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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 |