, a Gram-positive anaerobic bacterium, is widespread in the environment and commonly found in the intestines of animals, including humans. C. perfringens strains are classified into five toxinotypes (A, B, C, D and E) based on the production of four major toxins (α, β, ε, ι). However the toxins (theta, delta, lambda and enterotoxin) are also synthesized by C. perfringens strain. Many attempts to purify the toxins produced by C. perfringens have been proposed. In this review we discuss the purification methods used to isolate toxins from C. perfringens reported in last four decades.

Uniterms:Clostridium perfringens.Purification. Toxin. Downstream

RESUMO

Clostridium perfringensé uma bactéria anaeróbia Gram-positiva, amplamente distribuída no meio ambiente e comumente encontrada no intestino de animais, incluindo o homem. As espécies de C. perfringensestão classificadas em cinco tipos toxigênicos (A, B, C, D, E) em função da produção de quatro toxinas (α, β, ε, ι). Entretanto, as toxinas teta, delta, lambda e enterotoxina são também sintetizadas por outras espécies dessa bactéria. Muitas metodologias para purificação das toxinas produzidas por C. perfringens têm sido propostas e, portanto, nesta revisão foram apresentados e discutidos os métodos e resultados de purificação dessas toxinas relatados nas últimas quatro décadas.

Unitermos:Clostridium perfringens.Purificação.Toxina. Processos.

INTRODUCTION

Recent advances in genetic engineering, DNA recombinant technology, cellular fusion technology, and biotechnology in general, make possible the commercial production of new active products as pharmaceuticals, vaccines and hormones. However, the general purification technology for these products has been developed slowly mainly as compared to the production technology. Purification is troublesome because of system complexity and the need to retain biological activity. Usually, biological material has been purified by precipitation with salts or organic solvents, and by using various chromatographic techniques, all of them having enormous difficulties in large-scale applications (Alves et al., 2000). Besides, according to Diamond and Hsu (1992), 50-90% of the biological products production costs are determined by the purification strategy.

Many attempts to purify toxins from Clostridium perfringens have been reported (Krug, Kent, 1984; Moreau, Jolivet-Reynaud, Alouf, 1986; Stephen, 1961; Ito, 1968; Smyth, Arbuthnott., 1974; Mitsui, Mitsui, Hase, 1973; Möllby, Wadström, 1973; Bird, Low, Stephen, 1974; Takahashi, Sugahara, Ohsaka, 1974; Yamakawa, Ohsaka, 1977; Jolivet-Reynaud, Moreau, Alouf, 1988) However the separation is rather difficult since the variety of toxins produced from this microorganism is high (12 toxins are known) (Hirata et al. 1995). Generally, the main methods used for protein purification are carried out by chromatography as ion exchange, affinity, hydrophobic interaction, and gel filtration. These different types of processes provide the choice for selective fractionation and concentration of macromolecular commodities and the new expanding portfolio of bioproducts (Lyddiatt, 2002).

C. perfringens is a Gram-positive anaerobic bacterium, and able to form spores. It is widespread in the environment, commonly found in the intestines of animals and humans, and can be pathogenic. In humans, it can cause gangrene and gastrointestinal diseases (for instance: food poisoning and necrotic enteritis), whereas in other animals, gastrointestinal and enterotoxemic diseases occur more frequently (Petit, Gibert, Popoff, 1999; Gkiourtzidis et al., 2001).

C. perfringens strains are classified into five toxinotypes (A, B, C, D and E) based on the production of four major toxins (α, β, ε, ι) (Table I) (Petit, Gibert, Popoff, 1999). C. perfringens type A is the most common toxinotype in the environment, is ubiquitous, and responsible for gangrene in humans, mediated primarily by α-toxin, secondarily by θ-toxin and hydrolytic enzymes. This strain is also associated with food poisoning in humans, because synthesizes an enterotoxin (CPE) that is responsible for the gastrointestinal symptoms. Unlike other toxins, CPE is only produced during sporulation (McClane, 1996). The enterotoxin (CPE) is also produced by toxinotype B, C, D and E (Table II) (McClane, 1996). C. perfringens types B produce α-toxin, β-toxin and ε-toxin, but the higher production is β-toxin, which causes enterotoxemia and necrotic enteritis in lambs, piglets and calves (Table I). In humans, the β-toxin is a known cause of necrotic enteritis (Steinthorsdottir et al.,1995). C. perfringens types C produce α-toxin and β-toxin (Table I). C. perfringens type D produces α-toxin and ε-toxin, however its higher production is of ε-toxin (Table I). The C. perfringens type E produces ι-toxin, which has been implicated in fatal calf, lamb, and guinea pig enterotoxemia (Table I) (Bosworth, 1943; Madden, Horton, McCullough, 1970). C. perfringens type B, E and some by type D, produce λ-toxin, which cause enteritis and enterotoxemia in domestic animals (Table II) (Bidwell, 1950; Hatheway, 1990; Rood, Cole, 1991). This λ-toxin may contribute to the pathogenesis by activating other potent toxins, such as the ε- and ι-toxins produced by these type strains (Jin et al., 1996).

C. perfringens type C and some by type B, produce δ-toxin, which is one of the three extracellular hemolytic toxins that can be released, along with many other exotoxins and exoenzymes (Brooks, Stern, Warrack, 1957; Glenny et al., 1933; Oakley, Warrack, 1953; Orlans, Jones, 1958; Smith, 1979; Sterne, Warrack, 1964).

In this review, the purification methods of toxins, produced by C. perfrigens, are presented and discussed. The information here presented can help the production of antigens aiming industrial application, and the comprehension of their structures or effects.

