Construction of Larval cDNA Expression Library and Immunological Identification of Positive Clones in Tick Haemaphysalis qinghaiensis

A primary cDNA expression library with a titer of 5.0 × 105 PFU mL-1 was constructed from mRNA extracted from larval Haemaphysalis qinghaiensis ticks in order to identify certain genes,which would then be used as candidate molecules for development of effective vaccines to control this parasite.Totally 11 positive clones,which designated as HqL01-11,were obtained by immunoscreening of the library using a polyclonal antibody generated in rabbit with larval tick protein extract.Results of sequence analysis from BLASTN searching revealed that 6 of them had no significant homology with the adult H.qinghaiensis ticks’ known genes,4 of them had no significant homology with all genes deposited in GenBank database.HqL07,HqL08,HqL09,and HqL11 were deposited to GenBank database,and accession numbers were EF605263,EF605264,EF605265,and EF605266,respectively.Subsequently,HqL07 and HqL09 were expressed in vitro and the molecular weights of the corresponding expressed products were 60 and 70 kDa,respectively.Western blot analyses showed that HqL07 and HqL09 had immunogenicity.This study laid the foundation for future production of genetically engineered vaccines for the immunological control of H.qinghaiensis.

Optimization of Suspension Culture Conditions for Hairy Roots of Aconitum coreanum(Levl.) Raipaics

[ Objective] This study aimed to establish an optimized suspension culture for hairy roots of Aconitum coreanum （Levi.） Raipaics. [ Method ] Hairy root of Aconitum coreanum （ Levl. ） Raipaics A0489 was selected as experimental material, to screen the optimal medium for suspension culture. [ Result] Aconitum coreanum （Levl.） Raipaics hairy roots showed different growth conditions in various liquid medium; salt concentration, nitrogen content, sugar content, rotation speed and room temperature had great influences on the growth of Aconitum coreanum （Levi.） Raipaics hairy roots. [ Conclusion] The optimal suspension culture system for Aconitum coreanum （ Levi. ） Raipaics hairy roots was established ： B5 was used as basic medium, culture temperature was 20℃ under dark conditions, rotation speed was 140 r/m in the first 15 d and 110 r/m in the latter 15 d.

Safety of a Live Attenuated Infectious Bovine Rhinotracheitis Vaccine IBRV LNM Strain

The paper was to evaluate the vaccine safety,and to prevent public health risk due to virus spread,the approach vaccination of was adopted in this research; and neck intramuscular injection of IBRV LNM attenuated vaccine strain was carried out. Blind passage for three generations in animal has tested the reversion risk to virulence. A total of 14 healthy and weaning cows at 6- 8 month old were divided into three groups. The 1st reversion of virulence trials used 105. 0TCID50/mL neck intramuscular injection of IBRV LNM attenuated vaccine strain. Then,the nose swab samples were collected for continuous 14 days. After passed through 0. 45 μm filter membrane,nasal swabs mixture was prepared as the virulence test inoculum for next generation. The body temperature was detected and clinical observation was carried out for continuous 14 days after inoculation. The inoculation dose was 1ml / cattle. Blood was collected on the 0 and 14 thdays of animal vaccination. After serum isolation,it was used for the antibody detection of serum. Research results showed that no virus was isolated from the nasal swabs from the F2 generation; vaccinated animals did not show any clinical signs of IBR; serological testing of IBRV antibody was negative,which indicated that the strain-inoculated animals did had reversion of virulence in all three generations.

Heterogeneity of <i>Pestivirus</i>Species in Asia

Pestivirus are responsible for cosmopolitan diseases affecting cattle, pigs and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. Understanding genomic characteristic and virus taxonomy is fundamental in order to sustain control and prophylactic programs. Given the recent various studies reporting a relatively high number of new strains, in particular from Asian countries, in the present study, six hundred-fifty-one genomic sequences have been considered applying the palindromic nucleotide substitutions method for genotyping. Based on the secondary structure analysis of the 5’ untranslated region of RNA, sequence characteristics among Asian genomic clusters within the different Pestivirus species suggested geographic segregation and occurrence of micro-evolutive steps in the genus evolutionary history. This aspect was particularly evident in atypical sequences originated from China or Turkey, indicating risk of diffusion by animals and products trade or contamination of biological products as bovine calf serum, with potential diagnostic and control difficulties.

