Emergence of suspected type D botulism in ruminants in England and Wales (2001-2009), associated with exposure to broiler litter.

Scanning surveillance by the Veterinary Laboratories Agency revealed the emergence of suspected botulism in ruminants in 2003, presented as flaccid paralysis. From 2003 to 2009, 168 cattle and 19 sheep incidents were recorded, with mortality between 5 and 80 per cent. All sheep incidents and 95 per cent of cattle incidents had proximity to broiler litter. From July 2006, the gut contents collected from 74 affected cattle and 10 affected sheep were tested for Clostridium botulinum toxins using mice bioassays and for organisms by culture. Type D toxin was identified in 32 per cent of cattle and 18 per cent of sheep samples. C botulinum type D organisms were identified in 40 per cent of cattle and 30 per cent of sheep samples, but broth from one sample reacted with C and D antisera. Type C botulism has previously been reported more commonly than type D in the UK and has been associated with the use of poultry litter as fertiliser, bedding or feed. The almost exclusive association with C botulinum type D toxins or organisms in the gut contents in this survey suggests a change in the source or epidemiology of botulism in the UK. The source of C botulinum type D was uncertain. Broilers may carry C botulinum type D in their gut flora subclinically. The emergence of a new type D strain, or changes in broiler husbandry and nutrition, medication and other enteric infections may have affected colonisation with C botulinum. Further investigation of poultry and farm environments for sources of type D awaits the development of tests for C botulinum toxins that do not require the use of mic

Review of the potential health impact of β-casomorphins and related peptides: Report of the DATEX working group on β-casomorphins

Proteins are a very diverse family of large organic compounds involved in many important biological processes. Following their enzymatic hydrolysis during food processing or digestion, proteins may release fragments from their primary amino acid sequence. These fragments are called peptides, and many of them are known to be physiologically active. The possible beneficial effects of bioactive peptides have attracted increasing interest in recent years. On the other hand, there are also reports suggesting that some food-derived peptides might adversely affect human health. Among these, \u392-casomorphin-7 (BCM7), a peptide sequence present in the milk protein \u392-casein, has been suggested to contribute to an increased risk for certain non-communicable diseases, such as autism, cardiovascular diseases and type I diabetes. Some literature reports have proposed possible mechanistic explanations for such associations Recognising the alleged negative effect of BCM7 on human health, EFSA deemed it necessary to perform a comprehensive review of the published scientific literature in order to assess the relationship of this peptide and related peptides with non-communicable diseases. The review covers the following aspects: possible sources of \u392-casomorphins (BCMs) and related peptides, polymorphism of \u392-casein, presence of BCMs and related peptides in food before digestion, formation of BCM7 and related peptides during human digestion and the possible molecular interactions of these peptides with the host environment. Furthermore, it covers the absorption and fate of these peptides, including their possible transfer mechanisms across the intestinal epithelium, transport in the blood stream and transfer across the bloodbrain barrier. Finally, possible and suggested organ- and system-specific effects are reviewed, with specific emphasis on the gastrointestinal, central nervous and cardiovascular systems and the suggested link with type 1 diabetes mellitus. This review recognises that proteins, including those present in the diet, are a potential source of a wide range of biologically active peptides, including some with affinity to opioid receptors. The latter are also known as opioid peptides. Opioid peptide sequences have been characterised in animal and plant proteins. To date much work has focused on characterising opioid peptides derived from milk proteins, in particular the caseins. Beta-casomorphins are a group of opioid peptides which can be released from \u392-casein. The \u392-casein derived peptide with the sequence Tyr60-Pro61-Phe62-Pro63-Gly64-Pro65-Ile66 is known as \u392-casomorphin-7. The release of BCM7 through enzymatic digestion of bovine \u392-casein is dictated by different amino acid sequences of this protein. The sequences vary genetically between cow breeds. The amino acid present in position 67 of the sequence in \u392-casein appears to be critical for the release of BCM7. In the A2 variant of \u392-casein a Proline residue occurs at position 67, whereas the A1 and B variants of \u392-casein have a Histidine residue at this position. In the case of the variants containing Proline the enzymatic hydrolysis of the Ile66-Pro67 bond does not occur or occurs at a very low rate. The proportion of the different protein variants expressed in the milk, including those of \u392- casein, is related to their allele distributions in the various cattle breeds and populations. Changing