Alpha-toxin ( α)

Alpha toxin is one of the most important lethal and dermonecrotic toxins produced by C. perfringens, known as phospholipase C (PLC). It is produced in different amounts by all types (A, B, C, D, E) of C. perfringens and is considered as a primary virulence factor involved in clostridial myonecrosis (Williamson, Titball, 1993; Award et al., 1995). In C. perfringens type A, the alpha-toxin is the unique lethal protein produced during vegetative growth. Owing to its role in gas gangrene disease, food poisoning and animal enterotoxemia, C. perfringens type A strains, particularly the alpha-toxin, have been the subject of intense investigations over the past 60 years (McDonel, 1986). Alpha-toxin, produced by C. perfringens, is a metalloenzyme with molecular weight of 43 kDa (Takahashi, Sugahara, Ohsaka, 1974; Hale, Stiles, 1999) and LD₅₀ of 40 ng/mL^-1 (Naylor, Martin, Barker, 1997), and catalyses the hydrolysis of lecithin and phospholipids (Saint-Joanis, Garnier, Cole, 1989; Hale, Stiles, 1999).

C. perfringens produces many different biologically active proteins, thus complicating the characterization of any one specific toxin without laborious purification procedures (Macchia, Bates, Pastan, 1967). Various methods of purification have been proposed for alpha-toxin (Mitsui, Mitsui, Hase, 1973a; Bangham, Dawson, 1962; Macchia, Bates, Pastan, 1967; Shemanova et al., 1968; Diner, 1970; Sugahara, Ohsaka, 1970; Ispolatovskaya, 1971; Casu et al., 1971; Stahl, 1973). They include conventional or classical procedures such as precipitation with salts or organic solvents, electrophoresis, gel filtration, and adsorption or ion exchange chromatography. The first purification of alpha-toxin was achieved by precipitation with mixture sodium sulfate and ammonium sulfate (MacFarlane, Knight, 1941), and subsequently by precipitation with ammonium sulfate (Saint-Joanis, Garnier, Cole, 1989). The crude toxin was precipitated with different concentrations of ammonium sulfate, until to achieve the desired degree of ammonium sulfate saturation. After, one the table was elaborated to determine the exact concentrations of ammonium sulfate (McDonel, 1980; Saint-Joanis, Garnier, Cole, 1989). The ammonium sulfate precipitation was often used to prepare a crude toxin for application to gel filtration chromatography (Ito, 1968; Mitsui, Mitsui, Hase, 1973a; Diner, 1970; Ikezawa, Yamamoto, Murata, 1964; Katsaras, Hartwigk, 1979) or electrofocusing (Smyth, Arbuthnott, 1974). These purification steps provided poor recovery yields, such as 54% (van Heyningen, 1941), 56% (Bangham, Dawson, 1962), 14% (Roth, Pillemer, 1953), and 56.6% (Smyth, Arbuthnott, 1974) (Table III). During the ammonium sulfate precipitation, the alpha-toxin suffers considerable biochemical modifications, with possible denaturation of the molecular structure (Odendaal, 1987).

Stephen (1961) was one of the first author to employ high-pressure ultrafiltration to obtain alpha-toxin in high concentration, and the method used to purify was zone electrophoresis and immunoelectrophoresis (Method 1) (Table III). The efficiency of high-pressure ultrafiltration for purification of alpha-toxin was evaluated by Odendaal (1987), and its use was compared in 4 different purification methods (Table III). The results obtained with 3 different ultrafiltration membranes followed by gel filtration showed that by using the membranes Millipore^TM PSED OHV10 and Amicon^TM XM-100 filter (method 7), a three-hundred-and-fivefold purification could be achieved as against a twelve-fold increase obtained with ammonium sulphate/acetone precipitation (method 5). The ultrafiltration process is less time-consuming, easier to perform and less laborious than ammonium sulfate and acetone precipitations. Method 5 was easy and relative fast, but showed low purification factor (12.7-fold); method 6 showed to be easy to perform, less laborious, less time-consuming, provided high purification factor (200-fold), but was of high cost. Method 8 showed to be easy to perform, less laborious, less time-consuming, but showed low purification factor (24-fold) (Table III).

Alpha-toxin has been successfully purified through chromatography column, specifically ion exchange. It was purified by the method 2, in batchwise, and reached a 200-fold increase in specific activity (Möllby, Wadström, 1973) (Table III). Yamakawa and Ohsaka (1977) purified alpha toxin by using method 4; the final product was purified in approximately 15.5-fold, and 17% the toxin was recovered (Table III). In comparison with the others methods described, this method was inefficient of the point of view of purification factor and recovery.

C. perfringens alpha-toxin was also purified by affinity chromatography described for method 3 (Table III). The purification factor encountered was 200-fold, with a recovery yield of about 60% of enzymatic, lethal or hemolytic activity. The purified toxin was homogenous in polyacrylamide gel electrophoresis, ultra-centrifugation and immunodiffusion. By isoelectric focusing of the purified toxin, three major molecular forms were encountered (Takahashi, Sugahara, Ohsaka, 1974) (Table III).

The alpha-toxin from recombinant Bacillus subtilis cells was purified after three steps: ammonium sulfate precipitation (60% saturation), which was followed by affinity chromatography (Sepharose 4B-linked egg yolk lipoprotein column), and then by chelating chromatography (Sepharose Fast Flow gel column). The three purification steps yielded increasingly pure toxin. The final step resulted in a homogeneous product that was purified approximately 130-fold, and 23% of the toxin was recovered (Hirata et al.,1995).

Comparing all described aforementioned methods, it can be observed that the use of chromatography methods (size exclusion molecular, affinity chromatography, ion exchange) is very important to obtain pure toxins. The combined utilization of size exclusion chromatography and utlrafiltration membranes provided higher purification factor, but this procedure its of high cost.

Beta-toxin ( β)

Beta-toxin is produced only by C. perfringens types B and C (Table I), and causes enterotoxemia and necrotic enteritis in lambs, piglets and calves. In humans, this toxin causes necrotic enteritis. In spite of the importance of beta-toxin in veterinary medicine, the biological activity of this protein is poorly defined (Steinthorsdottir et al., 1995; Gkiourtzidis et al., 2001). Its molecular weight is 35 kDa (Hsieh et al., 1998), LD₅₀ is 310 ng/kg^-1 (Jin et al., 1996).

According to Hauschild (1971) the combined effects of beta- and epsilon-toxins are responsible for the diseases caused by C. perfringens type B, such as lamb dysentery and enterotoxemia of foals, goats, sheep and calves.

