Electrophoretic characterization for detection of Clostridium perfringens enterotoxin type ( A ) in meat.

Assiui Vet. Med. J. Voi. 53 No. 115 October 2007

Microbial Toxins Research Unit
Animal Health Research Institute, Dokki, Giza

_ ELECTROPHORETIC CHARACTERIZATION FOR
DETECTION OF CLOSTRIDIUM PERFRINGENS

ENTEROTOXIN TYPE (A) IN MEAT
(With 2 Tables and One F igure)

By
A.M.O, EL-MAHROUK
(Received at 18/8/2007)

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SUMMARY

A total of 100 buffalo meat samples were collected from different markets in
Giza govemorate. They were subjected to physical and bacteriological
examination. Thirty nine percentage of the samples tested were found to be
contaminated with Clostridium perfringens type A. The most probable
number (MPN/gram) values ranging from 0 to 35. Also, the spore count of
Clostridium perfringens type A at 100°C at different time interval was
investigated. Electrophoretic analysis and immunobloting for detection of
Clostridium perfringens enterotoxin type (A) was carried out for its
characterization. The immunogenic band was determined with molecular

Key words: Electrophoresis Cl. perfringens, enterotoxin, meat

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Assiut Vet. Med. J. Vol. 53 No. 115 October 2007

INTRODUCTION

Most species of the genus Clostridium are saprophytes that
normally grow in soil, water and decomposing plant and animal matter,
playing an important part in the process of putrefaction.

Clostridium perfringens type A isolates can carry the enterotoxin
gene (cpe) on either their chromosome or a plasmid, but food poisoning
isolates usually have a chromosomal cpe gene. This linkage between
chromosomal cpe isolates and food poisoning has previously been
attributed; Jihong and Bruce, 2006; Lin McClane, 2006.

In vitro toxin production is an important tool not only for
diagnostic purposes but also for the study of pathogenesis of Clostridium
perfringens infections; Fernandez et al., 2007.

The prevalence of the enterotoxin gene in a well-characterized
collection of 71 Clostridium perfringens strains from 36 separate food-
poisoning cases or outbreaks was analyzed with the polymerase chain
reaction (PCR); Ridell et al, 1998.

Meat and fish are sensitive to contamination and support growth
of microorganisms. Anaerobic bacteria constitute an important group of
microorganisms responsible for many health hazards to consumer from
consumption of processed meat and fish products where oxygen
availability is limited .The most important species of anaerobic bacteria
are Cl. botulinum and Cl. perfringens, but some other species are also
known including Cl butyricum, Cl sordellii, Cl. bifermentans, Cl.
sporogenes and Cl. Barati; Mead, 1992.

Clostridium perfringens type A is one of the four most important
bacterial agents causing food poisoning. Differential biochemical
characterization appears to be important because of certain confounding
species. Both the heat sensitive and resistant spore forming strains cause
food poisoning; Narayan, 1982.

Cl. perfringens carried in the human and animal intestine, soil,
dust and flies, ruminant meat are often contaminated. They have
resistant spores and are thus able to survive well in such type of
environment; Barnes, 1985,

The isolation of Cl. botulinum from foods is generally considered
to be of Jess significance than the detection of the toxin; Hobbs et al,
1982. Cl. botulinum causes a food borne intoxication known as botulism.

Seven types of CL botulinum (A, B, C, D, E F and G) are

a on the basis of antigenic Specificity of their toxins; Pierson ef
ai., :

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Assiut Vet. Med J. Vol. 53 No, 115 October 2007

The objective of this study was to investigate the role of several
methodological variables that might be encountered during the study and
to fulfil the following items:
1-Isolation and identification of Clostridium perferingens type A from
meat.

2-Detection of the ability of of Clostridium perferingens type A to
produce toxins in culture medium.

3-Detection of the of Clostridium perferingens type A in foods of
Reversed passive latex agglutination (PET- RPLA) test using a
commercial kit.

