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Manifestations of Clostridium perfringens and related bacterial enteritides in broiler chickens

by 5m Editor
6 February 2006, at 12:00a.m.

By J. Wilson, University of Guelph; G. Tice, Elanco Animal Health; M. L. Brash, Elanco, Division Eli Lilly Canada Inc., and S. St. Hilaire, Idaho State University and published by Lohmann animal Health - Well known as the cause of necrotic enteritis, C. perfringens (CP) is now recognized as causing a spectrum of effects including subclinical infection, mild disease with focal intestinal necrosis, diarrhoeal illness and liver disease, as well as the classic form of acute fulminant necrotizing enteritis.

Manifestations of Clostridium perfringens and related bacterial enteritides in broiler chickens - By J. Wilson, University of Guelph; G. Tice, Elanco Animal Health; M. L. Brash, Elanco, Division Eli Lilly Canada Inc., and S. St. Hilaire, Idaho State University and published by Lohmann animal Health - Well known as the cause of necrotic enteritis, C. perfringens (CP) is now recognized as causing a spectrum of effects including subclinical infection, mild disease with focal intestinal necrosis, diarrhoeal illness and liver disease, as well as the classic form of acute fulminant necrotizing enteritis.

Abstract

The mild and subclinical forms of infection appear to be widespread, and, possibly increasing in incidence. Furthermore, bacterial enteritis is increasingly being recognized less as an invasion by pathogenic organisms per se and more as an expression of the normal process by which the intestinal bacterial population changes over time in response to changes in the intestinal environment. Such shifts in enteric bacterial populations have been referred to as dysbacteriosis. Management of the microbial ecology of the intestinal tract is therefore an important element of preventing disease, enhancing performance, and preventing foodborne illness. Diagnosis of emerging forms of bacterial enteritis including CP infection can be challenging and involves a comprehensive analysis of flock history and condemnation records, clinical signs, gross and histopathology, bacterial culture and empirical response to treatment, augmented by molecular techniques where available. Control measures are at present based on extensions of validated approaches to the control of the classical form of the disease: managing known risk factors for necrotic enteritis (coccidiosis, diet and litter quality) and the use of approved antimicrobial agents with proven efficacy against CP.

Introduction

First described by Parish in 1961, Clostridium perfringens (CP) is the causative agent of necrotic enteritis (NE) - one of the most economically important enteric pathogens of broiler chickens (Ficken and Wages, 1997). It is now understood that CP causes a spectrum of illness, which includes subclinical disease (Stutz et al., 1983) (infection that affects performance without causing clinical signs), mild clinical infection (including diarrhoea) (Kaldhusdal and Hofshagen, 1992), and liver disease, as well as the more commonly recognized fulminant infection that can result in outbreaks with substantial mortality.

It is likely that the economic importance of this pathogen has been underestimated as reduced growth and increased condemnations due to liver pathology are seldom included in assessments (Lovland and Kaldhusdal, 2001). A spectrum of clinical expression is well recognized for a variety of enteric pathogens. For example, in humans, E. coli 0157:H7 infection can cause death but the organism can also be found in clinically healthy individuals (Wilson et al., 1996). The same is true of coccidiosis in chickens (McDougald, 2003). Many factors may influence the severity of disease associated with enteric pathogens, including virulence, host susceptibility and immune status, infettive dose and diet (Barker and Van Dreumel, 1993).

The purpose of this paper is to describe the currently recognized manifestations of CP and related bacterial infections in broilers. Particular emphasis is placed on subclinical or mild disease, with low mortality but potentially significant impacts on performance. Clinical manifestations, pathogenesis and epidemiology of CP and related bacterial enteritides in broilers

CLASSICAL FORM OF NE

NE typically occurs as outbreaks characterized by depression, ruffled feathers, diarrhoea, huddling, anorexia and, frequently, high mortality. The duration of clinical signs may be very short and sudden death occurs commonly with no premonitory signs (Long, 1973; Tsai and Tung, 1981; Shane et al., 1985; Ficken and Wages, 1997). Gross and microscopic lesions generally occur in the small intestine, in particular in the jejunum and ileum and, less commonly, the caeca. They include focal to diffuse acute mucosal coagulatioe necrosis (Ficken and Wages, 1997), usually with little haemorrhage. The affected mucosa may be covered by fibrinonecrotic material which, if the animal survives, may form depressed areas of ulceration. These may undergo full repair including re-epithelialization or, in chronic cases, may undergo scarring and result in intestinal stricture. Histologically, the early intestinal lesion consists of acute coagulation necrosis of the mucosa which may be superficial to full thickness. Large numbers of gram-positive bacilli can be seen within the necrotic debris. In peracute cases there is little inflammatory cell infiltrate although, if the animal survives, there is a progression to heterophil and mononuclear cell infiltration followed by fibrosis (Long, 1973; Bernier et al., 1974b; Tsai and Tung, 1981; Shane et al., 1985; Ficken and Wages, 1997).

CP has been shown to be the causative agent of NE based upon identification of the organism in field cases (Long, 1973; Tsai and Tung, 1981) and through the successful reproduction of the disease by administration of the organism in feed (Truscott and AISheikhly, 1977; Prescott et al., 1978; Cowen et al., 1987; Brennan et al., 2001a;), intravenously (Bernier et al., 1977), in the crop (George et al., 1982) or intra-duodenally (Al-Sheikhly and Truscott, 1977a,b,c).

The pathology associated with CP is a result of the alpha and beta toxins produced by the bacteria within the intestinal tract where it replicates readily (Al-Sheikhly and Truscott, 1977a,b). Intraduodenal inoculation of bacteria-free toxin is capable of producing the disease experimentally (Al- Sheikhly and Truscott, 1977b). Death is a result of absorption of these toxins and the products of tissue necrosis, as well as intraluminal fluid loss and circulatory collapse (Al-Sheikhly and Truscott, 1977b). Outbreaks of NE have been reported from most areas of the world in which poultry are produced (Long, 1973; Tsai and Tung, 1981; Frame and Bickford, 1986; Kaldhusal and Skjerve, 1996; Ficken and Wages, 1997). Naturally occurring disease affects chickens between two weeks and six months of age, with the majority of reports occurring in broilers in the two to five week range raised on litter (Long, 1973). An outbreak of NE and coccidiosis has been reported in 12-16 week old cage-reared replacement layer pullets (Frame and Bickford, 1986). NE has also been reported in turkeys (Gazdzinski and Julian, 1992).

Relatively little information is available in the peer-reviewed literature regarding morbidity and mortality rates associated with NE. Long (1973) reported that the daily mortality rate in outbreaks rises rapidly to 0.1 to 0.5% per day. A study of 75 field outbreaks in Connecticut found that mortality occurred for approximately one week in most outbreaks and that daily mortality rarely exceeded 1 % (Long, 1973). Tsai and Tung (1981) reported on an outbreak in which daily mortality averaged 1.7%. In a study of necropsy submissions to Ontario veterinary diagnostic laboratories between 1969 and 1971, Long (1973) found that NE constituted 7.7% of all broiler submissions. The frequency of submission of NE cases was highest during the summer months. NE constituted 28% of all diagnoses of enteric disease in broilers submitted to the Veterinary Laboratory Services Branch, Guelph, between 1994 and 1996 (Ontario Ministry of Agriculture, Food and Rural Affairs, 1996).

