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Title: First report of swarming character of Clostridium botulinum types C and D
Author: Saeed EMA Böhnel H, Gessler F
ID: 29638-2011
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4.REPORT: A novel chance finding is claimed re
swarming observed in C botulinum colonies. This is useful information if
confirmed by others, and for technicians working with the organism. The
findings are supported by the methods used and photographs.ABSTRACT Could be
shortened and convey same message see text. INTRODUCTION. Not appropriate, most
of it describes botulism which is not a purpose of this work please delete as
shown in text. The second paragraph dealing with laboratory assays for C.
botulinum has ben modified please check. Materials/Methods:s The
materials and methods were standard and well described with necessary detail to
reproduce results.
A
ABSTRACT
This study aimed at isolation and identification of
Clostridium botulinum from samples of botulism-suspected cases of various
animal species and their environment. The standard mouse bioassay, magnetic
bead-ELISA and PCR were used to identify the isolates. Out of 74 samples investigated,
only three isolates were confirmed to be C. botulinum. Two of them were type C
and one type D. The two type C isolates were from intestinal contents of two
broiler chickens from a farm with botulism outbreak and the type D isolate was
from a liver of a bovine species. The character of these isolates which was not
found reported before is that their cells are swarming if grown on normally
dried plates (refrigerated plates dried for 30 min at 37 °C). Their swarming
growth was not seen by the naked eyes in the primary plates but observed by
hand lens on the subsequent sub-culturing plates as very thin film
contaminating colonies that were picked from the primary plates as C.
botulinum-like colonies. The C. botulinum-like colonies were found to be non-C.
botulinum by the three test methods used after being separated from the
swarming growth on plates with increased agar percentage while the discrete
colonies of the swarming growth were C. botulinum. The significant importance
of this finding is that the swarming growth on anaerobic plates should be
considered in isolation of C. botulinum.
Key words: Clostridium botulinum; swarming growth; isolation; mouse bioassay;
magnetic bead-ELISA; PCR
See here
Clostridium botulinum was isolated and identified in samples from botulism-suspected cases of various animal species and environment, by standard mouse bioassay, magnetic bead-ELISA and PCR. From 74 samples, only 3 isolates were confirmed C. botulinum. Two were type C and 1 type D. The 2 type C isolates were taken from intestinal contents of 2 broiler chickens from a farm with a botulism outbreak and the type D isolate was from a bovine liver. These isolates revealed swarming when grown on normally dried plates (refrigerated plates dried for 30 min at 37 °C). The swarming growth was not observed by the naked eyes in the primary plates but observed by hand lens on the subsequent sub-culturing plates as very thin film contaminating colonies that were picked from the primary plates as C. botulinum-like colonies. The C. botulinum-like colonies were not C. botulinum tested by 3 methods after separation from swarming growth on plates with increased agar percentage. However, the discrete colonies of the swarming growth were C. botulinum. Swarming on anaerobic plates should now be considered when isolating C. botulinum.
to
be deleted, disease description etc...not relevant
Botulism is a
non-febrile highly fatal disease of man and animals caused by neurotoxins of
Clostridium botulinum. It is characterized by partial or complete flaccid
paralysis of the muscles of locomotion, mastication and deglutition due to
inhibition of the release of the neurotransmitter acetylcholine by botulinum
neurotoxins (designated as types A through G) at cholinergic nerve endings. It
occurs sporadically, but in intensively farmed animals, it is responsible for
high mortalities (Trueman et al., 1992). Animal botulism can be a public health
problem, since humans can be intoxicated by ingestion of contaminated meat. The
bacterium is almost ubiquitous and is found in soils and organic matter
worldwide, but it may not always be toxic, perhaps helping to explain the
sporadic nature of the outbreaks. C. botulinum is occasionally present in the
animal gut and can be an opportunist due to gut stasis (Hunter et al., 1999).
