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Enteric infection of swine with Clostridium perfringens
types A and C
J. Glenn Songer, PhD; Robert D. Glock, DVM, PhD
JGS, RDG: Department of Veterinary Science and Microbiology, University
of Arizona, Tucson, Arizona 85721; Email gsonger@u.arizona.edu
This diagnostic note has not been refereed.
Songer JG, Glock RD. Enteric infection of swine with Clostridium perfringens
types A and C. Swine Health Prod. 1998;6(5):223-225.
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Summary
Hemorrhagic necrotic enteritis in piglets, induced by Clostridium
perfringens type C, is a well-known disease syndrome that remains a
problem in spite of long-term availability of inexpensive, generally-effective
toxoids. Type A infections are now recognized with increasing frequency
in neonatal and weaned pigs, and approaches to diagnosis and prophylaxis
are both different and more complex than those for type C infections. Careful
documentation of the clostridial etiology of these cases, including exclusion
of other enteric pathogens, can provide the basis for effective strategies
for prevention and control.
Clostridium perfringens is the etiologic
agent of multiple syndromes in domestic animals, which represent some of
the most important conditions confronting producers and veterinary practitioners.
The pathogenesis of C. perfringens infections is mediated by one
or more of nearly 20 potent exotoxins. Indeed, the species is divided into
types on the basis of production of [alpha], [beta], e, and i toxins, the
so-called major toxins (Table 1). A fifth toxin,
enterotoxin (CPE), is important in disease in humans and probably in domestic
animals.
The four major toxins are elaborated during growth of vegetative cells,
but CPE production is co-regulated with sporulation, and the toxin is released
upon lysis of vegetative cells. It can be produced by strains of any of
the toxigenic phenotypes (Table 1).
Widespread, long-term availability and use of toxoids has not eliminated
clostridial enteric disease in pigs. Improper administration of immunoprophylactic
products and nonresponding sows may be a small part of the problem, but
evidence increasingly suggests that strains of C. perfringens type
A may be common causes of enteric disease, in pigs and other animals.
Practitioners and diagnosticians have traditionally been reluctant to
accept C. perfringens type A as a cause of enteric disease, probably
because strains of type A are part of the normal intestinal flora of virtually
all warm-blooded animals. However, consideration of the information in Table 1 reveals that the only criteria for membership
in type A are that the strain:
- produce [alpha] toxin, and
- fail to produce [beta], e, and i toxins.
Thus, it seems reasonable (even likely) that there exist groups of strains,
within what we now call type A, that produce heretofore-undescribed virulence
attributes (such as toxins) and that cause specific disease syndromes in
domestic animals.
We were recently asked to examine a large group of isolates of C.
perfringens obtained from piglets with classical hemorrhagic, necrotic
enteritis to confirm that they were type C. In fact, 95% were type A, and
considering that true type-C cases yield pure cultures of type C, it seems
unlikely that these were "normal flora" type-A strains picked
inadvertently from a background of disease-causing type-C strains. Elucidation
of the role of type-A strains will require amassing epidemiologic and microbiologic
data, as well as genotyping and phenotyping the isolates. For the present,
the differential diagnosis for swine enteric disease should include both
type A and type C.
Clinical manifestations and pathologic lesions
Type-C infections are common in newborn animals of many species, perhaps
because this organism is able to colonize rapidly in the absence of well-established
normal intestinal flora. Damage to microvilli and terminal capillaries occurs
prior to adhesion to the jejunal mucosa and progressive mucosal necrosis.
Bacterial invasion, and further multiplication and toxin production, follows
desquamation of epithelial cells. Vegetative cells and spores can be observed
on the mucosa.
Neonatal pancreatic secretion deficiencies and ingestion of protease
inhibitors in colostrum favor the action of [beta] toxin. Intestinal lesions
are usually extensive and severe, but death is probably due ultimately to
toxemia. Peracute disease in piglets 1-2 days of age is characterized by
diarrhea and dysentery, with blood and necrotic debris in feces. Hemorrhagic
necrosis of the mucosa, submucosa, and muscularis mucosa is extensive, with
gas accumulation in tissue and hemorrhagic exudate in the lumen. Morbidity
is 30%-50% and the case fatality rate is 50%-100%. Older piglets often develop
nonbloody, yellowish diarrhea, with jejunal mucosal necrosis. Sows may be
the major source of infection for newborn pigs.
Strains of type A are prevalent in the intestines of warm-blooded animals,
and in the environment. However, isolates of this phenotype are also consistently
associated with enteric disease (Table 1).