selection targets in the last decades has resulted in changes in bovine breed composition in most European countries. While no detailed information is available, it is likely that these changes in breed composition have had an impact on milk composition, including protein variants. Taking into account the lack of detailed knowledge in milk protein variant composition and the diverse geographical origin of dairy products and ingredients across Europe, insufficient information is currently available on the exposure of individual consumers to different \u392-casein variants. It would appear that fresh raw/unprocessed milk obtained from healthy cows does not contain BCM7 or related peptides. By contrast, there is a substantial body of evidence indicating that different proteolytic systems involved in fermented milk or cheese manufacture can potentially hydrolyze \u392-casein to BCM7 or other BCMs and further degrade these peptides to shorter-chain peptides and even amino acids. However, little or no information is currently available about the actual BCM levels which different proteolytic systems may generate in fermented or enzyme-treated milk products. Furthermore, the stability of these peptides in the food products, once generated, is variable. Technological conditions, such as heat treatments, applied in industrial dairy processing do not seem to influence the occurrence of BCMs in final products or influence their formation during subsequent digestion. Moreover, limited information is available on the occurrence of BCMs in commercial infant formulas. The role of proteolytic systems in the release of BCMs during simulated gastrointestinal digestion (SGID) or in vivo digestion, has not been fully clarified. No current studies report quantitative values for the formation of BCMs during in vivo digestion of dairy products. However, there are indications that the sequential action of several digestive enzymes may be involved and the formation of certain BCMs after SGID (with multiple enzyme activities) has been demonstrated. Animal data clearly indicate that BCMs, including BCM7, can act as opioid receptor agonists, probably acting via \ub5-type opioid receptors. However, in most if not all animal studies to date, in vivo opioid effects for BCM7 and related milk-derived peptides have only been observed following intra-peritoneal (i.p.) or intra-cerebro-ventricular (i.c.v.) administration. In comparison to medicinal and endogenous opioids, bovine BCM7 does not seem to be a very potent opioid ligand. A prerequisite for opioid activity after oral ingestion is that the peptides must pass the intestinal epithelial barrier. In addition, subsequent biotransformation in the liver and stability in plasma may be factors determining the ultimate biological activity. Finally, passage through the blood-brain-barrier is in principle needed for an activity in the central nervous system. Relatively little is known on the mechanisms of transfer of intact peptides longer than 3 amino acids across the intestinal barrier. If this transport occurs, then the extent is very low and passive diffusion is the most likely transfer mechanism. The presence of \u392-casomorphin immunoreactive material has been reported in blood in two studies with neonatal dogs and calves. However, the presence of intact \u392-casomorphin molecules in blood after intake of milk or casein has not been established in in vivo studies. Opioid peptides, including \u392- casomorphin 4, -5 and -7 are highly sensitive to hydrolysis by dipeptidyl peptidase IV thereby strongly limiting or preventing the transfer of these peptides in an intact form across the intestinal mucosa and the blood-brain barrier. Available data suggest that in principle, transport of food-derived peptides and proteins across the human intestinal mucosa is possible. However, quantitative data on this phenomenon are lacking. In certain cases such as in neonates and adults with specific diseases, intestinal permeability has been reported to be significantly increased. In general, the review did not find any quantitative data on the absorption of intact bioactive peptides for adults, except in the case for di- and tripeptides with reported antihypertensive properties. Food-derived peptides, including casomorphins, can have different effects in the intestinal lumen and the intestinal mucosa, such as regulatory effects on gastro-intestinal motility and on gastric and pancreatic secretions. Many studies report effects of \u392-casomorphins on the central nervous system (CNS) following i.p. or i.c.v. administration in animals. A possible link between BCM intake and sudden infant death syndrome (SIDS) has been suggested in some publications. However, no clear evidence for such a relationship was found during the review. The mechanisms proposed were considered rather speculative and partly contradictory. Similarly, a link between casein-derived peptides and autism in subjects with increased intestinal permeability has been suggested. However, recent data do not provide any support for such a relationship. It has been suggested that BCM7 might be atherogenic through an oxidative action on LDL. This mechanism was originally proposed in a single report; however, it has not been confirmed by later studies. By