To learn the brain immunopathogenesis in mice infected with Toxoplasma gondii PRU strain with the cytokines in the tissue,51 mice were grouped into the infected and the control.Thirty-three ICR mice were infected intraperitoneally with cysts,and 10 cysts in each,18 mice were injected with PBS as control.Five mice from the infected and three from the control were sacrificed under anesthesia on 5d,10 d,15d,20 d,30d,and 90dpost-infection(p.i.),and the brain tissues were collected for hematoxylin-eosin(HE)staining and RNA extraction followed by examination of IFN-γ,IL-4,IL-6,and TNF-αwith real-time PCR.The rest of the tissues were subjected to ELISA for the cytokines detection.Comparing with the control,IFN-γin the infected mice began to rise on 5d,decreased to the lowest on 10 d,and then began to raise again p.i.TNF-αbegan to decrease obviously from 10 dto 15dp.i,and reached its highest level on 30 dp.i.IL-6increased on 5dp.i,and then decreased to the lowest on 10 dp.i,then rose slightly and reached the highest on 30 dp.i.No remarkable alteration of IL-4was noted.ICR mice infected with T.gondii PRU strain presented an obviously down-regulated expression of IFN-γ,IL-6and TNF-αfrom 10 dto 15dp.i,which might help the parasites survive from the host immune challenge.Additionally,the timing phase of cytokines expression may be responsible for the cyst formation and latent T.gondii infection.

Thirty-two Zhangzhou game chicken(DJ)were randomly selected and incubated at the same stage. Some immune indexes of chickens at 12 weeks of age were detected. PCR-SSCP technique and sequencing method were applied to detect SNPs in exon 9 of Nramp1 gene,then analyze relationship between the polymorphisms of Nramp1 gene and immune indexes. The results showed that 2 samesense mutations at C315 G and C357 T loci were found in exon 9 of Nramp1 gene of DJ,and three genotypes were detected. The heterophil/lymphocyte(H/L)value,the content of Ig G and Ig M of different Nramp1 genotypes in DJ showed significant differences(P0.05). H/L value,the content of Ig G and Ig M of AA genotype were significantly higher than those of BB genotype(P0.05),the contents of Ig M and Ig G of AA were significantly different from that of AB genotype,while there were no differences between other immune indexes of AA and AB genotypes. The results of preliminary study revealed that the general immune performance of AA genotype was superior to that of BB and AB genotype,and could be used as high resistance genotype.

To investigate the protective effect of fluconazole and itraconazole for the reactivated toxoplasmosis in mice,ICR mice infected with Toxoplasma gondii(T.gondii)for two months were grouped as follows:cyclophosphamide(CTX) treatment;cyclophosphamide combined with fluconazole(CTX+F),cyclophosphamide with itraconazole(CTX+I),and cyclophosphamide with azithromycin(CTX+A).Normal mice only given with CTX and mice with T.gondii infection were set up as control.The CTX doses mouse got was 100mg/kg·d by intraperitoneal injection except the infection(In)group.At the same time,mice from CTX+F and CTX+I were administrated fluconazole or itraconazole at various concentrations of 20mg/kg·d,30mg/kg·d and 40mg/kg·d,respectively,and the concentration of azithromycin got by mice from CTX+A group was 250mg/kg·d.The medicines were administrated for continuous 14days.At the end of the experiment,the mice from CTX group were dead;however,the mice from CTX+A group,CTX group and In group were survival,and the survival rate between CTX+A and CTX was significant deviation(P=0.016).Survival deviation of mice was very significant,which came from CTX group,CTX +F group,CTX+I group,and CTX+A group(P=0.000).Azithromycin was the best in the three drugs,among which fluconazole effect was better than itraconazole(P=0.000).However,survival rate resulted in by different fluconazole or itraconazole dose was not significant deviation.Compared CTX group with others,the number of cysts in the brain were obvious increased(P=0.001),but the number of cysts was no statistical difference among CTX+A,CTX+ I,CTX+F,and In groups.Although there were different cysts in three concentrations of CTX+F and CTX+I groups,there was no statistically significant.It's suggested that fluconazole could make some protection for the reactivated acute toxoplasmosis in mice,and it could be an alternative to T.gondii infected patients who were immunity suppression or with opportunistic fungal infection.