[alpha] toxin probably does not cause lesions or fluid loss, so it is likely
that yet-undescribed virulence attributes are involved in the pathogenesis
of swine enteric disease. However, intravascular hemolysis, capillary damage,
inflammation, platelet aggregation, shock, and fatal cardiac effects in
lambs and calves with enterotoxemia are consistent with the action of a
hemolytic toxin, such as [alpha] toxin, in circulation, and these possible
systemic effects in any domestic species should not be ignored.
Type A enteritis in neonatal pigs is most often characterized by mild
necrotizing enterocolitis and damage to the tips of the villi, affecting
primarily the jejunum and ileum. A form of the disease has been reproduced
by inoculation of conventional and gnotobiotic, colostrum-deprived pigs,
in which enteropathy followed substantial adherence and multiplication of
an [alpha]-toxigenic C. perfringens in the gut. [alpha] toxin administered
alone to neonatal piglets caused mild enteritis and villous edema, with
epithelial and vascular damage.
There is an increasing awareness of the possible role of enterotoxin-producing
strains of C. perfringens (mainly of type A) in swine enteric disease.
Clinical materials are not routinely examined for CPE, nor are isolates
of C. perfringens from animals, but the CPE gene has been commonly
found in isolates from horses, cattle, sheep, swine, poultry, and other
species. CPE may be found in stools of diarrheic pigs with superficial mucosal
necrosis and villous atrophy, although not consistently. Spore counts are
often two logs higher in pigs with diarrhea than in normal pigs. Inoculation
of hysterectomy-derived, colostrum-deprived (HDCD) piglets with enterotoxigenic
strains of type A resulted in disease ranging from profuse, bloody diarrhea,
enteritis, and death, to nonbloody diarrhea and intestinal gas accumulation,
with low mortality. The latter syndrome resembles the experimental infection
in conventionally weaned pigs. CPE caused fluid accumulation in ileal loops,
and, when administered intragastrically to HDCD piglets, led to transient
diarrhea. Diarrhea and death in HDCD pigs were prevented by giving serum,
milk, or colostrum from sows immunized against enterotoxin, and conventional
pigs born to the same sows were protected against oral challenge with an
enterotoxigenic strain. Parenteral inoculation with CPE does not stimulate
protective antibodies, but pigs naturally infected with enterotoxigenic
strains produce anti-CPE antibodies. Furthermore, protection of neonatal
pigs by colostrum from immunized dams suggests that commercial products
for immunization of pigs against clostridial enteric disease might benefit
from inclusion of some form of CPE toxoid.
Diagnosis
Diagnosis should begin with a thorough evaluation of herd history and
current clinical signs. Typically affected, untreated pigs should be submitted
for postmortem examination. Clinical signs and gross lesions allow a tentative
diagnosis in type-C infections, but diagnosing type-A infections is more
complex (see below). Appropriate specimens (Table
2) should be fixed in formalin for histopathologic examination or submitted
fresh for microbiologic examination.
Bacteriologic culture for C. perfringens is straightforward. Scrapings
obtained from the mucosa with a sterile microscope slide are first examined
by Gram staining. It is frequently possible to see large numbers of Gram-positive
or Gram-variable rods, and it is not uncommon to observe spores. Clostridium
perfringens organism is perhaps the most oxygen-tolerant of the pathogenic
clostridia, and growth can occasionally be observed on aerobically-incubated
plates in the presence of extensive growth of facultative organisms. Thus,
it is not absolutely essential that solid media be freshly prepared. Typically,
cultures would be made on an agar medium containing 5% bovine or ovine blood,
incubated in an oxygen-free atmosphere in a jar or anaerobic chamber. The
atmosphere may be generated by commercially-available reagent envelopes
or by evacuation and refilling with oxygen-free gases. Identification of
C. perfringens is facilitated by the nearly invariate production
of a double zone of hemolysis. About 2% of strains do not produce q toxin,
and, thus, have no clear inner zone of hemolysis. Lack of the outer zone
of hemolysis, which is produced by [alpha] toxin, is extraordinarily rare.
Gram stains of colonies should reveal Gram-positive or Gram-variable rods
(depending upon the age of the culture), rarely with spores.
There is little evidence that quantitation of C. perfringens is
useful as a diagnostic aid. However, culture of dilutions of mucosal scrapings
will allow isolation of the strain of C. perfringens that is present
in highest numbers. In disease caused by type A, this will increase the
chances of isolating the etiologic strain, rather than a "normal flora"
strain. Given the current state of knowledge of pertinent virulence attributes
within type A, this may be most important if the producer or practitioner
wishes to prepare and use an autogenous toxoid.
Demonstration of toxins by in vivo assays is no longer a common practice.