Animals are mostly affected by C. botulinum types C or D toxins and rarely by
type A or B (Schocken-Iturrino et al., 1989).
Modify here
see below
Laboratory
diagnostics include the conventional culture methods to isolate the organism,
mouse bioassay and molecular and immunological methods. C. botulinum is
difficult to isolate; it is extremely fastidious, strict anaerobic, there is no
selective medium for all its groups, and it can lose its toxicity during the
isolation process (Smith and Sugiyama, 1988) or even in pure form (Collins and
East, 1998). The mouse bioassay method was described as the standard method for
detecting, identifying, and typing of botulinum neurotoxins (BoNTs) (CDC, 1998,
Smith and Sugiyama, 1988). However, it is not suitable for examination of test
samples containing other lethal substances (Dezfulian and Bartlett, 1985).
Sensitive immunoassays such as enzyme-linked immunosorbent assay (ELISA)
(Ferreira et al., 2003) and molecular methods such as PCR-based methods (Szabo
et al., 1993) were developed for diagnosis of botulism. Immunoassays, unlike
the mouse bioassay, can detect the toxin either in active or inactive form.
However, ELISA methods are most often less sensitive than the mouse bioassay
(Trueman et al., 1992). PCR is important if the specimen contains viable cells
and no detectable toxin by the bioassay or immunoassay. But, false-negative
results were also reported (Fach et al., 1996). Thus, combination of culture
method, bioassay, immunoassay and PCR seems to be important for better
diagnosis of botulism.
Several Clostridium species can swarm and thus complicated the isolation of C.
botulinum (Hernández-Chavarría et al., 2001, Jousimies-Somer et al., 2002,
Sharma and Anand, 2002). C. novyi type A, the related Clostridium to group III
C. botulinum (types C and D, and C. novyi type A) (Eklund and Poysky, 1974), is
known of its swarming character (Sharma and Anand, 2002). However, it was not
found reported that C. botulinum can swarm. Toxins of group III C. botulinum
organisms are each encoded on separate pseudolysogenic bacteriophages (Eklund
et al., 1972). Cultures of toxigenic strains can be cured of their prophages
and stop producing toxins and can be converted to toxigenic state by
reinfection by phages (Oguma et al., 1986). Type C strains and C. novyi type A
can be reinfected by either C or D bacteriophages, but strains of type D are
infected only by the homologous phage (Eklund et al., 1974).
The purpose of this study was to isolate and identify C. botulinum from samples
of classic botulism-suspected cases by the standard mouse bioassay, magnetic
bead-ELISA and PCR. The specific objective of this manuscript is to report for
the first time a swarming growth character for C. botulinum types C and D.
See Here
C.
botulinum is difficult to isolate; is extremely fastidious, strict anaerobic,
there is no selective medium for all its groups, and loses its toxicity during
the isolation process (Smith and Sugiyama, 1988), or even in pure form (Collins
and East, 1998). The mouse bioassay method was described as the standard method
for detecting, identifying, and typing of botulinum neurotoxins (BoNTs) (CDC,
1998, Smith and Sugiyama, 1988). However, it is not suitable for examination of
test samples containing other lethal substances (Dezfulian and Bartlett, 1985).
Sensitive immunoassays such as enzyme-linked immunosorbent assay (ELISA)
(Ferreira et al., 2003) and molecular methods such as PCR-based methods (Szabo
et al., 1993) were developed for diagnosis of botulism. Immunoassays, unlike
the mouse bioassay, can detect the toxin either in active or inactive form.
However, ELISA methods are most often less sensitive than the mouse bioassay
(Trueman et al., 1992). PCR is important if the specimen contains viable cells and
no detectable toxin by the bioassay or immunoassay. But, false-negative results
were also reported (Fach et al., 1996). Thus, combination of culture method,
bioassay, immunoassay and PCR seems to be important for better diagnosis of
botulism.