CP is a common inhabitant of the chicken intestinal tract, with no apparent impact on the host (Dutta and Devriese, 1980; Niilo, 1980; Benno et al., 1988; Ficken and Wages, 1997). It can also be found in faeces, soil, dust and contaminated feed or litter. In outbreaks of NE, both contaminated feed and litter have been incriminated as sources of infection (Wicker et al., 1977; Ficken and Wages, 1997). Coccidia infection is a well documented predisposing factor for NE (Shane et al., 1985; Frame and Bickford, 1986; Baba et al., 1992). Colonization of the small intestine by Eimeria spp. leads to mucosal damage, which can provide a surface for CP to proliferate (Al-Sheikhly and Al-Saieg, 1980; Shane et al., 1985). Even mild forms of coccidiosis may predispose birds to NE (Shane et al., 1985) and, as a result, coccidiosis and NE prevention strategies are often applied simultaneously.

Diets high in wheat and barley, as opposed to corn, have been associated with an increased risk of NE in experimental and epidemiologic studies (Truscott and AI-Sheikhly, 1977; Branton et al., 1987; Riddell and Kong 1992; Hofshagen and Kaldhusdal, 1992; Kaldhusdal and Hofshagen, 1992). Dietary fishmeal has also been identified as a predisposing factor for NE (Truscott and Al-Sheikhly, 1977). Manipulation of the diet (for example, through the addition of wheat or barley or their constituents) can affect the numbers of CP in the intestinal tract (Hofshagen and Kaldhusdal, 1992; Branton et al., 1996). It is hypothesized that dietary changes may alter the intestinal micro-environment in a manner which promotes clostridial overgrowth or stimulates toxin production in the intestinal lumen (Bernier et al., 1974a; AI-Sheikhly and Truscott, 1977b; Kaldhusdal and Skjerve, 1996). Recently, it was proposed that diets high in wheat and barley increased mucus production in the intestal tract, which resulted in a shift in the microflora in favour of mucolytic bacteria, and in particular CP (Collier et al., 2003). Some diets may also alter the viscosity of digesta and increase transit time in the gut, both of which could lead to changes in the gut microflora. It is also possible that these diets, if not properly digested, physically damage the mucosal surface, which would, again, lead to an increased risk of clostridia) disease. Physical damage to the intestinal mucosa is a recognized predisposing factor for NE. For example, litter high in fibre content (Truscott and AI-Sheikhly, 1977) has been associated with the disease.

CLINICALLY MILD CP AND RELATED ENTERIC INFECTIONS

During the past few years CP has been associated with a variety of forms of infection that produce clinical signs that are milder than the classical form of NE described by Parish in the 1960's (Stutz et al., 1983; Kaldhusdal and Hofshagen, 1992; Hoitink, 1997; Eshuis et al., 1998; Lovland and Kaldhusdal, 1999; Lovland and Kaldhusdal 2001; Brennan et al., 2001a; Brennan et al., 2001b; Pattison, 2002).

Mild focal ulceration of the small intestine accompanied by impaired growth performance with or without clinical signs of infection has been described by a number of researchers in experimental challenge studies (Brennan et al., 2001a; Brennan et al., 2001 b) and following natural challenge (Kaldhusdal and Hofshagen, 1992). Grossly, these ulcers are equivalent to the areas of mucosal necrosis seen in the fulminant form of NE but are smaller-in the range of I to 5 mm in diameter. Affected birds often recover clinically so, presumably, areas of ulceration can heal completely. Kaldhusdal and Hofshagen (1992) described very mild histologic changes in affected birds consisting of superficial ulceration of the apex of the villus accompanied by heterophil infiltration and focal aggregates of clostridia. The gross changes described by these and other authors (Brennan et al., 2001 a; Brennan et al., 2001 b) suggest that the lesion can extend to the level of the lamina propria. Kaldhusdal and Hofshagen (1992) also described sticky droppings adherent to the cloaca in affected birds.

In the late 1990's two veterinarians in the Netherlands described naturally occurring field outbreaks of clinically mild disease consistent with CP infection. Hoitink in 1997 described a condition in Dutch broilers between 5 and 10 days of age characterized principally by necrosis of liver accompanied by small areas of necrosis in the small intestine of some birds. Gram-positive bacteria were identified in stained smears of liver and small intestine and CP was cultured from affected birds. Mortality in affected flocks was low, but growth rate was reduced and a high degree of liver condemnation occurred at slaughter.

Eshuis and co-authors m 1998 described broiler flocks in their practices experiencing decreased feed intake beginning at 15 days of age accompanied by normal water intake and resulting in wet litter. Rates of gain and feed conversion were reduced. Although there was no associated mortality, at necropsy some affected birds had a layer of gray material over the intestinal mucosa which microscopically consisted of necrosis of the intestinal epithelium consistent with NE. In other cases, no abnormalities were observed in the intestinal tract. Eshuis considered the two forms of the disease to be different expressions of CP infection and suggested that the more mild form comprises a form of dysbacteriosis, a condition characterized by abnormal populations of bacteria within the intestinal tract with minimal intestinal pathology.

Focal intestinal ulceration would be expected to result in localized fluid and loss and failure of nutrient absorption as well as permitting absorption of products of tissue necrosis and toxic materials present in the gut content. This would account for depressed growth and feed intake and clinical depression observed in field cases and experimental studies (Kaldhusdal and Hofshagen, 1992; Hoitink, 1997; Eshuis et al., 1998). Diarrhoea in such cases could result from a combination of fluid loss from localized inflammation and decreased fluid absorption due to disruption of the epithelial barrier (Barker and Van Dreumel, 1993). Diarrhoea has been identified as a common clinical sign in a recent global survey of attitudes and beliefs relating to CP infection among poultry professionals (Carrier, 2000). This same survey estimated that mild forms of clostridia) disease cost $0.05 per bird globally.

It is likely this is an underestimate, given the difficulty in diagnosing mild forms of CP infections (Lovland and Kaldhusdal, 2001). Diarrhoea and wet litter have been reported as significant problems following restrictions on the use of antibiotic growth promoting agents in Europe (Dudley-Cash, 2003). Pattison (2002) described a condition in broilers beginning in 2000 characterized by thinning and ballooning of the small intestine accompanied by gut content that is viscous or watery and frequently full of bubbles. The condition begins at about 21 days of age and results in wet litter. Treatment with tylosin is effective in controlling outbreaks although relapses can occur. The etiology of the condition is unclear, however the fact that it apparently began following restriction on the use of antibiotic growth promoters and that it responds to the use of an antibiotic with known efficacy against enteric bacteria including CP suggests that the condition may be, at least in part, bacterial in origin. Pattison (2002) considers it to be a form of dysbacteriosis.

The intestinal flora of healthy broilers has been studied in some detail by molecular and conventional techniques (Barnes et al., 1972; Salanitro et al., 1978; Mead 1989; Apajalahti et al., 1998; Lee, 2002; Zhu et al., 2002). The results of such studies demonstrate the complexity of the normal flora and serve as a reminder that the full range of microorganisms within the avian gut - both commensal and pathogenic - remains incompletely characterized. Ambiguity over the etiology of diarrhoeal illness observed in broilers in the field has prompted the use of molecular techniques to attempt to correlate bacterial population shifts with clinical signs. Although in the early stages, these studies demonstrate that shifts do occur in the intestinal flora in cases of otherwise unspecified diarrhoeal illness (Panneman, 2001, 2002), a situation that is paralleled in humans and other species.

