IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 Effect of Yogurt and Bifidobacterium on Cryptosporidium parvum Infection In Experiential Infected Mice S.T. Mohamme d, K. Jabur, H.A. Aja Deparment of Biology, Colle ge of Science, Unive rsity of Al-Mustanserria Received in:1 July 2010 Accepte d in:16 June 2011 Abstract This study aimed to collected 150 feces sample from calves suffering sever d iarrhea ,then there were isolation and p urification of Cryptosporidium parv um oocyst from samples contain it. These oocy sts were used to induce exp erimental infection in the Immunosupp eressed mice, then the mice treated by Bifidium bacteria , its sup ernatant and local y ogurt were used for bacterial isolation. This study showed that t he local y ogurt was the best treatment which led to stop oocyst shedding from the mice in the 8 th day after treatment under sufficient t reatment (89.72%) , at the same time the mice treated with bacteria st opp ed the oocy st shedding in the 9 th day with sufficient t reatment (81.40 %) , while its sup ernatant were stopp ed the oocy sts shedding in the 10 th day with sufficient t reatment (43.4%). Key words: Cryptosporidium parv um - Bifidobacteria - Lactobacilli - p robiotic Introduction Cryptosporidium parv um is water -borne p rotozoan parasite that causes acute diarrh ea in humans and domest ic animals [1]. Infection occurs when oocyst s in food water, or the environment are in gested,the exp osure to reduce conditions or p ancreatic enzy me & bile salts causes oocy st to excyst and release four sp orozoites that invade the hosts intestinal ep ithelium and undergo both asexu al and se xual multip lication cycles. The life cycle p roduces new oocysts t hat are shed in large quantities in the feces [2].M anagement of cry p tosp oridiosis , t he disease caused by C. parv um infection, has been hamp ered by the look of effective drugs. Bifidobacterium are normal inhabitants of healthy human and animal intest inal tracts .Tissier was the first to p romote the therap eutic use of Bifidobacteria for treating infant diarrhea by giving them large doses of Bifidobacteria orally. Probiotics are viable non p athogenic microorganism that ,when ingested have beneficial effects in the p revention and treatment of p athological condition [3] . Lactobacilli and Bifidobacteria are lactic acid bacteria (LBA) commonly found in fermented dairy p roducts such as y ogurt which exhibit p robiotic prop erties [4,5] . Bifidobacteria p roduce antimicrobial comp ounds can inhibit growt h of st rains of E.coli , IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 Pseudomonas spp and Sallmonella [6,7] . Bifidobacteria have also been found to aid in anti tumor activity in the host by st imulating the host s immune resp onse, lowering cholest erol level, sy nthesize vitamins ,t hiamin as well as folic acid. [8] . The aim of this research was to st udy the effect of bacterial cell-free sup ernatants from Bifidoba cterium and yogurt on C. parv um in immune def iciency mice. Materials and Methods Feces S amples One hundred fifty feces samples were collected from calves suffering from severe diarrhea, fro m Al-Eshaki calves farm in Al-Eshaki dist rict . Fecal samples of animal’s rectum were collected directly and save it in tightened p last ic cold container till arriv e to t he lab. Microscopic exami nati on Glass slides were p repared from each fecal samp le by direct smear method, t he positive slide fixed by methanole and st