Evy Goossens

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.

Toxocara vitulorum in American bison (Bison bison) calves

Toxocara vitulorum is a common parasite of cattle, zebu and water buffalo in primarily tropical regions (Akyol 1993); the helminth causes high mortality in buffalo calves in South Asia (Roberts 1992, Gundran and More 1999). Following infection with embryonated eggs, adult cows do not develop patent infections, but instead larvae remain dormant in liver, muscle and lymph node tissues. One to eight days before parturition, the larvae are reactivated and migrate to the udder, where they are passed into the colostrum and milk until 10 to 18 days after parturition. Following this galactogenic transmission, patent infections with adult T vitulorum occur in the small intestines of calves younger than three months, with high burdens leading to diarrhoea, colic, intestinal obstruction, emaciation and sometimes death (Roberts 1990b). This short communication describes T vitulorum infection in farmed American bison (Bison bison) calves in Belgium. Increased mortality had been observed on the farm since 1998 in calves younger than three months. The causes of death were not identified, but adult ascarids had occasionally been observed at postmortem examination. The bison herd had been established in 1991 from animals from the USA, and since then a few animals had been imported from other European farms, mainly in France. In 2003, four herds of 33 to 43 animals were rotated on ten pastures of approximately 25 ha each. Between March and June that year, 147 births were recorded. Routine helminth control consisted of a single treatment of 200 μg/kg bodyweight ivermectin (Ivomec 1 per cent; Merial) or doramectin (Dectomax 1 per cent Injectable; Pfizer Animal Health) of all animals in December and of calves at approximately three months of age, when the calves were caught for ear tagging. The efficacy of this control programme had not been evaluated. Toxocarosis has never been described in bison herds in the USA and Canada (Haigh 1996, Dies and Coupland 2001), but anecdotal reports have mentioned the occasional findings of T vitulorum in bison in France and the UK. In Belgium, T vitulorum infections occur sporadically in cattle (Thienpont and others 1977). The farm was therefore investigated for the presence of, and morbidity due to T vitulorum infection in the bison calves. In June 2003, rectal faecal samples were collected from 82 calves from three of the herds (76 per cent of the calves), aged from 23 to 73 days (mean [sd] 45[3] days). Faecal egg counts were measured within 24 hours by a modified McMaster method with a sensitivity of 50 eggs per gram of faeces (epg) (Thienpont and others 1979). Faecal consistency (normal, loss of faecal consistency or watery diarrhoea) and body condition (good, intermediate or poor) were scored and correlated with different levels of faecal egg counts by calculating odds ratios (Thrusfield 1995). In all cases, P<0·05 was taken to indicate significance. Eggs of T vitulorum were observed in 50 of the 82 faecal samples (61·0 per cent) (Fig 1). The finding of T vitulorum eggs in calves as young as 23 days corresponds with the age of first appearance of eggs, 22·3 days, described in buffalo calves by Roberts (1990a). The mean (sd) faecal egg count was 10,144 (5250) epg. The average age corresponding with the 16 highest faecal egg counts (20 per cent of the 82 samples) was 40 (6) days, with a mean peak egg output of 47,334 (17,822) epg. This is similar to the age of peak faecal egg output in buffalo calves (35·7 [2·6] days) reported by Roberts (1990a). Thirty-five of the 50 calves that passed T vitulorum eggs in the faeces had normally formed faeces; in 12 animals there was loss of consistency of the faeces, and three calves had watery diarrhoea. One of the diarrhoeic animals was a 31-day-old calf whose faecal egg count was 135,250 epg. An odds ratio of 4·476 (95 per cent confidence interval 1·566 to 12·800) indicated a correlation between loss of faecal consistency and faecal egg counts above 2000 epg. No correlation between body condition score and egg counts could be found. Two of five calves that died before the age of 55 days were examined postmortem; no adult T vitulorum were recovered. The cause of death of these calves could not be determined. Faecal egg counts between 2000 and 30,000 epg are considered to be pathogenic in buffalo calves (Srivastava and Sharma 1981, Roberts 1990b). However, in the present study loss of faecal consistency was only marginally associated with high egg counts, no illthrift was seen among the infected bison calves, and calf mortality in 2003 was low (4·9 per cent) compared with that of the four previous years (11·0 per cent). The possibility that the moderate clinical effects of toxocarosis in bison calves observed in this study are caused by a higher resilience of bison compared with buffalo, or by management factors including routine anthelmintic treatments, cannot be ruled out. Higher resilience of the bison herd could be innate, or could be caused by a better general condition of the animals, as a result of a better plane of nutrition compared with that of buffaloes in the tropics. The routine anthelmintic treatment of the bison calves at three months old is unlikely to have had a significant effect on the transmission of T vitulorum, because most eggs are excreted between 30 and 70 days of age. In addition, a lack of efficacy of ivermectin against adult T vitulorum has been reported in buffalo calves (Hassanain and Degheidy 1990). Data on the efficacy of doramectin against T vitulorum are limited. A single treatment with pyrantel or levamisole at the age of 10 to 14 days is more appropriate, as this was shown to be highly effective for controlling the infection in water buffalo calves (Roberts 1992). In conclusion, toxocarosis was found to be common in this large bison herd in Belgium; however, the clinical consequences for the calves were minor, and toxocarosis could not be associated with calf mortality in the herd. Veterinary Record (2007) 160, 556-557