While a shift in the gut microflora in favour of organisms that do not normally predominate is known as dysbacteriosis (Schoorel et al., 1980; Dumitrasco et al., 1980; Sidorchuk and Bondarenko, 1984; Klemparskaya et al., 1987), an excess of microorganisms within the small bowel is known as small intestinal bacterial overgrowth (SIBO). Though not well documented in chickens, small intestine bacterial overgrowth has been described in humans, cats and dogs (Johnston, 1999). In humans, identified causes include changes that lead to reduced peristaltic movement (Roussel, 1994; Schippers et al., 1996), changes in gastric acidity, decreases in the production of bacteriostatic peptides by the pancreas (Floch et al., 1972; Rubinstein et al., 1985; Simpson et al., 1990), altered mucus production (Johnston, 1999; Collier et al., 2003), malnutrition (Gracey et al., 1977), and reduced Immunoglobulin A secretion into the lumen of the gastrointestinal tract (Lichtman et al., 1986).

Depending on the predominant gut microflora, the relationship between these organisms and the host may be synergistic. Some benefits that the small intestinal microflora provide include protein breakdown by proteolytic enzymes, protection against pathogenic bacteria such as Salmonella spp. and E. coli, and immune stimulation (Johnston, 1999). When abnormal bacterial populations predominate, they may produce effects that are deleterious to the host through mechanisms such as the production of toxic metabolites, competition with the host for nutrients, and stimulation of enterocyte turnover (Johnston, 1999; Anderson et al., 2000) (see Subclinical CP Infection, below).

SUBCLINICAL CP INFECTION CAUSING GROWTH SUPPRESSION

There is a significant body of scientific literature and field experience which supports a role of intestinal bacteria in growth suppression in poultry and other species. It has long been recognized, for example, that germ-free chicks grow more rapidly and have higher feed efficiencies than conventionally reared birds (Feighner and Dashkevitz, 1987). Furthermore, many antibiotic growth promoters have no effect on growth in germ-free chicks (Feighner and Dashkevicz. 1987), and infecting germ-free animals with normal gut microflora has a growth suppressive effect (Coates, 1980).

It has been shown that CP can reduce the growth rate of germ-free chickens and that penicillin suppresses the replication of CP and overcomes this growth depressing effect in conventionally reared chickens while in germ-free chickens it has no effect (Stutz et al., 1983). A variety of other antimicrobials such as tetracylines, virginiamycin, bacitracin, and avoparcin have been shown to reduce intestinal levels of CP in broilers while improving growth rate, prompting numerous authors to suggest a role of this organism in growth suppression (Stutz and Lawton, 1984; Hofshagen and Kaldhusdal, 1992).

A variety of mechanisms have been suggested to explain the growth suppressing effect of intestinal bacteria including CP. One is through the production of toxic metabolites that irritate the gut mucosa, thereby inhibiting nutrient absorption. Various authors have suggested that the production of ammonia from urea by intestinal flora may be responsible for growth suppression, although the results of these studies are somewhat contradictory (Visek, 1978; Feighner and Dashkevicz, 1987; Anderson et al., 2000).

Phenolic and aromatic compounds are produced from the breakdown of protein by bacteria in the gut and excreted by the kidneys (Deichmann and Witherup, 1943). Studies measuring phenol excretion in piglets supplemented with antibiotic-treated feed showed an increase in weight gain and a reduction in phenol excretion in comparison with piglets not given antibiotics (Anderson et al., 2000), suggesting that these compounds may suppress growth. Many microbes, including CP, also hydrolyze and deconjugate bile salts (Cole and Fuller, 1984; Johnston 1999; Anderson et al., 2000), which impair lipid absorption by the host (deSomer et al., 1963; Floch et al., 1972) and directly damage enterocytes (Johnston, 1999). Several researchers have demonstrated an inverse relationship between hydrolase activity in the small intestine and growth in chickens fed antibiotics (Feighner and Dashkevicz, 1987). Another mechanism by which enteric organisms decrease growth efficiency may be through the utilization of nutrients that would otherwise be available to the host (Anderson et al., 2000. Bunyan et al. (1977) have suggested that achievement of maximum growth is dependant on colonization of the crop and upper intestine with lactobacilli and that the growth enhancing effect of antimicrobials might be to inhibit organisms which compete for growth with lactobacilli.

The presence of enteric bacteria is also associated with an increase in intestinal weight. Germ-free rodents and other animals have thinner intestinal walls than their conventional equivalents (Abrams et al., 1963). This is in part due to a decrease in lymphoid tissue in the lamina propria (Stutz et al., 1983), which results from a decrease in antigenic stimulation of the mucosa (Gaskins, 1996). The gut flora, then, promote thickening of the intestinal mucosa which reduces its absorptive capacity. Several authors have shown that administration of antibiotics in the feed significantly decreases intestinal weight (Stutz and Lawton, 1984; Henry et al., 1986; Henry et al., 1987; Roura et al., 1992) compared to untreated controls. It has been suggested that this may lead to increased absorptive capacity of various nutrients including amino acids, fatty acids, vitamins and minerals (Davison and Freeman, 1983) and hence increased rate of growth.

Finally, certain sub-populations within the gut flora may actually cause subclinical disease. Reduction in villus height and crypt depth has been reported in humans with small intestinal bacterial overgrowth (Toskes et al., 1975) suggesting that certain gut microbes may damage enterocytes directly in this context. Visek (1978) points out that the most relevant intestinal bacteria may be anaerobes, which have received relatively little study and suggests that inhibition of disease causing microbes may be of great significance in the growth enhancing activity of some antimicrobials.

LIVER DISEASE ASSOCIATED WITH CP

Hepatic lesions have been described in birds with both classical and mild forms of CP infection. (Hutchison and Riddell, 1990; Onderka et al., 1990; Lovland and Kaldhusdal, 1999). In the classical form (necrotic enteritis) the liver lesion consists of multifocal hepatic necrosis. Histologically, there is acute coagulative hepatic necrosis with little associated haemorrhage or inflammatory response (Ficken and Wages, 1997).

CP infection has also been linked to a form of hepatitis observed in processing plants that is a significant cause of carcass condemnation in many parts of the world. Grossly, affected livers may appear pale and firm, with or without small whitish foci. Histologically, there is bile duct hyperplasia, fibrosis, and focal granulomatous inflammation (Onderka et al., 1990; Lovland and Kaldhusdal, 1999). CP can be isolated from affected livers (Lovland and Kaldhusdal 1999, Sasaki et al., 2000) and can produce classical and mild NE when fed to chicks (Hutchison and Riddell, 1990). Experimental inoculation of CP into the bile duct results in lesions typical of field cases from which CP can be reisolated (Onderka et al., 1990).

