Download DAUBE_1996_Am-J-Vet-Res_496

Published in: American Journal of Veterinary Research (1996), vol.57, pp. 496-501
Status: Postprint (Author’s version)
Hybridization of 2,659 Clostridium perfringens isolates with gene probes for
seven toxins (α, β, ε, ι, θ, µ, and enterotoxin) and for sialidase
Georges Daube, DVM, DVSc; Patricia Simon; Bernard Limbourg, DVM; Christophe Manteca, DVM; Jacques
Mainil, DVM, DVSc; Albert Kaeckenbeeck, DVM
From the Department of Bacteriology, Faculty of Veterinary Medicine, University of Liege, Sart-Tilman, Bat
B43, Liege, B-4000 (Daube, Simon, Manteca, Mainil, Kaeckenbeeck), and Fédération de Lutte contre les
Maladies Animales, 604 Chaussée de Marche, Er-pent, B-5101 (Limbourg), Belgium.
Supported by Institut pour l'encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture grant
No. 5313, 5452, and 5551 and by a grant from the Department of Agriculture of the Région Wallonne.
This report represents a portion of a thesis submitted by the senior author to the university as partial fulfillment
of the requirement for the Docteur en Sciences Vétérinaires degree.
The authors thank L. A. Devriese for providing Clostridium perfringens isolates and M.-P. Marot, K. Renier, N.
Collin, and V. Pirson for technical assistance.
Objective — To genetically characterize Clostridium perfringens isolates for association of pathologic type with
various diseases.
Design — Prospective study.
Sample Population — 2,659 C perfringens isolates from various nonhuman animals species, human beings, and
foods.
Procedure — Colony hybridization with DNA probes for 7 toxin (α, β, ε, ι (subunits a and b), θ, µ, and
enterotoxin) genes and 1 sialidase gene were performed to group the isolates by pathologic type.
Results — Enterotoxin-negative type-A isolates were the most common (2,575/2,659), were isolated from all
sources, and were separated into 5 pathologic types. In cattle and horses with enterotoxemia, essentially only
these pathologic types were identified. The enterotoxin-negative isolates of types C or D each had a single
pathologic type. Type-C isolates were isolated only from swine with necrotic enteritis and type-D isolates from
small ruminants with enterotoxemia, except that 1 type-D isolate was also found from a healthy fish. Type-B or E isolates were not found. Among the 47 enterotoxin-positive isolates, 5 isolates from sheep or deer were type D
and the other 42 were type A. These 42 isolates were grouped into 3 pathologic types: 1 type was isolated from
samples of almost all origins, but the other 2 types were found in only 5 fish, 4 human beings, and 1 dog.
Conclusions and Clinical Relevance — Genetic characterization of these isolates allowed identification of 11
different pathologic types. This approach may be useful in molecular diagnosis and prophylaxis of clostridial
disease. (Am J Vet Res 1996;57:496-501)
Published in: American Journal of Veterinary Research (1996), vol.57, pp. 496-501
Status: Postprint (Author’s version)
In recent years, rapid advances in the genetics of Clostridium perfringens virulence factors1 have allowed
performance of extensive surveys of the strains isolated in various diseases. 2 The 4 major lethal toxins (α, β, ε,
and ι) and enterotoxin are the most important factors in inducing C perfringens-related disease,3 but the other 7
minor toxins (δ, θ, Κ, λ, µ, v, and sialidase) also may participate in specific disease syndromes.4-6 However,
except for θ-toxin,7 direct evidence for pathogenesis has not been reported. Type-A C perfringens produces αtoxin; causes gas gangrene in human beings, colitis in horses, and necrotic enteritis in birds; and is the more
common type isolated from soil and sewage. The enterotoxigenic type-A isolates induce food poisoning in
human beings. Type-B isolates, producing α-, β-, and ε-toxins, and type-C isolates, producing α- and β-toxins,
are responsible for necrotic enteritis in young animals of most species and in human beings, in which
enterotoxigenic type-C isolates are involved.8 Type-D isolates, producing α- and ε-toxins, cause enterotoxemia,
especially in small ruminants, but this type also has been described in other species. Type-E isolates, producing
α- and ι-toxins, have rarely been isolated and have been involved in necrotic enteritis in cattle. The purpose of
the study reported here was to examine the pathologic types of clinical isolates of C perfringens via colony
hybridization with 9 probes specific for the major toxins.
