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Intestinal Integrity and the Impact of Losing It
Frederic J. Hoerr, DVM, PhD
State of Alabama
The digestive tract is a tube lined by specialized epithelial cells that are continuous with the epithelial layers covering the skin. Thus, the digestive tract is open to the external environment and to potential exposure to organisms and toxic agents that are introduced by ingestion. Along the length of the tract, the epithelial cells differentiate into a variety of cells with special functions that include the secretion of various fluids, electrolytes, enzymes, and in the gizzard, physical disruption of particulate digesta. The cells form a semipermiable surface that selectively allows passage of fluid, electrolytes, and dissolved nutrients. Regardless of its specialized function, every epithelial cell in the digestive tract is part of a continuous physical barrier to protect against the entry of foreign materials and organisms into the bloodstream and gaining access to other viscera. The integrity of this protective barrier is broken when organisms and toxic agents damage epithelial cells. This epithelial lining continually sheds cells into the center of the digestive tube (lumen) with ongoing regeneration of new cells that will differentiate to assume the functions of those lost. The surface area of intestinal lining (mucosa) is greatly expanded from that in a simple tube due to extensive microscopic folding to form a carpet of somewhat flattened, finger-like projections called villi. In the avian gut, villi exist throughout the length of the small and large intestine, steadily decreasing in height along the way. The luminal surface of each villus is, in turn, increased by many microvilli to facilitate absorption on the surface of the cells.
Each villus is lined with epithelial cells (enterocytes) that are differentiated according to location on the villus to absorb fluids and nutrients (tip), secrete electrolytes and fluids (side and crypt), and to regenerate and replace damaged cells or those lost to normal attrition (crypt).
Mucus and Fluid Secretion
Mucus that is secreted onto the epithelial surface lubricates movement of digesta along the digestive tract. It is secreted by specialized epithelial cells arranged into glands in the mouth and esophagus, and by individual goblet cells in the proventriculus and intestine. Mucus is not secreted in the crop or gizzard, however, digesta arrives in those organs softened and lubricated by the previous upstream site. Mucus is a viscous material composed of water and glycoprotein. It protects the mucosal cells in the stomach and intestine from autodigestion by gastric acid, pepsin and other digestive enzymes. The protective effect of mucus is further evidenced by increased secretion on the mucosal surface and goblet cell hypertrophy in response to noxious stimuli. Mucus is one of the barriers to bacterial and fungal invasion. Virulent strains of Candida albicans, the agent of thrush, have a mucinolytic enzyme that dissolves the mucin barrier to enhance adherence to and penetration of epithelial cells.
Helicobacter pylori, the agent causing gastric ulcers in humans, secretes urease that breaks down the protective layer of gastric mucus. Poultry feeds with underheated soy meal may contain urease. In addition to mucus, the gut secretes large amounts of water mixed with electrolytes. It is estimated that for every gram of food ingested, the gut secretes about 2 grams of water that facilitates digestion and absorption. The excessive water in the lumen is reabsorbed in the lower small intestine, cecum, and colon. The fluid in the upper small intestine, however, is protective in that it keeps bacteria in suspension and washes them downstream.
The Lamina Propria
The epithelial lining of the gut is supported by the lamina propria, which contains the connective tissue that underlies the specialized surfaces, the vascular and lymphatic channels, and the immune system, or gut-associated lymphoid tissue (GALT). The blood vessels going into and away from the tip of the villus form a countercurrent mechanism that creates a hyperosmolar condition to facilitate absorption of fluid. Throughout the gut, the rich vascular supply serves to rapidly dilute and carry away any agents or chemicals (endogenous or exogenous) that may breach the mucosal barrier.
Agents that directly damage the components of blood vessels can interfere with intestinal integrity by causing ischemic injury to the mucosa (infarction), or leakage of blood from the vascular channel (hemorrhage). Virulent forms of Newcastle disease and avian influenza, invasive candidiasis, coccidiosis caused by Eimeria tenella, and the more pathogenic forms of salmonellosis are examples of diseases that can injure the gut vascular system.