To study the model that Toxoplasma gondii inapprent infection mice is reactivated to acute toxoplasmosis by cyclophosphamide(CTX),20ICR mice were injected with cysts intraperitoneally,and a mouse was got 10cysts of T.gondii Prugniaud strain(PRU).10ICR mice were injected with partes aequales sterile PBS.After two months,all mice were injected intraperitonelly with CTX,100mg/kg.d for 14days.The body weight,number of WBC and T.gondii gene in the blood on alternate days were monitored,at the same time,the liver,spleen and lung from the mice on day 10post injection were made for HE staining and immunofluorescence staining.As a result,the infected mice appeared the symptoms of acute toxoplasmosis after 8days post injection.Compared with the mice befor experimental,the body weight and the number of blood WBC were descendent obviously,and T.gondii genes were seen on day 10post injection.T.gondii gene could be tested in liver,spleen and lung of the dead mice after 10days post injection.The histopathological damages were observed by HE staining,and T.gondii antigen could be detected by immunofluorescence in the mice on day 10post injection.Cyclophosphamide could make successfully inapparent infection mice reactivate acute toxoplasmosis.

The PCR-SSCP technology and DNA sequencing was conducted to detect single nucleotide polymorphisms(SNPs) of exon 8 in glypican-1(GPC1) gene in twelve breeds.Five immune traits and their associations with SNPs were also detected at 56 days of age in Wenchang,Rugao and Anka chickens.1 non-synonymous SNP,g.155 CA,were found,resulting in amino acid sequence altered from Threonine to Arginine.In all 12 breeds,the average frequencies of AA,AB and BB genotypes were 0.259,0.545,and 0.211,respectively,and the average value of observed heterozygosis and Shannon's index were 0.4658 and 0.6710.Except Taihe chicken and Dagu chicken,the other populations were all in Hardy-Weinberg equilibrium at this locus(P0.05).The dendrograms divided the 12 chicken populations into 3 clusters,which reflected the resistance difference between these 12 breeds.IL-1 concentrations and H / L values of AB genotype individuals was significantly differ from that of BB genotype individuals,rather than that of AA genotype individuals in Rugao and Anka chickens.There was no significance in SRBC antibody titer,ND antibody and AI antibody titer between three genotypes.In general,the disease resistance of Rugao and Wenchang were better than that of Anka chicken.

【Objcetive】A study on small RNA and its binding protein in poultry was carried out in order to provide a basic foundation for the study of genetic characters,occurrence and disappearance mechanisms of small RNAs in avain and transgenic chicken in avian.【Mehtod】The sequences of piRNAs and CDS of the Piwil1 gene were acquired from the testis of quail by constructing cDNA library and TA cloning and sequencing.Next,the expressions of piRNAs and Piwil1 in quail tissues of different stages of life were analyzed by Q-PCR.【Result】Ithis study,we obtained Thirty piRNAs cloned sequences and CDS of the Piwil1 gene were obtained from the testis of quail for the first time.The results of Q-PCR showed that piRNAs and Piwil1 genes had a variable expression in quail tissues of different stages of life.【Conclusion】The results showed that the length of quail piRNAs was similar to mammals.In quail,Piwil1 gene had high homology with that of the red jungle fowl,whose protein contains the PAZ and Piwi domain,and has no signal peptide splice site,suggesting that it belong to Piwi family and might play a regulatory role by reducing the capacity of binding RNA or affecting the affinity between other transcription factors and the promoter of structural gene.In testis,the expression of piRNAs and Piwil1 gene increased with the development of individuals,so it was inferred that piRNAs might regulate the process of spermatogenesis through interacting with Piwi proteins in poultry.

A total of 72 Rugao chicken(RG) and 55 Recessive White chicken(RW),which belong to Chinese native chicken breeds and introduced breeds respectively,were selected randomly and incubated at the same stage.The value of immune traits was detected.PCR-SSCP technique was applied to analyze the correlation between the polymorphisms of Nramp1 gene and immune traits in RG and RW.The results showed thatthe heterophil/lymphocyte(H/L) value,lymphocyte transformation rate and the content of IgM of RG and RW were significantly different(P0.05).H/L value of AA genotype was significantly lower than that of BB genotype and AB genotype,while the AA genotype's lymphocyte transformation rate and IgM were significantly higher than those of the BB genotype in both RG and RW.The results demonstrated that the general immune performance of RG was superior to that of RW.The general immune performance of AA genotype was superior to that of BB genotype and AB genotype.