Materials and Methods
Gene probe — Six DNA probes were derived from multicopy recombinant plasmids carrying toxin- or sialidaseencoding genes (Table l).2 Plasmid DNA was isolated by ultracentrifugation in cesium chloride gradients as
described.9 The other 3 gene probes were obtained by polymerase chain reaction. 2 The fragment of the subunit b
gene of the ι-toxin was obtained with the primers GATGAGAGCTGTGTTGAACTT and TAGAAGTTGAAGGATCCTTTG. The DNA template was extracted from the strain NCIB 10748, according to the method
previously described.2,10
Clostridium perfringens isolates and DNA hybridization — Isolates of C perfringens (n = 2,659) from the
collection of our laboratory were studied. The isolates had been collected between 1989 and 1993, and most of
them were isolated from human beings or nonhuman animals having digestive tract disorders (Table 2). Most
bovine, equine, or small ruminant isolates were from animals that had died of enterotoxemia. Samples from
human beings, dogs, cats, and swine had been obtained from diagnostic laboratories; most of them had been
submitted because the human being or animal had diarrhea, although clostridial disease had not been suspected.
Eight isolates from 8 cases of necrotic enteritis in pigs also were included.a Isolates from fish and fowl were
obtained from healthy animals, except for 6 chickens with diarrhea. The 45 food isolates were cultured from
samples of foodstuffs of animal origin, especially meat, fish, or shellfish, without any association with an
episode of food poisoning.
One to 17 isolates (mean = 3) were purified from each of the 769 samples. Isolates of C perfringens were
identified on blood agar platesb after incubation for 18 hours in anaerobic conditions (80% N2, 10% CO2, 10%
H2). On the basis of colony appearances, hemolysis profile, and gram-stained smears, colonies considered to be
C perfringens were sub-cultured for 24 hours anaerobically on agarc for lecithinase (α-toxin) production and
sulfite reduction. Identification of the sulfite-reducing colonies was confirmed by disk method, using disks d to
detect urease and indol production and glucose, lactose, sucrose, trehalose, and mannose fermentation. The toxin
type of 133 isolates was obtained by use of classical mouse lethality assays.11,e
The DNA colony hybridizations were performed as previously described. 2,f Nineteen C perfringens strains were
used as controls in hybridization.2
Published in: American Journal of Veterinary Research (1996), vol.57, pp. 496-501
Status: Postprint (Author’s version)
Table 1—Derivation of the 9 DNA probes used to classify isolates of Clostridium perfringens
Probe
Origin
Endonuclease*
Size (base pairs)
Reference No.
α
β
pTOX5†
PCR
fragment
pGDS7†
PCR
fragment
PCR
fragment
pLWL10†
pRT1B†
pNAG-S3†
Unnamed†
BamH1 and HindIII
NA
950
1,025
17
2,21
HindIII
NA
505
297
24
2,49
NA
331
49
Kpnl and HindIII
Accl
HindIII
Kpnl and HindIII
504
736
1,300
1,400
50
51
52
53
ε
ιa
ιb
E
θ
µ
s
*Used to derive the gene probe fragment from the plasmid; †plasmid.
PCR = polymerase chain reaction; NA = not applicable; E = enterotoxin; and S = sialidase.