contrast, numerous in vitro studies indicate that many food derived peptides/hydrolysates display antioxidant activity. The possibility that BCM7 could contribute to an increased risk for atherosclerosis has also been suggested from a study in a rabbit model. This study concluded that \u392-casein A1 would be atherogenic, in contrast to \u392- casein A2. However, during the review the validity of the experimental model and the extrapolation to atherosclerosis in humans were not regarded as convincing. Further speculations for an association between BCM7 intake and cardiovascular disease mortality have been raised as a result of ecological studies. However, these ecological studies did not account for several confounding factors. In addition, recent large cohort studies led to opposite conclusions. Two human intervention studies comparing diets containing \u392-casein A1 and A2 did not show a correlation between the estimated \u392-casein A1 consumption and development of certain biomarkers for cardiovascular disease (CVD). A limitation of these studies was the small number of subjects and the short intervention period. Overall, this review process did not find any strong evidence for a link between the consumption of \u392- casein A1 and an increased risk for CVD in humans. Insulin dependent diabetes mellitus (IDDM) is recognised as a multifactorial autoimmune disease; however, its pathogenesis is unclear. Autoantibodies have been found in IDDM patients, but the role of autoantibodies in type 1 diabetes is currently not known. These autoantibodies have not been proven to be directly involved in either tissue destruction or disease progression. The development of IDDM is the result of a combination of genetic predisposition and environmental risk factors where genetic predisposition is a necessary condition. Many dietary risk factors of IDDM have been indicated, including short duration of breast feeding, as well as administration of gluten, soy, cow's milk and solid foods at young age. Several different milk proteins or peptides derived from these proteins have been proposed as possible diabetogenic factors. The mechanism most often suggested is immunological. The diabetogenicity of \u392-casein A1, A2 and B has been evaluated in animal studies and ecological studies on humans. Animal studies have presented contradictory results. Some ecological studies have linked the intake of BCM7 with IDDM. Ecological studies have the shortcoming of being unable to establish a cause-effect association and they cannot adjust for possible confounding factors. They are at best indicating a hypothesis but cannot provide a proper base to demonstrate a cause-effect relationship. The correlations suggested by such studies may become very weak when taking into account the uncertainties in individual consumption, in the \u392-casein variant composition, and in some countries also the IDDM incidence rate. Moreover, the difference in content of \u392-casein A1+B in milk produced in countries with high or low prevalence of IDDM appears relatively small and does not explain, from an immunological point of view, the difference in incidence of IDDM across countries. Based on the present review of available scientific literature, a cause-effect relationship between the oral intake of BCM7 or related peptides and aetiology or course of any suggested non-communicable diseases cannot be established. Consequently, a formal EFSA risk assessment of food-derived peptides is not recommended

Scientific Report of EFSA prepared by a DATEX Working Group on the potential health impact of \u3b2-casomorphins and related peptides

Proteins are a very diverse family of large organic compounds involved in many important biological processes. Following their enzymatic hydrolysis during food processing or digestion, proteins may release fragments from their primary amino acid sequence. These fragments are called peptides, and many of them are known to be physiologically active. The possible beneficial effects of bioactive peptides have attracted increasing interest in recent years. On the other hand, there are also reports suggesting that some food-derived peptides might adversely affect human health. Among these, \u3b2-casomorphin-7 (BCM7), a peptide sequence present in the milk protein \u3b2-casein, has been suggested to contribute to an increased risk for certain non-communicable diseases, such as autism, cardiovascular diseases and type I diabetes. Some literature reports have proposed possible mechanistic explanations for such associations Recognising the alleged negative effect of BCM7 on human health, EFSA deemed it necessary to perform a comprehensive review of the published scientific literature in order to assess the relationship of this peptide and related peptides with non-communicable diseases. The review covers the following aspects: possible sources of \u3b2-casomorphins (BCMs) and related peptides, polymorphism of \u3b2-casein, presence of BCMs and related peptides in food before digestion, formation of BCM7 and related peptides during human digestion and the possible molecular interactions of these peptides with the host environment. Furthermore, it covers the absorption and fate of these peptides, including their possible transfer mechanisms