4-Western Blotting or immunoblotting for identification of
Enterotoxigenic isolates of Cl perfringense type A

MATERIALS and METHODS

Samples: A total of 100 buffalo meat samples were collected from
different markets in Giza governorate (types of sample, site of
collection, date) were reported on each sample. The samples (10g) were
collected aspectically in clean plastic bag and kept in ice box where
transferred to the laboratory of animal Health Research Institute (AHRI),
Dokki, Giza, Egypt. The samples were immediately subjected to
physical and bacteriological examination without delaying..

Cultural conditions: Processing of each food sample started with a
homogenization step using sterilized surgical scissors. Ten milliliters of
sterile fluid thioglycolate (FTG) medium were then added to the 50 ml
flask containing the minced meat. An aliquot (1 ml) of each FTG meat
suspension was added to each of two tubes containing 10 ml of sterile
FTG. To enrich for any Cl perfringens spores present in the meat
sample, one of those two tubes was heat shocked at 72°C for 20 min
before incubation at 37°C for 18 to 24 h. The other tube was directly
incubated at 37°C for 18 to 24 h to enrich primarily for Cl. perfringens
vegetative cells present in that meat sample.

Each FTG enrichment culture showing growth was streaked onto
one plate of tryptose-sulfitecycloserine agar containing 10% egg yolk
(TSC with egg yolk) and a second plate of brain heart infusion agar
containing 10% .sheep -blood and 40 Ug /ml neomycin . Both plates
were then incubated for 18 h at 37°C in an anaerobic jar. When a meat
sample did grow presumptive Cl. perfringens, those colonies were
inoculated into 10 ml of FTG medium, which was then incubated for an
overnight at 37°C. To confirm the identity of those presumptive FTG
cultures as Cl. perfringens, standard methods were used; Food and Drug

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Assiut Vet. Med. J. Vol. 53 No. 115 October 2007

Administration, 1998. A loopful of each culture was stabbed into a tube
of motility nitrate and lactose-gelatin media. Those tubes were then
incubated at 37°C for 18 to 24 h. Toxin type of the isolates were
determined by neutralization test in mice; Stern and Batty, 1975.

Determination of MPN of CL. perfringens per gram in meat:

A three-lube most probable number (MPN) method was used to
investigate Cl. perfringens levels in meat samples; Lin and Labbe, 2003.
Briefly, a 10 g aliquot of a meat suspension (prepared as described
above) was diluted by 10 fold increments (from 10-' to 10-5) in FTG,
and then 1 ml aliquots of each dilution from a single sample were
inoculated into three tubes containing 10 ml , pf differential reinforced
clostridial broth medium (DRCM). After incubation at 37°C for 24 h,
cultures testing positive for Cl perfringens produced a unique black
precipitation in this DRCM.

Statistical analyses were performed; Koburger, 1975.
Determination of spore heat resistance for CPE-positive
Cl. perfringens type A meat isolates.

To evaluate the heat resistance of Cl perfringens meat isolates,
the spore count was measured at 100°C for each isolate; Sarker ef ai,,
2000.

Briefly, sporulating cultures of C/ perfringens were prepared by
inoculating a 0, 2 ml aliquot of a FTG culture into 10 ml of Duncan-
Slrong (DS) sporulation medium. After an overnight incubation at 37°C,
the presence of sporulating cells in each DS culture was confirmed by
phase contrast microscope. Those DS cultures were then heat shocked at
72°C for 20 min to kill any remaining vegetative cells and to facilitate
spore germination. A 0.1 ml aliquot of each heat-shocked DS culture
was then serially diluted with sterile FTG medium to obtain dilutions
ranging from 107 to 107. Two mi aliquots of each dilution were
duplicate plated onto BHI agar plates in order to establish the number of
viable spores present per milliliter of DS. cultures at the start of heating
(i.e., at the zero time point of the experiment).