Beta-toxin is responsible for the diseases caused by C. perfringens type C which including Struck of sheep and enterotoxemia of lambs, calves and piglets and necrotic enteritis of man and fowls (Hauschild, 1971). Most of the conditions in which beta-toxin plays the major role are characterized by ulceration of the intestines or acute hemorrhage enteritis (Worthington, Mülders, 1975). The purification of this toxin is necessary to study the mechanism of action.

The beta-toxin was purified after four steps (method 1), as presented in Table IV, and the final purification factor was of 28-fold (Worthington, Mülders, 1975). The 2^nd purification method of beta-toxin involved immunoaffinity chromatography (Table IV). The toxin was purified about 340-fold from the culture supernatant of C. perfringens type C with a yield of about 24%, in terms of biologically active beta-toxin. Purity of the toxin was checked by polyacrylamide gel electrophoresis; a purified toxin gave a single band (Sakurai, Duncan, 1977).

The comparison between the aforementioned methods indicates that immunoaffinity chromatography to purify beta-toxin should be utilized, since the purification factor was of 340-fold and the yield of 24%.

Epsilon-toxin ( ε)

Epsilon-toxin produced by C. perfringens type B and D is the third most potent clostridial toxin, after botulinum and tetanus toxins. This toxin is produced as a relatively inactive prototoxin with molecular weight of ~33 kDa (Payne, Oyston, 1997; Subramanyam et al., 2001; Parreiras et al., 2002), and as an active toxin with molecular weight of 32 kDa (Payne, Oyston, 1997) their LD₅₀ is 70 ng/kg^-1 (Miyamoto et al., 2000). It is responsible for the pathogenesis of fatal enterotoxemia in domestic animals. This toxin exhibits toxicity toward neuronal cells via the glutamatergic system (Miyamoto et al., 1998; Miyamoto et al., 2000) or extravasation in the brain (Finnie, Blumbergs, Manauis, 1999). It has been suggested that is a pore-forming toxin based on the following observations: (i) ε-toxin can form a large complex in the membrane of Madin-Darby canine kidney cells, and permeabilizes them (Petit et al. 1997; Nagahama, Ochi, Sakurai, 1998); (ii) the large complex formed by ε-toxin is not dissociated by SDS-treatment, which is a common feature of pore-forming toxins (Petit et al. 1997); and (iii) the CD spectrum of ε-toxin shows that it mainly consists of β-sheets (Habeeb, Lee, Atassi, 1973), as observed for pore-forming β-barrel toxins. A characteristic feature of ε-toxin is its potent neurotoxicity, which is not observed for other structurally well-defined pore-forming toxins (Miyata et al., 2001). Another characteristic of ε-toxin is the activation of the inactive precursor (ε-protoxin) by proteases such as trypsin, chymotrypsin (Hunter et al., 1992), and λ-protease produced by C. perfringens. This activation is accompanied by removal of both N- and C-terminal peptides (Jin, et al. 1996; Minami et al., 1997).

Epsilon-protoxin has been purified by methanol precipitation (Verwoerd, 1960), and by ion exchange chromatography (Thomson, 1963; Orlans, Richards, Jones, 1960; Hauschild, 1965; Habeeb, 1969). Protoxin prepared by Habeeb (1969) presented higher toxicity than protoxin prepared by other workers above but was not electrophoretically homogeneous.

Highly purified C. perfringens type D epsilon-protoxin was prepared from culture filtrate of C. perfringens by ammonium sulfate precipitation and ion exchange chromatography (DEAE-cellulose). The purification factor was approximately 77-fold (Worthington, Mülders, Van Rensburg, 1973).

Theta-Toxin ( θ)

Theta(θ)-toxin is one of the toxins produced by Clostridium perfringens type A, a causative pathogen for gas gangrene (Ispolatovskaya, 1971). The toxin is cytolytic (hemolytic) and lethal, belonging to a group of oxygen-labile or SH-dependent hemolysin (Bernheimer, 1974; Bernheimer, 1976). In the same group are found streptolysin O, cereolysin, tetanolysin and listeriolysin produced by Streptococcus pyogenes, Bacillus cereus, Clostridium tetani and Listeria monocytogenes, respectively. These hemolysins share common properties (Bernheimer, 1974; Bernheimer, 1976).

Many workers have attempted to purify theta-toxin from culture filtrate of Clostridium perfringens but have failed to obtain the toxin in good yield because of its instability (Roth, Pillemer, 1955; Mitsui, Mitsui, Hase, 1973b; Smyth, 1975; Soda, Ito, Yamamoto, 1976), probably because this toxin is formed by isoforms (Smith, 1975) and due the presence of reduced form and oxidized form toxins (Yamakawa, Ito, Sato, 1977).

The theta-toxin of C. perfringens was purified by a series of methanol precipitation and subsequent high-speed centrifugation. The purified preparation was free of kappa (κ)-toxin and hyaluronidase but contained traces of alpha(α)-toxin. Electrophoretic patterns indicated that the (θ)-toxin constituted about 80-90% of the total protein of purified preparation (Roth, Pillemer, 1955). Authors as Habermann, 1959 and Habermann, 1960, purified theta-toxin by methanol precipitation, starch-gel electrophoresis, and ion exchange chromatography (DEAE-cellulose). The purified preparations were free of alpha and kappa-toxins, and hyaluronidase. This purified preparation produced a single spot and single precipitin line in membrane electrophoresis and in immunoelectrophoresis, respectively.

By purification method using ammonium sulfate precipitation and electrofocusing (method 1 - Table V), four isoforms of theta-toxin were found. The overall recovery was 86.8% and the purification factor varied, depending of isoform, of 1060 to 1800-fold (Smyth, 1975).

This toxin was purified by Hauschild, Lecroisey, Alouf, (1973) by ammonium sulfate precipitation and successive chromatography steps, described method 2 (Table V). The total recovered theta-toxin was about 20% of the crude extract. The toxin losses, during the four purification steps were about 30, 20, 25, and 5%, respectively. This purification method was developed to produce pure theta-toxin on large scale. The authors did not relate purification factor, but tests realized indicated that the toxin is pure.