across the intestinal epithelium, transport in the blood stream and transfer across the bloodbrain barrier. Finally, possible and suggested organ- and system-specific effects are reviewed, with specific emphasis on the gastrointestinal, central nervous and cardiovascular systems and the suggested link with type 1 diabetes mellitus. This review recognises that proteins, including those present in the diet, are a potential source of a wide range of biologically active peptides, including some with affinity to opioid receptors. The latter are also known as opioid peptides. Opioid peptide sequences have been characterised in animal and plant proteins. To date much work has focused on characterising opioid peptides derived from milk proteins, in particular the caseins. Beta-casomorphins are a group of opioid peptides which can be released from \u3b2-casein. The \u3b2-casein derived peptide with the sequence Tyr60-Pro61-Phe62-Pro63-Gly64-Pro65-Ile66 is known as \u3b2-casomorphin-7. The release of BCM7 through enzymatic digestion of bovine \u3b2-casein is dictated by different amino acid sequences of this protein. The sequences vary genetically between cow breeds. The amino acid present in position 67 of the sequence in \u3b2-casein appears to be critical for the release of BCM7. In the A2 variant of \u3b2-casein a Proline residue occurs at position 67, whereas the A1 and B variants of \u3b2-casein have a Histidine residue at this position. In the case of the variants containing Proline the enzymatic hydrolysis of the Ile66-Pro67 bond does not occur or occurs at a very low rate. The proportion of the different protein variants expressed in the milk, including those of \u3b2- casein, is related to their allele distributions in the various cattle breeds and populations. Changing selection targets in the last decades has resulted in changes in bovine breedcomposition in most European countries. While no detailed information is available, it is likely that these changes in breed composition have had an impact on milk composition, including protein variants. Taking into account the lack of detailed knowledge in milk protein variant composition and the diverse geographical origin of dairy products and ingredients across Europe, insufficient information is currently available on the exposure of individual consumers to different \u3b2-casein variants. It would appear that fresh raw/unprocessed milk obtained from healthy cows does not contain BCM7 or related peptides. By contrast, there is a substantial body of evidence indicating that different proteolytic systems involved in fermented milk or cheese manufacture can potentially hydrolyze \u3b2-casein to BCM7 or other BCMs and further degrade these peptides to shorter-chain peptides and even amino acids. However, little or no information is currently available about the actual BCM levels which different proteolytic systems may generate in fermented or enzyme-treated milk products. Furthermore, the stability of these peptides in the food products, once generated, is variable. Technological conditions, such as heat treatments, applied in industrial dairy processing do not seem to influence the occurrence of BCMs in final products or influence their formation during subsequent digestion. Moreover, limited information is available on the occurrence of BCMs in commercial infant formulas. The role of proteolytic systems in the release of BCMs during simulated gastrointestinal digestion (SGID) or in vivo digestion, has not been fully clarified. No current studies report quantitative values for the formation of BCMs during in vivo digestion of dairy products. However, there are indications that the sequential action of several digestive enzymes may be involved and the formation of certain BCMs after SGID (with multiple enzyme activities) has been demonstrated. Animal data clearly indicate that BCMs, including BCM7, can act as opioid receptor agonists, probably acting via \u3bc-type opioid receptors. However, in most if not all animal studies to date, in vivo opioid effects for BCM7 and related milk-derived peptides have only been observed following intra-peritoneal (i.p.) or intra-cerebro-ventricular (i.c.v.) administration. In comparison to medicinal and endogenous opioids, bovine BCM7 does not seem to be a very potent opioid ligand. A prerequisite for opioid activity after oral ingestion is that the peptides must pass the intestinal epithelial barrier. In addition, subsequent biotransformation in the liver and stability in plasma may be factors determining the ultimate biological activity. Finally, passage through the blood-brain-barrier is in principle needed for an activity in the central nervous system. Relatively little is known on the mechanisms of transfer of intact peptides longer than 3 amino acids across the intestinal barrier. If this transport occurs, then the extent is very low and passive diffusion is the most likely transfer mechanism. The presence of \u3b2-casomorphin immunoreactive material has been reported in blood in two studies with neonatal dogs and calves. However, the presence of intact \u3b2-casomorphin molecules in blood after intake of milk or casein has not been established in in vivo studies. Opioid peptides, including \u3b2- casomorphin 4, -5 and -7 are highly