الفهرس 
ِAcknowledgement
Historical backgroundOnly 14 pages are availabe for public view
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Abstract
summary:
Nakano et al., (1990) studied the Incidence of Clostridium botulinum
in honey of various origins. C. botulinum spores were present in 6 of 58
Japanese honey samples (10.3%), 9 of 76 Chinese honey samples (12%),
and 3 of 15 honey samples from Argentina (20%). Incidence was higher in
samples taken from drums (18%) soon after import, and from apiaries in
Osaka (23%), than in honeys from shops (5%).
In United States of America and Argentina infant botulism is the most
frequent form of botulism with an average of 100 and 26 cases per year
respectively. Australia, Italy, and Canada reported the next highest number
of infant botulism casesKoepke et al., (2008).
3. Comparison of 2 methods for detection of C. botulinum spores in
honey:
Honey is a complex material for microbiological investigation Nevas
et al., (2002). High sugar content, low pH (3.9), high viscosity, low water
activity (0.5- 0.6) White, (1978) and existence of hydrogen peroxide inhibits
microorganisms from growth Gross et al., (2004). Only microorganisms
present are spores of certain bacteria as genus Clostridium and yeasts
Snowdon and Cliver (1996).
Therefore, in the present study two methods for detection of C.
botulinum spores in honey were evaluated. SF method was found to be more
efficient than DC method. By using the DC method in preparing honey
samples; some false negative results were obtained.
This was evident from the results obtained in Table (10) which shows
the detection of 13 isolates by using the SF method. This is in contrast to 7
isolates only were obtained by using the DC method. This may be attributed
to the presence of C. botulinum spores in the supernatant which was
collected to isolate the organism from it. While, the precipitate contains
inhibitory substances that affect bacterial growth as hydrogen peroxide,
flavonoids, lysozyme, phenolic acids and terpenes Nevas (2006).
Supporting this study, other investigators found that SF method was
more efficient than DC method. Nevas (2006) mentioned that SF method in
pre-processing of honey samples provided sensitivity sufficiently high to
detect 1 spore of C. botulinum in 1 gram of honey. This may be considered
adequate in determining whether honey is a potential cause of infant
botulism, as the number of spores in honey samples associated with infant
botulism has been reported to vary from 5 to 80 spores/gram Midura et al.,
(1979).
In contrary, Özlem et al., (2006) preferred DC method for isolation of
C. botulinum spores in honey and failed to detect C. botulinum spores by
using SF method.
4. Genotyping of isolated C. botulinum in honey samples for
neurotoxins genes types (A, B, E and F) using Multiplex PCR:
The use of Multiplex PCR in screening for the presence of botulinal
spores in honey samples shorten the detection time, lower the costs, and
avoid ethical concerns associated with laboratory animal testing Lindström
et al., (2001).
Culture enrichment under anaerobic condition followed by a PCR
procedure has been recommended by Dahlenborg et al., (2001) because it
provides higher number of target bacteria and reduces interference caused
by food components.
It is clear from Table (8) and Figure (1) that the number of isolated
strains were 5 for C. botulinum type A (1 (10%) in Orange honey, 2 (20%)
in Clover honey, 1(10%) in Nabak honey and 1(10%) in Black cumin
honey) and 10 for C. botulinum type B (2 (20%) in Orange honey, 1(10%) in
Medicinal plants honey, 3 (30%) in Clover honey, 3 (30%) in Nabak honey
and 1 (10%) in Black cumin honey. On the other hand, the number of
isolated strains of C. botulinum type E and C. botulinum type F were 0.
Moreover, 2 honey samples (1 orange honey and the other was from Nabak
honey) were positive for both types of C. botulinum type A and type B.
Supporting this study, Dario et al., (2009) described a useful
Multiplex PCR method for detection of C. botulinum type A, B, E, and F
neurotoxin genes in clinical, food, and environmental samples. They
detected 55 positive samples for C. botulinum in 234 examined honey
samples. Of these 55 positive samples, 48 were positive for C. botulinum
type A and 7 were positive for C. botulinum type B.
Nevas (2006) studied the prevalence of C. botulinum spores in 448
honey samples. They were 214 honey samples from Finland, 115 from
Norway, 61 from Sweden, 58 from Denmark. The prevalence of C.
botulinum spores was 12% in all honey samples studied. Of the 214 Finnish
honey samples 18 (8%) were found to be positive for C. botulinum. Spores
of C. botulinum types A, B and E were detected by PCR in 17, 40 and 5
honey samples respectively and on nine occasions, types A and B were
detected in the same sample.
The results in the present thesis is in general agreement with the
results obtained by Vu, (2006) who studied the presence of viable C.
botulinum spores in honey samples and infant foods purchased from retail
supermarkets in Germany and from retail stores and supermarkets in
Vietnam. Twenty of 179 (11.2%) infant food samples and 8 of 99 (8.1%)
honey samples collected from Vietnam were PCR positive for C. botulinum.
On the other hand, eight of 46 samples collected in Germany harbored
spores of C. botulinum type B. Of these 8 positive samples two honey
samples were also positive for type E.
Mäde et al., (2000) applied PCR after enrichment for detection of C.
botulinum in honey. The amplified products were verified by DNA
sequencing. The procedure was found easy to conduct without using
laboratory animals experiment. However, only C. botulinum types A, B, E,
F, and G were detected in this study.
In the present study, all C. botulinum strains isolated from honey
samples were proteolytic strains grew on CMM at 37 °C and at 42 °C.
Proteolytic strains of C. botulinum Types (A, B and F) in cooked meat
media caused blackening of the meat, decomposing it, reducing its volume
with the formation of foul smelling products Solomon et al., (2001).
This result is in general agreement with the results recorded by Eric et
al., (2005) as they found that all cultureswere proteolytic but varied
markedly in proteolysis, as evaluated by digestion of meat particles in
Trypticase peptone glucose broth with cooked meat particles. Also, all
strains showed lipase activity on Clostridium botulinum isolation agar.
Arnon, (1998) reported that most infant botulism cases are due to C.
botulinum types A and B. Proteolytic C. botulinum strains are more
frequently related to infant botulism than non-proteolytic strains.
II) Fish products samples
1. Prevalence of C. botulinum in fish products samples:
C. botulinum is ubiquitous in aquatic environments and has been
isolated from water, ocean sediments, intestinal tract of fish and the gills and
viscera of crabs and other shellfish Huss, (1981).
C. botulinum type E which is most common in fish and fishery
products is of particular concern because it grows at temperature as low as
3- 5 °C and produces little noticeable or no evidence of spoilage Frerk,
(2000).
Ingrid (2008) reported that even though a fish might be cleaned,
gutted and packaged, risk of botulism still existed. This risk existed because
C. botulinum spores could adhere to the surface of fishes and find their way
into muscle tissue during processing. Muscle tissue below the surface of fish
could provide an anaerobic environment, where outgrowth of vegetative
cells and toxin production could occur if time and temperature permitted
this.
In the present study the total number of positive samples for C.
botulinum in fish products samples was 19 in a percentage of (19 %) as
shown in Table (11).
Data presented in Table (11) and Figure (4) revealed that the number
of isolated C. botulinum were 4 in canned sardine, 3 in canned tuna, 7 in