Leen Timbermont

Quantification of gut lesions in a subclinical necrotic enteritis model

Currently Clostridium perfringens-induced necrotic enteritis is a major problem in broiler flocks. In the present study, broilers were inoculated with a combination of Eimeria maxima or overdose coccidial vaccine (one inoculation) with C. perfringens (repeated inoculations). Single C. perfringens, E. maxima or an overdose of live coccidial vaccine inoculations did not result in grossly visible necrotic gut lesions, while combined inoculation resulted in typical necrotic lesions at approximately 4 days after inoculations with C. perfringens in approximately one-half of the inoculated animals. Semi-quantitative histological lesion scoring was done to evaluate gut damage in gut sections of animals in which no gross necrotic lesions were detected. This included scoring of hyperaemia, haemorrhages, the amount of red blood cells and protein precipitate in the lumen, villus fusion and epithelial defects. The villus length and the villus length/crypt depth ratio were also analysed. This approach proved to be discriminative between single E. maxima infection, overdose of coccidial vaccine or C. perfringens inoculations and the non-inoculated control group, and between the double-inoculated and single-inoculated groups. In general, the highest histological scores for gut lesions were observed in the double-inoculated groups, but the single-inoculated groups had higher scores than the control group. It was concluded that oral inoculation of broilers with an overdose of live coccidial vaccine in combination with multiple oral C. perfringens inoculations is a suitable model for necrotic enteritis without inducing mortality of the animals. C. perfringens and Eimeria act synergistically in inducing grossly visible gut damage.

Control of Clostridium perfringens-induced necrotic enteritis in broilers by target-released butyric acid, fatty acids and essential oils

The efficacy of target-released butyric acid, medium-chain fatty acids (C6 to C12 but mainly lauric acid) and essential oils (thymol, cinnamaldehyde, essential oil of eucalyptus) micro-encapsulated in a poly-sugar matrix to control necrotic enteritis was investigated. The minimal inhibitory concentrations of the different additives were determined in vitro, showing that lauric acid, thymol, and cinnamaldehyde are very effective in inhibiting the growth of Clostridium perfringens. The in vivo effects were studied in two trials in an experimental necrotic enteritis model in broiler chickens. In the first trial, four groups of chickens were fed a diet supplemented with butyric acid, with essential oils, with butyric acid in combination with medium-chain fatty acids, or with butyric acid in combination with medium-chain fatty acids and essential oils. In all groups except for the group receiving only butyric acid, a significant decrease in the number of birds with necrotic lesions was found compared with the infected, untreated control group. In the second trial the same products were tested but at a higher concentration. An additional group was fed a diet supplemented with only medium-chain fatty acids. In all groups except for that receiving butyric acid in combination with medium-chain fatty acids and essential oils, a significant decrease in the number of birds with necrotic lesions was found compared with the infected, untreated control group. These results suggest that butyric acid, medium-chain fatty acids and/or essential oils may contribute to the prevention of necrotic enteritis in broilers.

Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers.

Since the ban on growth-promoting antibiotics in animal feed in the European Union, necrotic enteritis has become a major cause of mortality in broiler chickens. Despite the importance of the disease, the pathogenesis is still not completely understood. In the current study, Clostridium perfringens strains isolated from healthy flocks and isolates from outbreaks of necrotic enteritis were evaluated for the ability to cause gut necrosis in an intestinal loop model in laying hens and in an experimental infection model in broilers. High, intermediate and low alpha toxin producing strains were chosen from each isolation source. Only the isolates from field outbreaks induced necrotic gut lesions, independent of the amount of alpha toxin produced in vitro. It was also shown that alpha toxin producing isolates from calf hemorrhagic enteritis cases were not able to induce necrotic enteritis in poultry. These results suggest the presence of host specific virulence factors in C. perfringens strains, isolated from chickens with intestinal necrotic enteritis lesions.

Intra-species growth-inhibition by Clostridium perfringens is a possible virulence trait in necrotic enteritis in broilers

Necrotic enteritis in broiler chickens is associated with Clostridium perfringens type A, carrying the NetB toxin. C. perfringens type A is also a member of the normal intestinal microbiota of broilers. Clinically healthy chickens carry several different C. perfringens clones in their intestine. In flocks suffering from necrotic enteritis, however, mostly only one single clone is isolated from the gut of all the diseased animals. Selective proliferation of these clinical outbreak strains in the gut and spread within the flock seems likely, but an explanation has not yet been given. The hypothesis that necrotic enteritis associated C. perfringens strains might suppress the growth of normal microbiota C. perfringens strains, was therefore tested. Twenty-six C. perfringens strains isolated from healthy broilers and 24 clinical outbreak isolates were evaluated for their ability to induce intra-species growth-inhibition in an in vitro setup. A significantly higher proportion of the C. perfringens clinical outbreak strains inhibited the growth of other C. perfringens strains compared to C. perfringens strains isolated from the gut of healthy chickens. It is proposed that, in addition to toxin production, intra-species growth-inhibition may be a virulence trait that contributes to the ability of certain C. perfringens strains to cause necrotic enteritis in broilers.

