- Open Access
The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells
© Verherstraeten et al.; licensee BioMed Central Ltd. 2013
Received: 15 April 2013
Accepted: 7 June 2013
Published: 19 June 2013
Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells.
Since the ban on antimicrobial growth promoters in Europe, necrohemorrhagic enteritis emerged as a major cause of mortality in veal calves in Belgium, causing important economic losses [1–3]. Bovine necrohemorrhagic enteritis, also known as enterotoxaemia, is most typically characterized by sudden death and macroscopic post-mortem findings are necrotic and hemorrhagic lesions in the small intestine [4, 5]. Microscopically, necrosis of the intestinal mucosa and hemorrhages are observed [1, 5, 6]. Clostridium perfringens is considered as the causative agent [1, 7]. This spore-forming, Gram-positive, anaerobic bacterium is often found as a normal inhabitant of the intestine of most animal species and humans [6, 8, 9]. For reasons that are not yet fully understood, C. perfringens can, under certain predisposing conditions, proliferate rapidly, concurrently produce toxins and cause disease. Stress, for example, is considered to be such a predisposing factor, and also the feed composition is believed to be of major importance for the development of the disease [7, 10, 11]. The presence of a causative toxin determines the potential of a C. perfringens strain to cause lesions in several animal species. Classification of C. perfringens strains is based on the production of four major toxins, namely alpha, beta, epsilon and iota toxin. In addition to the major toxins, other toxins can be secreted, such as beta2-toxin and perfringolysin [5, 12].
There has been controversy on the toxins responsible for bovine necrohemorrhagic enteritis. Some studies proposed epsilon toxin as a possible causative toxin [13–16]. This toxin is produced by type B and D strains and plays a key role in the pathogenesis of sheep and goat enterotoxaemia. Filho et al. could indeed induce clinical signs and lesions, when a type D strain was inoculated intraduodenally in a calf . In an intestinal loop assay comparing alpha and epsilon toxin, only epsilon toxin was able to cause severe oedema and hemorrhages in the lamina propria . These results point to epsilon toxin as causative toxin. More recently, beta2-toxin (CPB2) has been linked to necrohemorrhagic enteritis in calves and cows [2, 6, 17–19]. Manteca et al. showed that inoculating a beta2-positive type A strain into a bovine ligated intestinal loop caused hemorrhages of the intestinal wall and necrosis . The strain however also produced high levels of alpha toxin, so a synergistic action between both toxins was proposed. Moreover, allelic variants of the CPB2-gene have been identified and strains from cattle mostly carried the atypical CPB2-gene that is not expressed, in contrast to the “consensus” variant [2, 6, 17]. In addition, a more recent study isolated only beta2-negative type A strains from calves with necrohemorrhagic enteritis and these strains were also able to induce pathological changes in inoculated intestinal loops . We recently developed an experimental intestinal loop model that mimics the typical lesions of calf necrohemorrhagic enteritis both macroscopically and microscopically . In this model, it was demonstrated that type A strains from bovine and non-bovine origin, including both beta2-negative and -positive strains, were able to induce necrohemorrhagic lesions . These results suggest that the causative toxin should be present in all the tested C. perfringens strains. This puts forward a potential role for alpha toxin and perfringolysin, since both are produced by nearly all C. perfringens strains . The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis is not reported before. In gas gangrene or clostridial myonecrosis, perfringolysin is involved in the pathogenesis in synergy with alpha toxin [22–24]. Additionally, Valgaeren et al. observed that the early pathogenesis of calf necrohemorrhagic enteritis in an experimental loop assay is characterized by congestion of the capillaries resulting in hemorrhages and finally necrosis of the mucosa . This points to the endothelium as a possible target.
Therefore, the objective of the present study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis and its synergism with alpha toxin in calves, by using a calf intestinal loop assay and endothelial cell cytotoxicity assays.