Culture supernatant fluids or eluates from gut contents (trypsin-treated
or untreated, neat or mixed with antiserum) are examined in mice (injected
intravenously) for lethality or guinea pigs (injected intradermally) for
dermonecrosis. Detecting toxins in clinical specimens does not necessarily
confirm the existence of disease, and false negatives may occur due to the
lability of these proteins, especially [beta] toxin. CPE can be detected
by cytotoxicity assays and immunoassays, and the latter are now commercially
available. The commercial reverse-passive latex agglutination test is apparently
subject to false positives, but the enzyme immunoassay is significantly
more accurate. The missing element in the array of diagnostic tools is a
simple in vitro method by which to detect [alpha], [beta], and e toxins.
Use of polymerase chain reaction (PCR) to genotype field isolates has
provided new opportunities to understand the natural history of porcine
clostridial enteritis, and provides a powerful tool to generate information
on which to base prevention and control strategies. Template DNA is obtained
from isolates of C. perfringens, and segments of the genes for the
major toxins, plus CPE, are amplified. Detection of specific products allows
placement of an isolate into one of the five genotypes. Silent toxin genes
are apparently rare in isolates from domestic animals, and the correlation
of genotype (presence of toxin genes) with phenotype (production of toxins)
is nearly 100%. An exception is type-E strains, which are occasionally isolated
from calves with hemorrhagic enteritis, and which carry a silent copy of
CPE. The availability of PCR genotyping is increasing.
If isolates of genotype C are obtained from pigs with typical lesions
in nonvaccinated herds, diagnosis is unequivocal. Current evidence suggests
that commercial type-C toxoids are effective in preventing enteritis and
enterotoxemia associated with this toxigenic type, but the possibility of
intragenotype variability in virulence attributes makes it wise to retain
isolates against the possible need to produce an autogenous toxoid.
If isolates of genotype A are obtained, if clinical signs and lesions
are compatible, and if other etiologies (viruses, parasites, such as Isospora
suis, and other bacteria, such as Escherichia coli) are ruled
out by concurrent microbiologic examination, a diagnosis of type-A enteritis
should be considered. A positive enzyme immunoassay for CPE in gut contents
is supportive, but most type-A isolates causing enteritis do not produce
CPE. Unfortunately, limitations in current knowledge and available diagnostic
technology do not allow positive confirmation of the type-A etiology; thus
one must take a Sherlock Holmes-like approach to finding an answer by eliminating
the alternate possibilities. Perhaps the most meaningful confirmation of
the occurrence of type-A enteritis is decreased incidence in the face of
vaccination with a carefully-prepared autogenous toxoid. However, the overall
utility of autogenous toxoids can be difficult to evaluate, since other
management changes are often made concurrently.
Prophylaxis and therapy
Type-C infections can usually be prevented by vaccinating sows with a
commercial toxoid, and there is anecdotal evidence that autogenous toxoids
prepared against type-A strains are useful. Immunization of sows must be
linked to efforts to maximize the quality and quantity of colostrum uptake.
Clostridium perfringens is susceptible to many antimicrobials, and
prophylaxis of type-C disease in swine has been achieved by use of feed-grade
bacitracin (at 250 g per ton of feed), lincomycin, and others, from 2 weeks
prefarrowing through the course of lactation. One would predict a similar
positive effect against type-A infections. Treating individual piglets with
oral or injectable antimicrobials has often been successful, but the high
rate of recovery in untreated pigs makes it difficult to interpret anecdotal
reports.
Beyond this, heightened attention to sanitation is important, including
cleaning the sow prior to farrowing, and routinely disinfecting farrowing
houses and crates.
References
1. Collins JE, Bergeland ME, Bouley D, Ducommun AL, Francis DH, Yeske
P. Diarrhea associated with Clostridium perfringens type A enterotoxin
in neonatal pigs. J Vet Diagn Invest. 1989;1:351-353.
2. Estrada-Correa AE, Taylor DJ. Porcine Clostridium perfringens
type A spores, enterotoxin, and antibody to enterotoxin. Vet Rec.
1989;124:606-610.
3. Jestin A, Popoff MR, Mahe S. Epizootiologic investigations of a diarrheic
syndrome in fattening pigs. Am J Vet Res. 1985;46:2149-2151.
4. Johannsen U, Menger S, Arnold P, Köhler B, Selbitz HJ. Experimental
Clostridium perfringens type A enterotoxaemia in unweaned piglets.
Monatsh für Veterinarmed. 1993; 48:267-273.
5. Meer RR, Songer JG. Multiplex PCR method for genotyping Clostridium
perfringens. Am J Vet Res. 1997;58:702-705.
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