This study was conducted to analyze the polymorphisms of chicken Toll-like receptors 4(TLR4) gene and aimed to provide a theoretical foundation for a further research on correlation between chicken TLR4 gene and disease resistance. Genetic variations at exon 2 of TLR4 gene in 14 chicken breeds and the red jungle fowl were detected by PCR-SSCP method and two alleles and three genotypes were found, Tibetan chicken and red jungle fowl only had BB genotype, while the others presented three genotypes of AA, BB and AB. Sequencing results showed two mutations, G114A and G142A, located at exon 2 of TLR4 gene. The results of Chi square test showed that all populations, except Xianju chicken, were in accordance with Hardy-Weinberg equilibrium at this locus (P > 0.05). According to analysis of population genetic variation, all the populations were at moderate polymorphism (0.25 < PIC < 0.5) except red jungle fowl and Tibetan chicken (PIC = 0). The study demonstrated that there were differences of normal anti-disease ability in Chinese indigenous chicken breeds and appeared no significant correlation with body size, product type and geographical location. The associated analysis of results showed that the SNPs of TLR4 gene in the study were not linked with potential major loci or genes affecting some resistant traits.

The aim of the present study was to detect single nucleotide polymorphism(SNP) in some areas of IL-1β gene of chicken to find genetic marker influencing on IL-1 content in serum.Two pairs of primers were designed according to the sequence of IL-1β gene of red jungle fowls,and the SNP of Rugao chicken(RG),Wenchang chicken(WC),and Anka chicken(AK) were analysed by PCR-SSCP,meanwhile,the IL-1 content in serum were detected at 56 days with reagent kit by ELISA.The results showed that the IL-1 content in serum of AK was significantly higher than that of RG(P0.01) and WC(P0.001).The mutation were found at four sites in all three breeds,and they were mutation G→A at 476 site,mutation T→C at 482 site,mutation C→T at 1 215 site and mutation T→C at 1 507 site.Then,the correlation between polymorphic sites and content of IL-1 were analysed,and only 1 215 site had relationship with IL-1 content in serum,which showed that chicken with TT genotype had significantly higher IL-1 content in serum than those with CC genotype.This study indicated that the 1 215 polymorphic site could be a genetic marker for IL-1content in serum.

Wenchang,Anka and Zang chickens were used for SNPs discovery in exon 9 of Nrampl gene using PCR-SSCP and sequencing methods.Two SNPs at C315G and C357T,and four genotypes of GG,GT,TT and AA were detected in three chicken populations.Allele T was the dominant allele in Wenchang and Zang chicken populations,while allele G was the domi-nant allele in Anka chicken population.All the populations were in Hardy-Weinberg equilibrium(P0.05).There were no sig-nificant differences in three chicken populations(P0.05).

[Objective]The SNPs of IL-1β gene and their correlations with some immune traits were tested. [Method]A pair of primers were designed as the sequence of IL-1 β of red jungle fowls,and the SNPs of intron 1 and 2 of IL-1β gene in Rugao Chicken(RG),Wenchang Chieken(WC),and Anka Chicken(AK) were detected using PCR-SSCP.Meanwhile,concentration of IL-1,H/L value,SRBC andbody filer,ND antibody tiler and AI antibody filer were detected at 56 days of age.Then,the correlation between SNP sites and immune traits were analyzed. [Results]Totally 6 SNP sites were detected at T107G,T130A,C143T,A208C,T241G and T274C.Furthermore,the mutation at 107,208,241 and 274 sites were found association with immune traits (P＜0.05 or P＜0.01),and some genotype combinations significantly affected H/L value and ND antibody tiler (P＜0.05).[Conclusion]The mutation at 241 site may be selected as a new candidate mark in disease resistance breeding because of its better performance in three immune traits than that of wild type chickens.Selecting breeder chickens by genotype combinations had more effect than by single.genotype of SNPs.