GALT may collectively represent the largest secondary immunological organ in the body. In the chicken, the immunological tissues are distributed in specific sites at the junction of the proventriculus and gizzard, and cecal tonsils; at concentrated ectopic locations; diffusely scattered through lamina propria; and as scattered individual intraepithelial lymphocytes. GALT is composed of B and T lymphocytes, plasma cells, macrophages (mobile cells that engulf foreign materials and infectious agents), and dendritic cells (resident, antigen-processing macrophages). One important function of GALT is the secretion of secretory antibody (IgA) onto the mucosal surface in response to the heavy exposure of the gut to foreign antigens on infectious agents and ingesta.
Since the gut is continuously and heavily exposed to foreign materials, the lamina propria is quite active due to the responsiveness of GALT, and is actually in a normal state of reactivity and mild inflammation. The transition from normal, mild inflammation to subclinical disease is an important consideration in poultry, not only from an economic standpoint, but also as a predecessor to fatal enteric disease. Multiple insults and disease mechanisms may occur simultaneously in production environments.
Infectious bursal disease, chicken infectious anemia, Mareks disease, and hemorrhagic enteritis of turkeys potentially impact secondary lymphoid tissues of chickens and turkeys. It is common to have clinical coccidiosis in broilers during and immediately following the onset of acute bursal disease. Lymphocytolytic mycotoxins such as T-2 toxin and diacetoxyscirpenol can rapidly deplete GALT.
Impaired Integrity of the Intestinal Epithelium
Intestinal integrity is compromised when the mucus layer is degraded; epithelial cells are effaced or destroyed, the vascular supply is interrupted, or the immune system is compromised. Intestinal integrity as considered specific to the epithelial layer, can be damaged by viruses, bacteria, fungi, myriad parasites, and toxins; reviewed by Moon, 1997.
This section will focus on specific types of injury to intestinal epithelium.
Viruses: Infection and replication of an enteric virus usually kills an epithelial cell. In contrast to bacteria, viruses do not produce toxins. Each of the intestinal viruses has a tropism for cells in a specific state of differentiation along the villus. Viruses preferentially infect cells in the crypts, on the tips and sides of the villus, or only on the tip, respectively. The severity of the clinical disease and the course of the uncomplicated viral infection are a reflection of target cells destroyed by the virus. For a virus that destroys cells on the tip of the villus, the absorptive function of the gut is lost and surviving epithelium is secretory. Watery diarrhea occurs until the villi are repaired with mature functional cells on the tips. A torovirus-like virus isolated from turkeys with Poult Early Mortality Syndrome (PEMS) does not cause death of the host cell (intestinal epithelial cells) (Dr. Akbar Ali, personal communication). Rather, the cell is stimulated to release messengers (cytokines) that interact with the immune system, and perhaps a more complex series of reactions with the inflammatory response, and nerves in the wall of the intestine. Each of these, in turn, releases lymphokines or mediators that produce cyclic amplification of responses by the intestinal epithelium. The result is increased fluid secretion in the gut that overrides its absorptive capacity, causing diarrhea and decreased digestive efficiency.
Bacteria: Bacteria damage epithelium by producing toxins, attaching to, and invading the cell. The host can ingest preformed bacterial toxin present in contaminated feed. Staphylococcus, Clostridium and Bacillus are some of the likely producers of enterotoxins in feed. The host may ingest bacteria that produce toxins once they begin to multiply in the gut. Escherichia coli, Clostridium perfringens, Clostridium colinum, and Campylobacter spp. produce enterotoxins that are polypeptides capable of causing diarrhea. Some enterotoxins cause increased fluid secretion and others are cytotoxic, causing cell lysis and death. Some strains of E. coli attach and adhere to the surface of the enterocyte and secrete a toxin that disturbs the water regulation of the cell. This causes a net secretion of chloride, loss of water, and the development of diarrhea. Necrotic enteritis caused by Clostridium perfringens is a severe manifestation of toxin-induced cell necrosis (death) that can result in mucosal destruction and death of the host. Other bacteria are capable of proliferating, invading and destroying intestinal epithelium. In the mildest case, certain E. coli attach and damage the microvilli on the luminal border of the enterocyte. This elicits the release of cytokines, development of inflammation, a net increase in secretion, and diarrhea. More enteroinvasive bacteria may proliferate within cells after invasion, causing cell death, and enabling cell-to-cell spread. Salmonella invades and passes through epithelial cells to invade to lymphatic and blood vessels, inciting inflammation in the gut, and then disseminating to other organs.