Yamakawa, Ito, Sato, (1977) purified theta-toxin 3300 fold from culture filtrate of culture C. perfringens by initial ion exchange chromatography (DEAE-Sephadex A-50), and gel filtration chromatography (Sephadex G-150) (method 3 - Table V). The specific activity was of 105,000 U/mg, with recovery of 50.4%. The electrophoresis of the solution containing the purified toxin showed two distinct bands, and indicated the presence of two forms of toxin.

Iota-Toxin ( ι)

The iota-toxin is produced only by C. perfringens type E and has been implicated in fatal calf, lamb and guinea pig enterotoxemias (Bosworth, 1943; Madden, Horton, McCullough, 1970). Its biological activity is dermonecrotic and lethal. Studies suggested that iota-toxin is a binary toxin dependent on two non-linked proteins. The molecular weight of iota-toxin is 47.5 kDa and of iota b is 71.5 kDa (Stiles, Wilkins, 1986).

The iota-toxin was purified by ammonium sulfate precipitation followed by ionic exchange chromatography (DEAE-Sepharose CL-6B), isoeletric focusing, and gel filtration chromatography (Sephadex G-100). In the first chromatography, the recoveries were of 88 and 74% for Iota_a e Iota_b , respectively (Stiles, Wilkins, 1986).

Delta-toxin ( δ)

C. perfringens delta-toxin is one of the three extracellular hemolytic toxins released by a number of type C strains and also possibly by type B strains (Brooks, Stern, Warrack, 1957; Glenny et al., 1933; Oakley, Warrack, 1953; Orlans, Jones, 1958; Smith, 1979; Sterne, Warrack, 1964). The molecular weight of delta-toxin is 42 KDa (Alouf, Jolivet-Reynaud, 1981). Previous investigations established that this toxin was immunogenic and lytic for the erythrocytes of even-toed ungulates (sheep, cattle, goats and swine) but not for the erythrocytes of other species such as humans, rabbits and horses (Brooks, Stern, Warrack, 1957; Oakley, Warrack, 1953; Willis, 1970). This difference can be owing to the low sensitivity or number of the specific receptors (possibly G_M2 ganglioside). Besides, the receptors can be hidden, as suggested by experiments performed with binded radiolabeled toxin onto cells. The cells might also differ in their relative distributions around the lipids and proteins of the membrane (Alouf, Jolivet-Reynaud, 1981).

Delta-toxin was purified from culture supernatant by ammonium sulfate precipitation, thiol-Sepharose gel chromatography, isoelectric focusing, and gel filtration (Sephadex G-75). 200-fold purification was achieved by using a four-step process descriptive above with recovery of 16%. Some denaturation occurred during the isoelectric focusing (Alouf, Jolivet-Reynaud, 1981). The purified preparation showed a specific activity of 320,000 hemolytic units per mg of protein.

Lambda-toxin ( λ)

The lambda-toxin is produced by most type B and E strains and some type D strains of C. perfringens. It causes enteritis and enterotoxemia in domestic animals (Madden, Horton, McCullough, 1970; Bidwell, 1950; Hatheway, 1990). Studies indicated that this toxin is a zinc metalloprotease with molecular weight 36 kDa (Jin et al., 1996) and contributes to the pathogenicity by degrading certain protein components of host cells. Alternatively, the toxin may contribute to the pathogenesis by activating other potent toxins, such as the epsilon- and iota-toxins, produced by these type strains (Rood, Cole, 1991). Lambda-toxin affects the vascular permeability, the same mechanism of action shown by the epsilon-toxin (Jin et al., 1996).

Table VI

The lambda-toxin can be purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrofobic interaction chromatography. By using this sequence of purification the enrichment factor and recovery yield of the lambda-toxin were estimated to be 87-fold and 30.5%, respectively (Jin et al., 1996).

Enterotoxin (CPE)

C. perfringens produces an enterotoxin that is responsible for human food poisoning in which diarrhea and abdominal cramps are associated with the ingestion of food contaminated. The enterotoxin is a single polypeptide of 35 kDa that is produced and accumulated intracellularly during sporulation of many strains of this microorganism, and is released upon sporangial lysis (Labbe, 1989).

Due to the importance of this protein, several methods have been developed to purify the enterotoxin (Bartholomew, Stringer, 1983; McDonel, McClane, 1988; Barnhart et al., 1976; Enders, Duncan, 1978; Granum, Whitaker, 1980; Stark, Duncan, 1972; Uemura et al., 1985). They include chromatography, such as affinity chromatography, size exclusion molecular and high performance liquid chromatography, polyacrylamide gel electrophoresis. The most commonly employed enterotoxin purification method involves a two-step ammonium sulfate precipitation followed by gel filtration on Sephadex G-100 (Granum, Whitaker, 1980).

The enterotoxin was also purified by using ammonium sulfate precipitation followed by gel filtration chromatography (Sephacryl S-200). The purification factor was 5.5-fold, and the recovery yield was 50%. The time (less than 48 h) and effort required by this purification method are far less than any previously reported procedure. Besides, the method may be useful for those laboratories needing to produce significant amounts of purified enterotoxin (Heredia, Garcia-Alvorado, Labbé, 1994).

CONCLUSIONS

Purification is troublesome because of system complexity and the need to retain biological activity. The purification procedures of different toxins produced by Clostridium perfringens, and presented in this review, involve multiple steps of the classical methodologies, such as chromatography and precipitation with salts or organic solvents. Comparing the results reported in the literature, and presented in the tables of this paper, generally, the purification factors were satisfactory for laboratory and industrial scale, but the results, obviously, depend on the type of toxin. The clear differences between purification factors of the same or different toxins, is owing to the characteristics of each individual protein. Besides, minor changes in upstream processes may require changes in the purification method to be applied. It is clearly observed that the main objective of the downstream processing of toxins is to attain high purification factors and recoveries, at low cost. Therefore, depending on the target toxin, it is necessary to include some adaptations of the current purification methods.