The remainder of each heat-shocked DS culture was then heated
at 100°C for time periods ranging from 1 min to 2 h- At each time point,
the boiled DS culture was mixed, and a 0.1-ml aliquot was withdrawn
and diluted (dilution range, from 107 to 10’) with sierilo rTG medium.
Each dilution was then duplicate plated onto BHI agar plates, which
were incubated anaerobically at 37°C for 18 h. Colonies developing
from germinated spores that survived heating, were then counted to
Assiut Vet. Med. J. Voi, 53 No. 1i5 October 2007

determine The number of viable spores present at th ime point per
milliliter of each healed DS culture,

Detection of enterotoxins producing isolates by reversed passive
latex agglutination (PET-RPLA Kit)

A porlion of the sporulated culture (about 5 m!) was centrifuged
for 15 rnin at 10,000 xg and cell free culture supernatant was tested for
enterotoxin by using RPLA Kit; Food and Drug Administration, 1998.
SDS-PAGE and Western Blotting; Vernon et al., 1996.

Solubilized cells (0.4 gm/ml) were mixed with an equal volume
of 4X sample buffer (3 % Tris, 20% b-mercaptoethanol, 10% SDS,
0.02% bromophenol blue and 40% glycerol; PH6.8) heated at 95°C for 3
min, and then centrifuged to remove any remaining insoluble material.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) was performed. Protein marker contains 5 proteins with
molecular weight ranged from 94-20 KDa used as molecular weight
standards (Sigma-Aldrich).

After electrophoresis, the separated proteins were transferred to
microcellulose membranes at 360 mA for 4 hours at 4°C. Membranes
were then blocked overnight with a blocking buffer including 1% (W/V)
gelatin in Tris-buffered saline (TBS: 20 mM Tris-HCL PH 7.4, 0.5 M
Nacl). The membranes were rinsed three times with TBS containing
0.05% Tween 20 and then incubated for 3 hours with either preimmune
serum or polyclonal antiserum obtained after infection. Each serum
sample was diluted (1:500) in blocking buffer. Memmbranes were rinsed
three times and incubated with goat anti-rabbit IgG peroxidase conjugate
(diluted 1:2,500 in blocking buffer) for 3 hours (Sigma, St. Louis, Mo.).
Immunoreactivity was detected by incubating blots with TBS containing
HO) and 4-chloro-1-napthol.

RESULTS
Table 1: Prevalence of Cl. perfringens in buffalo meat

aus No. of samples
No. of No. of positive ; .
total vegetative cell | No. (%) of spore MPN/g tested with RPLA kit
samples of cell of E range i
examined | C. perfringens | perfringens ~ A —
: . OX:
100 39 (39.0) 4 (4.0) 0-35 39 4

Assiut Vei. Mea. J. Voi_33 No. 115 October 2007

Table 2: Spore count of Cl. perfringens type A after heating at 100°C at
different time interval.

Isolates 0 30 min. 60 min. 90min. | 120 min.

I a | 3.4 | 0 0 10

2 2 3.31 22 1S id

3 17 | 4.0 LI 10 0
14 ig [3.19 [0 10 0 “4

Western Blot analysis of CPE, Lane A: Molecular weight marker; Lane
‘ B: Immunogenic band (35 KDa)

DISCUSSION

Clostridium perfringens isolates are commonly classified into
five types (types A to E) based on the production of four typing toxins
(alpha, beta, epsilon, and iota toxins); Songer, 1996. Type A isolates, the
most abundant toxinotype, produce alpha toxin, but not beta, epsilon, or
iota toxin; Immerseel e¢ al., 2004. Some type A isolates also produce
another toxin, Cl. perfringens enterotoxin (CPE). These enterotoxigenic
type A strains cause several human enteric diseases, including Cl.
perfringens type A food poisoning, which is among the three most
common food-borne illnesses in the United States, and some cases of
non-food-borne human gastrointestinal disease, including antibiotic-
associated diarrhea and sporadic diarrhea; Carman, 1997.

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To date, there are at least two explanations for the strong
association between type A isolates carrying a chromosomal cpe gene
and food poisoning. First, a recent study; Wen and McClane, 2004 that
evaluated the presence of cpe-positive isolates in American retail foods
showec that all 13 epe-positive type A isolates recovered from the foods
surveyed had a chromosomal cpe gene. The findings indicated that, at
least in part, chromosomal cpe isolates are the predominant cause of
food poisoning because they are the cpe-positive type A isolates that are
most often present in food.