Fungi: Fungal infections of the intestinal mucosa are not significant in poultry. Mycotoxins produced by fungi growing in grains and feeds are significant and damage the muscosa and interfere with intestinal digestive functions. Trichothecene mycotoxins cause caustic injury to the mucosa, destroying cells on the tips of villi, and radiomimetic injury to rapidly dividing crypt epithelium. Aflatoxin impairs digestion by decreasing bile secretion from the liver, and bicarbonate secretion from the pancreas. Other toxins that can influence intestinal health include fumonisins, sterigmatocystin, ochratoxin, and undefined toxins of Penicillium.
Protozoa and Other Parasites: Coccidia are the principal pathogens of the intestinal tract in poultry, invading and destroying epithelial cells, and for some species of coccidia, the lamina propria. This results in increased mucus secretion, reduced absorption, hemorrhage and fluid leakage from damaged mucosa, and a dynamic immune and inflammatory response. Coccidia can affect all levels of the small and large intestine. Coccidial injury to the mucosa enhance the adherence of pathogenic bacteria, such as Clostridium perfringens and Salmonella typhimurium, and decrease the adherence of nonpathogens such as Bacteroides vulgatus and Bifidobacterium thermophilum Ascarids are the principal nematode in the intestine of commercial poultry. In general, the inflammatory response to nematodes brings a greater involvement of mast cells and the mediators of acute inflammation, which are linked to neurological responses as well.
Toxic Injury: Oxidized, rancid fats produce free radicals that can cause sublethal injury to cells throughout the body, including gut epithelium. The most obvious clinical evidence of fat rancidity is vitamin E deficiency expressed as encephalomalacia. Biogenic amines, generated by bacterial spoilage of improperly handled fish and rendered substrates, target gastroenteric tissues.
Feed Passage and Diarrhea
Feed passage is the presence of undigested particles of feed in the feces of poultry and implies decreased digestive efficiency with economic consequences (feed conversion, growth, carcass yield, cost of production). Feed passage usually occurs with diarrhea, which is an increase in the mass of feces, the frequency of fecal passage, and/or the fecal fluidity. Feed passage represents a malabsorption/maldigestion syndrome that shares many of the same causes ofdiarrhea. For the purposes of this discussion, four underlying mechanisms of diarrhea and feed passage will be examined, according to the scheme of Crawford, 1997
Secretory Diarrhea:This involves the excessive secretion of fluid from the intestinal mucosa, relative to the fluid absorption capacity of the intestine. This is caused by viruses that destroy mature enterocytes on the tips of the villus, leaving functional secretory enterocytes in the crypt and on the side of the villus. It is also caused by bacterial enterotoxins that affect mediators of intestinal electrolyte transport, involving the mucosal epithelial cells, immune and mesenchymal cells, enteric neurons, and hormonal and central nervous system. In general, the mediators act by increasing chloride secretion from the crypts and decreasing NaCl uptake from the tips of villi. As water follows these electrolytes, the net result is fluid overload in the lumen of the intestine.
Osmotic Diarrhea:This involves excessive osmotic forces exerted by luminal solutes. Poultry diets high in salt are one cause, such as occurs with diets formulated with certain lots of bakery byproduct meal. Osmotic factors may be involved in digestive problems associated with antinutritional factors (non-starch polysaccharides, NSP) in barley, rye, and wheat, and other ingredients, reviewed by Iji, 1999. These complex carbohydrates, typically hexoses and pentoses, are resistant to digestive enzymes, create a viscous environment within the intestinal lumen, increase the mass of luminal digesta, and produce moist sticky droppings.
Malabsorption: This is the output of voluminous, bulky feces with increased osmolarity owing to unabsorbed nutrients and, in man, excessive fat. It occurs because of defective intraluminal digestion due to ineffective enzymes or a lack of enzymes. Defective absorption could occur with the loss of mature enterocytes that have been replaced with immature cells lacking full absorptive function. Again, NSP may be involved with malabsorption because the gelatinous luminal content blocks the access of enzymes to otherwise digestible nutrients. This problem is also associated with increased mitotic activity and increased crypt depth in the gut mucosa, suggestive of an increased turnover of enterocytes, and a relative increase in secretory enterocytes or a decrease of mature enterocytes. Bile acid concentration within the digesta is also diluted, possibly impairing lipid absorption. The decreased bile concentration is associated with increases in luminal aerobic and anaerobic bacteria.