In a study of CP associated hepatitis (CPH) in Norway, flocks having a high level of CPH had significantly lower 35 day live weight, feed conversion and contribution margins and significantly higher mortality, downgrades and condemnations than flocks with a low level of CPH (Lovland and Kaldhusdal, 2001). A second epidemiologic study in Norway (Lovland and Kaldhusdal, 1999) showed a strong temporal correlation between the incidence of CPH and NE. The mechanism by which the liver is affected by CP in CPH is poorly understood. It has been suggested that bacterial toxins, tissue breakdown products, and the bacteria themselves may be transported to the liver via the portal blood (Bernier et al., 1974b; Long and Truscott, 1976; Tsai and Tung, 1981; Shane et al., 1985; Ficken and Wages, 1997).

There are several reports of hepatic and cholangiohepatic damage associated with intestinal bacterial overgrowth in other species, including humans (Lichtman et al., 1990). The mechanism by which this occurs is not clearly defined and may depend on the type of bacteria that dominate the gut microflora. Suggestions as to the etiology of hepatic and heptobiliary abnormalities associated with small intestinal bacterial overgrowth include release of endotoxins (Utili et al., 1976) and peptidoglycanpolysaccharide polymers (Wahl et al., 1986), deconjugation of bile salts (King and Toskes, 1979), nutritional deficiencies, and endogenous alcohol production (Barona et al., 1986).

Diagnosis: Necrotic Enteritis

A presumptive diagnosis of NE can be made on the basis of typical clinical signs (depression, huddling, diarrhoea and sudden death - often as outbreaks) and gross lesions consisting of acute necrosis of the small intestine, generally with little haemorrhage. The gross lesion may be confused with ulcerative enteritis, caused by C. colinum, and coccidiosis (Long et al., 1974; Porter, 1998). Confirmation of gross findings by histologic and microbiologic examination of affected tissues is thus recommended.

In culture, CP bacteria are short to intermediate spore-forming Gram positive rods, and are readily grown on blood agar plates incubated anaerobically at 37°C. Colonies produce a characteristic inner zone of haemolysis and outer zone of partial haemolysis. Identification is made on differential media. Most strains ferment glucose, maltose, lactose and sucrose, do not ferment mannitol and variably ferment salicin. Principal products of fermentation are acetic and butyric acids. Gelatin is hydrolyzed, milk is digested and there is no indole production. Growth on egg yolk demonstrates the presence of lecithinase and absence of lipase production.

Subculturing on egg yolk agar plates, one-half of which have been spread with CP antitoxin and incubating anaerobically overnight will produce a zone of precipitation around colonies on control sides of the plate and little or no precipitation on sides spread with the antitoxin (Ficken and Wages, 1997).

A presumptive diagnosis can also be made on the basis of examination of Gram stained impression smears of intestinal mucosa (Ficken and Wages, 1997).

CLINICALLY MILD CP AND RELATED ENTERIC INFECTIONS

Definitive diagnosis of the clinically mild forms of CP infection is challenging at the present time due to the non-specific nature of the clinical signs and lesions and because characterization of the disease syndrome is still in the early stages.

Gross examination of birds at necropsy may reveal changes consistent with mild CP infection such as focal intestinal or hepatic necrosis (Kaldhusdal and Hofshagen, 1992). Based on field reports, histologic examination of affected tissues would be expected to reveal focal necrosis of affected gut and/or liver with little associated inflammatory cell infiltration. It is also possible that more subtle histologic changes can occur which have not yet been described in the literature. Anaerobic culture of intestinal swabs and Gram stained impression smears of intestinal mucosa may be useful as a diagnostic aid. It is important to note, however, that the mere presence of CP in intestinal material is not in itself adequate evidence for a definitive diagnosis since the organism is a normal inhabitant of the intestinal tract (Ficken and Wages, 1997). Some researchers have suggested monitoring faecal counts of CP (Kaldhusdal et al., 1999). A history of episodes of fulminant CP infection in previous flocks may suggest a diagnosis of mild CP infection on a given premises, particularly in the presence of other evidence such as mild intestinal lesions or diarrhoea. The incidence of CPH at slaughter may also be an effective indirect means of monitoring the incidence of other forms of CP infection. CPH has been shown to correlate well with episodes of NE (Lovland and Kaldhusdal, 1999).

Diagnosis of dysbacteriosis faces similar challenges since the condition is still incompletely characterized in broilers at the present time. Factors suggestive of dysbacteriosis in broilers include a history of diarrhoeal illness or wet droppings and gross lesions consisting of thinning and ballooning of the small intestine accompanied by watery or viscous intestinal content (Pattison, 2002). Diarrhoea itself can be challenging to identify on a flock basis. Monitoring litter quality should assist producers in identifying changes in faecal water content. Mild changes may be difficult to detect, depending on the litter quality and type, but litter boxes have been used successfully to measure the water content of bird droppings and alert producers to early signs of diarrhoea (Mortimer, 2002).

Molecular techniques such as 16S rRNA terminal restriction fragment length polymorphism (T RFLP) (Marsh 1999; Panneman, 2001) have been used to characterize the gut microflora and identify potentially pathogenic shifts in microbial populations, however these remain largely experimental at present.

Empirical response to therapy with antibacterial agents with known efficacy against CP and other enteric pathogens can be considered a useful diagnostic indicator for both CP infection and dysbacteriosis. Tylosin has been used successfully for this purpose (Pattison, 2002). Elimination of other potential causes of diarrhoea and wet litter is another important component of the diagnostic process. There are numerous causes of diarrhoea in chickens. Infectious causes include coccidiosis, ulcerative enteritis and a variety of viral infections such as infectious bursal disease and infectious bronchitis virus and others which are not yet well characterized. Non-infectious causes of diarrhoea include heat and other forms of stress and a variety of dietary factors (Porter, 1998; Hoppe, 1999).

SUBCLINICAL CP INFECTION CAUSING GROWTH SUPPRESSION

Subclinical CP infection should be considered in flocks with reduced growth performance and either historical or current episodes of fulminant NE or other events referable to CP. These events may include clinically mild CP infection or liver disease as described previously, particularly in flocks which are not receiving feed additives for the control of NE. By necessity, this is a diagnosis of exclusion or inference, as reduced growth performance can be caused by a wide range of factors. Monitoring faecal counts of CP could, in principle, be of value (Kaldhusdal et al., 1999). However the methodology is not yet available for routine diagnostic purposes. In the future it may also be possible to determine infection with CP by monitoring birds for antibodies against the bacteria. An Enzyme linked Immunosorbent Assay (ELISA) is under development for this purpose (Kaldhusdal and Lovland, 2002). Empirical response to therapy may also be a useful diagnostic approach for this condition.

LIVER DISEASE ASSOCIATED WITH CP

CP associated hepatic disease is readily detected through gross examination of livers at slaughter. The gross lesions are reasonably distinctive and readily distinguished from other common causes of liver condemnation at slaughter such as congestive heart failure (ascites) and perihepatitis (Hutchison and Riddell, 1990). Gross findings can be confirmed histologically or by culture of CP. This method of monitoring is retrospective but, as clostridia) problems often persist on a given premises, it may be of assistance in managing future flocks. Acute hepatic necrosis associated with fulminant or clinically mild forms of CP can be readily identified on the basis of gross and histologic examination of livers and anaerobic culture (Ficken and Wages, 1997).