Materials and methods
Bacterial strains and culture conditions
Description of strains and toxin activities in supernatants of an overnight culture
Plasmid-encoded toxin gene(s)a
Plc activity(μg mL-1)c
PFO activity (log2(titre))c
12.5 ± 2.5
4.8 ± 0.2
JIR325 pfoA::ermB plcΩpJIR1774, suicide plasmid
pfoA-complemented ΔpfoA Δplc
4.1 ± 0.3
plc-complemented ΔpfoA Δplc
6.8 ± 0.7
double-complemented ΔpfoA Δplc
8.0 ± 0.5
3.5 ± 0.1
Filter-sterilized supernatant of an overnight culture of each strain was assayed for perfringolysin activity by the doubling dilution hemolytic assay with horse red blood cells as described previously (Table 1) . The perfringolysin activity was expressed as the reciprocal of the last dilution which showed complete hemolysis. The perfringolysin assay was performed in triplicate. For the detection of alpha toxin in the supernatant, the Bio-X Alpha Toxin Elisa Kit (Bio-X Diagnostics, Jemelle, Belgium) was used according to the instructions of the manufacturer (Table 1). The amount of alpha toxin was calculated as described previously in μg mL-1. The alpha toxin ELISA was performed in triplicate.
Assessing the role of perfringolysin by using an intestinal loop model
The animal studies were undertaken with approval (EC2012_056) of the Ethical Committee of the Faculty of Veterinary Medicine (Ghent University). Three 11-week-old Holstein Friesian calves, originating from a commercial veal herd, were used. Intestinal loop assays were conducted as described by Valgaeren et al. . Briefly, the calves were anesthetized and the small intestine was exteriorized. Intestinal loops were ligated and injected with 20 mL logarithmic cultures followed by an injection of 10 mL 25% commercial milk replacer (Vitaspray, Vitamex®, Drongen, Belgium) suspended in sterile 0.9% NaCl solution. Because the gas gangrene strain JIR325 could induce lesions comparable to the bovine strains, this strain was used in combination with its isogenic mutants (Table 1) . Each strain was injected in quintuplicate and as a control BHI was injected in five loops instead. After injection of the loops, the abdomen was closed and the calves were maintained under anesthesia. At 6-h post-inoculation the animals were euthanized and samples were taken. Samples were fixed in 4% phosphate buffered formaldehyde. They were embedded in paraffin wax, sectioned and stained with hematoxylin-eosin by the conventional method for histological examination. The sections were evaluated for presence of necrotic lesions (Leica DM L2 microscope with Leica DFC320 camera and LAS software).
Isolation of bovine umbilical vein endothelial cells
Primary bovine umbilical vein endothelial cells (BUVEC) were isolated from umbilical cord veins by an adaptation of the method of Jaffe et al. . Umbilical cords obtained from calves born by caesarean section, were transported in 0.9% HBSS-HEPES buffer (pH 7.4, Gibco, Grand Island, NY, USA) supplemented with 500 U/mL penicillin (Sigma-Aldrich) and 50 μg/mL streptomycin (Sigma-Aldrich). The umbilical vein was cannulated and flushed with pre-warmed (37 °C) HBSS (Gibco, Grand Island, NY, USA). 0.5% collagenase type IV (Sigma-Aldrich) suspended in HBSS was infused into the lumen of the clamped shut umbilical vein and it was incubated for 30 min at 37 °C. After gently massaging the umbilical vein, the cells were flushed from the vein by perfusion with 10 mL HBSS containing 20% fetal calf serum (FCS, Bockneck Labs Inc., Toronto, Canada). After rinsing (250 × g, 5 min), a single cell suspension was obtained by filtration through a 70 μm cell strainer (BD Labware, San Jose, CA, USA). The pelleted (250 × g, 10 min) cells were resuspended in endothelial cell growth medium containing 20% FCS (EGM-2; Lonza, Basel, Switzerland) and seeded in 25 cm2 plastic tissue culture flasks. After 24 h incubation at 37 °C in the presence of 5% CO2, the cell medium was changed. Bovine umbilical vein endothelial cells were grown to confluence. The endothelial origin and purity was verified by immunocytochemistry using anti-mouse CD31 antibody (Dako, Heverlee, Belgium).