sensitive to hydrolysis by dipeptidyl peptidase IV thereby strongly limiting or preventing the transfer of these peptides in an intact form across the intestinal mucosa and the blood-brain barrier. Available data suggest that in principle, transport of food-derived peptides and proteins across the human intestinal mucosa is possible. However, quantitative data on this phenomenon are lacking. In certain cases such as in neonates and adults with specific diseases, intestinal permeability has been reported to be significantly increased. In general, the review did not find any quantitative data on the absorption of intact bioactive peptides for adults, except in the case for di- and tripeptides with reported antihypertensive properties. Food-derived peptides, including casomorphins, can have different effects in the intestinal lumen and the intestinal mucosa, such as regulatory effects on gastro-intestinal motility and on gastric and pancreatic secretions. Many studies report effects of \u3b2-casomorphins on the central nervous system (CNS) following i.p. or i.c.v. administration in animals. A possible link between BCM intake and sudden infant death syndrome (SIDS) has been suggested in some publications. However, no clear evidence for such a relationship was found during the review. The mechanisms proposed were considered rather speculative and partly contradictory. Similarly, a link between casein-derived peptides and autism in subjects with increased intestinal permeability has been suggested. However, recent data do not provide any support for such a relationship. It has been suggested that BCM7 might be atherogenic through an oxidative action on LDL. This mechanism was originally proposed in a single report; however, it has not been confirmed by later studies. By contrast, numerous in vitro studies indicate that many food derived peptides/hydrolysates display antioxidant activity. The possibility that BCM7 could contribute to an increased risk for atherosclerosis has also been suggested from a study in a rabbit model. This study concluded that \u3b2-casein A1 would be atherogenic, in contrast to \u3b2- casein A2. However, during the review the validity of the experimental model and the extrapolation to atherosclerosis in humans were not regarded as convincing. Further speculations for an association between BCM7 intake and cardiovascular disease mortality have been raised as a result of ecological studies. However, these ecological studies did not account for several confounding factors. In addition, recent large cohort studies led to opposite conclusions. Two human intervention studies comparing diets containing \u3b2-casein A1 and A2 did not show a correlation between the estimated \u3b2-casein A1 consumption and development of certain biomarkers for cardiovascular disease (CVD). A limitation of these studies was the small number of subjects and the short intervention period. Overall, this review process did not find any strong evidence for a link between the consumption of \u3b2- casein A1 and an increased risk for CVD in humans. Insulin dependent diabetes mellitus (IDDM) is recognised as a multifactorial autoimmune disease; however, its pathogenesis is unclear. Autoantibodies have been found in IDDM patients, but the role of autoantibodies in type 1 diabetes is currently not known. These autoantibodies have not been proven to be directly involved in either tissue destruction or disease progression. The development of IDDM is the result of a combination of genetic predisposition and environmental risk factors where genetic predisposition is a necessary condition. Many dietary risk factors of IDDM have been indicated, including short duration of breast feeding, as well as administration of gluten, soy, cow\u2019s milk and solid foods at young age.Several different milk proteins or peptides derived from these proteins have been proposed as possible diabetogenic factors. The mechanism most often suggested is immunological. The diabetogenicity of \u3b2-casein A1, A2 and B has been evaluated in animal studies and ecological studies on humans. Animal studies have presented contradictory results. Some ecological studies have linked the intake of BCM7 with IDDM. Ecological studies have the shortcoming of being unable to establish a cause-effect association and they cannot adjust for possible confounding factors. They are at best indicating a hypothesis but cannot provide a proper base to demonstrate a cause-effect relationship. The correlations suggested by such studies may become very weak when taking into account the uncertainties in individual consumption, in the \u3b2-casein variant composition, and in some countries also the IDDM incidence rate. Moreover, the difference in content of \u3b2-casein A1+B in milk produced in countries with high or low prevalence of IDDM appears relatively small and does not explain, from an immunological point of view, the difference in incidence of IDDM across countries. Based on the present review of available scientific literature, a cause-effect relationship between the oral intake of BCM7 or related peptides and aetiology or course of any suggested non-communicable diseases cannot be established. Consequently, a formal EFSA risk assessment of food-derived peptides is not recommended