Table 2—Origin of isolates of C perfringens
Species
No. of isolates
No. of samples
Cattle
Human beings
Sheep
Dogs
Cats
Fish
Swine
Goats
Fowl
Deer
Horses
Other†
Food‡
Total
1,443
224
219
200
171
77
63
55
42
29
25
66
45
2,659
390
46
63
35
22
16
48
20
40
8
8
41
32
769
Associated disease*
(No. of samples)
Enterotoxemia (360)
Diarrhea (37)
Enterotoxemia (63)
Diarrhea (14)
Diarrhea (4)
None
Diarrhea (15), necrotic enteritis (10)
Enterotoxemia (19)
Diarrhea (6)
Enterotoxemia (8)
Enterotoxemia (8)
NA
NA
…
*Other samples were obtained from animals without signs of clostridial disease. †Other animal species or unknown origin. ‡Of animal origin
for human consumption.
Table 3—Distribution of probe genes among isolates of C perfringens
Origin
Cattle (n = 1,443)
Human beings (n = 224)
Sheep (n = 219)
Dogs (n = 200)
Cats (n = 171)
Fish (n = 77)
Swine (n = 63)
Goats (n = 55)
Fowl (n = 42)
Deer (n = 29)
Horses (n = 25)
Other* (n = 66)
Foodt (n = 45)
Total (n = 2,659)
Hybridizing probe (%)
α
β
ε
ιa
ιb
E
θ
µ
S
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0
0
0
0
0
0
6.4
0
0
0
0
0
0
0.2
0
0
9.1
0
0
1.3
0
20
0
6.9
0
3
0
1.4
0
0
0
0
1.2
0
0
0
0
0
0
0
0
0.1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.4
5.8
2.3
2.5
1.25
13
1.6
3.6
0
6.9
4
0
0
1.8
91.1
71.9
95
66.5
84.2
62.3
84.1
100
90.5
100
96
92.4
91.1
86.9
92.9
94.2
96.8
88
86.5
92.2
100
100
100
100
100
98.5
93.3
93.2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
*Other animal species or unknown origin. tOf animal origin for human consumption.
Published in: American Journal of Veterinary Research (1996), vol.57, pp. 496-501
Status: Postprint (Author’s version)
Table 4—Comparison of mouse lethality assay and DNA-DNA hybridization results in 133 isolates of C
perfringens
Probe
α
β
ε
ιa
ιb
θ
µ
E
S
Total
Nonlethal
14
0
0
0
0
14
13
0
14
14
Type A
96
0
0
1
0
88
93
8
96
96
Lethal
TypeC
4
4
0
0
0
4
4
0
4
4
TypeD
19
0
19
0
0
19
19
2
19
19
Values are No. of isolates that hybridized with each probe.
Results
All 2,659 isolates hybridized with α-toxin and sialidase probes (Table 3). Moreover, those probes hybridized
with lethal and nonlethal isolates (Table 4). Only 4 isolates from swine with necrotic enteritis hybridized with
the β-toxin probe; those isolates were lethal for mice. Only isolates from sheep, goats, and deer hybridized with
the ε-toxin gene probe; all of those tested isolates were lethal for mice. Two isolates from a healthy cat
hybridized with the ιa-toxin (ADP-ribosyltransferase) probe, but not with the ιb-toxin probe. One of those isolates
was classified as toxin type A by use of the lethality test.
Enterotoxin probe hybridized with isolates from most origins, but such hybridization was more common in
isolates from human beings or from fish than in those from other species. None of the 45 human food isolates
hybridized with the enterotoxin gene probe.
The θ-toxin gene was detected in 2,311 of the 2,659 isolates. Isolates that did not hybridize with this probe were
more commonly from carnivorous (cats and dogs), aquatic (fish), or omnivorous (human beings and swine)
species than from herbivorous animals (ruminants and horses). The same tendency was observed for the µ-toxin
gene, but was less marked.