Assessing the cytotoxic effects of perfringolysin on bovine endothelial cells
In order to visually assess cytotoxicity, BUVEC were seeded on 13-mm-circular glass slides (VWR International BVBA, Leuven, Belgium) in a 24-well plate at a concentration of 1 × 105 cells/mL and were incubated at 37 °C in the presence of 5% CO2. After 36 h, the cells were exposed to 3% filter-sterilized supernatant of an overnight culture diluted in serum-free endothelial cell growth medium (SFM). The strains used for producing the supernatants were the wild-type strain JIR325 and its isogenic mutants (Table 1). SFM was used as a control. After 1.5 h incubation, the cells were rinsed three times with HBSS containing Ca2+ and Mg2+. They were fixed with 100% methanol and stained with Haemacolor-stain (Merck, Darmstadt, Germany). The glass slides were mounted on a microscope slide and observed microscopically.
To quantify the cytotoxicity, BUVEC were seeded in a 96-well plate at a concentration of 1 × 105 cells/mL and incubated for 36 h as described above. 1% or 6% of the above mentioned supernatants in SFM was added to the cells. SFM was used to determine reference values. After 1.5 h incubation, a Neutral Red Uptake Assay was performed . Briefly, Neutral Red Medium (EGM-2 medium containing neutral red (Merck N.V./S.A., Overijse, Belgium)) was added to the cells and the plates were incubated at 37 °C. After 3 h of incubation, the cells were rinsed with HBSS containing Ca2+ and Mg2+ and treated with extracting solution (50% absolute ethanol, 49% distilled water and 1% glacial acetic acid) for 15 min at room temperature on a shaker to extract the neutral red from the cells. The absorbance was determined at 550 nm and cell viability was expressed as the percentage of viable cells compared to untreated cells (negative control) and cells treated with the supernatant of the wild-type (positive control). Each culture supernatant was tested in duplicate in three independent assays.
Significant differences in the number of loops with necrosis between the wild-type and the mutants were determined using a two-tailed Fisher Exact test (P < 0.05). Significant differences between the relative viability of the mutants and 0%, which corresponds with the relative viability of the wild-type strain, were investigated using a two-tailed Wilcoxon signed rank test (P < 0.05). A one-way analysis of variance (ANOVA) with the post hoc Tukey-Kramer multiple-comparison test was used to identify significant differences in the relative viability of cells after contact with the supernatants of the mutants (P < 0.001). All statistical analyses were performed using GraphPad Prism Software 5.0 (GraphPad Software, Inc., USA).
Assessing the role of perfringolysin by using an intestinal loop model
Assessing the cytotoxic effects of perfringolysin on bovine endothelial cells
The presence of a causative toxin in a C. perfringens strain determines its potential to cause lesions and subsequently diseases in several animal species. However, there is still controversy on the toxin responsible for bovine necrohemorrhagic enteritis. In a previous study, it was demonstrated that type A strains from bovine and non-bovine origin can induce necrohemorrhagic lesions in a calf intestinal loop assay . These results suggest that the causative toxin is one of the toxins produced by all C. perfringens type A strains. In the present study, it was shown that perfringolysin and alpha toxin are involved in the induction of necrohemorrhagic lesions. Indeed, a plc-complemented ΔpfoA Δplc mutant (or pfoA-deficient strain) and a pfoA-complemented ΔpfoA Δplc mutant (or plc-deficient strain) of a gas gangrene strain had a decreased ability to induce necrohemorrhagic lesions in a calf intestinal loop assay. The gas gangrene strain JIR325 was used, because perfringolysin- and alpha toxin-deficient mutants of this strain were already available and because this strain was able to induce mucosal hemorrhages and necrosis of the villus tips comparable to the lesions induced by bovine strains in a calf intestinal loop model . In previous studies, it was suggested that epsilon toxin and beta2-toxin are essential in the development of necrosis. Filho et al. induced lesions in a calf intraduodenally inoculated with a type D strain . The authors proposed epsilon toxin as causative toxin. However, based on our results, alpha toxin and perfringolysin may also have been involved in lesion development, since both toxins are produced by type D strains as well. In an intestinal loop assay, comparing alpha and epsilon toxin, only epsilon toxin was able to cause severe oedema and hemorrhages in the lamina propria , but these authors did not observe necrosis, in contrast to our results which showed necrohemorrhagic lesions comparable to field cases. Manteca et al. stated that beta2-toxin was an essential toxin, because inoculation of a beta2-positive type A strain into a bovine ligated intestinal loop caused necrohemorrhages of the intestinal wall . However, the strain also produced a high level of alpha toxin and the authors proposed a synergistic effect of alpha toxin and beta2-toxin. Since nearly all type A strains produce perfringolysin as well, this toxin may also have been important in the development of the lesions. In addition, our results agree with a recent study in which only beta2-negative type A strains were isolated from calves with necrotic enteritis and these strains were able to induce pathologic changes when inoculated in intestinal loops . It is still possible that beta2-toxin or other toxins can have supplementary effects.