The effect of commonly used anticoccidials and antibiotics in a subclinical necrotic enteritis model

Necrotic enteritis poses an important health risk to broilers. The ionophore anticoccidials lasalocid, salinomycin, maduramicin, narasin and a combination of narasin and nicarbazin were tested in feed for their prophylactic effect on the incidence of necrotic enteritis in a subclinical experimental infection model that uses coccidia as a predisposing factor. In addition, drinking water medication with the antibiotics amoxicillin, tylosin and lincomycin was evaluated as curative treatment in the same experimental model. The minimal inhibitory concentrations (MICs) of all antibiotics and anticoccidials were determined in vitro against 51 Clostridium perfringens strains isolated from broilers. The strains examined appeared uniformly susceptible to lasalocid, maduramicin, narasin, salinomycin, amoxicillin and tylosin, whereas an extended frequency distribution range of MICs for lincomycin was seen, indicating acquired resistance in 36 isolates in the higher range of MICs. Nicarbazin did not inhibit the in vitro growth of the C. perfringens strains even at a concentration of 128 µg/ml. Supplementation of the diet from day 1 onwards with lasalocid, salinomycin, narasin or maduramicin led to a reduction in birds with necrotic enteritis lesions as compared with the non-medicated infected control group. A combination product of narasin and nicarbazin had no significant protective effect. Treatment with amoxicillin, lincomycin and tylosin completely stopped the development of necrotic lesions.

Variable protection after vaccination of broiler chickens against necrotic enteritis using supernatants of different Clostridium perfringens strains.

Necrotic enteritis is an economically important disease of chickens caused by Clostridium perfringens. Immunity to necrotic enteritis is not fully characterized yet, but previous reports indicate that immunoprotective potential is present in the secreted component of C. perfringens. This study aimed to compare the vaccine potential of the supernatants of eight chicken strains of C. perfringens differing in origin, level of alpha toxin production and presence of netB gene. The supernatant of only one strain provided full protection, while one other strain provided partial protection against a severe infection challenge. Our results indicate that the protective characteristics of the supernatants are not solely based on the presence of NetB or alpha toxin.

Antimicrobial resistance in Clostridium perfringens isolates from broilers in Belgium

The antimicrobial susceptibility of Clostridium perfringens strains isolated from Belgian broilers between May and September 2007 was investigated. All 39 tested isolates were sensitive to enrofloxacin, erythromycin, tylosin, florfenicol and bacitracin. Twenty-six (66%) and 24 (61%) out of the 39 tested isolates showed acquired resistance to tetracycline and lincomycin, respectively. The C. perfringens isolates were also screened by PCR for the presence of the resistance genes tet(K), tet(L), tet(M), tetB(P), tet(O), tet(W), lnu(A) and lnu(B). In 22/26 tetracycline resistant strains and 7/24 lincomycin resistant strains, resistance could be attributed to one or more of these genes. An extended frequency distribution range of MICs was seen for ampicillin. These data are consistent with data derived from studies carried out in 1980 and in 2004, indicating that no changes in antimicrobial resistance patterns have taken place during time in C. perfringens isolates from broilers in Belgium.

The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

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.

Generation of Single-Copy Transposon Insertions in Clostridium perfringens by Electroporation of Phage Mu DNA Transposition Complexes

ABSTRACT Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn916-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens.

Intestinal clostridial counts have no diagnostic value in the diagnosis of enterotoxaemia in veal calves

Enterotoxaemia is an important cause of sudden death in veal calves. This study aimed to evaluate intestinal Clostridium perfringens counts as a diagnostic tool for enterotoxaemia. Field necropsies were conducted on 48 sudden death cases in Belgian Blue veal farms. In 31/48 suddenly deceased calves, the diagnosis of enterotoxaemia was made based on haemorrhagic lesions in the small intestines, while in seven of these cases, no clear-cut diagnosis could be made based on macroscopic appearance of the gut. In the 10 remaining calves, a definitive cause of death other than enterotoxaemia could be identified. Samples of the intestinal content were taken for quantification of C perfringens. After matching cases and controls for diet, and the interval between death and sampling, no significant differences could be detected between the mean C perfringens counts of the small intestines in enterotoxaemia cases and counts in the matching segments in the control group. These results indicate that intestinal C perfringens counts cannot be advised as a discriminative postmortem diagnostic tool for enterotoxaemia in veal calves, not even when sampled within three hours after death.