As shown in Table (1) 39.0% of meat samples tested in the
present study were found to be contaminated with CI perfringens
isolates, similar result were reported by Abd El-Rahman ef ai., 1995 who
mentioned that C/. perfringens is present in high incidence in meat and
meat product. Table (1) showed that low contamination frequency is
consistent with MPN results indicating that the meat samples tested in
this study had MPN / gram values ranging from 0 to 35, this observation
were in agreement with that mentioned by Lin and Labbe 2003 and Wen
and McClane 2004 who tested MPN/gram in American retail foods and
found that its value ranged from 0 to 32. About 4.0% of C1. perfringens
from meat samples grew after heat shocking obviously indicative of they
restrained spores.

In the provisional, 39.0% of meat samples grew Cl. perfringens
only in the absence of heat shocking It is hypothetically achievable that
some of these samples also contained C] perfringens spores that
spontaneously germinated in the absence of heat shocking. However, the
large difference in Ci. perfringens observed between heat-shocked
versus non heat -shocked meat samples strongly suggests that most of
the non-heat-shocked food samples growing Cl. perfringens had been
contaminated with vegetative cells which were killed by heat shocking,
rather than spores. In most of the non -heat -shocked samples yielding
Cl. perfringens had contained spores that spontaneously germinated into
vegetative cells, those samples also tested positive for C.perfringens
after heat shocking and this agree with that mentioned by Varga et al,
2004 who suggests that the strong association between type A isolates
carrying a cpe gene and Cl. perfringens type A food poisoning is
attributable (at least in part) to the exceptional heat resistance of those
isolates, which should favor their survival in incompletely cooked or
improperly held foods

Little is known about the mechanisms responsible for the specific
heat resistance of chromosomal cpe isolates, although a recent study;

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Assiut Vet. Med. J. Vol. 53 No. 115 October 2007

Raju and Sarker, 2005 showed that Gi) the heat resistance of
chromosomal cpe isolates is not dependent on the presence of a
functional cpe gene and (ii) the heat sensitivity of plasmid cpe isolates is
not dependent on the presence of a cpe plasmid.

With the ultimate goal of better controlling Ci. perfringens type
A food poisoning, workers have begun investigating why this food-
borne illness is so strongly associated with type A isolates carrying a
chromosomal cpe gene; Jihong et al., 2006. CPE is clearly responsible
for the symptoms of Ci. perfringens type A food poisoning; Sarker et ai.,
1999, but the relationship between food poisoning and chromosomal cpe
isolates does not appear to involve isolates that produce either a more
potent CPE or larger amounts of CPE than plasmid cpe isolates produce;
Collie et al, 1998, and McClane, 2001. Since temperature abuse is the
leading factor responsible for Cl perfringens type A food poisoning
outbreaks; McClane, 2001.

The enteropathogenic effects of CPE are primarily mediated
through a multi step cytotoxic action, which initiates when CPE hinds to
a pertinacious receptor (s); Cornillot et al., 1995 Clostridiuin perfringens
uses its potent arsenal of 14 toxins to cause enteric and histotoxic
infections in humans and domestic animals.

Deaths from C perfringens type A food poisoning are not
common but do occur in the elderly and debilitated. CPE toxin is both
necessary and sufficient for the enteric virulence of Ci. perfringens type
A food poisoning isolates; Sarker ef al., 1999, Ingestion of purified CPE
by human volunteers was determined to be sufficient for reproducing the
cramping and diarrheic symptoms of the natural food poisoning;
Skjelkvale and Uemura, 1977.

Results from the present study provide an explanation for the
association between C/. perfringens type A isolates and cpe gene and for
this purpose all samples proved to toxigenic by RPLA were undergo
investigation using immunoblott and it is worthy to mention that all the
isolates proved to be toxigenic by RPLA was also toxigenic by
immunoblott with an additionally 2 strains which give a clear idea about
the sensitivity and accuracy of the test and this observation in agreement
with that mentioned by Guennadi et al., 2004.

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