Control: Classical NE

Management of disease associated with CP should focus on prevention and, in cases where this fails, early detection and treatment. Prevention strategies should include minimizing exposure to known risk factors such as coccidiosis (Al-Sheikhly and Al-Saieg, 1980; Shane et al., 1985; Baba et al., 1992), diets high in wheat, barley, rye, and fishmeal (Truscott and Al-Sheikhly, 1977; Branton et al., 1987; Riddell and Kong, 1992; Hofshagen and Kaldhusdal, 1992; Kaldhusdal and Hofshagen, 1992) and litter high in fibre (Truscott and Al-Sheikhly, 1977). The prudent use of feed additives with demonstrated efficacy against CP is also recommended and, in some cases, essential for the prevention of NE or treatment of outbreaks when they occur. A variety of authors have described the susceptibility of CP to various antimicrobial agents in vitro including bacitracin, penicillin, virginiamycin, lincomycin, lasalocid, tylosin, avilamycin and narasin (Bernier, 1974x; Dutta and Devriese, 1980; Kondo, 1988; Benno et al., 1988; Watkins et al., 1997).

Stutz and Lawton (1984) quantified CP in intestinal content of chicks fed diets containing bacitracin, lincomycin, penicillin or virginiamycin. All medicated feeds resulted in decreased numbers of CP in intestinal content compared to unmedicated controls.

The efficacy of certain drugs in the prevention of NE has been demonstrated including penicillin (Long and Truscott, 1976) bacitracin (Prescott et al., 1978) virginiamycin (George et al., 1982), lincomycin (Truscott and Al-Sheikhly,1977; Hamdy et al., 1983), and narasin (Brennan et al., 20016). However, few studies have evaluated medicated feed or water in the treatment of NE once clinical signs and intestinal lesions have become apparent in affected birds. Recently, Brennan et al. demonstrated that tylosin is effective in reducing morbidity and mortality among broiler chickens when administered after the onset of an NE outbreak (2001a).

CLINICALLY MILD AND SUBCLINICAL CP INFECTION, HEPATITIS AND DYSBACTERIOSIS

Control of other forms of CP infection is currently based on extrapolation of measures known to be effective in the control of fulminant NE. A rational approach to the control of these conditions would thus include a combination of reducing risk factors such as diet and litter quality, coccidiosis control and the prudent use of feed additives with demonstrated efficacy against CP. Presumed cases of dysbacteriosis in broilers have been controlled using antibiotics (Pattison, 2002).

Conclusions

This review confirms that CP is a highly significant cause of enteric disease in broiler chickens. Long known to cause serious clinical illness characterized by outbreaks of sudden death, a pattern is now emerging in which CP is seen as causing a spectrum of effects including subclinical infection, mild disease with focal intestinal necrosis and liver disease, as well as the classic form of acute fulminant necrotizing enteritis (Stutz et al., 1983; Hutchison and Riddell, 1990; Kaldhusdal and Hofshagen, 1992; Ficken and Wages, 1997). The mild and subclinical forms of infection appear to be widespread, significant and, possibly increasing in incidence (Lovland and Kaldhusdal, 2001; Carrier, 2000).

Furthermore, bacterial enteritis is increasingly being recognized less as an invasion b Y pathogenic organisms per se and more as an expression of the normal process by which the intestinal bacterial population changes over time in response to changes in the intestinal environment (Fukata et al., 1991; Apajalahti et al., 1998; Netherwood et al.. 1999; Panneman, 2002; Collier et al., 2003). When this process results in mild disease, characterized, for example, by the production of gas and fluid within the intestinal lumen (Pattison, 2002), it is referred to as dysbacteriosis - a phenomenon that is well recognized in humans and other animal species. Studies of the intestinal microflora in both normal and clinically affected broilers suggest that CP may be one of a variety of organisms that, when they proliferate within the intestinal lumen, can impact on performance. Given the dramatic changes in poultry management that have occurred over the past 30 years, it is perhaps not surprising that shifts in the relative abundance of different bacterial species should occur and that their clinical manifestations might also change over time. For example, restrictions on the use of antibiotics that prevent NE appear to have resulted in an increase in the incidence of clinical NE, mild forms of CP infection and conditions consistent with dysbacteriosis, at least under some circumstances (Pattison, 2002; Casewell et al., 2003; Dudley-Cash, 2003).

Maintenance of intestinal integrity is a critical component of modern poultry management. By integrity, we mean not only the prevention of intestinal lesions. Rather, we are referring to an optimization of the process of digestion and absorption of nutrients and protein turnover, the physical and physiologic integrity of the epithelial barrier and the microbial ecology of the intestinal content. The latter is important not only from the perspective of preventing disease and enhancing performance, but is also a critical element of the process of preventing foodbome disease (McMeekin et al., 1997).

Although the diagnosis of fulminant NE is straightforward, diagnosis of subclinical and mild CP infection presents challenges to poultry practitioners and field personnel. This is because the conditions themselves are not yet fully characterized, clinical signs associated with these conditions are relatively non-specific, diagnostic tests to characterize the total gut flora in a meaningful manner are not yet readily available and because the presence of CP in gut content is not it itself definitive evidence of an etiologic role of that organism in the absence of significant mucosal necrosis.

Diagnosis of these conditions thus consists of a comprehensive analysis of flock history and condemnation records, clinical signs, gross and histopathology, culture of CP and empirical response to treatment. Where available, these traditional methods can be augmented with molecular techniques (Ficken and Wages, 1997; Lovland and Kaldhusdal, 2001; Panneman, 2002).

Similarly, recommendations for control and treatment of mild and subclinical NE are at present empirical and based on extensions of validated approaches to the control of the classical form of the disease. These consist of obtaining an accurate diagnosis, identifying and managing known risk factors for NE (coccidiosis, diet and litter quality) (Truscott and Al-Sheikhly, 1977) and the use of approved antimicrobial agents with proven efficacy against CP (Brennan et al., 2001x; Brennan et al., 20016).

