Prophylactic role of lactic acid prepared from lactobacillus acidophilus on cryptosporidiosis

C ryptosporidium has considered as a prominent enteropathogen of humans and animals. Until recently, there is no totally effective therapy other than a healthy intact immune system. Probiotics reported to stimulate both innate and acquired immunity at mucosal and systemic levels. It has been suggested that probiotics inhibit infection by excretion of substances harmful to one of the parasite’s developmental stages and possibly offer new therapeutic factors for the treatment of cryptosporidiosis .The aim of the present study was to detect the prophylactic effect of lactic acid prepared by Lactobacilli acidophillus against the infection with Cryptosporidium in an rabbits model, by estimating hematological and immunological criteria. The results showed that the using of probiotics induced significant reduction in parasite burden, lymphocyte numbers were increased significantly in infected and treated animals. There is a significant increase in the level of IgG ,IgM , C3 and C4 in


Introduction
Cryptosporidiosis is one of the most important diarrheal pathogens affecting people in the worldwide [1].In the developing world it is leading to death .The mortality rate is 5-10 million deaths cases each year [2].Although Cryptosporidium was first discovered in 1907, it was not until 1976 that this parasite was identified as a cause of human infection [3].In 2004, cryptosporidiosis was added to the World Health Organization's reports as a 'Neglected Diseases Initiative', which includes diseases affecting people mainly in low-resource settings [4].The apicomplexan protozoal parasite Cryptosporidium, an intracellular extra cytoplasmic protozoal parasite, has aparasitic life cycle that involves both asexual and sexual reproductive cycles, which is completed within an individual host.Mode of transmission from one host to another involves direct fecal-oral transmission often involving ingestion of oocystcontaminated food and water [5].Cryptosporidium is related to direct zoonotic and this parasite infects human directly from animals [6].The transmission form is a robust, environmentally resistant oocyst, excreted in the stool, which can exist for long periods of time in the environment.Because animals, in particular domesticated livestock, are its primary host, human infection is usually zoonotic [7].As many prevention methods for diarrhea have adverse effects, scientists are now turning to probiotics in hope of using it as a supplement to treat acute diarrhea as prophylactic agent [8].Lactobacillus acidophilus wich called lactic acid bacteria LAB occurs naturally in the human and animal gastrointestinal tract and mouth.Some strains of L. acidophilus may be considered to have probiotic characteristics and these strains are commercially used in many dairy products [9].The present work is to study the prophylactic efficacy of consumption of L. acidophilus on experimental cryptosporidiosis using male rabbits model, with estimate some hematological and immunological criteria.

I. Materials and methods
A. The parasite Cryptosporidium parvum oocysts (isolated from fecal samples of infected calves) were purified from the feces material of infected calves by sodium chloride (NaCl) and cesium chloride (CsCl) gradient density centrifugation, then maintained in rabbits.Briefly, rabbits were placed in metabolic cages, orally infected with 10 3 oocysts, and monitored daily for oocyst shedding by modified Ziehl Neelsen stain of prepared fecal swabs [11].All positive feces were collected daily and concentrated by the sugar centrifugal floatation method after being stored in 2.5% potassium dichromate solution at 4°C to be used within one month [12,13].

B. Experimental animals
A total of twenty one albino rabbits (ages 10-18 months) and (weights 1000-1800 gm), and free from intestinal parasitic infections were used in this study.C. Bacteria strain Lactobacillus acidophillus was used for this study which originally isolated from human feces.Lactobacilli (MRS) broth was used for inoculation of frozen cultures and propagation of Lactobacillus [14].This lactic acid was also prepared from isolated bacteria for experimental inoculation [14] .

D. Test for adhesion of Lactobacillus to crop epithelial cells
An overnight culture of Lactobacillus acidophillus in MRS was centrifuged and the bacteria resuspended in phosphate buffered saline (PBS ( at 3-7 pH .Crop epithelial cells were collected from starved rabbits by scraping the crop wall with the edge of a glass slide and suspending the scraping in PBS .The suspension were mixed to give a final ratio of 50 bacteria to one epithelial cell .After rotating (16rev./minat 37C for 30 min , a sample was withdrawn and examined by Gram stain and the number of bacteria attached to each of ten epithelial cells was counted