Several Clostridium species can swarm and complicate isolation of C. botulinum
(Hernández-Chavarría et al., 2001, Jousimies-Somer et al., 2002, Sharma and
Anand, 2002). C. novyi type A, the related Clostridium to group III C.
botulinum (types C and D, and C. novyi type A) (Eklund and Poysky, 1974), is
known for its swarming character (Sharma and Anand, 2002). However, C.
botulinum swarming has not been reported. Toxins of group III C. botulinum
organisms are each encoded on separate pseudolysogenic bacteriophages (Eklund
et al., 1972). Cultures of toxigenic strains can be cured of their prophages
and stop producing toxins and can be converted to toxigenic state by
reinfection by phages (Oguma et al., 1986). Type C strains and C. novyi type A
can be reinfected by either C or D bacteriophages, but strains of type D are
infected only by the homologous phage (Eklund et al., 1974).
The purpose of this study was to isolate and identify C. botulinum from samples
of classic botulism-suspected cases by the standard mouse bioassay, magnetic
bead-ELISA and PCR. The specific objective of this manuscript is to report for
the first time a swarming growth character for C. botulinum types C and D.
MATERIALS AND METHODS
Minor
see suggestion below
Test samples: A total of 74 samples from botulism-suspected cases and their
environment were investigated for presence of C. botulinum. The samples were
obtained from the samples routinely sent to the reference laboratory for
diagnosis of botulism in animals at the Institute for Tropical Animal Health,
Georg-August-University, Göttingen. The sources of samples were mainly bovine
(intestinal contents, faeces, and few tissue samples) and soil samples; in
addition to few samples from horses (faeces), chickens (intestine and liver)
and animal feed.
Culture methods and isolation: The medium Fastidious Anaerobic Agar (Quelab,
Montreal) with 5 % egg yolk emulsion (Becton Dickinson, Sparks) or 5 % horse
blood (Oxoid, Wesel) and its broth was selected and used at pH 7.0±0.2.
Streaking of the plates, heat or alcohol treatments of samples were done either
directly or after liquid medium enrichment of each sample. Culture and
isolation were conducted in an aerobic biosafety class 2 cabinet
(Stoltenberg-Lerche, Düsseldorf), which is with facilities to keep the inside
environment sterile. A single temperature for growth (37 °C) was used. The
anaerobic conditions (90 % N2, 5 % H2, 5 % CO2) were fixed automatically using
Anoxomat® System, Mart, Lichtenvoorde. Inoculated broth medium was incubated
for 3-5 days and plates for 2 days. Gram-stained smears directly from the
specimens, enrichment broth culture and from the suspected colonies were
prepared and examined. Colonies with characteristics consistent with C.
botulinum were picked. Selected colonies were purified and their culture
supernatants were used for testing.
Mouse bioassay: The standard method for BoNT detection, the mouse bioassay was
used according to CDC (1998). White mice of the institute’s breeding station
weighing 18-25 g were used for testing toxicity of culture supernatants and
toxin neutralization. Broth cultures of isolates were centrifuged at 4,000 rpm
for 20-30 min. To test for toxicity, mice were injected i.p. with 0.5 ml of
each isolate culture supernatant and mice were observed for four days for signs
of botulism. Toxin neutralization tests were carried out for lethal isolates.
Toxin dilution was made in gelatine phosphate buffer (pH 6.2). Polyvalent
antitoxins type ABE (Aventis Behring, Marburg), and monovalent antitoxins types
C and D (Onderstepoort Veterinary Institute [OVI], Onderstepoort) were mainly
used. In some instances, monovalent antitoxin types A to E (ID-DLO, Lelystad)
and types B and E (Institute for Applied Biotechnology [IBT], University of
Göttingen) were used. Types F and G were not tested. Antitoxins were rehydrated
and used according to manufacturer’s instructions.