The 2,659 isolates could be classified into 11 different pathologic types (Table 5). The most common pathologic
type (2,093/2,659 isolates) was characterized by hybridization with only the α-, θ-, and µ-toxins and sialidase
probes. This pathologic type corresponded to the common nonenterotoxigenic type-A strains. This toxin type
also included 4 other pathologic types, 3 with different θ- and µ-toxin results and 1 that hybridized with the ιatoxin gene probe. The nonenterotoxigenic type-C and -D isolates hybridized with the θ- and µ-toxin probes.
The enterotoxigenic isolates were divided into 4 pathologic types: toxin type-A or -D isolates hybridizing with
the θ- and µ-toxin probes; type-A isolates hybridizing with the µ-toxin probe, but not with the θ-toxin probe; and
type-A isolates not hybridizing with θ- and µ-toxin probes.
In cattle, even those with enterotoxemia, only pathologic types corresponding to toxin type A were isolated
(Table 6). Enterotoxigenic isolates were obtained from only 4 of the 390 studied cattle. In small ruminants and
deer with enterotoxemia, the pathologic types corresponding to toxin type D were common (24/90 isolates). The
enterotoxigenic type-D isolates were only from sheep and deer. In swine, dogs, cats, horses, and fowl, except for
4 type-C isolates from swine with necrotic enteritis, all isolates were toxin type A. The isolates of
enterotoxigenic type A that did not hybridize with the θ-toxin probe were obtained only from human beings, fish,
and 1 dog, whereas isolates of that type that hybridized with the θ-toxin probe were obtained from nearly all
species.
Published in: American Journal of Veterinary Research (1996), vol.57, pp. 496-501
Status: Postprint (Author’s version)
Table 5—Distribution of pathologic types among the 2,659 isolates of C perfringens
Hybridizing probes (%)
Origin
Cattle (n = 1,443)
Human beings (n = 224)
Sheep (n = 219)
Dogs (n = 200)
Cats (n = 171)
Fish (n = 77)
Swine (n = 63)
Goats (n = 55)
Fowl (n = 42)
Deer (n = 29)
Horses (n = 25)
Other* (n = 66)
Foodt (n = 45)
Total (n = 2,659)
α,S
1.1
2.2
0.5
4.5
0
2.6
0
0
0
0
0
0
0
1.2
α,θ,S
6.1
3.1
2.7
7.5
13.5
5.2
0
0
0
0
0
1.5
6.7
5.5
α,µ,S
7.8
23.7
4.6
28.5
15.8
24.7
15.9
0
9.5
0
4
7.6
8.9
11.4
α,θ,µ,S
84.6
65.2
82.6
57
68.4
53.2
76.2
76.4
90.5
89.7
92
87.9
84.4
78.7
α,ιa,θ,µ,S
0
0
0
0
1.2
0
0
0
0
0
0
0
0
0.1
α,E,S
0
0.4
0
0
0
0
0
0
0
0
0
0
0
0.04
α,µ,E,S
0
1.8
0
0.5
0
10.4
0
0
0
0
0
0
0
0.5
α,θ,µ,E,S
0.4
3.6
0.5
2
1.2
2.6
1.6
3.6
0
3.4
4
0
0
1.1
α,β,θ,µ,S
0
0
0
0
0
0
6.3
0
0
0
0
0
0
0.2
α,ε,θ,µ,S
0
0
7.3
0
0
1.3
0
20
0
3.4
0
3
0
1.2
α,ε,θ,µ,E,S
0
0
1.8
0
0
0
0
0
0
3.4
0
0
0
0.2
See Table 3 for key.
