Effect of probiotic acidophilus plus against infection with secondary hydatid disease in BALB /c mice

The current study investigated the effect of acidophilus plus probiotic in the immune activities in mice to infestation with the cystic echinococcosis. Two dilutions of the probiotic bacteria 9*106/0.1 ml, 30*103/0.1 ml CFU were used, by intraperitoneally injection in the experimental animals, pre and post infections with protoscoleces of Echinococcus granulosus. Before infection remedy comprised injection by acidophilus plus twice with 72 hours interval, on the seventh day, animals were injected with protoscoleces intraperitoneally, after infection remedy implicated injection of animals with protoscoleces of Echinococcus protoscoleces first, next to 72 hours, probiotics were inoculated intraperitoneally. Many criteria were taken into consideration including, numbers, weights, diameters and percentage reduction of hydatid cysts of treated mice in contrast to the animals infested with only. The study showed a decline in cysts including their diameter, weighting, digit, accompanied by increasing the percentage reduction of hydatid cysts in treated mice, the highest percentage reduction was 98.03%, at both dilutions, 6 months post infection, and the minimum cysts number was 0.8 in comparison with the control group 39.4, with significant difference, in the same experiment. The minimum cysts weight was 0.0104 gm, 6 months post infection at the dilution 9*106/0.1 ml CFU, compared with the control group 0.442 gm. The smallest cysts diameter was 0.057 mm in comparison with the control group 0.882 mm at dilution 9*106/0.1 ml CFU, 6 months post infection. Acidophilus plus it may well be deduced that probiotic bacteria can be used as medicinal and remedial method against infection with hydatidosis.

. It is neglected tropical areas disease, which is the most important reason of health deficiency and economic losses. China is account as a considerable part of the global loss (2). Cystic echinococcosis is causing by Echinococcus cestoda, the common species, E. granulosus. Humans are accidentally infected by ingesting the parasite eggs which are raised from specific hosts (e.g. wolves, dogs, foxes, and jakals) (3), by fecal-oral stream. Parasite ova ingested in the human digestive systems, hatched in duodenum, growing up as larvae and passing through the gut wall into liver with blood route. About 75% of cases affect liver (2,4,5). The taxonomy of genotypes/species, leading to cystic echinococcosis, includes the subdivided E. garnulosus senso lato complex groups, E. granulosus sensu srtricto, E. equines, E. ortleppi, E. Canadensis, and E. felids (lion strain) (6,7). In medicine, it is difficulty for drugs to permeate into the multiple layers of the hydatid cyst, but chemotherapy and operation are still the most utilized methods for therapy of cystic Echinococcosis (8). Albendazole drug is ovicidal, larvicidal and vermicidal, but operation is still the main treatment (3,9). So, researchers, in recent years, have developed new alternatives in an attempt for elimination of some parasitic infections. The most important is the use of probiotic bacteria, probiotics represent microbiota of different sources endogenous and exogenous, of which are most important for human and animals if administrated on adequate amount (10). The prophylactic activity of this type of bacteria may be by competition, seclusion and impedance to harmful bacteria of the intestine. Other technique involves synthesis of compounds against bacteria, includes H2O2, bacteriocin, and immunomodulation (11). Several studies proved the efficacy of probiotics for prevention against many diseases such as, cancer, allergies, inflammatory, intestinal and autoimmune disease (12). Probiotics effect on other eukaryotic pathogens, explained the efficacy of Lactobacillus in the prevention contra extra intestinal parasites, such as Plasmodium, Babesia (13). The effects of useful microorganisms, probiotics became interesting for their protection or therapeutic applications against diseases including worm diseases (14) like zoonotic Schistosomiasis which is caused by genus Schistosoma (15). Other probiotics strains such as L. acidophilus, L. plantarum and L. delbrueckii revealed an obvious activity versus ancylostomiasis in normally affected canine with a disease. Elevation of white blood cells has been observed (16). Researchers found that L. casei is the firstly strain among other strains of Lactobacilli has anthelmintic effect of about 75-100% prevention, another strain is L. plantarum with efficacy of 90% against Trichenilla spiralis (17).
The aim of the current study is to use probiotic bacteria, for the first time, against infection with secondary hydatid disease as an antiparasitic immunomodulator in BALB/c mice, depending on many criteria including numbers, weights, diameters, and percentage reduction of hydatid cysts in the experimental animals, in comparison with the control group.