Exudative Diseases: This is characterized by frequent defecation of variable volume, but with the presence of blood or detritus from necrosis and inflammation. In chickens, this could be observed by severe coccidiosis, clinical salmonellosis, necrotic enteritis, or histomoniasis. In this situation, the gut experiences severe insult involving the necrosis and loss of enterocytes, possible loss of fluid, electrolytes and plasma from the damaged mucosa, and a major inflammatory response. The host must contend with the escape of the primary or secondary pathogens into the vascular system and dissemination to the liver, and possibly beyond. If the disease is not fatal, anorexia and diversion of nutrients for inflammation and repair will reduce growth and yield, impair feed conversion, and increase the cost of production.
Commercial poultry production during the last 50 years has benefited from pharmaceutical and biological products that enabled flock size to increase, genetic potential and improved nutritional formulations to be realized, and overall production to increase. Food animal agriculture is providing wholesome poultry meat protein for consumption in the human diet at unprecedented levels. The regulatory actions of the past decade have withdrawn, or are likely to withdraw, many products that assist in food animal production, ostensibly for the public health and well-being. As a result, once-controlled enteric diseases such as necrotic enteritis of poultry have returned, adversely affecting the health and well-being of the birds and the economics of poultry meat production. In order to effectively respond to this challenge, poultry health professionals will need to re-evaluate those factors that form the critical triad of infectious disease expression: the level of exposure/dose/titer of the pathogen, the virulence of the pathogen, and the relative susceptibility or resistance of the host.
Decreasing Exposure to Pathogens: Commercially, poultry will never be raised in the absence of infectious pathogens, despite biosecurity efforts to minimize pathogen exposure. Multiple disease stressors are likely to be experienced by commercial chickens or turkeys, even under good management and nutritional practices. Under the best of situations, including the recent emphasis on returning to alternative, noncommercial farms, feed ingredients come from biological systems with inherent stresses and quality control issues. Grain markets and geographic availability dictate that more problematic feedstuffs will need to be utilized in the best manner possible, or may actually be economically desirable under least-cost formulations. Commercial poultry housing is not, and likely will never be, a sterile environment. In the US, flock management remains mostly all-in, all-out. However, each successive flock is raised on the litter containing the pathogens of the preceding flock. Together, these issues dictate that birds will be exposed to infectious and toxic agents through the feed and environment. Aerobic and anaerobic bacteria, toxin-producing fungi, and protozoan parasites are neither easily eliminated nor likely to disappear from poultry production. Actions to reduce pathogen exposure must meet biological and economic justification. Suppressing a pathogen with antibiotics or providing competition for the pathogen once it is in the bird requires strategic use of a diminishing list of pharmacological,and possibly biological products. Responsible use of these products is paramount.
Decreasing Virulence of the Pathogen: This is the least practical of the three control points of disease. Bacteria regulate the expression of many virulence factors but these are elusive and impractical control points at this time.
Improving Host Resistance: Genetic selection for resistance to disease with limited assistance from pharmacological and biological products is of increasing importance, as is control of immunsuppressive diseases. With regard to emerging diseases such as necrotic enteritis, it is clear that feeding corn is advantageous to grains with higher NSP such as wheat, barley or rye. Resolution of the biochemical issues of NSP in the gut lumen and on the mucosal surface, and NSP as a matrix for the proliferation of aerobic and anaerobic bacteria is key to the future health of poultry in many areas of the world. Feed enzymes offer promise. New strategies for developing effective oral vaccines to protect mucosal surfaces are under investigation, involving vaccine adjutants to protect the antigen for delivery to GALT.
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Dr. Hoerr is the Director of the State Diagnostic Laboratories in Alabama and has provided poultry diagnostic services for the Alabama poultry industry since 1980. Alabama is currently the third ranking state for broiler production in the US, with weekly chick placements of about 20 million. Dr. Hoerr coordinates poultry diagnostic services with the central diagnostic laboratory in Auburn and three branch laboratories located in broiler production areas. This laboratory system processes about 7,000 cases for the broiler industry including disease investigations, serological surveillance, and chick quality testing. Dr. Hoerr holds a faculty appointment at the Auburn University College of Veterinary Medicine where he teaches avian diseases and conducts research on inherited resistance to disease in broilers. Dr. Hoerr earned his DVM, MS, and PhD degrees from Purdue University, and is a Diplomate of the American College of Veterinary Pathology and the American College of Poultry Veterinarians.