Protective efficacy of Eglin C from Hirudo medicinalis against Eimeria papillata-induced coccidiosis.

The current study aimed to find a new therapeutic agent from Hirudo medicinalis for murine coccidiosis. Ion-exchange chromatography was performed to separate different fractions of HEA (hirudo extract antigens). Eight different fractions were experimentally tested against murine eimeriosis induced by Eimeria papillate. The oocysts output was counted to determine the most effective fractions. For the five most effective fraction groups, jejunal histological examination and goblet cells count as well as mRNA expression of MUC2 gene using RT-PCR were performed. The data indicated that these fractions significantly decreased the oocysts output and the number of parasite developmental stages, while the goblet cell numbers and the expression of MUC2 were increased. 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, Eglin C protein can be used as a target for therapeutic treatment or as an anti-inflammatory agent for coccidiosis infection.

Therapeutic effects of Hirudo medicinalis extract antigens on modulation of CD4+CD25+Foxp3 T cell activity in murine eimeriosis.

Eimeriosis is a common parasitic disease in the chicken industry. The aim of this study was to assess the protective role of Hirudo extract antigens (HEA) against murine eimeriosis induced by Eimeria papillate. The oocyst output, developmental stages, goblet cells and oxidative stress, were investigated. Immunohistochemistry was used to detect anti-apoptotic Bcl2 marker and the number of both CD4+ and CD25+ cells in jejunal tissue, while ELISA was used to quantify TGF-ß, IL-10 and IL-22 in jejunal tissue homogenate. Real-time PCR was also used to detect mRNA expression of mucin 2 (MUC2), inducible nitric oxide synthase (iNOS), IL-1ß, IFN-γ, TNF-α, IL-6, and FoxP3. The most effective dose (5 µg/mice) reduced the oocyst output by 82.95 ± 1.02% (P ˂ 0.001). Similarly, the same dose reduced the jejunal developmental stages by 66.67 ± 0.49% (P ˂ 0.001). Furthermore, HEA therapy increased the number of jejunal goblet cells by 12.8 ± 1 (P ˂ 0.001) and the expression of MUC2 by 0.83 ± 0.06 (P ˂ 0.001). In contrast, TNF-α, IFN-γ, IL-6, iNOS, and IL-1ß expression as well as apoptosis were reduced. The number of CD4+ and CD25+ in the jejunal tissue was increased (14.6 ± 1.2 (P ˂ 0.001), 6.84 ± 1 (P ˂ 0.01), respectively) after HEA therapy. The molecular analysis showed an increased expression of intestinal Foxp3 (3.2 ± 0.13 (P ˂ 0.001), while IL-22 was reduced (124 ± 10 (P ˂ 0.001)) versus an increase in TGF-ß (250 ± 17 (P ˂ 0.01)) and IL-10 (236 ± 16 (P ˂ 0.001)) after HEA treatment in comparison to the non-treated infected group. With respect to the infected group, HEA reduced lipid peroxidation (LPO) (15.7 ± 1.12 (P ˂ 0.001)) and nitric oxide (NO) (13 ± 1.3 (P ˂ 0.001)) but increased reduced glutathione (GSH) (3.7 ± 0.26 (P ˂ 0.001)). In conclusion, HEA therapy protected against intestinal tissue damage by activation of CD4+CD25+Foxp3 cells which showed anti-inflammatory action. Hence, HEA can be recommended as a therapeutic treatment for eimeriosis.

Immunoprotective responses against murine sarcocystosis by ß - Irradiated sporocysts.

This study aimed to induce protective immunity against infection with Sarcocystis muris in experimental mice using ß-irradiated sporocysts. Mice were vaccinated with 50 sporocysts of S. muris which were exposed to 1.84 µSv ß-irradiation for 2, 4 and 8 h. After challenge infection, different samples were taken for evaluation. Serum and intestinal wash were assayed for IFN-γ and IgA, respectively. Mesenteric lymph nodes (MLNs) and spleen were investigated for CD4+ and CD8+ T cells using immunohistochemistry. For liver, the morphological changes in parasitic stages and the count of infiltrated CD8+ T, NK1.1+ and FasL+ cells were also investigated. Real time (RT) - PCR was used for detection of liver MHC I, CD1d, IFN-γ, perforin and FasL as well as the parasite 18S ribosomal(r) RNA in liver and muscle tissues. Alterations of liver parasitic stages as well as a decrease in the infection with the parasite in both of liver and muscle tissues were dependent on radiation exposure time. An investigation for the mechanism of immunoprotection showed an increase in liver NK1.1+ & FasL+ cells, serum IFN-γ and intestinal IgA, while CD4+ and CD8+ T showed a remarkable increase in MLNs and spleen. FasL expression increased in the liver dependently on radiation exposure time, while perforin, MHC I and CD1d were not. ß-irradiated sporocysts with 1.84 µSv for 8 h s could induce the highest protection against infection with Sarcocystis. This could be largely relied on the increased infiltration of NK cells and associated higher expression of FasL in the liver.

Anticoccidial activities of Chitosan on Eimeria papillata-infected mice.

Eimeria spp. multiply within the intestinal tract causing severe inflammatory responses. Chitosan (CS), meanwhile, has been shown to exhibit anti-inflammatory activities in different experimental models. Here, we investigated the effect of CS on the outcome of inflammation caused by Eimeria papillata in the mouse intestine. Investigations were undertaken into the oocyst output in feces and developmental stages and goblet cells in intestinal tissue. Assays for lipid peroxidation, nitric oxide (NO), and myeloperoxidase (MPO) were also performed. T cells in intestinal tissue were counted using immunohistochemistry while total IgA in serum or intestinal wash was assayed using ELISA. In addition, mRNA expression of tumor necrosis factor alpha (TNF-α), transforming growth factor ß (TGF-ß), interleukin (IL)-10, and IL-4 were detected using real-time PCR. The data indicated a reduction in both oocyst output and in the number of parasite developmental stages following CS treatment, while the goblet cell hypoplasia in infected mice was also inhibited. CS decreased lipid peroxidation, NO, and MPO but did not alter the T cell count or IgA levels in comparison to the infected group. The expression of TNF-α and TGF-ß decreased but IL-10 and IL-4 increased after CS treatment in comparison to the non-treated infected group. In conclusion, CS showed anti-inflammatory and protective effects against E. papillata infection.

Redescription of Eimeria zarudnyi Alyousif & Al-Shawa, 2003 as Choleoeimeria zarudnyi n. comb. (Apicomplexa: Eimeriidae).

Coprological examination of the worm lizard Diplometopon zarudnyi Nikolskii revealed the presence of oöcysts of Choleoeimeria zarudnyi (Alyousif & Al-Shawa, 2003) n. comb. in five (17%) of the 30 lizards examined. Sporulated oöcysts were found in the faeces and the gallbladder contents. These are tetrasporocystic, ellipsoidal, 25-32 × 18-25 (mean 27 × 22) µm, with a smooth bi-layered wall. The dizoic sporocysts are ovoidal, 10-13 × 6-9 (mean 11 × 7) µm, with a granulated sporocyst residuum. Sporozoites are banana-shaped with an average size of 13 × 3 µm. Endogenous stages (meronts, gamonts and gametes) are confined to the gallbladder epithelium and the infected cells were hypertrophied. Based on the morphological features of the exogenous stages and the endogenous development of the present parasite, its generic affiliation is revised and Eimeria zarudnyi Alyousif & Al-Shawa, 2003 is transferred to the genus Choleoeimeria.