ained by modified cold Zehil Ne ilsen st ain according to Beaver and Jun g methods [9], then examined under oil immersion(X1000) to detect the p resence of oocy st . Isol ation and purification of Cryptosporidium oocyst . The oocyst were isolated and p urified from the p ositive sample by using sucrose flotation method followed by Ungar et.al. [10] as a followed:- -Emulsify 1gm of feces in 10ml of normal saline. -centrifuge for 1minte. -add 10 ml of saturated sucrose solution. -centrifuge then collected the sup ernated -washing by normal saline sever al times. -. p urified oocy sts were kept at 4C ْ◌ until use. Crypto spo ridium oocyst preparation . About 10 ml of C. oocyst susp ension which was isolated as described abov e , were p ut in clean centrifuge tube and centrifuged at (700 x) for 10 min, and then the sup ernatant was decanted . - The p ellet of oocysts were suspended in PBS and oocysts were counted and the number were adjusted at ( 1 x10 4 ) oocyst / ml and then inoculated orally by using st omach tube to cause infection into mice. - Preparation of Bifidobacterium and supernatants. Bifidoba cterium was isolated from probiotic yogurt available in lo cal markets. Re- detected accordin g to Wood and Holzaptel method [11]. - Then (1x10 8 ) cell/ml of Bifidobacterium susp ension and its sup ernatants were p rep ared accordin g to Contreras et.al . methods [12]. - Ani mals : M ale white Swiss mice were obtained from medical college –Al-Nahrein university Their ages were between 11-16 weeks with weights between 22-25gm . M ice feces was examined befor e starting exp eriment to insure the intest inal vacancy of p arasitic infections. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 - Ex perime ntal Design Forty eight mice were Immunosupp eressed by which injection with (0.1)ml\mice\day with dexamethazone accordin g to Regh [13] .Then divid ed into 4 group s each containin g 6 mice . * Group 1 ( G1): The mice of the first group were given 0.1ml of susp ension of Bifidoba cterium whi ch contain (1x10 8 ) cell/ml for (10) days by using st omach tube . The dose is determined accordin g to Alak et.a l [14] . Then inoculated with (1x10 4 ) oo cyst inoculation with Bifidoba cterium continued till the end of exp erimental p eriod . *Group 2 (G2) : The mice of the 2nd group were inoculated with o.1ml of bacterial sup ernatants. for (10) days. Then each mouse inoculated with (1 x10 4 ) oocyst . Inoculation with bacterial sup ernatants continued till the end of exp erimental p eriod. *Group 3 (G3) : Each mouse was inoculated with o.1ml of p robiotic y ogurt which was used p reviously in bacterial isolation for (10) days . T hen each mouse was inoculated with (1 x 10 4 ) oocyst ,their feeding with y ogurt continued till the end of exp erimental p eriod. * Group 4 (G4) : M ice in this group were inocu lated with 0.1 ml of PBS for (10) days , then inoculated with susp ension C. oocyst which contain ( 1 x 10 4 ) oo cyst /ml inoculation the PBS continued till the end of exp erimental period as a control group . - During exp eriment t he following d ata had been noticed. 1-recording any clinical clear si gn . 2-recording in perpant p eriod. 3-feces examination to calculate of oocyst in one gram , accordin g to Ry an . et.al. method.[15] . 