Valgaeren et al. also found indications that endothelial damage may be involved in the early stages of intestinal lesion development . Our results suggest perfringolysin-induced cytotoxic effects on endothelial cells may play a potential role in the development of necrohemorrhagic enteritis. We showed that the plc-complemented ΔpfoA Δplc mutant (or pfoA-deficient strain) was significantly less cytotoxic for bovine umbilical cord endothelial cells (BUVEC). Endothelial cells form a vital barrier that controls the exchange of cells, macromolecules and fluids between the vascular lumen and the surrounding tissue. They also maintain the normal blood flow due to their antiplatelet, anticoagulant and fibrinolytic properties. Disruption of the endothelial barrier leads to increased vascular permeability along with tissue edema and hemorrhage. Furthermore, it augments local coagulation and vascular thrombosis, and subsequent hypoxic tissue necrosis . In order to confirm that the endothelium is the target cell of perfringolysin or alpha toxin in bovine necrohemorrhagic enteritis, it would be interesting to localize the toxin in lesions, as already been done for beta toxin in necrotic enteritis in piglets and in a human case [31, 32]. Beta toxin has been shown to induce porcine endothelial cell damage in vitro and to bind to endothelial cells, and not to epithelial cells, in the gut of diseased animals, suggesting that disruption of endothelial cells plays a role in type C enteritis [30, 31, 33].
Additionally, our results show that perfringolysin and alpha toxin act synergistically in inducing BUVEC cytotoxicity and necrohemorrhagic lesions in a calf intestinal loop model. In gas gangrene, perfringolysin and alpha toxin also act synergistically [22–24, 34]. Alpha toxin is a phospholipase C that hydrolyzes phosphatidylcholine and sphingomyelin, both of which are important constituents of eukaryotic cell membranes. Perfringolysin is a cholesterol-dependent cytolysin and oligomerizes upon contact with cholesterol-containing membranes to form large transmembrane pores by inserting a beta-barrel into the membrane . It has been stated that the ability of perfringolysin to perforate the membrane of target cells, is determined by the amount of free cholesterol molecules present [35, 36]. Moe and Heuck found that alpha toxin cleaves the phosphocholine headgroup of phosphatidylcholine, increasing the number of free cholesterol molecules in the membrane and by doing so, facilitating the interaction of perfringolysin and cholesterol . This concerted action of alpha toxin and perfringolysin may contribute to the synergistic effect between both toxins in gas gangrene and in bovine necrohemorrhagic enteritis.
In gas gangrene, perfringolysin modulates the host inflammatory response by upregulating leukocyte and endothelial adhesion molecules. This causes leukocyte accumulation within the blood vessels and inhibits the normal influx of phagocytic cells into infected host tissue, reducing inflammation [22, 23, 37]. Additionally, alpha toxin enhances the expression of platelet adhesion molecules, contributing to the formation of freely moving intravascular aggregates of platelets, fibrin and neutrophils. This leads to the obstruction of the vessels and contributes to a decreased blood flow . Gas gangrene is characterized by tissue necrosis, thrombosis and a lack of leukocyte infiltration at the site of infection. On the contrary, bovine necrohemorrhagic enteritis is associated with congestion of the capillaries, hemorrhages and inflammation [7, 10, 20]. So perfringolysin and alpha toxin appear to be involved in both diseases, but may act in a different way. The use of mutants deficient in the production of alpha toxin or perfringolysin in a mouse myonecrosis model showed that alpha toxin is essential for thrombosis formation [22, 23]. Furthermore, when rabbits were treated intravenously with recombinant perfringolysin a vasodilatory effect and a reduced systemic vascular resistance was observed. On the other hand, in rabbits treated with recombinant alpha toxin the vascular resistance was maintained and the arterial pressure was reduced [23, 38, 39]. Altered vascular integrity, but not vascular occlusion, seems to be in accordance with the role of perfringolysin in bovine necrohemorrhagic enteritis. This may also explain partly the inflammation present in necrohemorrhagic enteritis as opposed to the lack of leukocyte infiltration in gas gangrene.