Table 6—Main associations between pathologic types and origin of isolates of C perfringens
Disease status
Origin
Enterotoxemia Cattle (n = 360)
Sheep (n = 63)
Goats (n = 19)
Deer (n = 8)
Horses (n = 8)
Necrotic
Swine (n = 8)
enteritis
None
Human beings (n
suspected
= 46)
Fish (n = 16)
Dogs (n = 35)
Cats (n = 22)
Cattle (n = 30)
Swine (n = 40)
Fowl (n = 40)
Food* (n = 32)
α,S
2.5
1.6
0
0
0
0
Hybridizing probes {% of cases)
Type A
TypeC
α,θ,S α,µ,S α,θ,µ,S α,ιa,µ,S α,E,S α,µ,E,S α,θ,µ,E,S α,β,θ,µ,S
15
15.3 94.4
0
0
0
1.1
0
7.9
11.1 85.7
0
0
0
1.6
0
0
0
52.6
0
0
0
5.3
0
0
0
100
0
0
0
12.5
0
0
12.5 100
0
0
0
12.5
0
0
0
60
0
0
0
0
40
TypeD
α,ε,θ,µ,S α,ε,θ,µ,E,S
0
0
15.9
4.8
47.4
0
12.5
12.5
0
0
0
0
8.7
8.7
39.1
73.9
0
2.2
8.7
10.9
0
0
0
12.5
8.6
0
0
0
0
0
18.8
31.4
31.8
0
0
0
3.1
68.8
54.3
54.5
13.3
26.3
10
12.5
81.3
88.6
95.5
86.7
76.3
90
87.5
0
0
4.5
0
0
0
0
0
0
0
0
0
0
0
31.3
2.9
0
0
0
0
0
12.5
2.9
4.5
0
2.6
0
0
0
0
0
0
0
0
0
6.3
0
0
0
0
0
0
0
0
0
0
0
0
0
*Of animal origin for human consumption.
Discussion
Until recently, only phenotypic studies had been used to evaluate virulence factors in C perfringens.4-6 Since
1980, studies about virulence-based taxonomy of C perfringens have not been reported; however, the prevalence
of the enterotoxin gene was determined in isolates from human food or feces in association with episode of food
poisoning,12 from swine with diarrhea,13 and from various healthy animals.14 Genomic organization of virulence
genes has been described in some reference strains of C perfringens.15,16 Pathologic typing of C perfringens
isolated from animals with various clostridial diseases could help researchers in associating virulence factors
with diseases or in detecting the reservoirs of some pathologic types.
In this study, the major lethal toxins, except α-toxin, were always found by use of the mouse lethality test when
the corresponding genes were detected by DNA-DNA hybridization. The C perfringens α-toxin possesses both
phospholipase C and sphingomyelinase activities and is an important virulence factor.7,17,18 The α-toxin gene is
located on the chromosome near the putative origin of replication. 15 All the isolates we evaluated hybridized
Published in: American Journal of Veterinary Research (1996), vol.57, pp. 496-501
Status: Postprint (Author’s version)
with the α-toxin gene probe, but had different in vitro α-toxin activity, as revealed by the lethality test or
observation of hemolysis. Variations in gene expression or in toxin activity could explain variations in the
virulence of type-A isolates in human gas gangrene and in animals with enterocolitis. 19,20
The β-toxin gene was recently cloned and sequenced,21 but the gene location has not yet been determined. Only a
few isolates from swine with necrotic enteritis hybridized with the β-toxin gene probe. Other isolates, classified
primarily as toxin type C, from similar swine did not hybridize with this probe and were classified as toxin type
A with the mouse lethality test. Type-C strains have been responsible for cases of necrotic enteritis in young
animals in most species, especially in young swine.22 However, necrotic enteritis was rarely described in animals
in our study, and most studied swine were not neonates.
Isolates that hybridized with the ε-toxin gene probe were almost exclusively from small ruminants. Although
pulpy kidney disease is characteristic of sheep and goats, type-D isolates in other species have been described,
especially in cattle.23 In this study, the 1,443 bovine isolates did not hybridize with the ε-toxin probe. The ε-toxin
gene is located on an extrachromosomal element,16,24 and thus, the factors determining the host preference are
probably located on the same genetic support. Our study revealed that type-D C perfringens was isolated only
from sheep, goats, or deer with enterotoxemia and from 1 fish, but never from cattle with enterotoxemia.