4- sufficient treatment for bacterial Bifidobacterium sup ernatant and y ogurt was measured accordin g to Xiao et.al. method [16]. sufficient treatment %=(mean number of oocyst in 1gm)_( mean number of oocyst in 1gm) of feces of control group of feces of treated group X100 mean number of oocyst in 1gm of feces of control group S tatistical anal ysis Data are rep orted as mean ± . Standard deviation . IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 Result and Discussion In this st udy we didn’t record any clinical si gns in infected and treated group , while p rep ant p eriod recorded in all infected mice was between (3-4) day s for all group s. The results in Table -1- showed that t he numbers of oocyst in G1 and G3 were similar which reach (400) oo cyst /gm at first day , while diff erent in G2 and G4 which reach (516.66) and (633.33) oocyst / gm resp ectively. Re-detected Table -2-showed the sufficient treatment for three group s ,noticed that the sufficient treatment for y ogurt was high which reached (89.72%),followed by Bifidium bacteria that reached (81.43%).while the sup ernatant sufficient treat was the lowest reachin g (43.4%) only. We should notice that the sufficient treatment calculated depended on the average shedding oocyst through the treatment p eriod without taking in mind the time used by substance to stop shedding the oocy st totally . In this st udy , we invest igated the effect of administration of p robiotic bacteria on the development and p rogression of exp erimental infection in mice were f ed daily with (1x10 8 ) cell/ml of Bifidium bacteria , its sup ernatant and yogurt st arting (10 days) before the infection until the sp ontaneous clearance of the parasite . Our results showed that daily administ ration of y ogurt and Bifidium bacteria was able to eradicate the p arasite in mice , but the y ogurt was more efficient than bacteria because the y ogurt contains many types of bacteria like B ifidium and lactic acid bacteria (LAB)( Lactobacillus acidophilus and L.v euteri ) which exh ibit p robiotic prop erties [17],also M icrobial balan ce is an imp ortant factor in the maintenance of intest inal homeostasis and y ogurt or fermented milk sup p lementation has been p rop osed to control diarrheal disease [18] In fact , Lactobacillus acidoph ilus and L.v euteri have shown to reduce the duration and number ofC.parv um oocy st shed in feces of exp erimentally infected mice [19,20,21]. which suggested that certain LAB may p ossess p otential therap eutic p rop erties against C.parv um [22],and that may make clear the high suffi cient t reatment of y ogurt. Fast er et al. [23] found that Lactobacilli and Bifidobacterium & their sup ernatant effect on the viability of C. parv um oocy sts . These bacterium may still have p otential for therap eutic use against C. parv um as suggested by the ability undiluted B.longum sup ernatant to significantly reduce oocy st viability [24]. Administ ration of mixture of Bifidoba cterium spp or L. acidophilus to p ig lets p ositively affected the elimin ation of cryptosp oridium oocyst from feces and imp roved sy stemic metabolism and comp osition of the intestinal micro flora [25]. The p rotective mechanism that these Bifidobacterium confer against cryptosp oridium and the nature and identity of the factors involved requires furt her study both invitro and invivo. Re ferences 1- Heine , J.j; Pohlenz , J.F ; M oon , H.W.and Woode , G.N. (1984). Enteric lesions and diarrhea in gnotobiotic calves monoinfected with Cryptosporidium sp ecies .J.Infect Dis.150,768-775. 2- Pereira , S.J.; Ramirez , N.E ; Xiao .L ; Word , L.A. (2002). p athogenesis of human and bovine Cryptosporidium parv um in gnotobiotic pigs . J.infect .D.S .186,715-718. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 3-Duggan, C.; Gannon , J.; Walked , W.A. (2002). p rotective nutrients and functional foods for the gastrointest inal tract, Am.J. Clin. Nut rc.75, 789-808. 4-Tejada – Simon, M .V.; Lee, J.H.; Vst unal , Z.; Peska , J.J. (1990). Ingestion of y ogurt containin g lactobacillus acidoph ilus and Bifidobacterium to p otential immuno globulin are sp onses of cholera toxin in mice. J.Dairy Sci .82,649-66. 5- Canganella , F.; Ovidi , M .; Paganini, S.and Trovatelli,L.D. (1998). Surv ival of und esirable microorganisms in fruit, y ogurts during st orage at different temp erature. food . microbial ,15 ,71-77. 6- O’Riordan,k.; Fitzagerald , G.F. (1998). Evaluation of bifidobacteria for the p roduction of antimicrobial comp ound and assessment of p erformance in cott age cheese at refrigeration temp erature .J.App le . M icrobial.85,103-114. 7- Searle ,L.,E; Best , A. ; Nunez , A.; Johnson , L., Colley, W.A.; wood ward ,M .J .and Robert. , M . (2009). A mixture containin g galactooligosaccharid , p rodused by the enzy mic activity of Bifidobacterium bifidum , reduce Sa lmonella enterica serov ar Typhimurium infedtion in mice. J.M ed.M icrobiol 8-Seid avi,A.R.;M irhosseini,S.Z.;Sh ivazad,M .;Chamani,M .andSdeghi,A.A. (2008). App lication of aduplex PCR for the sp ecific and si multaneous detection of Bifidobacterium spp Lactobacillus spp.in duodenum ,jejunum,ileum and cecum of bro ilers.J.of Rapid M ethods and Automation in M icrobiology .16,100-112. 9- Beaver ,P.C. and Jun g , R.C . (1985).Animal A gent and Vectors of Human Disease .5 th ed. Lea and Fib iger, pp . 294. 10-Vangar ,B.L.P ; Soave , R. and Fay er , R. (1986).Enzyme immunoassay detection of immnoglobin M and G antibodies Cryptosporidium in immunocorp otant and immunoco mprised persons. J. Infection. Dis , 153 (3) :570-578. 11- Wood,B.J.and Holzapfel,W.H.. (1995).The gener a of lactic acid bacteria .The genus Bifidio bacterium in the genera of lactic acid bacteria. Edited by Wood,B.J.and Holzapfel,W.H..P440-665. 12- Contrerus,B.G.L.;Vuy st, L.; Devreese, B.;Busan ;B asman ,F Ray maekers , J . ; Yovok.; Sablon, E and Vandamme, E.J. (1997).Isolation,p urification and amino acid sequence of laction A,one of bacterio cine produced by Lactobacillus amylovorus LM GP.1319. App l. Environ. M icrobiol.,63(1):13-20. 13- Regh, J.E. (1996). Effect of interferony in exp erimental C. parv um infection .J. Infect. Dis .,174:22 -32. 14-Alak, J.I ; Wolf , B.W.; M durvwa , E.G. ; Pimentel – Smith G. E.and Adey emo . O. (1997). Effect of lactobacillus reuteri on intest inal resist ance of Cryp tosporidium parv um Infection in a mur ine model of acquired immunodeficiency sy ndrome .J. Infect. Dis. 175, 218-221. 15- Ry an ,M ; Carol, C.; Tim ,A. and Olson , D. (1999). Duration of naturally acquired giardiosis and cryp tosp oridiosis in dairy calves and their association with diarrhea. J.Amer .Vet.M ed.Assoc.,214(3); 391-396. 16- Xiao , L ; Saeed , K.and Rings , D . (1996). Efficacy of albendazole and Fenbendazole against Giardia infection in cattle. Vet. Parasitol. 61:165-170. 17- Canganella , F; Ovidi , M . ; Pagan in , S.; Vett raino , A.M. and Trovatelli , L.D. (1998). Survival of und esirable microor ganisms in fruit y ogurts during st orage at different temp erature . Food. M icrobial. 15,17-77. 