Next to the toxic effect of perfringolysin on endothelial cells, other effects on the gastro-intestinal mucosa are most likely of importance in the development of necrohemorrhagic enteritis. Indeed, before perfringolysin can target the endothelial cells, it has to cross the epithelial barrier. While cytotoxic effects of perfringolysin and alpha toxin on intestinal epithelial cells cannot be excluded, also other C. perfringens toxins, enzymes or other molecules could affect the intestinal integrity. Intestinal integrity disturbances can also be caused by C. perfringens independent factors in the field, such as viral and parasitological pathogens. The most well-known example of a predisposing pathogen for necrotic enteritis is coccidiosis in broilers [40, 41], but also in calves several infectious agents, such as coccidia, enteropathogenic bacteria, corona- and rotaviruses can affect the intestinal barrier integrity [40–44]. In addition, certain feed components can act as predisposing factors for the induction of gut lesions. These include high non-starch polysaccharide containing diets and high protein diets, the latter most likely feeding the auxotrophy C. perfringens has for many amino acids [45, 46]. The diet can have a direct effect on the virulence of C. perfringens, but it can as well affect the intestinal tract. In calves fed with milk replacing proteins an increase in permeability of the intestinal mucosa was observed, which caused leakage of macromolecules from the gut into the tissues [41, 47–49]. This might also facilitate the uptake of toxins through the epithelial barrier.
In conclusion, our study indicates that perfringolysin is involved in the pathogenesis of bovine necrohemorrhagic enteritis and acts synergistically with alpha toxin. We hypothesize that both toxins may induce intestinal lesions by targeting the endothelial cells.
The authors thank J.I. Rood for providing the bacterial strains used in this study. The authors acknowledge support from all veterinary surgeons from the division of anesthesia for their assistance in the intestinal loop assays, the PhD students from the pathology-department who assisted in the sampling and the department of obstetrics, reproduction and herd health for the supply of bovine umbilical cords. The authors would like to thank Christian Puttevils, Delphine Ameye and Astra Dhanijns for the technical assistance. This project was supported by the Institute for Science and Technology, Flanders under contract number 090910.
- Manteca C, Daube G, Pirson V, Limbourg B, Kaeckenbeeck A, Mainil JG: Bacterial intestinal flora associated with enterotoxaemia in Belgian Blue calves. Vet Microbiol. 2001, 81: 21-32. 10.1016/S0378-1135(01)00329-7.View ArticlePubMedGoogle Scholar
- Muylaert A, Lebrun M, Duprez JN, Labrozzo S, Theys H, Taminiau B, Mainil J: Enterotoxaemia-like syndrome and Clostridium perfringens in veal calves. Vet Rec. 2010, 167: 64-65. 10.1136/vr.b4869.View ArticlePubMedGoogle Scholar
- Pardon B, De Bleecker K, Hostens M, Callens J, Dewulf J, Deprez P: Longitudinal study on morbidity and mortality in white veal calves in Belgium. BMC Vet Res. 2012, 8: 26-10.1186/1746-6148-8-26.PubMed CentralView ArticlePubMedGoogle Scholar
- Valgaeren BR, Pardon B, Verherstraeten S, Goossens E, Timbermont L, Haesebrouck F, Ducatelle R, Deprez PR, Van Immerseel F: Intestinal clostridial counts have no diagnostic value in the diagnosis of enterotoxaemia in veal calves. Vet Rec. 2013, 172: 237-10.1136/vr.101236.View ArticlePubMedGoogle Scholar
- Songer JG: Clostridial enteric diseases of domestic animals. Clin Microbiol Rev. 1996, 9: 216-234.PubMed CentralPubMedGoogle Scholar