Perfringolysin O: The Underrated Clostridium perfringens Toxin?

The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis.

Rethinking the role of alpha toxin in Clostridium perfringens-associated enteric diseases : a review on bovine necro-haemorrhagic enteritis

Bovine necro-haemorrhagic enteritis is an economically important disease caused by Clostridium perfringens type A strains. The disease mainly affects calves under intensive rearing conditions and is characterized by sudden death associated with small intestinal haemorrhage, necrosis and mucosal neutrophil infiltration. The common assumption that, when causing intestinal disease, C. perfringens relies upon specific, plasmid-encoded toxins, was recently challenged by the finding that alpha toxin, which is produced by all C. perfringens strains, is essential for necro-haemorrhagic enteritis. In addition to alpha toxin, other C. perfringens toxins and/or enzymes might contribute to the pathogenesis of necro-haemorrhagic enteritis. These additional virulence factors might contribute to breakdown of the protective mucus layer during initial stage of pathogenesis, after which alpha toxin, either or not in synergy with other toxins such as perfringolysin O, can act on the mucosal tissue. Furthermore, alpha toxin alone does not cause intestinal necrosis, indicating that other virulence factors might be needed to cause the extensive tissue necrosis observed in necro-haemorrhagic enteritis. This review summarizes recent research that has increased our understanding of the pathogenesis of bovine necro-haemorrhagic enteritis and provides information that is indispensable for the development of novel control strategies, including vaccines.

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.

The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis

Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens and leads to sudden death. Alpha toxin, together with perfringolysin O, has been identified as the principal toxin involved in the pathogenesis. We assessed the potential of alpha toxin as a vaccine antigen. Using an intestinal loop model in calves, we investigated the protection afforded by antisera raised against native alpha toxin or its non-toxic C-terminal fragment against C. perfringens-induced intestinal necrosis. Immunization of calves with either of the vaccine preparations induced a strong antibody response. The resulting antisera were able to neutralize the alpha toxin activity and the C. perfringens-induced endothelial cytotoxicity in vitro. The antisera raised against the native toxin had a stronger neutralizing activity than those against the C-terminal fragment. However, antibodies against alpha toxin alone were not sufficient to completely neutralize the C. perfringens-induced necrosis in the intestinal loop model. The development of a multivalent vaccine combining the C-terminal fragment of alpha toxin with other C. perfringens virulence factors might be necessary for complete protection against bovine necrohemorrhagic enteritis.