The present study compared some immune traits to evaluate the disease resistance of different chicken breeds.Random samples of 80 chickens from each population of Rugao Chicken (RG),Wenchang Chicken (WC),and Anka Chicken(AK) were partitioned into A and B groups.Each group had an injection of 0.5 mL and 1 mL of 25% suspension of sheep red blood cell (SRBC) at 49 days of age,respectively.The value of heterophil/lymphocyte(H/L) at 41,56,71and 89 days of age,SRBC antibody titer at 56,71and 89 days old,and the antibody of Newcastle disease (ND) and avian influenza(AI) at 30,41,56,71 and 89 days of age were detected,respectively.The results showed as follows:The difference of H/L value existed between different breeds in the same days of age,but it became smaller along with time.H/L value in RG changed less than that of other breeds,however,WC and AK had obvious change from high to low level.The SRBC antibody titer of three breeds got the highest at 56 days,the highest with WC and the lowest AK(P0.05).Moreover,there were significant differences between them.The sensitivities to different dosages of breeds were different except RG.ND antibody titer of three breeds had the change,variable,though the AK's antibody titer was in the mid of RG and WC before the third immunity,it was extremely significantly higher than the other two breeds of chicken after that period (P0.01).There were insignificant difference of AI antibody titer between RG and WC in all test time except 56 day,meanwhile antibody titer of AK was extremely significantly lower than that of the other two breeds in all test time(P0.001).This study showed that the immune traits were different between chicken breeds,and Chinese chicken breeds had the some advantage of outside breeds.

Hybridization between different populations is the most common means to enrich genetic variation,and also is one of the methods commonly used in the protection of wild endangered species.Studies on genetic differentiation level and mechanism after hybridizations are crucial to protect and evaluate animal genetic resources.In this paper,genetic differentiation mechanism of wild Japanese quail distributed in China,domestic quail and their hybrid offspring,F1 and F2,were studied by using microsatellite DNA markers.The results showed that,F1 hybrids had 4.14 alleles per locus and its values of PIC and were 0.5584 and 0.6265,respectively,which indicated more rich genetic diversity than its parents.However,F2 hybrids had less genetic diversity with 3.57 alleles per locus,and 4 alleles were lost in cross because of genetic drift.F1 and F2 hybrids presented different trends in genetic differentiation from their forefathers.The coefficient of gene differentiation between F2 was the highest level of 0.0712,while the lowest of 0.0275 were shown between F1 hybrids and wild Japanese quail.All above results presented that genetic differentiations of hybrid offspring were affected by genetic drift in dispersive process.So we can infer that hybridization played a key role in population astatic differentiation and loss of genetic diversity.

随机抽取如皋、文昌和安卡鸡各80只,经新城疫和禽流感免疫后分别于30、41、56、71和89日龄检测抗体水平变化。结果表明,如皋鸡公母抗体水平无明显差异,而文昌鸡和安卡鸡在性别之间差异显著。文昌鸡新城疫抗体在所有检测日龄均低于如皋鸡和安卡鸡,而安卡鸡禽流感抗体在所有检测日龄均极显著低于如皋鸡和文昌鸡(P<0.01)。本研究初步表明,就新城疫和禽流感而言,不同鸡种以及性别之间的免疫性能存在明显差异。

Genetic diversity of two wild quail species distributed in China,wild Japanese quail and wild Common quail,were studied with gene frequency of 10 loci in viscera and muscle in 9 quail populations at home and abroad,thereinto,the data of 6 quail populations were cited from other researchers.According to the comparison of corresponding genetic index of quail populations,such as heterozygosity of each locus,mean heterozygosity,genetic differentiation coefficients and multivariate analysis method,the results were shown as follows,in contrast to wild quail populations across Japanese Islands,wild quail in China possessed of rich biological diversity,and mean heterozygosity of wild Japanese quail and wild common quail were 0.388 and 0.298,respectively;Moreover,the two wild quail species had no significant difference in genetic diversity and differentiation with kinds of domestic quail populations.In addition,the two wild quail was also closer to domestic quail populations in phylogenetic relationship than wild quail across Japanese Islands based on fuzzy cluster.

By the technology of polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP),genetic polymorphism of exon 4 and exon 5 of κ-casein gene in southern Chinese Holstein cattle were studied.The results showed as follows: exon 4 and exon 5 of κ-CN gene were polymorphic caused by a AC substitution in exon 4(g.13100 AC) and a TC substitution in exon 5(g.5153 AC).The allele frequencies of A,B of exon 4(AA,AB) and exon5(AA,AB,BB)of κ-CN gene were 0.9379,0.0621 and 0.6512,0.3488,repetively.The genotype distribution of the two exons both fitted Hardy-Weinberg Law(P0.05). Exon 4 locus showed low polymorphism(PIC=0.1097) while Exon 5 locus showed normal polymorphism(PIC=0.3511).The heterozygosity and number of effective alleles were 0.1165,1.1318 and 0.4543,1.8324,respectively in exon 4 and exon 5 of κ-CN gene of southern Chinese Holstein cattle.