Recebido para publicação em 11 de agosto de 2003.

References

• ALOUF J. E.; JOLIVET-REYNAUD, C. Purification and Characterization of Clostridium perfringens delta-toxin. Infect. Immun, v. 31, n.2, p. 536-546, 1981.
• ALVES, J. G. L. F.; CHUMPITAZ, L. D. A.; SILVA, L. H. M.; FRANCO, T. T., MEIRELLES, A. J. A. Partitioning of whey proteins, bovine serum albumin and porcine insulin in aqueous two-phase systems. J. Chromatogr. B, v. 743, p. 235-239, 2000.
• AWARD, M. M.; BRYANT, A. E.; STEVENS, D. L.; ROOD, J. I. Virulence studies on chromosomal α-toxin and θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens mediated gas gangrene. Mol. Microbiol, v. 15, p. 191-202, 1995.
• BANGHAM, A. D.; DAWSON, R. M. C. Electrokinetic requirements for the reaction between C. Perfringens alpha-toxin (phospholipase C) and phospholipid substrates. Biochem. Biophys. Acta, v. 59, p. 103-115, 1962.
• BARNHART, H M.; BULLERMAN, L. B.; BALL, E. M.; WAGNER, F. W. Isolation and purification of Clostridium perfringens enterotoxin by affinity chromatography. J. Food Sci., v. 41, p. 903-905, 1976.
• BARTHOLOMEW, B. A.; STRINGER, M. F. Observations on the purification of Clostridium perfringens type A enterotoxin and the production of a specific antiserum. FEMS Microbiol. Lett., v. 18, p. 43-48, 1983.
• BERNHEIMER, A. W. Interactions between membranes and cytolytic bacterial toxins. Biochem. Biophys. Acta, v. 344, p. 27-50, 1974.
• BERNHEIMER, A. W. Sulfhydryl activated toxins. In: BERNHEIMER, A. W., ed. Mechanism in bacterial toxinology New York: Wiley Medical, 1976. p. 85-97.
• BIDWELL, E. Proteolytic enzymes of Clostridium welchii Biochem. J, v. 46, p. 589-598, 1950.
• BIRD, R. A.; LOW, M. G.; STEPHEN, J. Immunopurification of phospholipase C (alpha-toxin) from Clostridium perfringens FEBS Lett., v. 44, n. 3, p. 279-281, 1974.
• BOSWORTH, T. J. On a new type of toxin produced by Clostridium welchii J. Comp Pathol, v. 53, p. 245-255, 1943.
• BROOKS, M. E.; STERN, M.; WARRACK, G. H. A re-assessment of the criteria used for type differentiation of Clostridium perfringens J. Pathol. Bacteriol, v. 74, p.185-195, 1957.
• BUNTING, M.; LORANT, D. E.; BRYANT, A. E.; ZIMMERMAN, G. A.; McINTYRE, T. M.; STEVENS, D. L.; PRESCOTT, S. M. Alpha toxin from Clostridium perfringens induces proinflammatory changes in endotelial cells. J. Clin. Invest, v. 100, n. 3, p. 565-574, 1997.
• CASU, A.; PALA. V.; MONACELLI, R.; NANNI, G. Phospholipase C from Clostridium perfringens: purification by electrophoresis on acrylamide-agarose gel. Ital. J. Biochem., v. 20, n. 5, p. 166-178, 1971.
• DIAMOND, A. D. and HSU, J. T. Aqueous two-phase systems for biomolecule separation. Adv. Biochem. Eng, v. 47, p. 89-135, 1992.
• DINER, B. A. Purification and properties of Clostridium welchii phospholipase C. Biochem. Biophys. Acta, v. 198, p. 514-522, 1970.
• DIXON, M. A nomogram for ammonium sulphate solutions. Biochem. J, v.54, p. 457-458, 1953.
• ENDERS, G. L. JR.; DUNCAN, C. L. Preparative polyacrylamide gel electrophoresis purification of Clostridium perfringens enterotoxin. Infect. Immun, v. 17, p. 425-429, 1978.
• FINNIE, J. W.; BLUMBERGS, P. C.; MANAVIS, J. Neuronal damage produced in rat brains by Clostridium perfringens type D epsilon toxin. J. Comp. Pathol, v. 120, n. 4, p. 415-420, 1999.
• GIBERT, M.; JOLIVET-REYNAUD, C.; POPOFF, M. R. Beta 2 Toxin, a novel toxin produced by Clostridium perfringens Gene, v. 203, n. 1, p. 65-73, 1997.
• GKIOURTZIDIS, K.; FREY, J.; BOURTI-HATZOPOULOU, E.; ILLIADIS, N.; SARRIS, K. PCR detection and prevalence of α,β,β2,ε,ι and enterotoxin genes in Clostridium perfringens from lambs with clostridial dysentery. Vet. Microbiol, v. 82, p. 39-43, 2001.
• GLENNY, A. T.; BARR, M.; LLEWELLYN-JONES, M.; DALLING, T.; ROSS, H. E. Multiple toxins produced by some organism of the Clostridium welchii group. J. Pathol. Bacteriol, v. 37, p. 53-74, 1933.
• GRANUM, P. E.; WHITAKER, J. R. Improved method for purification of enterotoxin from Clostridium perfringens type A. Appl. Environm. Microbiol, v. 39, p. 1120-1122, 1980 .
• HABEEB, A. F. S. A. Studies on E prototoxin of Clostridium perfringens type D. 1. purification methods: evidence for multiple forms of E prototoxin. Arch. Biochem Biophys, v. 130, p. 430-440, 1969.
• HABEEB, A. F.; LEE, C. L.; ATASSI, M. Z. Conformational studies on modified proteins and peptides. VII. Conformation of epsilon-prototoxin and epsilon-toxin from Clostridium perfringens Conformational changes associated with toxicity. Biochem. Biophys. Acta, v. 322, p. 245-250, 1973.