Results and discussion
This study showed that the infecting dose 4 ×10 3 oocyst /rabbit achieved 100% take up of infection.Clinical signs were noticed in infected animals (Gr. 2)such as diarrhea ,moderated fever and loose of appetite compared with the treated rabbits(Gr.3)that's agree with [2] The treated rabbits(Gr.3)showed significant (P<0.05)decrease in the number of oocysts shed on 3,5 , 7 and 11 days in comparison with the non-treated group (table1).These results confirm the effective prophylactic role of probiotic bacteria used in the present study against Cryptosporidium and are in agreement with [20].Probiotics are important to stimulate the proliferation of mucosal epithelial cells which are considered as the first line of defense against intestinal pathogens lik Cryptosporidium [21].
The infection of cryptosporidiosis is largely out of treatment especially in the immunocompromised patients ,Immunocompetent individuals can get rid of the infection because parasite eradication relies on innate and acquired immunity [17].So, in the preset study, probiotic was used as prophylactic factor to promote individual health due to their effects on luminal microbial ecology and immune modulation.Selection of L. acidophilus bacteria was based on many researches that use it in a variety of fermented dairy products [18].As well as their presence in the normal microflora of humans and their ability to resist stimulated small intestinal transit [19].(2).The result shows no significant differences in WBCs count between the three groups.lymphocyte numbers were increased significantly in infected Gr.2 and treated Gr.3 animal groups (4.69 × 10 9 /L and 3.39×10 9 /L)respectively comparing with Gr.1.Our results confirm previous reports that some strains of probiotic LAB can enhance several aspects of myeloid and lymphoid cell function in vivo [22], and shows that L. acidophilus is able to enhance immune function at both the systemic and local (intestinal) level [23].Significantly, the level of each of these immune responses was inversely related to the degree of pathogen translocation to the liver and spleen and others, with the exception of serum antibodies responses.Since T/B cell function, phagocytosis and local (intestinal) pathogen-specific antibody production have each been demonstrate the important in immune-mediated protection against Cryptosporidium [24].Granulocyte numbers decreased in treated rabbits (3.25 × 10 9 /L) comparing with the other two groups, while there are no significant differences between the animal groups according to the monocyte numbers .The findings of specific IgG and IgM and the complements C3 and C4 in the sera of non-treated , oocyst excreting rabbits comparing with treated animals were expected, and agreement with Riggs [25] who found that protection against this parasite has been largely associated with production of immunoglobulins and complements; a major player not only in cell-mediated immunity, but in early innate immune responses as well.In vitro studies have demonstrated that C3 and C4 directly prevents the parasite from invading host cells [26].
On the other hands, these results show the prophylactic role of probiotic.Many studies mentioned that probiotics induce modulation of the intestinal environment by having the capacity to control the proliferation of surrounding microorganisms [27].Wollowski et al [28] reported that probiotic bacteria increase the mechanisms of innate immunity and the activation of B cells for mucosal immunity important for protection against many pathogens, prevention of the penetration by foreign antigens, and maintenance of mucosal homeostasis.

1 . 2 .
[15]   E. Experimental infection Stored oocysts were washed with phosphate buffered saline (pH 7.4) by centrifugation at 1000g for 10 min just before inoculation.Infection was done by oral inoculation of 4× 10 3 oocysts/ rabbit in 0.1 ml PBS.DOocysts of Cryptosporidium parvum × 100 F. Animal groupsA total of twenty one New Zealand white rabbits were used in this study (ages 10-18 months) and (weights 1000-1800gm) , divided into three groups 7 rabbits in each, group; 1(Gr.1):control group (non infected) was inoculated with 1 ml PBS S\c , group 2 (Gr.2): infection group (infected with Cryptosporidium) and group 3 (Gr.3): was inoculated with prepared lactic acid and the orally inoculated daily dose / rabbit was freshly prepared and continued daily to end of experiment(14 weeks).adjusted to a concentration of 10 x10 3 CFU in 0.1 ml PBS twice day.This group then infected with Cryptosporidium (4 ×10 3 oocyst /ml) .II.Assement of prophylactic efficacy of the probiotic Determination of intensity of infection by Determination of Cryptosporidium oocysts count: fresh fecal samples from Gr 2 and Gr.3 were collected on days 3,5,7and 11 post infection (P.I.) Counting of shed oocysts in 10 microscopic fields (x400) of a modified Zeil Neelsen stained smear, calculation of the mean oocyst count and the percent reduction in each group was determined [11].Hematological tests: Anticoagulated blood samples were used to determine WBCs and differential blood count [16].3. Immunological tests: At the end of the seventh day P.I. period , exsanguinations of rabbits from the 3 groups were subjected for separation of sera.IgG and IgM levels and C3 and C4 ratio were determined by Radial Immunodiffusion Plates, commercially available (LTA / Italy).III.Statistical analysisData were computerized and statistically analyzed using the arithmetic mean and standard deviation, Chi square test and one way ANOVA.
b): means bearing different letter within the same day are significantly different The hematological findings between the groups are presented in table