Prevalence of Clostridium perfringens Alpha Toxin in Processed and Unprocessed Fish

Transcription

1 International Journal of Microbiological Research 3 (3): , 2012 ISSN IDOSI Publications, 2012 DOI: /idosi.ijmr Prevalence of Clostridium perfringens Alpha Toxin in Processed a Unprocessed Fish M.M. El-Shorbagy, Lamyaa M. Reda a H. Mona 1 Anaerobic Unit, Bact. Research Department, Animal Heal Research Institute, Dokki, Giza 2 Central Lab., Vet. Hospital, Faculty of Vet. Med., Zagazig Univ., Egypt 3 Vet Hospital, Faculty of Vet. Med., Zagazig Uni., Egypt Abstract: The current research was carried out on one hured a irteen raom fish samples (56 processed a 57 unprocessed) collected from different localities in El-Sharkia Governorate, Egypt to obtain a complete picture of C. perfringens. Bacteriological a biochemical examination was done on e isolates. The total incidence of positive samples from 56 processed fish samples were 32 isolates (57.1%) a from 57 unprocessed fish samples were 34 isolates (59.6%). The Nagler's test was applied on e recovered C. perfringens isolates. Typing of C. perfringens isolates revealed at e incidence of toxigenic a non-toxigenic isolates were 84.8 a 15.2% respectively. Typing of toxigenic strains of C. perfringens revealed at C. perfringens type A was e most predominant one comparing wi type D. Immune diffusion test showed at 17 a 11 toxins of types A a D gave identified reaction wi incidences of 47.2 a 55% respectively. ELISA test revealed at 22 out of 25 isolates of C. perfringens produced toxin. Key words: C. perfringens Fish Nagler s Test Immune diffusion Test INTRODUCTION collagenase, hyaluronidase a deoxyribonuclease [6]. The colonies of C. perfringens are smoo, rou, Fish are considered one of e most widely accepted glistening a surroued by double Revise zone of a valuable food in most countries. Egypt is currently hemolysis [7]. C. perfringens are large Gram-positive rods one of e fastest growing countries in e field of ( x µm), encapsulated, non-motile, spore aquaculture to solve protein shortage problem in forming, fermentative a catalase negative [8]. The Egypt.This increase in aquaculture has led to e furer spores of some Clostridia species are high heat resistant spread of diseases [1, 2]. The presence of microbial a may survive heat treatment of canned foods. If e paogens, especially ose of bacterial origin is one surviving spores germinate a e vegetative cells grow, of e most significant factors affecting fish culture spoilage will occur [9]. C. perfringens is divided into 5 [3]. Anaerobic bacteria are important groups of types (A, B, C, D a E) on e basis of e production of microorganisms which are responsible for reduction of 4 major toxins (,, a ), each type had been linked to grow rate, increased mortality a high costs of specific diseases [10]. So, e main goals of e present treatment wi antibiotics in addition to many public study were: heal hazards [4, 5]. C. perfringens is widely distributed in soil a Identification a typing of C. perfringens isolates intestinal contents of man a animals. It has a great from processed a unprocessed fish samples. effect on e human heal causing food poisoning. It Serological identification of C. perfringens toxins by also causes a number of human diseases ranging from toxin-antitoxin neutralization test, minimal leal dose necrotic enteritis to wou infection a gas gangrene. in mice a immune diffusion test. This paogenicity is associated wi leal extra cellular Detection of C. perfringens type A (alpha toxins) toxins which have been defined as enzyme activity as isolated from collected samples by ELISA test. Correspoing Auor: Lamyaa M. Reda, Central Lab., Vet. Hospital, Faculty of Vet. Med., Zagazig Univ., Egypt. 195