- Lebrun M, Filee P, Mousset B, Desmecht D, Galleni M, Mainil JG, Linden A: The expression of Clostridium perfringens consensus beta2 toxin is associated with bovine enterotoxaemia syndrome. Vet Microbiol. 2007, 120: 151-157. 10.1016/j.vetmic.2006.10.020.View ArticlePubMedGoogle Scholar
- Lebrun M, Mainil JG, Linden A:Cattle enterotoxaemia andClostridium perfringens: description, diagnosis and prophylaxis.Vet Rec. 2010, 167: 13-22. 10.1136/vr.167.1.12.View ArticlePubMedGoogle Scholar
- Rood JI: Virulence genes of Clostridium perfringens. Annu Rev Microbiol. 1998, 52: 333-360. 10.1146/annurev.micro.52.1.333.View ArticlePubMedGoogle Scholar
- Morris WE, Dunleavy MV, Diodati J, Berra G, Fernandez-Miyakawa ME: Effects of Clostridium perfringens alpha and epsilon toxins in the bovine gut. Anaerobe. 2012, 18: 143-147. 10.1016/j.anaerobe.2011.12.003.View ArticlePubMedGoogle Scholar
- Morris WE, Venzano AJ, Elizondo A, Vilte DA, Mercado EC, Fernandez-Miyakawa ME: Necrotic enteritis in young calves. J Vet Diagn Invest. 2011, 23: 254-259. 10.1177/104063871102300209.View ArticlePubMedGoogle Scholar
- Nowell VJK, Kropinsky AM, Songer JG, Macinnes JI, Parreira VR, Prescott JF: Genome sequencing and analysis of a type A Clostridium perfringens isolate from a case of bovine clostridial abomasitis. PLoS One. 2012, 7: e32271-10.1371/journal.pone.0032271.PubMed CentralView ArticlePubMedGoogle Scholar
- Songer JG, Miskimmins DW: Clostridium perfringens type E enteritis in calves: two cases and a brief review of the literature. Anaerobe. 2004, 10: 239-242. 10.1016/j.anaerobe.2004.05.001.View ArticlePubMedGoogle Scholar
- Niilo L, Avery RJ: Bovine “enterotoxemia” I. Clostridium Perfringens types isolated from animal sources in Alberta and Saskatchewan. Can Vet J. 1963, 4: 31-36.PubMed CentralPubMedGoogle Scholar
- Uzal FA, Kelly WR, Morris WE, Assis RA: Effects of intravenous injection of Clostridium perfringens type D epsilon toxin in calves. J Comp Pathol. 2002, 126: 71-75. 10.1053/jcpa.2001.0514.View ArticlePubMedGoogle Scholar
- Filho EJ, Carvalho AU, Assis RA, Lobato FF, Rachid MA, Carvalho AA, Ferreira PM, Nascimento RA, Fernandes AA, Vidal JE, Uzal FA: Clinicopathologic features of experimental Clostridium perfringens type D enterotoxemia in cattle. Vet Pathol. 2009, 46: 1213-1220. 10.1354/vp.08-VP-0304-U-FL.View ArticlePubMedGoogle Scholar
- Uzal FA, Rolfe BE, Smith NJ, Thomas AC, Kelly WR: Resistance of ovine, caprine and bovine endothelial cells to Clostridium perfringens type D epsilon toxin in vitro. Vet Res Commun. 1999, 23: 275-284. 10.1023/A:1006362819202.View ArticlePubMedGoogle Scholar
- Gibert M, Jolivet-Reynaud C, Popoff MR: Beta2 toxin, a novel toxin produced by Clostridium perfringens. Gene. 1997, 203: 65-73. 10.1016/S0378-1119(97)00493-9.View ArticlePubMedGoogle Scholar
- Bueschel DM, Jost BH, Billington SJ, Trinh HT, Songer JG: Prevalence of cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: correlation of genotype with phenotype. Vet Microbiol. 2003, 94: 121-129. 10.1016/S0378-1135(03)00081-6.View ArticlePubMedGoogle Scholar
- Manteca C, Daube G, Jauniaux T, Linden A, Pirson V, Detilleux J, Ginter A, Coppe P, Kaeckenbeeck A, Mainil JG: A role for the Clostridium perfringens beta2 toxin in bovine enterotoxaemia?. Vet Microbiol. 2002, 86: 191-202. 10.1016/S0378-1135(02)00008-1.View ArticlePubMedGoogle Scholar