PCR assay: To test for C. botulinum types A, B, E and F toxin genes, a
multiplex PCR assay, which was developed by Lindström et al. (2001), was
adopted. For C. botulinum type C toxin gene detection, a PCR method developed
by Gessler and Böhnel (2006) was used, and for detection of C. botulinum type D
toxin gene, a method established by Takeshi et al. (1996) was conducted.
Cultures for DNA isolation were grown in Fastidious Anaerobic Broth (FAB),
Reinforced Clostridial Medium (Merck, Darmstdt) or Cooked Meat (Difco, Detroit)
for 48 h at 37 °C. DNA was extracted by boiling for 10 min. A volume of 3
μl of each supernatant was used as template in the PCR. A programmable
thermal cycler (TGradient, Biometra, Göttingen, Germany) was used for the
cycling.
Magnetic bead-ELISA: A magnetic bead-enzyme-linked immunosorbent assay,
developed by IBT for detection of BoNT/C and D (Gessler et al., 2006) was
adopted. Only the isolates which were neutralized by botulinum antitoxins type
CD mixture with the mouse bioassay were tested. The isolates were grown in FAB
at 37 °C for 2-5 days and cultures were centrifuged at 4,000 rpm for 30 min.
Monoclonal mouse-antibody and polyclonal goat-antibody (biotinylated) against
type C and D BoNTs were used as capture and detecting antibodies, respectively
and to form toxin-antibody complex. Magnetic beads coated with sheep anti-mouse
IgG, as secondary capture antibody, were added and presence of specific
reactants was indicated by enzyme-substrate system (streptavidin-HRP-TMB).
RESULTS OK
Culture supernatants of all C. botulinum-like isolates were tested for toxicity
in mice and then toxin neutralization. The neutralization results of culture
supernatants of only three isolates were found reproducible and specific; two
type C and one type D. This was confirmed by the PCR and MB-ELISA, when only
these three isolates were positive by PCR (Figs. 2 and 3) and only the two type
C isolates were positive by MB-ELISA. The two type C isolates were from
intestinal contents of two broiler chickens from a farm with botulism outbreak
and the type D was from a bovine liver. The three isolates were short to long
slender Gram-positive rods with subterminal pulging spores, lipase-positive,
β-haemolytic (human, but not horse blood).
The type D isolate which was positive by both mouse bioassay and PCR and
negative by MB-ELISA was from one of the first few samples investigated. This
negative result has led to again sub-culture that isolate on blood and egg yolk
agar plates to again study its cultural characteristics and a hand lens was
used to help in that. During examination of the plates, a very thin film of a
swarming growth was observed. To separate this swarming bacterium, plates dried
for more than 30 min and 3% agar plates were used. In both types of plates,
discrete colonies were obtained for both the swarming bacteria and the original
isolate. The colonies were not exactly the same. Gram-stained smears from both
colony types were found to be consistent with C. botulinum cells. A part of a
colony of each of the two colony types and growth of reference strains of type
C and D was cultured on a normally dried and normal agar percentage plates. The
inoculum from the swarming bacteria was confirmed to be swarming and the others
were not swarming. The two colony types were retested by the three test
methods. The previous result (i.e. positive by both mouse bioassay and PCR and
negative by MB-ELISA) was given by the swarming bacterium and the other
bacterium was negative for all of the three tests. After this finding any
swarming growth (seen by naked eye or hand lens) was considered for testing.
Another two swarming isolates were found to be positive for C. botulinum type C
by the three methods. Some other swarming clostridia, mainly C. tetani were
also found in plates of other samples, but none of them was positive for C.
botulinum by any method.Slight morphological differences were observed in the
discrete colonies of the C. botulinum type C and D isolates (Fig. 1a-c). The
colony of one of the type C isolates is raised pyramidal with rhizoidal edge
and the other is flat or slightly raised with rhizoidal edge, while that of the
type D is raised with uneven edge.