Development of a quality of meals and meal service set of indicators for residential facilities for elderly

ObjectivesTo develop a content validated set of indicators to evaluate the quality of meals and meal service in residential facilities for elderly. Inadequate food intake is an important risk factor for malnutrition in residential facilities for elderly. Through better meeting the needs and preferences of residents and optimization of meals and meal service, residents’ food intake can improve. No indicators were available which could help to guide strategies to improve the quality of meals and meal service.DesignThe indicator set was developed according to the Indicator Development Manual of the Dutch Institute for Health Care Improvement (CBO). The working group consisted of three nurse researchers and one expert in gastrology and had expertise in elderly care, malnutrition, indicator development, and food quality. A preliminary list of potential indicators was compiled using the literature and the working group’s expertise. Criteria necessary to measure the indicator in practice were developed for each potential indicator. In a double Delphi procedure, the list of potential indicators and respective criteria were analyzed for content validity, using a multidisciplinary expert panel of 11 experts in elderly meal care.ResultsA preliminary list of 20 quality indicators, including 45 criteria, was submitted to the expert panel in a double Delphi procedure. After the second Delphi round, 13 indicators and 25 criteria were accepted as having content validity. The content validity index (CVI) ranged from 0.83 to 1. The indicator set consisted of six structural, four result, and three outcome indicators covering the quality domains food, service and choice, as well as nutritional screening. The criteria measure diverse aspects of meal care which are part of the responsibility of kitchen staff and health care professionals.ConclusionThe ‘quality of meals and meal service’ set of indicators is a resource to map meal quality in residential facilities for elderly. As soon as feasibility tests in practice are completed, the indicator set can be used to guide meal and meal service quality improvement projects in collaboration with kitchen staff and health care professionals. These improvement projects will help to improve food intake and reduce the risk of malnutrition among elders living in residential facilities.

Clostridium perfringens strains from bovine enterotoxemia cases are not superior in in vitro production of alpha toxin, perfringolysin O and proteolytic enzymes

BackgroundBovine enterotoxemia is a major cause of mortality in veal calves. Predominantly veal calves of beef cattle breeds are affected and losses due to enterotoxemia may account for up to 20% of total mortality. Clostridium perfringens type A is considered to be the causative agent. Recently, alpha toxin and perfringolysin O have been proposed to play an essential role in the development of disease. However, other potential virulence factors also may play a role in the pathogenesis of bovine enterotoxemia. The aim of this study was to evaluate whether strains originating from bovine enterotoxemia cases were superior in in vitro production of virulence factors (alpha toxin, perfringolysin O, mucinase, collagenase) that are potentially involved in enterotoxemia. To approach this, a collection of strains originating from enterotoxemia cases was compared to bovine strains isolated from healthy animals and to strains isolated from other animal species.ResultsStrains originating from bovine enterotoxemia cases produced variable levels of alpha toxin and perfringolysin O that were not significantly different from levels produced by strains isolated from healthy calves and other animal species. All tested strains exhibited similar mucinolytic activity independent of the isolation source. A high variability in collagenase activity between strains could be observed, and no higher collagenase levels were produced in vitro by strains isolated from enterotoxemia cases.ConclusionsBovine enterotoxemia strains do not produce higher levels of alpha toxin, perfringolysin O, mucinase and collagenase, as compared to strains derived from healthy calves and other animal species in vitro.

Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis

BackgroundBovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.ResultsAlpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model.ConclusionAlthough immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.

Prevalence and bacterial colonisation of fundic ulcerations in veal calves

The European veal industry is specialised in raising surplus dairy calves on a low iron milk diet to obtain white meat. Next to Holstein-Friesian (HF) dairy calves, also beef calves of local breeds, such as the Belgian Blue (BB) breed in Belgium, are raised as white veal calves. Marked differences in milk diet exist between these breeds, especially considering the quality of the protein (Pardon and others 2012). In a recent survey on mortality in veal calves in Belgium, perforating abomasal ulcerations, either fundic or pyloric, accounted for 3 per cent of mortality, with marked differences between HF (6.4 per cent) and BB (1.2 per cent) (Pardon and others 2012). Fatal ulcerations are only the tip of the iceberg, revealing a larger underlying health and welfare issue of non-fatal ulcerations. A recent study determined the within-herd prevalence of pyloric ulcerations in The Netherlands, France and Italy at 74.1 per cent on average, ranging from 31.7 per cent to 100 per cent (Brscic and others 2011), but no information on fundic ulcerations was mentioned. The only study on the prevalence of fundic ulcerations in the European veal production system is a Swiss study in a veal production system with exceptionally high welfare standards, resulting in a total of 23 per cent of fundic lesions in the standard production system, and 8 per cent in the naturafarm production system (Bahler and others 2010). In addition …