• HABERMANN, E. Zur toxikologie und pharmakologie des gasbrandgiftes (Clostridium welchii typ A) und seiner komponenten. Arch. Exp. Pathol. Pharm, v. 238, p. 502-524, 1960.
• HABERMANN, E. Über die Giftkomponrnten des gasbranderregers Clostridium welchii typ A. Arch. Exp. Pathol. Pharm, v. 235, p. 513-534, 1959 .
• HALE, M. L.; STILES, B. G. Detection of Clostridium perfringens alpha toxin using a capture antibody ELISA. Toxicon, v. 37, p. 471-484, 1999.
• HATHEWAY, C. L. Toxigenic clostridia. Clin. Microbiol. Review, v. 3, p. 66-98, 1990.
• HAUSCHILD, A. H. W. Clostridium perfringens toxins types B, C, D, and E. In: KADIS, S., MONTIE, T. C.; AJL, S. J., eds. Microbial toxins - Bacterial protein toxins. New York: Academic Press, 1971. v.2, p.200.
• HAUSCHILD, A. H. W. Separation of Clostridium perfringens type D toxins. J. Immunol, v. 94, p. 551-554, 1965.
• HAUSCHILD, A. H. W.; LECROISEY, A.; ALOUF, J. E. Purification of Clostridium perfringens type C theta-toxin. Canadian J. Microbiol, v. 19, p. 881-885, 1973.
• HEREDIA, N. L.; GARCIA-ALVORADO, J.; LABBÉ, R. G. Improved rapid method for production and purification of Clostridium perfringens type A enterotoxin. J. Microbiol. Methods, v. 20, p. 87-91, 1994.
• HIRATA, Y.; MINAMI, J.; KOYAMA, M.; MATSUSHITA, O.; KATAYAMA, S-I.; JIN, F.; MAETA, H.; OKABE, A. A method for purification of Clostridium perfringens phospholipase C from recombinant Bacillus subtilis cells. Appl. Environm. Microbiol, v. 61, p. 4114-4115, 1995.
• HSIEH, H. V.; STEWART, B.; HAUER, P.; HAALAND, P.; CAMPBELL, R. Measurement of Clostridium perfringens β-toxin production by surface plasmon resonance immunoassay. Vaccine, v. 16, n. 9-10, p. 997-1003, 1998.
• HUNTER, S. E.; BROWN, J. E.; OYSTON, P. C.; SAKURAI, J.; TITBALL, R. W. Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin and leukocidin of Staphylococcus aureus Infect. Immun, v. 61, n. 9, p. 3958-3965, 1993.
• HUNTER, S. E. C.; CLARKE, I. N.; KELLY, D. C.; TITBALL, R. W. Cloning and nucleotide sequencing of the Clostridium perfringens epsilon toxin gene and its expression in Escherichia coli Infect. Immun, v. 60, p. 102-110, 1992.
• IKEZAWA, H.; YAMAMOTO, A.; MURATA, R. Gel flitration of Clostridium perfringens alpha-toxin (phospholipase C). J. Biochem, v. 56, p. 480-, 1964.
• ISPOLATOVSKAYA, M. V. Type A Clostridium perfringens toxin. In: KADIS, S.; MONTIE, T. C.; AJL, S. J., eds. Microbial toxins - Bacterial protein toxins. New York: Academic Press, 1971. v.2, p. 109-158.
• ITO, A. Alpha toxoid of Clostridium perfringens I. Purification and toxoiding of alpha toxin of Clostridium perfringens Jap. J. Med. Sci. Biol, v. 21, p. 379-391, 1968.
• JIN, F.; MATSUSHITA, O.; KATAYAMA, S-I.; JIN, S.; MATSUSHITA, C.; MINAMI, J.; OKABE, A. Purification, Characterization, and Primary Structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease. Infect. Immun, v. 64, n.1, p. 230-237, 1996.
• JOLIVET-REYNAUD, C.; MOREAU, H.; ALOUF J. E. Purification of alpha toxin from Clostridium perfringens: phospholipase C. Meth. Enzymol, v. 165, p. 91-94, 1988.
• LABBE, R. G. Clostridium perfringens. In: DOYLE, M. P., ed. Foodborne bacterial pathogens New York: Dekker, 1989. p. 191-233.
• LESLIE, D.; FAIRWEARTHER, N.; PICKARD, D.; DOUGAN, G.; KEHOE, M., Phospholipase C and haemolytic activies of Clostridium perfringens alpha-toxin cloned in Escherichia coli: sequence and homology with a Bacillus cereus phospholipase C. Mol. Microbiol, v. 3, p. 383-392, 1989.
• LYDDIATT A. Process chromatography:current constraints and future options for the adsorptive recovery of bioproducts. Curr. Opin. Biotech, v. 13, p. 95-103, 2002.
• KATSARAS, K.; HARTWIGK, H. Reinigunng von toxinen des Clostridium perfringens typ A, mittels DEAI ionen austauscher und gel filtration. Zentralbl. Veterinärmed., Reihe B, v. 26, p. 623-633, 1979.
• KRUG, E.L.; KENT, C. Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme. Arch. Biochem. Biophys, v. 231, p. 400-410, 1984.
• MACCHIA, V.; BATES, R. W.; PASTAN, I. The purification and properties of a thyroid-stimulating factor isolated from Clostridium perfringens J. Biol. Chem, v. 242, n. 16, p. 3726-3730, 1967.
• MACFARLANE, M. G.; KNIGHT, B. C. J. G. The biochemistry of bacterial toxins. The lecithinase activity of Clostridium welchii Biochem. J, v. 35, p. 884-901, 1941.
• MADDEN, D. L.; HORTON, R. E.; MCCULLOUGH, N. B. Spontaneous infection in ex-germ-free guinea pigs due to Clostridium perfringens Lab. Animal Care, v. 20, p. 454-455, 1970.
• McCLANE, B. A. An overview of Clostridium perfringens enterotoxin. Toxicon, v. 34, n. 11-12, p. 1335-1343, 1996.
• McDONEL, J. L. Toxins of Clostridium perfringens type A, B, C, D and E. In: DORNER, F.; DREWS, J., eds. Pharmacology of bacterial toxins Oxford : Pergamon Press, 1986. p. 477-517.