Suggestion: MATERIALS AND METHODS
Test samples: A total of 74 samples from botulism-suspected cases and their
environment were investigated for presence of C. botulinum. The samples were
obtained from the samples routinely sent to the reference laboratory for
diagnosis of botulism in animals at the Institute for Tropical Animal Health,
Georg-August-University, Göttingen. The sources of samples were mainly bovine
(intestinal contents, faeces, and few tissue samples) and soil samples; in
addition to few samples from horses (faeces), chickens (intestine and liver)
and animal feed.
Culture methods and isolation: The medium Fastidious Anaerobic Agar (Quelab,
Montreal) with 5 % egg yolk emulsion (Becton Dickinson, Sparks) or 5 % horse
blood (Oxoid, Wesel) and its broth was selected and used at pH 7.0±0.2.
Streaking of the plates, heat or alcohol treatments of samples were done either
directly or after liquid medium enrichment of each sample. Culture and
isolation were conducted in an aerobic biosafety class 2 cabinet
(Stoltenberg-Lerche, Düsseldorf), which is with facilities to keep the inside
environment sterile. A single temperature for growth (37 °C) was used. The
anaerobic conditions (90 % N2, 5 % H2, 5 % CO2) were fixed automatically using
Anoxomat® System, Mart, Lichtenvoorde. Inoculated broth medium was incubated
for 3-5 days and plates for 2 days. Gram-stained smears directly from the
specimens, enrichment broth culture and from the suspected colonies were
prepared and examined. Colonies with characteristics consistent with C.
botulinum were picked. Selected colonies were purified and their culture
supernatants were used for testing.
Mouse bioassay: The standard method for BoNT detection, the mouse bioassay was
used according to CDC (1998). White mice of the institute’s breeding station
weighing 18-25 g were used for testing toxicity of culture supernatants and
toxin neutralization. Broth cultures of isolates were centrifuged at 4,000 rpm
for 20-30 min. To test for toxicity, mice were injected i.p. with 0.5 ml of
each isolate culture supernatant and mice were observed for four days for signs
of botulism. Toxin neutralization tests were carried out for lethal isolates.
Toxin dilution was made in gelatine phosphate buffer (pH 6.2). Polyvalent
antitoxins type ABE (Aventis Behring, Marburg), and monovalent antitoxins types
C and D (Onderstepoort Veterinary Institute [OVI], Onderstepoort) were mainly
used. In some instances, monovalent antitoxin types A to E (ID-DLO, Lelystad)
and types B and E (Institute for Applied Biotechnology [IBT], University of
Göttingen) were used. Types F and G were not tested. Antitoxins were rehydrated
and used according to manufacturer’s instructions.
PCR assay: To test for C. botulinum types A, B, E and F toxin genes, a multiplex
PCR assay, which was developed by Lindström et al. (2001), was adopted. For C.
botulinum type C toxin gene detection, a PCR method developed by Gessler and
Böhnel (2006) was used, and for detection of C. botulinum type D toxin gene, a
method established by Takeshi et al. (1996) was conducted. Cultures for DNA
isolation were grown in Fastidious Anaerobic Broth (FAB), Reinforced
Clostridial Medium (Merck, Darmstdt) or Cooked Meat (Difco, Detroit) for 48 h
at 37 °C. DNA was extracted by boiling for 10 min. A volume of 3 μl of
each supernatant was used as template in the PCR. A programmable thermal cycler
(TGradient, Biometra, Göttingen, Germany) was used for the cycling.
Magnetic bead-ELISA: A magnetic bead-enzyme-linked immunosorbent assay,
developed by IBT for detection of BoNT/C and D (Gessler et al., 2006) was
adopted. Only the isolates which were neutralized by botulinum antitoxins type
CD mixture with the mouse bioassay were tested. The isolates were grown in FAB
at 37 °C for 2-5 days and cultures were centrifuged at 4,000 rpm for 30 min.