18-Ivan, A.C.and Watter , J.D. (2000). Validation of p robiotic concept : lactobacillus reuter confers broad sp ectrum protection against disease in humans and an imals. M icro. Eco log. in Health and disease . V12, issue 4.p : 247-285. 19-Alak , J.I. ; Wolf , B. W. ; M alurvwa E.G. ; Kolavala , S.; Abdelrah man , H. and supp iramanian , V. (1999). Sup p lementation with Lactobacillus reuteri or L.acidophilus IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 reduced intest inal shedding of Cryp tosporidium parv um oocysts in immunodeficient C57BL/6 mice .Cell.M ol.Biol .45, 855-863. 20- Foster , J.C.; Glass ,M ., D.; Courtney . P.D.and Wood , L.A. (2003).Effect of lactobacillus and Bifidobacterium on Cryptosporidium parv um Oocy st . Viability . Food .M icrobial 20(3) :351-357 21-Hey man, M . (2000). Effect of lactic acid bacteria on diarrh eal disease .J.Am. Coll. Nut r.19(2supp l) .1375-1465. 22- Isolauri , E . (2001).Probiotics in human disease. Am. J.Cl in .Nutr 73:11425-11465. 23-Laura , E.J; Angus,B.; Alejandro, N.; Francisc , J. S. and Roberto , M .L. (2009). Amixture containin g galactoSacchar id , p roduced by the enzumic activity of Bifidobacterium bifidium reduces Salmonella enterica servor Typ himurium infection in mice. J.M ed. M icrobial .58,37- 84. 24- Rotkiewicz, T.; Rotkiewicz,z ; Depta , A ; and Kander , M . (2001). Effect of Lactobacillus acidoph illus and Bifidobacterium sp . On the course of Cryptosporidium parv um invasion in new – born p iglets, Bull.Vet.Inst.Pulawy .45,187-195. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 Table (1): Number of C. parvum oocystes in treatment & control groups ± stander deviation Day after treatment gro ups 1 2 3 4 5 6 7 8 9 10 Ba cteria Bifid ium G1 40 0 ± 63.24 1166.66 ± 103.27 25 00 ± 63 .24 30 50 ± 83.66 19 33.33 ± 103.27 1350 ± 83 .66 85 0 ± 83.66 166.66 ± 51.63 0 0 Superna tant G2 5166 ± 10 3.27 15 00 ± 63 .24 40 33.33 ± 18 6.18 49 50 ± 187.08 55 16.66 ± 116.90 41 66.66 ± 16 3.29 30 00 ± 14 1.42 58 00 ± 141.42 5150 ± 10 4.88 Yog urt G3 40 0 ± 63.24 85 0 ± 104.88 14 33.33 ± 10 3.27 15 00 88 5.166 ± 440.22 73 3.33 ± 10 3.27 51 6.66 ± 75.27 0 0 - Con trol G4 63 3.33 ± 98.31 2516.66 ± 116.90 56 16.66 ± 28 5.77 65 00 ± 141.42 82 33.33 ± 186.18 51 800 ± 86 33.33 55 700 ± 9283.33 97 50 ± 164.31 10 433.33 ± 25 0.331 Table (2): S ufficient treatment for treatment groups Type of treatme nt Use d Dose Percent of treatment effeciency (%) Bifidium bacteria (1 x 10 8 ) cell/ml 81.43 Supernatant 0.1 ml 43.40 yogurt 0.1 ml 89.72 2011) 3( 24المجلد مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة طفیلي االبواغ في Bifidobacteriumبكتریا اللبن المحلي و تاثیر في الفئران المصابة تجریبیا Cryptosporidium parvum الخبیئة سبأ طاھر محمد ، خولة جبر ، حمزیة علي عجة قسم علوم الحیاة، كلیة العلوم، الجامعة المستنصریة 2010 تموز 1:استلم البحث في 2011 حزیران 16 :قبل البحث في الخالصة أكیاس بیضعینة براز من عجول مصابة باإلسهال ،ثم تم عزل وتنقیة 150جمع تضمنت الدراسة إلحداث إصابة في أكیاس البیض واستعملت ، ات البراز الحاویة علیهامن عین Cryptosporidium parvumطفیلي y]ي حلوروا شحها واللبن الم Bifidiumعولجت الفئران المصابة ببكتریا . الفئران المثبطة مناعیا ogurt] الذي عزلت منه .البكتریا توقف الفئران عن طرح أكیاس البیض أظهرت هذه الدراسة أن اللبن المحلي هو األفضل في المعالجة، إذ أدى إلى Bifid بینما توقفت الفئران المعالجة بخالیا البكتریا، %)89.72(وبكفایة عالجیة بعد العالج في الیوم الثامن ium في بروا شح البكتریا عن طرح أكیاس البیض فتوقفت في الیوم الفئران المعالجة أما%) . 81.43(وبكفایة عالجیة الیوم التاسع %).43.40(لعاشر وبكفایة عالجیة ا Lactobacilli - p : الكلمات المفتاحیة robiotic - Bifidobacteria - Cryptosporidium parv um -