• McDONEL, J. L. Clostridium perfringens toxins (type A, B, C, D, E). Pharm. Ther, v. 10, p. 617-655, 1980.
• McDONEL, J. L.; MCCLANE, B. A. Production, purification and assay of Clostridium perfringens enterotoxin. Meth. Enzymol, v. 165, p. 94-103, 1988.
• MINAMI, J.; KATAYAMA, S.; MATSUSHITA, O.; MATSUSHITA, C.; OKABE, A. Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C- terminal peptides. Microbiol. Immunol, v. 41, n. 7, p. 527-535, 1997.
• MITSUI, K.; MITSUI, N.; HASE J. Clostridium perfringens exotoxins. 1. Purification and properties of alpha-toxin. Jap. J. Exp. Med, v. 43, p. 65-80, 1973.
• MITSUI, K.; MITSUI, N.; HASE J. Clostridium perfringens exotoxins. II. Purification and some properties of q-toxin. Jap. J. Exp. Med, v. 43, p. 377-391, 1973.
• MIYAMOTO, O.; MINAMI, J.; TOYOSHIMA, T.; NAKAMURA, T.; MASADA, T.; NAGAO, S.; NEGI, T.; ITANO, T.; OKABE, A. Neurotoxicity of Clostridium perfringens epsilon toxin for the rat hippocampus via the glutamatergic system. Infect. Immun., v. 66, n. 6, p. 2501-2508,1998.
• MIYAMOTO, O.; SUMITANI, K.; NAKAMURA, T.; YAMAGAMI, S-I.; MIYATA, S.; ITANO, T.; NEGI, T.; OKABE, A. Clostridium perfringens epsilon toxin causes excessive release of glutamate in mouse hippocampus. FEMS Microbiol. Lett., v. 189, n. 1, p. 109-113, 2000.
• MIYATA, S.; MATSUSHITA, O.; MINAMI, J.; KATAYAMA, S.; SHIMAMOTO, S.; OKABE, A. Cleavage of a C- terminal peptide is essential for heptamerization of Clostridium perfringens e-toxin in the synaptosomal membrane. J. Biol. Chem, v 276, n. 17, p. 13778-13783, 2001.
• MÖLLBY R.; WADSTRÖM, T. Purification of phospholipase C (alpha-toxin) from Clostridium perfringens Biochem. Biophys. Acta, v. 321, p. 569-584, 1973.
• MOREAU, H.; JOLIVET-REYNAUD, C.; ALOUF, J. E. Enzymatic and cytolic mechanism of Clostridium perfringens alpha-toxin. In: FALMAGNE, P., ALOUF, J. E., FEHRENBACH, F. J.; JELJASZEWICZ, J., eds. Bacterial protein toxins II. Oxford: Springer-Verlag, 1986. p. 65-73.
• NAGAHAMA, M.; OCHI, S.; SAKURAI, J. Assembly of Clostridium perfringens epsilon toxin on MDCK cell membrane. J. Nat. Toxins, v. 7, n. 3, p. 291-302, 1998.
• OAKLEY, C. L.; WARRACK, G. H. Routine typing of Clostridium welchii J. Hyg, v. 51, p. 102-107, 1953.
• ODENDAAL, M. W. Purification of the alpha toxin of Clostridium perfringens type A by ultra filtration and gel chromatography. Onderstepoort J. Vet. Res, v. 54, p. 39-43, 1987.
• ORLANS, E. S.; RICHARDS, C. B.; JONES, V. E. Clostridium welchii epsilon toxin and antitoxin. Immunology, v. 3, p. 28-44, 1960.
• ORLANS, E. S.; JONES, V. E. Studies on some soluble antigens of Clostridium welchii types B, C and D. Immunology, v. 1, p. 268-290, 1958.
• PERELLE, S.; GILBERT, M.; BOQUET, P.; POPOFF, M. R. Characterization of Clostridium perfringens iota toxin genes and expression in Escherichia coli. Infect. Immun., v. 61, p. 5147-5156, 1993
• PERELLE, S.; GILBERT, M.; BOQUET, P.; POPOFF, M. R. Characterization of Clostridium perfringens iota toxin genes and expression in Escherichia coli. Infect. Immun.,v. 63, p. 4967, 1995.
• PETIT, L.; GIBERT, M.; POPOFF, M. R. Clostridium perfringens: toxinotype and genotype. Trends Microbiol., v. 7, n. 3, p. 104-110, 1999.
• PETIT, L.; GIBERT, M.; GILLET, D.; LAURENT-WINTER, C.; BOQUET, P.; POPOFF, M. R. Clostridium perfringens epsilon toxin acts on MDCK cells by forming a large membrane complex. J. Bacteriol., v. 179, p. 6480-6487, 1997.
• ROOD, J. I.; COLE, T. Molecular genetics and pathogenesis of Clostridium perfringens Microbiol. Rev., v. 55, p. 621-648,1991.
• ROTH, F. B.; PILLEMER, L. Purification and some properties of Clostridium welchii type A theta-toxin. J. Immunol, v. 75, p. 50-56, 1955.
• ROTH, F. B.; PILLEMER, L. The separation of alpha-toxin from filtrates of Clostridium welchii J. Immunol, v. 70, n. 5, p. 533-537, 1953.
• SAINT-JOANIS, B.; GARNIER, T.; COLE, S. T. Gene cloning shows the alpha-toxin of C. Perfringens to contain both sphingomyelinase and lecithinases activities. Mol. Gen. Genetic, v. 219, p. 453-460, 1989.
• SAKURAI, J.; FUJII, Y. Purification and characterization of Clostridium perfringens beta toxin. Toxicon, v. 25, p. 1301-1310, 1987.
• SAKURAI, J.; DUNCAN, C. L. Purification of beta-toxin from Clostridium perfringens type C. Infect. Immun, v. 18, n. 3, p. 741-745, 1977.
• SHEMANOVA, G. F.; VLASOVA, E. V.; TSVETKOV, V. S.; LOGINOV, A. I.; LEVIN, F. B. Isolation and properties of lecithinase from Clostridium perfringens. Biokhimiya, v. 33, n. 1, p. 130-136, 1968.