2 MATERIALS AND METHODS Samples: A total of 113 fish samples were collected from large supermarket a retail fish shops in El-Sharkia Governorate. The samples were 56 processed fish samples (canned salmon, canned mackerel, salted sardine, renga a feisekh) a 57 unprocessed fish samples (Nile cat fish, Tilapia nilotica, danis, fresh water sardine, rossi, macaroni a bagha). Detection of C. perfringens Alpha Toxin by Iirect ELISA: The serum of e rabbit (50 µl/well) were diluted in 0.005% Tween 20 in PBS 1:16 were added to each well a e plate was incubated at 37 C for 2 hours. Rapid detection of C. perfringens type A (alpha toxin) was done by iirect ELISA technique. Alpha toxin isolated from fish used as antigen by [20]. RESULTS AND DISCUSSION Bacteriological Examination Isolation a Identification of C. perfringens: The surface of each sample was sterilized a bean size pieces were Clostridium perfringens is more widely spread an oer paogenic bacteria; its principle habitats are in e soil a e intestinal content of e man a animals. obtained from e deeper parts of each sample a There is much evidence at obligate anaerobic organisms inoculated separately into tubes of freshly prepared are probably e principal sources of infection in human cooked meat bro medium. All tubes were incubated beings, domestic animals a fish as mentioned by Hayed anaerobically in e anaerobic jar by using anaerobic gas [6]. In recent decades, many surveys have been generating kits (Oxoid) at 37 C for 48 hours. For isolation coucted on e incidence of C. perfringens in raw a of C. perfringens, a loopful from e previously incubated processed meat a poultry. This report iicates wide tube was streaked onto e surface of 10% sheep blood spread occurrence of is organism in processed a agar wi neomycin sulphate (200 µg/ml) a incubated unprocessed fish [21]. for 24 hours at 37 C in e anaerobic jar. The suspected colonies of C. perfringens were picked up a examined Incidence of C. perfringens Isolates in Processed Fish for eir morphological a culture characters [11], microscopical examination of stained films from e suspected colonies wi Gram s stain a biochemical tests were done [12]. Nagler s Test by Half Antitoxin Plate: This test was done as was previously described [13]. The attack of alpha toxin at is produced by all types of C. perfringens, on leciin is inhibited by alpha antitoxin [14]. Typing of C. perfringens Toxins by Dermonecrotic Test: Preparation of toxins a eir treatment [15] a e application of e dermonecrotic test [16, 17] were carried out. Serological Identification of C. perfringens Toxins: Toxin antitoxin neutralization test: This test was done as previously described [13]. Determination of Minimum Leal Doses (MLD) of e Prepared C. Perfringens Toxins: The test was done using e prepared toxins (alpha toxin of type A a epilson toxin of type D) as described previously [18]. Immune Diffusion Test: Agar gel immune diffusion test was used to determine e relationship between antigen a antibodies [19]. Samples: Table 1 shows at e prevalence of C. perfringens in 56 processed fish samples was 57.1%. The isolation of positivity was seen in feisekh, renga a salted sardine by percentages of 82.3, 80.0 a 75, respectively, but ere was no C. Perfringens isolates from canned products such as mackerel a salmon. Table 1: Incidence of C. perfringens in different types of processed fish samples Processed fish samples No. of + ve samples/total number Canned salmon 0/11 (0%) Canned mackerel 0/5 (0%) Salted sardine 6/8 (75%) Renga 12/15 (80%) Feisekh 14/17 (82.3%) Total 32/56 (57.1%) Table 2: Incidence of C. perfringens in different types of unprocessed fish samples Un-processed fish samples No. of +ve samples/total Number Nile cat fish 8/10 (80.0%) Tilapia nilotica 5/8 (62.5 %) Danis 4/8 (50.0 %) Sardine 3/8 (37.5%) Rossi 5/8 (62.5%) Macaroni 5/8 (62.5%) Bagha 4/7 (57.1%) Total 34/57 (59.6%) 196