Type-E isolates were not found in this study. The virulence of the 2 feline isolates that hybridized only with the
ιa-toxin gene probe is unknown. The ιb-toxin is involved in binding and internalization of the toxin in the cell.
Further study has revealed that these strains produce a nonlethal ADP-ribosyltransferase, and that lethality was
not recovered by complementation with C perfringens type E or with C spiroforme ιb, fraction.8 Our study
suggested these isolates are rare, although type-E isolates have been found in other parts of the world. 25,26
The number of enterotoxin-positive isolates was less than that found in another study.14 The rate of enterotoxinpositive isolates was 1% in our study vs 22% in that study for cattle, 13% vs 14% for horses, and 0% vs 10% for
poultry. However, sample origin, isolation procedures, and geographic origin were different in these studies.
Indeed, in the other study, the isolates were collected in Switzerland from healthy animals at slaughterhouses,
and the isolates were cultured on tryptose-sulphite-dextrose agar.
In this study, the human isolates were from fecal samples submitted to hospital laboratories, and were not
associated with episodes of collective food poisoning. The human carriage in this population was important,
approximately 22%. This high prevalence could have been associated with antibiotic use, 27 but specific data
were not available.
The 45 human food isolates did not hybridize with the enterotoxin probe, and thus none could have caused food
poisoning. Another study of C perfringens isolated from fecal or food samples in food-borne disease outbreaks
revealed that the prevalence of enterotoxigenic isolates was 66 and 57%, respectively.12 Thus, human foodstuffs
seem to be rarely contaminated with enterotoxigenic C perfringens. In our study, only fish, with a prevalence of
44%, were especially hazardous. Human carriers of enterotoxigenic C perfringens must also be considered as
potential sources of food poisoning.
The enterotoxigenic C perfringens isolates can carry an enterotoxin structural gene on a chromosomal fragment
or on an extrachromosomal element.16,28 Most of the food poisoning-associated isolates carried the gene on the
chromosome. The genetic support for the enterotoxin gene has not been determined in isolates of various origins.
Genetic location could be associated with variation in gene expression and, thus, in virulence.
All isolates hybridized with the sialidase gene probe in our study. The sialidase gene was located on the
chromosome in an instable region.15 The unique sialidase-negative C perfringens described is a laboratory strain,
JIR323, which has probably lost the sialidase gene.2 The lack of sialidase-negative wild isolates, as with the αtoxin gene, suggested that this factor is important for resistance against natural selection, but was not absolutely
necessary in vitro. This sialidase, unlike other neuraminidases, seems to be a cytoplasmic protein and, therefore,
probably has a minor role in pathogenesis.29
The θ- and µ-toxin genes are located on the same chromosomal fragment, with substantial variations but without
position changes.16 In one study,30 θ-toxin-negative isolates, without expression of this gene, were found. Two
regulation systems have been described.31-33
In this study, the lack of 1 or both genes in some isolates was detected, but the prevalence varied among species
of animals. This trend was especially obvious for the θ-toxin gene, with > 90% positive isolates in herbivore
Published in: American Journal of Veterinary Research (1996), vol.57, pp. 496-501
Status: Postprint (Author’s version)
species and only 62, 67, and 72% in fish, dogs, and human beings, respectively.
Characterization of these isolates allowed us to identify 11 different pathologic types or gene combinations. With
pathologic types corresponding to toxin types B and E and to the sialidase-negative toxin type-A strain,2 14
pathologic types were described with these 9 probes.