• SMITH, L. D. S. Virulence factors of Clostridium perfringens Rev. Infect. Dis, v. 1, p. 254-260, 1979.
• SMYTH, C. J. The identification and purification of multiple forms of q-haemolisin (q-toxin) of Clostridium perfringens type A. J. Gen. Microbiol, v. 87, p. 219-238, 1975.
• SMYTH, C. J.; ARBUTHNOTT, J. P. Properties of Clostridium perfringens type A alpha-toxin (phospholipase C) purified by electrofocusing. J. Med. Microbiol, v. 7, p. 41-66, 1974 .
• SMYTH, C. J. The identification of multiple forms of q-haemolysin (q-toxin) of Clostridium perfringens type A. J. Gen. Microbiol, v. 87, p. 219-238, 1975.
• SODA, S.; ITO, A.; YAMAMOTO, A. Production and properties of theta-toxin of Clostridium perfringens with special reference to lethal activity. Jap. J. Med. Sci. Biol, v. 29, n. 6, p. 335-349, 1976.
• STAHL, W. L. Phospholipase C purification and specificity with respect to individual phospholipids and brain microsomal membrane phospholipids. Arch. Biochem. Biophys, v. 154, n.1, p. 47-55, 1973.
• STARK, R. L.; DUNCAN, C. L. Purification and biochemical properties of Clostridium perfringens type A enterotoxin. Infect. Immun, v. 6, p. 662-673, 1972.
• STEINTHORSDOTTIR, V.; FRIDRIKSDOTTIR, V.; GUNNARSSON, E.; ANDRESSON, O. S. Expression and purification of Clostridium perfringens beta-toxin glutathione S-transferase fusion protein. FEMS Microbiol. Lett., v. 130, p. 273-278, 1995.
• STEPHEN, J. The isolation of the alpha-toxin of C. welchii type A by zone electrophoresis in vertical columns. Biochem. J, v. 80, p. 578-584, 1961.
• STERNE, M.; WARRACK, G. H. The types of Clostridium perfringens J. Pathol Bacteriol, v. 88, p. 279-283, 1964.
• STILES, B. G.; WILKINS, T. D. Purification and characterization of Clostridium perfringens iota-toxin: dependence on two nonlinked proteins for biological activity. Infect. Immun, v. 54, n. 3, p. 683-688, 1986.
• SUBRAMANYAM, K. V.; PANDURANGA RAO, V.; VIJAYAKRISHNA, S.; SUBBA RAO, M. V. Purification of epsilon toxin of Clostridium perfringens type D by Deae-cellulose chromatography. Indian Vet. J, v.78, p. 471-472, 2001.
• SUGAHARA, T.; OHSAKA, A. Two molecular forms of Clostridium perfringens alpha toxin associated with lethal hemolytic and enzymatic activities. Jap. J. Med. Sci. Biol, v. 23, n. 1, p. 61-66, 1970.
• TAKAHASHI, T.; SUGAHARA, T.; OHSAKA, A. Purification of Clostridium perfringens phospholipase C (a-toxin) by affinity chromatography on agarose-linked egg-yolk lipoprotein. Biochem. Biophys. Acta, v. 351, p. 155-171, 1974.
• THOMSON, R. O. The fractionation of Clostridium welchii antigen on cellulose ion exchangers. J. Gen. Microbiol, v. 31, p. 79-90, 1963.
• TWETEN, R. K. Cloning and expression in Escherichia coli of the perfringolysin O ( theta-toxin) gene from Clostridium perfringens and characterization of gene product. Infect. Immun, v. 56, n. 12, p. 3228-3234, 1988.
• UEMURA, T.; AOI, Y.; HORIGUCHI, Y.; WADANO, A.; GOSHIMA, N.; SAKAGUCHI, G. Purification of Clostridium perfringens enterotoxins by high performance liquid chromatography. FEMS Microbiol. Lett., v. 29, p. 293-297, 1985.
• VANDEKERCKHOVE, J. Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177. FEBS Lett., v. 225, n. 1-2, p. 48-52, 1987.
• VAN HEYNINGEN, W. E. The biochemistry of the gas gangrene toxins. 2. Partial purification of the toxins of C. welchii type A. Biochem. J, v. 35, p. 1246-1256, 1941.
• VERWOERD, D. W. Isolasie van die prototoksien van Clostridium welchii tipe D. J. S. Afri. Vet. Med. Ass, v. 31, p. 195-203, 1960.
• YAMAKAWA, Y.; OHSAKA, A. Purification and some properties of phospholipase C (a-toxin) of Clostridium perfringens J. Biochem, v. 81, p. 115-126, 1977.
• YAMAKAWA, Y.; ITO, A.; SATO, H. Theta-toxin of Clostridium perfringens, I. Purification and some properties. Biochem. Biophys. Acta, v. 494, p. 301-313, 1977.
• WILLIAMSON, E. D.; TITBALL, R. W. A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens also protects mice against experimental gas gangrene. Vaccine, v. 12, p. 1253-1258, 1993.
• WILLIS, A. T. In: WILLIS, A. T., ed. Clostridia of wound infection London: Butterworths, 1970. p. 41-156.
• WORTHINGTON, R. W.; MÜLDERS, M. S. G. The partial purification of Clostridium perfringens beta toxin. Onderstepoort J. Vet. Res, v. 42, n. 3, p. 91-98, 1975.
• WORTHINGTON, R. W.; MÜLDERS, M.S.G.; VAN RENSBURG, J. J. Clostridium perfringens type D epsilon prototoxin. Some chemical, immunological and biological properties of a highly purified prototoxin. Onderstepoort J. Vet. Res, v. 40, n. 4, p. 145- 152, 1973.

Publication Dates

• Publication in this collection
  23 Mar 2010
• Date of issue
  June 2004

History

• Received
  11 Aug 2003