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).
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