Laboratory animals
One month-aged experimental animals (Swiss mice) were utilized.

Cysts samples
Ovine cysts containing protoscoleces were obtained under sterilized conditions, alive protoscoleces, 20 µl of protoscoleces suspension were added to 20 µl of 0.1% of eosin pigment on a slid, then tested under light microscope. Bright green protoscoleces were considered as alive because of eosin exclusion by protoscoleces membranes, whereas red protoscoleces were regarded as dead because of acceptance of the pigment gained and counted according to authenticated manner (18,19,20).

Bacterial strains preparation and inoculation
One capsule of acidophilus plus provides lactobacilli culture, typically providing 2 billion of L. acidophilus, L. casei casei and L. casei rhamnosus. These lactobacilli strains were grown in nutrient broth medium for 24 hours then the culture was diluted in normal saline to contain 9*10 6 /0.1 ml CFU of acidophilus plus probiotics, the second dilution in normal saline contains 30*10 3 /0.1 ml CFU of acidophilus plus probiotics (16,20).

Experimental design
Animals were divided into seven groups, each group comprised of five animals. Group one: 30 Mice (5 mice for each experiment as control group) were injected with 2000 protoscoleces /20 gm body weight intraperitoneally by 0.1 ml of protoscoleces diluted in phosphate buffer saline (PBS). Group two: 5 Mice were injected intraperitoneally with the probiotic acidophilus plus 9*10 6 / 0.1 ml CFU, twice with interval 72 hours, on the 7 th day mice were injected with 2000 protoscoleces. Injection with probiotics continued for 50 days. Mice were dissected 2 months post infection. Group three: 5 Mice were injected intraperitoneally with probiotic acidophilus plus 9*10 6 / 0.1 ml CFU, twice with interval 72 hours, on the 7 th day mice were injected with 2000 protoscoleces intraperitoneally. Injection with probiotics continued for 110 days. Mice were dissected 4 months post infection. Group four: 5 Mice were injected intraperitoneally with probiotic acidophilus plus 9*10 6/ /0.1 ml CFU, for 170 days with 72 hours intervals, on the 7 th day mice were injected with 2000 protoscoleces intraperitoneally. Mice were dissected 6 months post infection. Group five: 5 Mice were injected intraperitoneally with 2000 protoscoleces, after 3 days the mice were injected intraperitoneally with acidophilus plus 30*10 3 /0.1 ml CFU for 50 days, with 72 hours intervals, mice were dissected after 2 months. Group six: 5 Mice were injected intraperitoneally with 2000 protoscoleces, after 3 days mice were injected intraretinally with acidophilus plus 30*10 3 /0.1 ml CFU for 110 days with 72 hours intervals, mice were dissected after 4 months. Group seven: 5 Mice were injected intraperitoneally with 2000 protoscoleces, after three days mice were injected with acidophilus plus 30*10 3 /0.1 ml CFU for 170 days with 72 hours intervals, mice were dissected after 6 months.

Statistical analysis
For testing the variances between the periods and dilution of bacteria acidophilus plus, a Complete Randomized Design (CRD) was performed. Then, the variances between group ' s mean values were tested by Duncan's multiple range test. The test periods, as a quantitively value, were compared by the Analysis of Variance (ANOVA). Two ready packages, Statistical Analysis System (SAS) and Minitab were applied (21).

