Integration site and expression of the exogenous DNA were investigated by genome walking analysis and Western blot. Specific primers were obtained according to E. Twelve insertion site sequencing were conducted.
Survey of coccidial infection of rabbits in Sichuan Province, Southwest China
Whole parasite soluble extracts were prepared from sporozoites as previously described Monoclonal antibody against His-tag ABclonal was applied. Jejunum and ileum were washed with cold HBSS, and smears were made by scraping the mucosa of the intestine. Fresh smears and sporulation process of newly collected oocysts were visualized under a confocal laser scanning microscopy SP5, Leica, Germany for observation of different developmental stages of fluorescent parasites. To explore the biological features of the transgenic strain, reproductivity and immunogenicity compared with the parental strain EmagWT were investigated.
Sixteen day-old rabbits housed one per cage were randomly distributed into four groups with four rabbits per group. The third group was not immunized and applied as an unimmunized challenged control. The fourth group was applied as an unimmunized and unchallenged control UUC. Daily oocyst outputs during patent period 6.
Oocyst outputs of all challenged rabbits were counted. Clinical signs of experimental animals were monitored every day, and body weight was measured twice a week during the whole experimental process. To investigate the exogenous protein-specific immunity stimulated by the transgenic parasites, fifteen day-old coccidia-free rabbits were randomly distributed into three groups.
Fourteen days later, all rabbits were euthanized. Splenocytes and mesenteric lymph nodes MLN lymphocytes were isolated, and single cell suspensions were prepared. A cell stimulation cocktail containing PMA Difference between groups were considered statistically significant when p values were less than 0.
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For in vitro transfection, fluorescent sporozoites in MDBK cell culture were observed 20 h after inoculation Figure 1 B. Meanwhile, RFP was found both in the nuclei and the cytoplasm indicating that the transfected plasmid can be employed by E. After several trials on in vivo transfection, we obtained a transgenic population at a transfection efficiency of 0.
Unfortunately, we did not obtain higher fluorescent rate in subsequent passages. Collectively, we obtained a transgenic E. By taking advantages of the fluorescent proteins expressed by the transgenic parasites, the morphological features of transgenic parasites during all developmental stages were observed Figure 2.
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Invading sporozoites as well as trophozoites ongoing nucleus division were found at 24 hpi Figures 2 A,B. Mature meronts of each generation were found at 48, 66, , and hpi, respectively Figures 2 C—M.
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Since EYFP was mainly expressed in the nuclei, both multinucleate and uninucleate merozoites were vividly distinguishable. Mature microgametocytes containing thousands of microgametes Figure 2 N and macrogametocytes whose EYFP was not only located within the large nucleus but also Golgi adjunct 21 Figure 2 O were discovered during — hpi.
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Figure 2. Exogenous proteins were expressed and targeted to different cellular compartments in endogenous developmental stages of EmagER. In addition, sporogony of the transgenic parasites was also observed Figure 3. Oocysts of the transgenic strain, freshly collected from fecal samples and purified by floatation with saturated salt water were applied Figure 3 A. The first nuclear division proceeded after contraction or shrinkage of the cytoplasmic mass Figures 3 B,C.
Completion of the second nuclear division and cytokinesis was evidenced by four sporoblasts symmetrically projected from the central cytoplasmic mass Figure 3 D and soon developed into four separated spheres and an oocystic residua Figure 3 E. Observation of all the developmental stages of the transgenic parasites demonstrated that the exogenous proteins were expressed and targeted to different cellular compartments.
Figure 3. Expression of exogenous proteins of EmagER during sporogony. Freshly collected oocysts of EmagER were incubated in 2. A A freshly collected unsporulated oocyst; B an oocyst during the process of spindle stage; C first nuclear division finished; D four separated sporoblasts projected from the central cytoplasmic mass during the second nuclear division; and E formation of four spheres of sporocysts and the oocystic residua.enter
金石堂網路書店－The Biology and Identification of the Coccidia (Apicomplexa) of Rabbits of the World
To investigate the biological feature of EmagER , reproductivity and immunogenicity were evaluated. First, daily oocyst outputs of rabbits infected with either EmagWT oocysts or EmagER oocysts were measured 6—12 dpi. Oocysts of both strains were first shed on 6. Robust immunity was evidenced, and no significant difference of body weight gain and oocyst reduction were observed compared with EmagWT Figures 4 B,C.
Figure 4. Reproductivity and immunogenicity of EmagER. Three groups of rabbits were inoculated with either oocysts of Eimeria magna wild-type EmagWT , transgenic E. A Daily oocyst output of individual rabbit during patent period were measured using a McMaster counting chamber.
Body weight gain B and total oocyst output C were measured in 14 days post challenge. The Th1 cytokine profile is a well-known indicator of cellular immunity elicited by intracellular pathogens including Eimeria spp. To determine whether the exogenous proteins expressed by the transgenic parasites can stimulate specific immunity, we investigated the mRNA transcriptional level of Th1 cytokines of lymphocytes of immunized rabbits after an in vitro stimulation.
This result indicated that EmagER stimulated exogenous protein-specific local immune response in rabbit. Figure 5. The transgenesis of parasites in the phylum Apicomplexa has made great progress in recent years. In chickens, at least two E. Much more detailed work in this area has been done with T. In rabbits, however, the only work done on transgenesis was developed by Shi et al. Here we report, for the first time, the construction of a transgenic line of E.
More importantly, the exogenous proteins expressed by this transgenic parasite stimulated a specific local immune response in our rabbits. This discovery in rabbits offers the prospect that transgenic rabbit coccidia might be candidates to transport other proteins as recombinant biological vaccines. Here, we used the regulatory sequences of E.
The biology and identification of the coccidia (apicomplexa) of rabbits of the world.
This reliability might result from the functional conservation of E. Additionally, Tang et al. We also found that it was efficient in our transgenic E. As noted earlier, successful transfection studies have been accomplished with two species of chicken coccidia. Liu et al. Later, Qin et al. The causative agent is a protozoan that has the ability to multiply rapidly and cause major damage in the intestinal wall, rupturing the cells of the intestinal lining.
The final stage, the oocyst, is extremely resistant to environmental stress and is difficult to completely remove from the environment. Oocysts are frequent contaminants of feed and water and when the sporulated oocysts are ingested by other animals, they start the life cycle over in the new host. With the demand for rabbits in scientific research and for rabbit meat for human consumption increasingly globally each year, rabbits are of epidemiologic significance for laboratory workers, university researchers, veterinarians, pet owners, and breeders.
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