3 Table 3: Typing of C. perfringens isolates recovered from fish samples Toxigenic C. perfringens isolates Non toxigenic Type of fish samples C. perfringens/ total isolates C. perfringens type A C. perfringens type D Total Processed 5/32 (15.6%) 17/32 (53.1%) 10/32 (31.32%) 27/32 (84.43%) Unprocessed 5/34 (14.7%) 19/34 (55.98%) 10/34(29.4%) 29/34 (85.32%) Total 10/66 (15.21%) 36/66 (54.5%) 20/66(30.3%) 56/66 (84.8%) These results coincide wi ose recorded by Kassem Results of Nagler s Test for Identification of [22] who stated at Clostridium spp. could be isolated C. perfringens Isolates: The obtained results revealed from e ree types of examined salted fish (feisekh, molouha a salted sardines), while canned product is safe a free from anaerobic microorganisms. The present study results also go ha in ha wi ose recorded by Richardson [23]. at 10 out of 66 isolates were toxin producer on egg yolk agar medium. These results go ha in ha wi ose recorded by Smi a Holdman [13] who applied Nagler's test using half antitoxin plate to detect leciinase activity of alpha toxin of different types of C. perfringens. Incidence of C. perfringens Isolates in Unprocessed Fish Samples: Table 2 shows at out of 57 unprocessed fish samples, 34 (59.6%) were positive for C. perfringens. The highest incidence of positivity was shown in Nile cat fish by a percentage of 80.0, while e lowest incidence of positivity appeared in sardine wi an incidence of 37.5%. These results are similar to at of Schoken et al. [24], Peterson et al. [25] a Marzouk et al. [26] who mentioned at C. perfringens need presence of high amount of organic pollutions of human a animal origin a strict anaerobic coition to grow a become paogenic. They also added at e high incidence of C. perfringens is due to e heavy use of poultry dropping a animal manure at usually contain C. perfringens. On e oer ha, Enany et al. [27] reported at Nile cat fish possess highest prevalence wi C. perfringens as Nile cat fish are highly exposed to subclinical infection wi different levels of C. perfringens toxin so at ey become immunized wiout showing symptoms of illness. Identification of C. perfringens Isolates: The suspected colonies of C. perfringens were Gram-positive, short plumb, rarely sporulated a non-motile bacilli when ey were stained wi Gram s stained. It was apparent at sheep blood agar wi neomycin sulphate (200 µg/ml) is a perfect medium for isolation of C. perfringens raer an oer Clostridium species a gave double zones of hemolysis. All e recovered strains in is work were fermentative to different sugars as glucose, maltose, lactose, sucrose a mannose wi production of acid a gases, gelatin liquefiers, litmus milk positive, catalase, oxidase a iole negative. Similar results were recorded by several auors as Vaikosen a Muller [11] a Assis et al. [28]. Typing of C. perfringens Isolates by Intradermal Injection of Guinea Pigs: The typing of C. perfringens by intradermal injection of guinea pig revealed at e incidence of toxigenic a non-toxigenic isolates were 84.8 a 15.2%, respectively as shown in table (3). The action of C. perfringens type A (alpha toxin) appeared as an irregular area of yellowish green necrosis tented to spread downward, while at of type D (epilson toxin ) appeared as a circular whitish green necrosis wi few small areas of purplish hemorrhagic necrosis [29]. Typing of e toxigenic C. perfringens isolates revealed at C. perfringens types A a D were e most predominant ones wi percentages of 54.5 a 30.3, respectively. Such fiing is similar to at of Enany et al. [27], Aschfalk a Muller [30] a Songer a Dale [31] who recorded presence of C. perfringens types A a D wi percentage of 50 a 25 respectively in Nile tilapia samples. Toxin Antitoxin Neutralization Test: All sixty six C. perfringens isolates were identified by toxin antitoxin neutralization tests. The results showed e protection of e injected albino guinea pigs because of neutralization of each toxin wi its specific antitoxin. Also, e obtained results revealed at C. perfringens type A was e most predominant one among e total recovered isolates as shown in table 3. These results are in accordance wi Joklik et al. [14] a Songer a Dale [31] who deserved at alpha toxin is e most important toxin produced by all types of C. perfringens. Results of MLD Test in Mice: All sixty six C. perfringens isolates were tested for MLD in mice a e obtained results showed at e minimum leal doses for 197