Discussion
In the recent years, the efficiency of useful probiotics to the body rely on the mechanization, through which they do their action by numerous processes implicating manufacture of microbe killer materials, modification the mucosa of the defense order, exchange gut microflora, boosting action of enzymes (22). Both dilution of bacteria probiotic (pre and post infection) exhibited significant effect on the infection in treated animals in comparison with the control group, and the effect is directly fit with period of treatment because treatment with bacteria continued nearly all the period of infection (2,6,8) months, respectively. In general, pre-infection treatment with bacteria 9*10 6 /0.1 ml (CFU) was stronger in effect that post infection treatment, this due to the stimulation of immune response of the treated experimental animals. Present study revealed antiparasitic effect of probiotic acidophilus plus (for both dilution), explained by significant decrease, of larval stage digit, weightiness, diameter, in addition increase in reduction proportion in hydatid cysts through the experiments period.
The primary route of bacterial activity to parasitic hitting possibly through boosting gut hurdle (23)(24)(25)(26). Helpful bacteria could also increase useful microbiota numbering as lactobacillus, that suppress harmful bacteria development via contesting on connection region on the gut endothelial layer. Another mechanics might imply excretion of some micro killer materials, mostly produced from lactic acid bacteria and could be hold potency against parasitic larvae (27). Moreover, bacterial output holds anthelmintic influence that might decrease maliciousness of numerous protozoa and helminthic illness (28). These results are similar to the study of Mohamed et al (29) and Zowail et al (30) who proved the protective and curative effects of useful bacteria in mice models against infection of Schistosoma mansoni (29)(30)(31). Many strains of probiotics, like Zymomonas mobilis 1*10 9 CFU/ml, when administrated by mouth provoked minor defense reaction, in mice against S. mansoni by providing prevention in treated animals (31,32). When Lactobacillus sporogenes used orally at a certain dose, for a long time,since the beginning of the infestation with S. mansoni, minimized impairment of DNA, improves liver and gut deterioration, therefore weakened adult together with ova numbers (29).
Results of the present study are also consistent with the Bsualdo et al (33) who assessed the protection effects of probiotic bacteria versus Toxocariasis within experimental animals. They provoked considerable shorthand in worm number in empirical animals processed with Enterococcus faecalis, further, E. faecalis in variable dosage revealed effect against larvae outside and inside the body (25). Likewise, another study on Tricuris muris infection in mice model, explained that oral feeding for alive L. rhamnosus potentially stimulated larval expulsion in Trichuris muris resistant C57BL/6 mice (34). A suspension of Lactobacillus acidohilus, L. delbruckii revealed potential leverage against canine ancylostomiasis (16). Researchers observed that L. casei has anthelminthic 75-100% prophylactic effect, and Lactobacillus plantarum P164 (17) revealed 90% protection against T. spiralis, ensured that these strains are safe as protective and curative probiotics (17,23,35,36), A suspension for different probiotics revealed an increased worthy preservation 90% against Anchylostoma caninum infection in dogs (16), otherwise, Bifidobacterium animalis 04450B dose 9*10 2 CFU showed less protection 33% decrease of adult worms and 21% decrease of egg production Strongyloids venesuelensis infection in mice (37). First of all, we used orally administration with probiotic bacteria in experimental animals, but we didn´t see significant or obvious results that ensure the effect of bacteria as stimulative, when compared with the control group, so we used intraperitoneally injection because larval stages (hydatid cysts) are extraintestinal, so that the effect of intraperitoneal injection was very effectively obvious than oral administration. When we compared our study with others, we saw that little studies only used the intraperitoneal injection with bacteria like Babesia, plasmodium (protozoa) (13). Differences in the effect of probiotics strains, may be due the variable study design, experimental animals, dependent doses, and manner of inoculation (14).

Conclusion
It may well be concluded that acidophilus plus probiotics possess have an obvious stimulative and curative effect against infection with secondary hydatid disease in mice, and may be applied as an alternative strategy against different parasitic infection.