- Valgaeren B, Pardon B, Goossens E, Verherstraeten S, Schauvliege S, Timbermont L, Ducatelle R, Deprez P, Van Immerseel F: Lesion development in a new intestinal loop model indicates the involvement of a shared Clostridium perfringens virulence factor in haemorrhagic enteritis in calves. J Comp Pathol. 2013, 149: 103-112. 10.1016/j.jcpa.2012.11.237.View ArticlePubMedGoogle Scholar
- Rood JI, Cole ST: Molecular genetics and pathogenesis of Clostridium perfringens. Microbiol Rev. 1991, 55: 621-648.PubMed CentralPubMedGoogle Scholar
- Ellemor DM, Baird RN, Awad MM, Boyd RL, Rood JI, Emmins JJ: Use of genetically manipulated strains of Clostridium perfringens reveals that both alpha-toxin and theta-toxin are required for vascular leukostasis to occur in experimental gas gangrene. Infect Immun. 1999, 67: 4902-4907.PubMed CentralPubMedGoogle Scholar
- Awad MM, Ellemor DM, Boyd RL, Emmins JJ, Rood JI: Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene. Infect Immun. 2001, 69: 7904-7910. 10.1128/IAI.69.12.7904-7910.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- O'Brien DK, Melville SB: Effects of Clostridium perfringens alpha-toxin (PLC) and perfringolysin O (PFO) on cytotoxicity to macrophages, on escape from the phagosomes of macrophages, and on persistence of C. perfringens in host tissues. Infect Immun. 2004, 72: 5204-5215. 10.1128/IAI.72.9.5204-5215.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Lyristis M, Bryant AE, Sloan J, Awad MM, Nisbet IT, Stevens DL, Rood JI: Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens. Mol Microbiol. 1994, 12: 761-777. 10.1111/j.1365-2958.1994.tb01063.x.View ArticlePubMedGoogle Scholar
- Awad MM, Bryant AE, Stevens DL, Rood JI: Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene. Mol Microbiol. 1995, 15: 191-202. 10.1111/j.1365-2958.1995.tb02234.x.View ArticlePubMedGoogle Scholar
- Zhang G, Darius S, Smith SR, Ritchie SJ: In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its alpha-toxin production. Lett Appl Microbiol. 2006, 42: 138-143. 10.1111/j.1472-765X.2005.01812.x.View ArticlePubMedGoogle Scholar
- Jaffe EA, Nachman RL, Becker CG, Minick CR: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973, 52: 2745-2756. 10.1172/JCI107470.PubMed CentralView ArticlePubMedGoogle Scholar
- Van Parys A, Boyen F, Verbrugghe E, Leyman B, Bram F, Haesebrouck F, Pasmans F: Salmonella Typhimurium induces SPI-1 and SPI-2 regulated and strain dependent downregulation of MHC II expression on porcine alveolar macrophages. Vet Res. 2012, 43: 52-10.1186/1297-9716-43-52.PubMed CentralView ArticlePubMedGoogle Scholar
- Gurtner C, Popescu F, Wyder M, Sutter E, Zeeh F, Frey J, Von Schubert C, Posthaus H: Rapid cytopathic effects of Clostridium perfringens beta-toxin on porcine endothelial cells. Infect Immun. 2010, 78: 2966-2973. 10.1128/IAI.01284-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Miclard J, Jäggi M, Sutter E, Wyder M, Grabscheid B, Posthaus H: Clostridium perfringens beta-toxin targets endothelial cells in necrotizing enteritis in piglets. Vet Microbiol. 2009, 137: 320-325. 10.1016/j.vetmic.2009.01.025.View ArticlePubMedGoogle Scholar
- Miclard J, Van Baarlen J, Wyder M, Grabscheid B, Posthaus H: Clostridium perfringens β-toxin binding to vascular endothelial cells in a human case of enteritis necroticans. J Med Microbiol. 2009, 58: 826-828. 10.1099/jmm.0.008060-0.View ArticlePubMedGoogle Scholar
- Schumacher VL, Martel A, Pasmans F, Van Immerseel F, Posthaus H:Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally inducedClostridium perfringenstype C enteritis in pigs. Vet Pathol. in press,Google Scholar
