الفهرس 
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Abstract
Coccidiosis is a seriously parasitic disease caused by a protozoan parasite of the genus Eimeria infecting animals. Eimeria species can infect their hosts’ digestive systems and rapidly proliferate in their cells. As a result of malabsorption from the digestive tract, Eimeria-infected animals produce less milk and meat, have lower body weight, dehydration, blood loss, and are more susceptible to other infections. Consequently, this disease causes significant economic losses across the world.
This study aimed to induce protection using Hirudo medicinalis extract antigens (HEA) as a therapeutic treatment against eimeriosis. Leech therapy is categorized as complementary or alternative medicine. Both leeching and the therapeutic ingredients in their saliva have been used in medicine. This is because the salivary glands of medicinal leeches produce over 100 biological active substances (BAS), compounds with thrombolytic, antithrombotic, antihypertensive, anti-atherogenic, anti-hypoxic, regenerative, anti-inflammatory, and analgesic properties. The original studies on the effect of hirudotherapy on macrophage functional activity in disorders accompanied by secondary immunodeficiency states (for example, emotional burnout syndrome) revealed that hirudotherapy activates pre-lowered phagocytic indices monocyte / macrophages to the lower limit of normal.
Mice were allocated into five groups five for each designed as follow; the first group was considered as control non infected group. The second group acted as infected group and received no HEA (μg/mice) but was inoculated with 103 E. papillate sporulated oocysts (0 HEA (μg/mice). Third, fourth and fifth groups were orally inoculated with 1.5×103 sporulated E. papillata oocysts and One hour after infection the latter groups received HEA ((5µg/mice referring as (5 HEA (μg/mice), (2.5µg/mice referring as 2.5 HEA (μg/mice), and 1µg/mice referring as 1 HEA (μg/mice) respectively) and the inoculation was continued daily for five successive
days. The oocyst output was estimated per gram of feces on day 5 p.i. Parts of jejunum had been subjected to paraffin processing to check on parasitic stages and goblet cells. Small pieces jejunum was subjected to immunohistochemistry for detection of CD4+, CD25+ and BcL2. In addition, small parts of jejunum tissue were investigated for mRNA expression of IFN-γ, TNF-α, IL-6, IL-1β, iNOS, Muc2, and Foxp3 using RT-PCR. Jejunal homogenate was used also evaluation of oxidative stress and antioxidant status as well as TGF-β, IL-10 and IL-22 were quantified using ELISA.
The obtained data revealed a significant decline in the total number of expelled oocysts of infected mice with different doses of HEA and 5µg/ mice of HEA was the best one. All the doses did significantly reduce the intracellular stages in comparison with the infected group, each dose of HEA showed a significant increase in the goblet cells number as well as up regulate its Muc2 expression, the highest dose was the most potent of them. The present data also was shown that mRNA expression of iNOS and inflammatory cytokines IL-1β, TNF-α, IFN-γ and IL-6 genes, as well as oxidative damage could reduce the comparing with the infected group after treated with all doses of HEA therapy. Jejunal CD4+ and CD25+ showed a significant increase in mice group treated with HEA with respect to the infected one. Also, HEA could significantly increase the expression BcL2 versus the infected group and upregulated the m-RNA expression of Foxp3 as well.
Concerning levels of IL-10, TGF-β and IL-22; HEA treated group showed a significant increase of TGF-β and IL-10 with respect to the infected group but not for IL-22.
After that, ion exchange chromatography was performed to separate different fractions of HEA. In mice, eight fractions obtained were evaluated against E. papillata infection. Five fractions could reduce oocysts count, jejunal parasitic
stages and jejunal histological injury score as well as improved goblet cells production and upregulate the expression of m-RNA of Muc-2. Effective fractions were subjected to SDS-PAGE and proteomic analysis for detection of their bioactive macromolecules.
The fractions reveled only a protein at 8 kDa while the results of spectroscopy and bioinformatics identified the protein as Eglin C. The pooled fractions containing Eglin C were tested in vitro to determine its stimulation for the intestinal lymphocyte proliferation and IFN-γ together with IL-6 release in the supernatant. The results showed that higher Eglin C concentrations reduced the stimulation index of lymphocyte proliferation as well as the stimulation index of IFN-γ and IL-6 production.
In conclusion, HEA appears to protect mice against E. papillata primary infection. This protective effect was attributable to HEA’s and its Eglin C anti-inflammatory, antioxidant, and anti-apoptotic properties, which resulted in less epithelial cell damage which makes the parasite entry into the host cell easier. Increased production of IL-10, TGF-β, and CD4+ CD25+ Foxp3 T cells, which aid in the normalization of goblet cell populations, was also beneficial. Hence, hirudotherapy can be considered as an effective remedy for coccidiosis and its potential to provide protection in veterinary species.