Monoclonal mouse-antibody and polyclonal goat-antibody (biotinylated) against
type C and D BoNTs were used as capture and detecting antibodies, respectively
and to form toxin-antibody complex. Magnetic beads coated with sheep anti-mouse
IgG, as secondary capture antibody, were added and presence of specific
reactants was indicated by enzyme-substrate system (streptavidin-HRP-TMB).
Figure 2: PCR detection of C. botulinum type C. Lane 1, standard DNA marker;
lane 2, positive control; lane 3 & 4, the two type C isolates; lane 6,
negative control.
Figure 3: PCR detection of C. botulinum type D. Lane 1, standard DNA marker;
lane 2, positive control; lane 4, negative control; lane 7, the type D isolate.
DISCUSSION check grammar etc .. otherwise supports findings
In the laboratory, botulism is diagnosed by detecting BoNT and/or C. botulinum
cells in the test sample. Cells could be isolated by culture technique and
identified by laboratory methods such as toxin and toxin gene detection assays.
However, C. botulinum isolation is known to be difficult (Smith and Sugiyama,
1988) due to its strict anaerobic character and high fastidiousness. It needs
good experience and special laboratory facilities, it can lose its toxicity
during sub-culturing, and above all, its isolation is especially difficult
because of the inhibitory effect of other microflora (Graham, 1978).
In this study, the isolates obtained by culture methods were tested by the
standard mouse bioassay, MB-ELISA and PCR. Only three isolates were confirmed
to be C. botulinum despite the sound clinical diagnosis of botulism in some of
the cases, the putative presence of the bacteria in healthy animals and the
wide distribution of C. botulinum in the environment, especially in soil.
However, this very little number of isolates was not unexpected and it could be
attributed to the difficulty to isolate the bacterium rather than to the
sensitivity of the tests used. The three isolates were two type C and one type
D. C. botulinum type D was isolated from a bovine case and type C from two
chicken cases and this association is consistent with the previous reports that
bovine botulism is mainly caused by types C and D (Böhnel, 1999) and avian
botulism is caused by type C (Smith and Sugiyama, 1988). Type D was not
detected by MB-ELISA but only by the bioassay and PCR. So, this result of
MB-ELISA is a true false-negative result, which could be due to an expected
difference in the antigenecity of the toxin of the strain used to raise the
antibodies used in this assay and that of this isolate. It was found that
within the type D strains, neurotoxins differ in molecular structure and
antigenecity (Moriishi et al., 1989).
As shown in the results, these three C. botulinum isolates were isolated as
swarming cells (short to long slender Gram-positive rods with sub-terminal
spores) and this swarming character was not found reported before. This
swarming growth, which was a very thin layer that was seen only by hand lens,
was separated and discrete colonies were obtained when sub-cultured on well
dried and high percentage agar plates. It was observed that the morphology of
the discrete colonies of these isolates was slightly different. During the
isolation and purification processes in the current study, swarming growth was
frequently observed, which greatly complicated the isolation of lipase-positive
colonies. The swarming cells encountered were mainly C. tetani as shown by
their characteristic shape of cells. Beside C. tetani, several other
Clostridium species were reported to swarm (Hernández-Chavarría et al., 2001,
Jousimies-Somer et al., 2002, Sharma and Anand, 2002); including C. novyi. Due
to its close relatedness to C. botulinum types C and D, C. novyi type A was put
as a member of group III C. botulinum (Eklund and Poysky, 1974). So, it might
not be unexpected to find swarming strains of types C and D C. botulinum. It is
known that group III members of C. botulinum (types C, D and C. novyi type A)
can be converted to each other if infected by the specific phage in vitro,
which was also thought to occur in nature (Eklund and Poysky, 1974). So, due to
this swarming character, these type C and D isolates may be C. novyi type A
strains naturally infected by type C and D specific phages. The significance of
this finding is that swarming growth in primary or subsequent plates should be
considered for isolation of C. botulinum.
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