The Relationship Between Nitric Oxide And Eimeria Tenella Infection In Chickens

In order to get evidence for differential iNOS activity, during a experimental infection with the E.tenella, the activity of iNOS was examined by spectrometer method, the relationships between iNOS activity in the cecal mucosa and dose of parasite or between iNOS activity and time points post inoculation (PI) were studied. The experimental results showed there were significant differences of iNOS activity among different time poins. The maximum iNOS activity appeared at 96h PI; In the 7 infective doses, the iNOS activity were various and siganificant higher than the control and appeared a dose-effective relationship between the iNOS activity and the infective doses from 0.5×104 to 2.5×104 oocysts per chicken. However, the activity was decreased when infective dose was equal to or higher than 3.0×104 oocysts per chicken. The distribution and expression of NOS in the cecum and spleens of chickens after infection(AI) with E.tenella were investigated by NADPH-diaphorase staining method. The results indicated the sub-mucosa and muscular layers appeared higher stained. The staining is supposed to be recognized as cNOS according to previous data and its expression characteristics. Epithelial cells and glands in cecal mucosa of infected chickens also appeared higher stained on 3th~5th d AI, but the staining was weaker on 7th AI. However, the above-mentioned parts of the control group chickens and also spleen of each experimental chicken appeared very weaker or no staining. The results suggested the staining of epithelial cells and glands in the cecal mucosa was supposed to be the expression of iNOS. The effects of exogenous NO on E.tenella oocysts were studied in the second parts of our research. The E.tenella oocysts treated in vitro by the well-known NO-donors, SNP, Sper/NO and GSNO. The results showed that among the 3 NO donors, only GSNO had siganificant effects on the percentage of sporulated oocysts, the inhibiting rate of purified sporulated oocysts was 93% to 97%, and reached 100% on the oocysts in the faces of chicken with E.tenella infection. GSNO also significantly decreased virulence of E .tenella oocysts; and the oocysts treated by 2-8 mmol/L GSNO almost lost their pathogenicity to chickens. Chickens were treated with tNOS stimulator-LPS and inhibitor-Dex by intraperitoneal injection, NO2" level of serum in chickens infected with E.letteita were detected by activated cadmium reduction method, and then chicken' s weight gain, immune organ parameters, OPG and cecaj lesion scores were also investigated, The results showed that the percentage of NBT positive cells of chickens in the group treated LPS and the infected group was higher than those of control group, the cecal lesion and the losing-weight caused by infection were alleviated with treatment by LI'S, The results indicated LPS had a protective role against EJenella in chickens; the serum NO/, the percentage of NBT positive cells, weight gain and immune organ parameters in chickens treated with Dex decreased significantly, but OPG and cecal lesion scores were significantly increased than those of the control group chickens with Eienella infection. The results suggested Dex may inhibit NO synthesis and then decrease the immunity to E.tenella infection in chicken. In order to study the relationships between NO and quantity of mast cells (MC) and content of histamine (HT) in cecal mucosa, and the role of NO synthesis in chickens during E.tenella infection, The effects of inhibiting the production of NO on the quantity of MC and the contents of HT in mucosa of chickens infected with E.tenella, and the pathology in cecum were investigated, The experimental chickens were divided into 5 groups and 18 chickens in each group. Except the uninfected control group, the other 4 groups were immunized and challenged with E.tenella, 3 of them were treated with AG (AG group), L-NAME (L-NAME group) and L-Arginine (the substrate of NOS,L-Arg group)repectively. The results of the experiments showed that the serum NO2-level in the chickens treated with AG was the lowest, but the content of HT in mucosa was the highest, and the quantity of MC was no significant differences between AG group and uninfected control group, but significantly higher than other groups. The immunized control group had the highest NO2-level, and the quantity of MC and contents of HT were higher than those of L-NAME group and L-Arg group (p<0.05); There were no significant differences in serum NO2-level, quantity of MC, contents of HT between L-NAME group and L-Arg group. The results of histologic pathology showed that, there were lots of E.tenella in various stage in chickens of AG group and L-NAME group, however, only a few E.tenella in cecum of chickens of L-Arg group and immunized control group. In vitro experiment showed histamine diphosphate monohydratecould significantly inhibit the concanavalin-A induced proliferation of lymphocyte in peripheral blood from chickens(p<0.01). The results of our experiments suggested there was no corelation between the quantity of MC and the content of HT in the cecum of chickens infected with E.tenella. AG and L-NAME inhibited NO synthesis in chicken, increased the parasite-burden in cecum of chicken in immunized control group, and enhanced degranulized-MC to release HT. The above-mentioned results indicated that the increasing synthesis of NO in chickens infected with E.tenella oocysts was an important protective reaction of chicken against E.tenella infection. The experiments of NO donors and the relationships between NO and quantity of MC and contents of HT has further proved that NO was an important effective molecule during chickens infected with E.tenella.