References

ABRAMS, G.D., BAUER, H. and SPRINTZ, H. (1963) Influence of the normal flora on mucosal morphology and cellular renewal. A comparison of germ-free and conventional mice. Laboratory Investigation 12: 355364.
AL-SHEIKHLY, F. and TRUSCOTT, R.B. (1977x) The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens. Avian Diseases 21(2): 256-263.
AL-SHEIKHLY, F. and TRUSCOTT, R.B. (1977b) The pathology of necrotic enteritis of chickens following infusion of crude toxins of Clostridium perfringens into the duodenum. Avian Diseases 21(2): 241-255.
AL-SHEIKHLY, F. and TRUSCOTT, R.B. (1977c) The pathology of necrotic enteritis of chickens following infusion of broth cultures of Clostridium perfringens into the duodenum. Avian Diseases 21(2): 230-240.
AL-SHEIKHLY, F. and AL-SAIEG, A. (1980) Role of coccidia in the occurrence of necrotic enteritis of chickens. Avian Diseases 24(2): 324-333.
ANDERSON, D.B., MCCRACKEN, V.J., AMINOV, R.I., SIMPSON, J.M., MACKIE, R.I., VERSTEGEN, M.W.A. and GASKINS, H.R. (2000) Gut microbiology and growth-promoting antibiotics in swine. Nutritional Abstracts and Reviews Series B.' Livestock Feeds and Feeding 70(2): 102-107.
APAJALAHTI, J.H.A., SARKILAHTI, L.K., MAKI, B.R.E., HEIKKINEN, J.P., NURMINEN, P.H. and HOLBEN, W.E. (1998) Effective recovery of bacterial DNA and percent-guanine-plus cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens. Applied and Environmental Microbiology, 64: 4084-4(188.
BABA, E., FULLER, A.L., GILBERT, J.M., THAYER, S.G. and MCDOUGALD, L.R. (1992) Effects of Eimeria brunetti infection and dietary zinc on experimental induction of necrotic enteritis in broiler chickens. Avian Diseases 36: 59-62.
BARKER, I.K. and VAN DREUMEL, A.A. (1993) Intestine. Pages I-318 in Pathology of Domestic Animals (4th Edition) K.V.F. Jubb. P.C. Kennedy and N. Palmer eds. Academic Press, Orlando FL.
BARNES, E.M., MEAD, G.C., BARNUM, D.A. and HARRY, E.G. (1972) The intestinal flora of the chicken in the period 2 to 6 weeks of age, with particular reference to the anaerobic bacteria. British Poultrv Science 13:311-326.
BARONA, E., JULKUNEN, R., TANNENBAUM, L. and LIEBER C.S. (1986) Role of intestinal bacterial overgrowth in ethanol production and metabolism in rats. Gastroenterology 90(1): 103-110.
BENNO, Y., ENDO, K. and MITSUOKA, T. (1988) Isolation of faecal Clostridium perfringens from broiler chickens and their susceptibility to eight antimicrobial agents for growth promotion. Japanese Journal of Veterinary Science 50(3): 832-834.
BERNIER, G., FILION, R. MALO, R. and PHANEUF, J-B. (1974x) Entérite nécrotique chez le poulet de gril 11. Caractères des souches de Clostridium perfringens isolées. Canadian Journal of Comparative Medicine 38: 286-291.
BERNIER, G., PHANEUF, J-B. and FILION, R. (1974b) Entérite nécrotique chez le poulet de gril, 1 Aspect clinico-pathologique. Canadian Journal of Comparative Medicine 38: 280-285.
BERNIER, G., PHANEUF, J-B. and FILION, R. (1977) Entérite nécrotique chez le poulet de grit. III Etude des facteurs favorisant la multiplication de Clostridiwn perfringens et la transmission expérimentale de la maladie. Canadian Journal ofComparative Medicine 41: 112-116.
BRANTON, S.L., REECE, EN. and HAGLER, W.M. (1987) Influence of a wheat diet on mortality of broiler chickens associated with necrotic enteritis. Poultry Science 66: 1326-1330.
BRANTON, S.L., LOTT, B.D., MAY, J.D., HEDIN, P .A., AUSTIN, F .W., LATOUR, M.A. and DAYS, E.J.(1996) The effects of nonautoclaved and autoclaved water-soluble wheat extracts on the growth of Clostridium perfringens. Poultry Science 75: 335-338.
BRENNAN, J., MOORE, G., POE, S.E., ZIMMERMANN, A., VESSIE, G., BARNUM, D.A. and WILSON, J. (2001 a) Efficacy of in-feed tylosin phosphate for the treatment of necrotic enteritis in broiler chickens. Poultry Science 80: 1451-1454.
BRENNAN, J., BAGG, R., BARNUM, D., WILSON, J. and DICK, P (2001 b) Efficacy of narasin in the prevention of necrotic enteritis in broiler chickens. Avian Diseases 45: 210-214.
BUNYAN, J., JEFFRIES, L., SAYERS, J.R., GULLIVER, A.L. and COLEMAN, K. (1977) Antimicrobial substances and chick growth promotion: the growth-promoting activities of antimicrobial substances including fifty-two used either in therapy or as dietary additives. British Poultry Science 18: 283-294.
CARRIER, D. (2000) Clostridial enteritis global impact assessment and global MIC survey - a review. Pages 56- 75 in Proceedings of the Poultry Enteritis Conference. Elanco Animal Health, Montreal Que.
CASEWELL, M., FRIIS, C., MARCO, E., MCMULLIN, P and PHILLIPS, I. (2003) The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. Journal of Antimicrobial Chemotherapy 52: 159-161.
COATES, M.E. (1980) The gut microflora and growth. Pages 175-188 in Growth in Animals. T. L. J. Lawrence. ed. Butterworths, Boston MA.
COLE, C.B. and FULLER, R. (1984) Bile acid deconjugation and attachment of chicken gut bacteria: their possible role in growth depression. British Poultry Science 25: 227-231.
COLLIER, C.T., VAN DER KLISS, J.D., DEPLANCKE, B., ANDERSON, D.B. and GASKINS, H.R. (2003). Effects of Tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis. Antimicrobial Agents and Chemotherapy 47: 3311-3317.
COWEN, B.S., SCHWARTZ, L.D., WILSON, R.A. and AMBRUS, S.I. (1987) Experimentally induced necrotic enteritis in chickens. Avian Diseases 31: 904-906.
DAVISON, T.F. and FREEMAN, B.M. (1983) Physiological aspects of growth promotion in poultry. Veterinary Research Communications 7: 59-68.
DEICHMANN, W.B. and WITHERUP, S. (1943) Phenol studies VI. The acute and comparative toxicity of phenol, o-, m-, and p-cresols for experimental animals. Journal of Pharmacology and Experimental Therapeutics 80: 223-239.
DUDLEY-CASH, W. (2003) Field research measures effect of removing GPA from broiler feeds. Feedstuffs 10- 12.
DUMITRASCO, D., GRIGORESCO, M., PARAU, N., SUCIU, A. and ERDOSY, S. (1980) Dysbacteriosis in enteropathies. Medicine Interne 18(3): 239-245.
DOTTA, G.N. and DEVRIESE, L.A. (1980) Susceptibility of Clostridium perfringens of animal origin to fifteen antimicrobial agents. Journal of Veterinary Pharmacology and Therapeutics 3: 227-236.
DESOMER, P., EYSSEN, H. and EVRARD, E. (1963) The influence of antibiotics on faecal fat in chicks. Pages 84-90 in Biochemical Problems of Lipids. A.C. Frazer, ed. Elsevier/North Holland Publishing Co., Amsterdam.
ESHUIS, J.W., VAN DOBBENBURGH, O.A., DELHAES, L.M., VAN DER MEIJ, D. and NIJLAND, J.T. (1998) Bacterie-regelaar Tylan an helpt tegen dysbacteriose. Pluimveehouderij 13.