4 C. perfringens types A a D were 1/16 a 1/8, 5. Damir, K., K. Bozirar a T. Emin, Differences in respectively. Stark a Duncan [32] fou at e minimum leal dose for C. perfringens is bacterial population in rainbow trout (oncorhynchus my kiss walbum) fry after transfere from incubator to pools. Food Technol. Biotechnol., 4: Results of Immune Diffusion Test: Immune diffusion test revealed at out of 36 C. perfringens type A toxin, 17, 7 a 12 toxin showed identity, non-identity a partial identity wi percentages of 47.2, 19.4 a 33.3, respectively. Furermore, out of 20 of C. perfringens type D toxin, 11, 3 a 6 showed identity, non-identity a partial identity wi percentages of 55.0, 15 a 30.0, respectively. These results go ha in ha wi ose recorded by Aloisi [33] a Mona [34] who reported at C. perfringens type A toxin showed identity, non identity a partial identity wi percentage of 46.2, 18.3 a 36.5, respectively. Furermore C. perfringens type D toxin showed identity, non-identity a partial identity wi percentages of 53.2, 17 a 29.8, respectively. Results of ELISA Test: A total of 25 rabbit serum samples were examined. There were 22 positive samples wi 88% specificity a 3 negative samples wi 12%. These results agree wi e fiing of Aschfalk a Muller [30] a El-Idrissi a Ward [35] who suggested at ELISA assay could be used to detect C. perfringens type A toxin in fish by a good rapid a simple manner a fair accuracy. In conclusion, in e present study e highest incidence of C. perfringens isolates was in feisekh a Nile catfish, while e lowest incidence was in canned salamon a mackerel. ELISA is a good a rapid technique for detection of alpha toxin of C. perfringens. REFERENCES 1. Bahnasawy, M.H., Effect of dietary protein levels on grow performance a body composition of Monosex Niletilapia, Oreochromis niloticus L. reared in fertilized tanks. Pakistan Journal of Nutrition, 5: Rajinikan, T., R. Ramasamy a V. Ravi, Efficacy of Probiotics, grow promotors a disinfectants in shrimp grow out farm. Am. Euras. J. Agric. Eviron. Sci., 3: Post, G.W., Textbook of Fish Heal. 2. T.F.H. pupl. Inc. Lt., pp: Samaha, H.A., Y.N. Hagga a M. Nadia, Brackish a Marine Water fish as a source of certain bacterial paogens to human beings. In e Proceedings of e 4 Sci. Conference for Vet. Med. Researches, Fac. Vet. Med., Alex. Univ., 2-4 October. 6. Hayes, P.R., Food Microbiology a Hygiene. 2 Ed. Elsevier Applied Science Loon, New York. 7. Quinn, P.J., B.K. Markey, M.E. Carter, W.J.C. Donnelly, F.C. Leonard a D. Maguire, Clostridium species. In: Clinical Veterinary Microbiology. 2 Eds Wolfe Publishing. Loon, pp: Cato, E.P., W.L. George a S.M. Finegold, Genus Clostridium. In: Bergey s Manual of Systematic Bacteriology. Vol.2. Edited by Snea, P.H.A., N.S. Mair, M.E. Sharpe a J.G. Hott. Williams a Willins Co., Baltimore, pp: Banwart, G.J., Basic Food Microbiology, 2 ed. VannoStra, Reinhold New York. 10. Petit, L.,M. Gibert a M.R. Popoff, 1999.C. perfringens toxinotype a genotype. Tres Microbiol., 7: Vaikosen, E.S. a W. Muller, Evaluating biochemical tests for isolation a identification of C. perfringens in faecal Samples of Small Ruminants in Nigeria. Bulletin of Animal Heal a Production in Africa, 49: Koneman E.W.,S.D. Allen,V.R. Dowell a H.W. Summers, 1992.Colour Atlasa Textbook of Diagnostic Microbiology. 4 Ed. J.B. Lippin Cott, New York, Loon. 13. Smi, L.D. a S. Holdman, The paogenic st anaerobic bacteria.1 Ed , Charles Thomas Publisher, USA. 14. Jokily, W.K., H.P. Willet a D.B. Amos, Zinsser Microbiology. 17 Ed. Appleton Century Crofts, New York. 15. Gadalla, M.S.A., I. Farrag a D. Sharaf, Effect of grow requirement on e improvement of clostridial vaccines. J. Egypt Vet.Med. Ass., 2: Oakley, C.L. a G.H. Warrack, Routine typing of C. Wechii. J.Hyg.Gamb., 51: Quinn, P.J., B.K. Markey, M.E. Carter, W.J. Donnelly, F.C. Leonard a D. Meguire, Veterinary microbiology a microbial disease. 2 Ed., Blackwell Science, pp: Eman, M.N., Studies on rapid meods for diagnosis of pulpy kidney a lamb dysentery, Ph. D. esis (Microbiology), Fac. Vet. Med., Cairo University. 19. Mary, L., Immunology a Serology in Laboratory Medicine. Seven Edition. Butter Wro- Heinemann Ltd. 198

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