The nonenterotoxigenic type-A isolates of this study could be grouped into 5 pathologic types. The most
common type corresponded to the toxin type A1, described by Niilo. 4 Strains positive for α- and µ-toxins and
positive or negative for θ-toxin were previously described.2 The other 2 pathologic types, negative for µ-toxin,
had not been reported. The nonenterotoxigenic type-C and -D isolates, each with 1 unique pathologic type,
corresponded to the toxin types C3 or C4 and D, respectively. 4
The enterotoxigenic isolates could be classified into 4 pathologic types. The first type, ε-toxin positive, was
isolated in 3 independent fatal cases of enterotoxemia in sheep and in 1 in deer. Enterotoxin could be important
in the pathogenesis of pulpy kidney disease by assisting ε-toxin transfer in the bloodstream, as has been shown
for enterotoxigenic type-C isolates.8 The relative importance of this type of isolate in small ruminants has not
been determined. Among the other 3 enterotoxin-positive pathologic types, 1 corresponded to the toxin type A2
and was the main enterotoxigenic type isolated in our study. 4 This pathologic type was isolated from samples of
almost all origins, unlike the other 2 pathologic types, which were found in only 5 fish, 4 human beings, and 1
dog. Because the same pathologic types also were observed in most of the reference food poisoning isolates, 2
these isolates are probably the most hazardous.
Although we associated the pathologic type with the origin of the isolates and with disease states, a causal
relationship could not be determined, because bacterial counts or phenotypic results (toxins in intestinal content)
were not available. Indeed, for most of the intestinal diseases caused by Clostridia, clinical signs are induced by
the exotoxins produced in situ in high concentrations by a large number of organisms. For example, for C
perfringens food poisoning in human beings, ingestion of many enterotoxigenic bacteria is responsible for
massive intestinal sporulation, with subsequent enterotoxin production.4,34 For pulpy kidney disease in small
ruminants, C perfringens type D must be present in high numbers for a period sufficient to produce enough εtoxin to cause the disease.35 In necrotic enteritis caused by C perfringens type C in young animals or human
beings, bacterial numbers also must rapidly increase to enable the β-toxin to induce lesions.36 In that disease,
nutritional factors were shown to be essential for disease expression.3,37 Other factors, especially stress, antibiotic
treatment, or intestinal motility modification, could induce intestinal multiplication of C botulinum, spiroforme,
difficile, or perfringens and toxin production.3,38-41 Detection of pathogenic Clostridia (eg, C botulinum, difficile,
tetani, spiroforme, or various toxin types of C perfringens) in animals without signs of clostridial disease has
been demonstrated.3,37,42-48 For all these reasons, the study of intestinal clostridial diseases by DNA-DNA colony
hybridization must be interpreted with bacterial counts in mind.
In addition, detection of toxigenic isolates by gene probes does not allow determination of toxin gene expression
or gene product activity in the intestine. Thus, epidemiologic surveys of nonclinical bacterial carriers, regulation
of toxin gene expression, and activity of expressed products are needed to determine, in each species and in each
disease, the best diagnostic tests. The colony hybridization method allowed ready characterization of many
isolates of known origin. Use of ELISA or easy in vitro neutralizing tests may more completely address the role
of C perfringens isolates in disease. For ethical and practical reasons, we do not think that the mouse lethality
test is a method for further use.
As in other bacterial species (eg, Escherichia coli), distinguishing between saprophytic or pathogenic isolates is
important. Moreover, such evaluation could help to assess the role of minor determinants in C per-fringensassociated diseases. Our approach may allow optimization of molecular diagnosis and prophylaxis in clostridial
disease.10
------------------------------------------a
Isolates obtained from MR Popoff, Institut Pasteur de Paris, Paris, France.
Anaerobic blood agar base with 10% cattle blood, Gibco-BRL, Paisley, Scotland.
c
Shahidi-Ferguson perfringens agar, Oxoid, Hampshire, United Kingdom.
d
Rosco disks, International Medical, Brussels, Belgium.
e
Clostridium welchii type A, C, D, E antitoxins, Wellcome Diagnostics, Dartford, England.
f
Whatman 541 filters, VWR Scientific, San Francisco, Calif.
g
Popoff MR, Institut Pasteur de Paris, Paris, France: Personal communication, 1992.
b
Published in: American Journal of Veterinary Research (1996), vol.57, pp. 496-501
Status: Postprint (Author’s version)
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