FEIGHNER, S.D. and DASHKEVITZ, M.P. (1987) Subtherapeutic levels of antibiotics in poultry feeds and their effects on weight gain, feed efficiency and bacterial cholytaurine hydrolase activity. Applied and Environmental Microbiology 53(2): 331-336.
FICKEN, M.D. and WAGES, D.P. (1997) Necrotic enteritis. Pages 261-264 in Diseases of Poultry 10th Edition. B. W. Calnek, ed. Iowa State Press, Ames.
FLOCH, M.H., BINDER, HJ., FILBURN, B. and GERSHENGOREN, W. (1972) The effects of bile acids on intestinal microflora. American Journal of Clinical Nutrition 25: 1418-1426.
FRAME, D.D. and BICKFORD, A.A. (1986) An outbreak of coccidiosis and necrotic enteritis in 16-week-old cage-reared layer replacement pullets. Avian Diseases 30(3):601-602.
FUKATA, T., HADATE, Y., BABA, E. and ARAKAWA, A. (1991) Influence of bacteria on Clostridium perfringens infections in young chickens. Avian Diseases 35: 224-227.
GAZDZINSKI, P and JULIAN, R.J. (1992) Necrotic enteritis in turkeys. Avian Diseases 36: 792-798.
GASKINS, H.R. (1996) Immunological aspects of host/microbiota interactions at the intestinal epithelium. Pages 537-587 in Gastrointestinal Microbiology (vol.2) Gastrointestinal Microbes and Host Interactions. R. I. Mackie. B. A. White, and R. E. Isaacson. eds. Chapman and Hall, New York.
GEORGE, B.A., QUARLES, C.L. and FAGERBERG, D.J. (1982) Virginiamycin effects on controlling necrotic enteritis infection in chickens. Poultry Science 61: 447-450.
GRACEY, M., CULLITY, G.J. and SUHARJONO, S. (1977) The stomach in malnutrition. Archives of Disease in Childhood 52:325-327.
HAMDY, A.H., THOMAS, R.W., YANCEY, R.J. and DAVIS, R.B. (1983) Therapeutic effect of optimal lincornycin concentration in drinking water on necrotic enteritis in broilers. Poultry Science 62: 589-591.
HENRY, P.R., AMMERMAN, C.B. and MILES, R.D. (1986) Influence of virginiamycin and dietary manganese on performance, manganese utilization, and intestinal tract weight of broilers. Poultry Science 65: 321-324.
HENRY, P.R., AMMERMAN, C.B., CAMPBELL, D.R. and MILES, R.D. (1987) Effects of antibiotics on tissue trace mineral concentration and intestinal tract weight of broiler chicks. Poultry Science 66:1014-1018.
HOFSHAGEN, M. and KALDHUSDAL, M. (1992) Barley inclusion and avoparcin supplementation in broiler diets. 1. Effect on small intestinal bacterial flora and performance. Poultry Science 71: 959-969.
HOITINK, H. (1997) Clostridium infection is changing appearance. Pluimveehouderij 34.
HOPPE, P. (September, 1999) Wet Litter in broilers. Poultry International 52-62.
HUTCHISON, T.W.S. and RIDDELL, C. (1990) A study of hepatic lesions in broiler chickens at processing plants in Saskatchewan. Canadian Veterinary Journal 31: 20-25.
JOHNSTON, K.L. (1999) Small intestinal bacterial overgrowth. Progress in Gastroenterology 29(2): 523-550
KALDHUSDAL, M. and HOFSHAGEN, M. (1992) Barley inclusion and avoparcin supplementation in broiler diets. 2. Clinical, pathological. and bacteriological findings in a mild form of necrotic enteritis. Poultry Science 71: 1145-1153.
KALDHUSDAL, M., HOFSHAGEN, M., LOVLAND, A., LANGSTRAND, A.H. and REDHEAD, K. (1999) Necrotic enteritis models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variable. FEMS Immunology and Medical Microbiology 24(3): 337-343.
KALDHUSDAL, M. and LOVLAND, A. (2002) Clostridial necrotic enteritis and cholangiohepatitis. Pages G3- G13 in Proceedings from The Elanco Global Enteritis Symposium.
KALDHUSDAL, M. and SKJERVE, E. (1996) Association between cereal contents in the diet and incidence of necrotic enteritis in broiler chickens in Norway. Preventive Veterinary Medicine 28: I-16.
KING, C.E. and TOSKES, P.P. (1979) Small intestine bacterial overgrowth. Gastroenterology 76: 1035-1055.
KLEMPARSKAYA, N.N., PINEGIN, B.V., SHAL'NOVA, G.A., KORSHUNOV, V.M., MAL'TSEV, V.N., GLAD'KO, I.A., KUZ'MINA, T.D., ULANOVA, A.M., DOBRONRAVOVA, N.N. and YERMOLOV, V.V. (1987) Normalizing effect of immunoglobulins in the treatment of endogenous infection and intestinal dysbacteriosis in irradiated mice. Journal of Hygiene, Epidemiology, Microbiology and Immunology 31(1): 91- 98.
KONDO, F. (1988) In vitro lecithinase activity and sensitivity to 22 antimicrobial agents on Clostridium perfringens isolated from necrotic enteritis of broiler chickens. Research in Veterinary Science 45: 337-340
LEE, M. (2002) Microbial dynamics of the broiler intestinal tract. Pages AI-A14 in Proceedings of the Poultry Enteritis Conference. Elanco Animal Health, Cambridge, England.
LICHTMAN, S., SHERMAN, P. and FORSTNER, G. (1986) Production of secretory immunoglobulins A in rat self-filling blind loops: Local secretory immunoglobulin A immune response to luminal bacterial flora. Gastroenterology 91: 1495-1502.
LICHTMAN, S.N., SARTOR, R.B., KEKU, J. and SCHWAB, J.H. (1990) Hepatic inflammation in rats with experimental small intestinal bacterial overgrowth. Gastroenterology 98(2): 414-423.
LONG, J.R. (1973) Necrotic enteritis in broiler chickens. I. A review of the literature and the prevalence of the disease in Ontario. Canadian Journal of Comparative Medicine 37: 302-308.
LONG, J.R., PETTIT, J.R. and BARNUM, D.A. (1974) Necrotic enteritis in broiler chickens. II. Pathology and proposed pathogenesis. Canadian Journal of Comparative Medicine 38: 467-474.
LONG, J.R. and TRUSCOTT, R.B. (1976) Necrotic enteritis in broiler chickens. 111. Reproduction of the disease. Canadian Journal of Comparative Medicine 40: 53-59.
LOVLAND A. and KALDHUSDAL, M. (1999) Liver lesions seen at slaughter as an indicator of necrotic enteritis in broiler flocks. FEMS Immunology and Medical Microbiology 24(3): 345-351.
LOVLAND, A. and KALDHUSDAL, M. (2001) Severely impaired production performance in broiler flocks with high incidence of Clostridium perfringens-associated hepatitis. Avian Pathology 30: 73-81.
MARSH, T.L. (1999) Terminal restriction fragment length polymorphism (T RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. Current Opinion in Microbiology 2: 323-327.
MCDOUGALD, L.R. (2003) Coccidiosis. Pages 974-991 in Diseases of Poultry I 1th Edition. Y.M. Saif, ed. Iowa State Press, Ames.
MCMEEKIN, T.A., BROWN, J., KRIST, K., MILES, D., NEUMEYER, K., NICHOLS, D. S., OLLEY, J., PRESSER, K., RATKOWSKY, D.A., ROSS, T., SALTER, M. and SOONTRANON, S. (1997) Quantitative microbiology: a basis for food safety. Emerging Infectious Diseases 3(4): 541-549.