- Hickey MJ, Kwan RY, Awad MM, Kennedy CL, Young LF, Hall P, Cordner LM, Lyras D, Emmins JJ, Rood JI: Molecular and cellular basis of microvascular perfusion deficits induced by Clostridium perfringens and Clostridium septicum. PLoS Pathog. 2008, 4: e1000045-10.1371/journal.ppat.1000045.PubMed CentralView ArticlePubMedGoogle Scholar
- Flanagan JJ, Tweten RK, Johnson AE, Heuck AP: Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding. Biochemistry. 2009, 48: 3977-3987. 10.1021/bi9002309.PubMed CentralView ArticlePubMedGoogle Scholar
- Moe PC, Heuck AP: Phospholipid hydrolysis caused by Clostridium perfringens alpha-toxin facilitates the targeting of perfringolysin O to membrane bilayers. Biochemistry. 2010, 49: 9498-9507. 10.1021/bi1013886.View ArticlePubMedGoogle Scholar
- Stevens DL, Tweten RK, Awad MM, Rood JI, Bryant AE: Clostridial gas gangrene: evidence that alpha and theta toxins differentially modulate the immune response and induce acute tissue necrosis. J Infect Dis. 1997, 176: 189-195. 10.1086/514022.View ArticlePubMedGoogle Scholar
- Asmuth DM, Olson RD, Hackett SP, Bryant AE, Tweten RK, Tso JY, Zollman T, Stevens DL: Effects of Clostridium perfringens recombinant and crude phospholipase C and theta-toxin on rabbit hemodynamic parameters. J Infect Dis. 1995, 172: 1317-1323. 10.1093/infdis/172.5.1317.View ArticlePubMedGoogle Scholar
- Stevens DL, Bryant AE: The role of clostridial toxins in the pathogenesis of gas gangrene. Clin Infect Dis. 2002, 35: S93-S100. 10.1086/341928.View ArticlePubMedGoogle Scholar
- Drew MD, Syed NA, Goldade BG, Laarveld B, Van Kessel AG: Effects of dietary protein source and level on intestinal populations of Clostridium perfringens in broiler chickens. Poult Sci. 2004, 83: 414-420.View ArticlePubMedGoogle Scholar
- Van Immerseel F, De Buck J, Pasmans F, Huyghebaert G, Haesebrouck F, Ducatelle R: Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathol. 2004, 33: 537-549. 10.1080/03079450400013162.View ArticlePubMedGoogle Scholar
- Lindsay DS, Dubey JP, Fayer R: Extraintestinal stages of Eimeria bovis in calves and attempts to induce relapse of clinical disease. Vet Parasitol. 1990, 36: 1-9. 10.1016/0304-4017(90)90088-S.View ArticlePubMedGoogle Scholar
- Chase CC, Hurley DJ, Reber AJ: Neonatal immune development in the calf and its impact on vaccine response. Vet Clin North Am Food Anim Pract. 2008, 24: 87-104. 10.1016/j.cvfa.2007.11.001.View ArticlePubMedGoogle Scholar
- Wei S, Gong Z, Che T, Guli A, Tian F: Genotyping of calves rotavirus in China by reverse transcription polymerase chain reaction. J Virol Methods. 2013, 189: 36-40. 10.1016/j.jviromet.2013.01.002.View ArticlePubMedGoogle Scholar
- Boyd MJ, Logan MA, Tytell AA: The growth requirements of Clostridium perfringens (welchii) BP6K. J Biol Chem. 1948, 174: 1013-1025.PubMedGoogle Scholar
- Fuchs AR, Bonde GJ: The nutritional requirements of Clostridium perfringens. J Gen Microbiol. 1957, 16: 317-329. 10.1099/00221287-16-2-317.View ArticlePubMedGoogle Scholar
- Barratt ME, Strachan PJ, Porter P: Antibody mechanisms implicated in digestive disturbances following ingestion of soya protein in calves and piglets. Clin Exp Immunol. 1978, 31: 305-312.PubMed CentralPubMedGoogle Scholar
- Kilshaw PJ, Slade H: Passage of ingested protein into the blood during gastrointestinal hypersensitivity reactions: experiments in the preruminant calf. Clin Exp Immunol. 1980, 41: 575-582.PubMed CentralPubMedGoogle Scholar
- Silva AG, Huber JT, Herdt TH, Holland R, Degregorio RM, Mullaney TP: Morphological alterations of small intestinal epithelium of calves caused by feeding soybean protein. J Dairy Sci. 1986, 69: 1387-1393. 10.3168/jds.S0022-0302(86)80545-8.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.