MEAD, G.C. (1989) Microbes of the avian cecum: types present and substrates utilized. Journal of Experimental Zoology Supplement 3: 48-54.
MORTIMER, I. (2002). The detection of dysbacteriosis. Pages D3-D7 in Proceedings from The Elanco Global Enteritis Symposium.
NETHERWOOD, T., GILBERT, HJ., PARKER, D.S. and O'DONNNELL, A.G. (1999) Probiotics shown to change bacterial community structure in the avian gastrointestinal tract. Applied and Environmental Microbiology 65:5134-5138.
NIILO, L. (1980) Clostridium perfringens in animal disease: A review of current knowledge. Canadian Veterinary Journal 21(5): 141-147.
ONDERKA D.K., LANGEVIN, C.C. and HANSON, J.A. (1990) Fibrosing cholehepatitis in broiler chickens induced by bile duct ligations or inoculation of Clostridium perfringens. Canadian Journal of Veterinary Research 54:258-290.
ONTARIO MINISTRY OFAGRICULTURE, FOOD AND RURALAFFAIRS (1996) Page 53 in Veterinary Laboratory Services Annual Report.
PANNEMAN, H. (2001) Microbial community profiling and characterization helps the vet. World's Poultry Science Journal 17(4): 34-36.
PANNEMAN, H. (2002) Concept: Microbial community profiling and characterisation (MCPC) - a comparison with other methods for the diagnosis of bacterial overgrowth in the duodenum of broiler chickens. Pages E3E7 in Proceedings from The Elanco Global Enteritis Symposium.
PARISH, W.E. (1961) Necrotic enteritis in fowl (Gallus gallus domesticus). 1. Histology of the disease and isolation of a strain of Clostridium welchii. Journal of Comparative Pathology 71: 377-393.
PATTISON, M. (2002) Some clinical and pathological features of enteritis in broilers - observations on treatment in the UK. Pages Cl - CIO in Proceedings of the Poultry Enteritis Conference. Elanco Animal Health, Cambridge England.
PRESCOTT, J.F., SIVENDRA, R. and BARNUM, D.A. (1978) The use of bacitracin in the prevention and treatment of experimentally-induced necrotic enteritis in the chicken. Canadian Veterinary Journal 19: 181183.
PORTER, R.E. (1998) Bacterial enteritides of poultry. Poultry Science 77: 1159-1165.
RIDDELL, C. and KONG, X-M. (1992) The influence of diet on necrotic enteritis in broiler chickens. Avian Diseases 36: 499-503.
ROURA, E., HOMEDES, J. and KLASING, K.C. (1992) Prevention of immunologic stress contributes to the growth-permitting ability of dietary antibiotics in chicks. Journal of Nutrition 122: 2383-2390.
ROUSSEL, A.J. (1994) Intestinal motility. Compendium of Continuing Education for the Practicing Veterinarian 16: 1433-1443.
RUBINSTEIN, E., MARK, Z., HASPEL, J., BEN-ARI, G., DREZNIK, Z., MIRELMAN, D. and TADMOR, A. (1985) Antibacterial activity of the pancreatic fluid. Gastroenterology 88: 927-932.
SALANITRO, J.P., BLAKE, I.G., MUIRHEAD, PA., MAGLIO, M. and GOODMAN, J.R. (1978) Bacteria isolated from the duodenum, ileum and cecum of young chicks. Applied and Environmental Microbiology 35: 782-90.
SASAKI, J., GORYO, M., OKOSHI, N., FURUKAWA, H., HONDA, J. and OKADA, K. (2000) Cholangiohepatitis in broiler chickens in Japan: histopathological, immunohistochemical and microbiological studies of spontaneous disease. Acta Veterinaria Hungarica 48(1): 59-67.
SCHIPPERS, E., WILLIS, S, RUCKDESCHEL, G. and SCHUMPELICK, V. (1996) Small intestinal myoelectrical activity and bacterial flora after Roux-en Y reconstruction. British Journal ofSurgery 83: 1271- 1275.
SCHOOREL, E.P., GIESBERTS, M.A., BLOM, W. and VAN GELDEREN, H.H. (1980) D-Lactic acidosis in a boy with short bowel syndrome. Archives of Disease in Childhood 55(10): 810-812.
SHANE, S.M., GYIMAH, J.E., HARRINGTON, K.S. and SNIDER, T.G. (1985) Etiology and pathogenesis of necrotic enteritis. Veterinary Research Communications 9: 269-287.
SIDORCHUK, I.I. and BONDARENKO, V.M. (1984) Selection of a biologically active mutant of Propionibacterium shermanii and the possibility of its use in complex therapy of enteral dysbacteriosis. Journal of Hygiene, Epidemiology, Microbiology and Immunology 28(3): 331-338,
SIMPSON, K.W., BATT, R.M., JONES, D. and MORTON, D.B. (1990) Effects of exocrine pancreatic insufficiency and replacement therapy on the bacterial flora of the duodenum in dogs. American Journal of Veterinary Research 59: 203-206.
STUTZ, M.W., JOHNSON, S.L. and JUDITH, AR. (1983) Effects of diet, bacitracin, and body weight restrictions on the intestine of broiler chicks. Poultry Science 62: 1626-1632.
STUTZ, M.W. and LAWTON, G.C. (1984) Effects of diet and antimicrobials on growth, feed efficiency, intestinal Clostridium perfringens, and ideal weight of broiler chicks. Poultry Science 63: 2036-2042.
TOSKES, P.P., GIANNELLA, R.A., JERVIS, H.R., ROUT, W.R. and TAKEUCHI, A. (1975) Small intestinal mucosal injury in the experimental blind loop syndrome; light and electron-microscopic and histochemical studies. Gastroenterology 68: 1193-1203.
TRUSCOTT, R.B. and AL-SHEIKHLY, F. (1977) Reproduction and treatment of necrotic enteritis in broilers. American Journal of Veterinary Research 38: 857-861.
TSAI, S.S. and TUNG, M.C. (1981) An outbreak of necrotic enteritis in broiler chickens. Journal of the Chinese Society for Veterinary Science 7:13-17.
UTILL R., ABERNATHY, C.O. and ZIMMERMAN, H. (1976) Cholestatic effects of Escherichia coli endotoxin on the isolated perfused rat liver. Gastroenterology 70: 248-253.
VISEK, W.J. (1978) The mode of growth promotion by antibiotics. Journal ofAnimal Science 46: 1447-1469.
WAHL, S.M., HUNT, D.A., ALLEN, J.B., WILDER, R.L., PAGLIA, L. and HAND, A.R. (1986) Bacterial cell wall induced heptic granulomas. Journal of Experimental Medicine 163: 884-902.
WATKINS, K.L., SHRYOCK, T.R., DEARTH, R.N. and SAIF, Y.M. (1997) In vitro antimicrobial susceptibility of Clostridium perfringens from commercial turkey and broiler chicken origin. Veterinary Microbiology 54:195-200.
WICKER, D.L., ISGRIGG, W.N. and TRAMMELL, J.H. (1977) The control and prevention of necrotic enteritis in broilers with zinc bacitracin. Poultry Science 56: 1229-1231.
WILSON J., SPIKA, J., CLARKE, R., MCEWEN, S., JOHNSON, R., RAHN, K., RENWICK, S., KARMALI, M., LIOR, H., ALVES, D., GYLES, C. and SANDHU, K. (1996) Verocytotoxigenic Escherichia coli infection in dairy farm families. Journal oflnfectious Diseases 174(5): 1021-1027.
ZHU, X.Y., ZHONG,T., PANDYA Y. and JOERGER, R.D. (2002) 16S rRNA-based analysis of microbiota from the cecum of broiler chickens. Applied and Environmental Microbiology 68: 124-137.

Source: World's Poultry Science Journal, Vol. 61 - September 2005