Study on the Culture Conditions for Hairy Roots of Aconitum coreanum( Levl.) Raipaics

[Objective]This study aimed to investigate the culture conditions for hairy roots of Aconitum coreanum（Levl.）Raipaics and optimize the amplification culture conditions in bioreactors,thus providing basis for large-scale production of A.coreanum hairy roots.[Method]The effects of different p H,culture modes and culture vessels on the growth of hairy roots were compared to obtain the optimal culture conditions for A.coreanum hairy roots.Moreover,the effects of medium volume and inoculation mode on bioreactor culture of A.coreanum hairy roots were investigated to analyze the feasibility of amplification culture of hairy roots in bioreactors.[Result]After autoclaving,medium p H was reduced slightly and the clarity of medium varied.To be specific,A.coreanum hairy roots cultured in medium at p H 5.80 exhibited the best growth.A.coreanum hairy roots cultured in liquid medium displayed more rapid growth than hairy roots cultured in solid medium.Medium p H and electrical conductivity（EC）varied with the extension of culture time.The yield and propagation coefficient of A.coreanum hairy roots were positively correlated with the size of culture vessels.The growth of hairy roots cultured in bioreactors were influenced by medium volumes and inoculation modes.[Conclusion]The growth of A.coreanum hairy roots were significantly influenced by medium p H,culture modes and culture vessels.A.coreanum hairy roots cultured in bioreactors exhibited high yield and large propagation coefficient.According to the results,the relationship between ventilation volume and hairy root density is crucial to the amplification culture of A.coreanum hairy roots.

Effects of dietary protein level on nutrients digestibility and reproductive performance of female mink(Neovison vison)during gestation

The objective of this study was to determine whether nutrient digestibility and reproductive performance of pregnant mink(Neovison vison) were affected by different dietary protein levels. One hundred and twenty female mink were randomly assigned to four groups, receiving diets of fresh material with different protein levels. The dietary protein levels,expressed as percentage of dry matter(DM),were 32,36, 40 and 44% respectively. These values corresponded to average 320, 360, 400 and 440 g protein/kg DM, respectively. Results were as follows. All of crude protein digestibility, nitrogen(N) intake, N retention increased along with dietary protein level increasing. Low protein level(32%) significantly reduced the above indicators(P < 0.05). DM digestibility and ether extract digestibility were not affected by dietary protein level. Results of mated females, barren females, kids per litter, live born kids per mated female, birth survival rate, and birth weight showed that mink achieved optimal reproductive performance when dietary protein level was 36%. In conclusion, dietary protein was anticipated to significantly influence some nutrients' utilization. Adopting the appropriate dietary protein level allow better reproduction performance. The most preferable reproductive performance was achieved when diet contained 275.5 g digestible protein per kg DM for female mink in gestation.

Effects of dry dietary protein on digestibility, nitrogen-balance and growth performance of young male mink

The experiment was to study the nutrient digestibility and metabolism performance of male minks,which were fed different protein level diets during growth period. Effects of protein quantity on growth and development of minks and feed conversion ratio(FCR) were also investigated. Sixty healthy male minks of 45 d were randomly allocated into six groups with ten replicates, which was one sable for each replicate. The minks in six groups were fed diets in which protein levels were 28, 30,32, 34, 36 and 38%,respectively. The six groups were denoted as P28, P30, P32, P34, P36 and P38. After 2 wk, all minks were weighed, average daily gains(ADG) were calculated, and the digestibility values of nutrients were determined. The results indicated that digestibility of calcium, nitrogen of fence, nitrogen deposition, net protein utilization(NPU), and biological value of protein(BV) were similar(P > 0.05), however, nitrogen intake greatly varied among groups(P < 0.01). Compared with group P28, groups of P34, P36 and P38 showed significant difference(P < 0.01) in ADG and FCR. In conclusion, it was recommended that adding34% protein to mink diet would optimize production parameters including ADG, digestibility of nutrition,and FCR, and negative result was observed when diet protein was lower than 28%.

An Attenuated Highly Pathogenic Chinese PRRS Viral Vaccine Confers Cross Protection to Pigs against Challenge with the Emerging PRRSV NADC30-Like Strain

A novel PRRSV strain was isolated in China that was genetically similar to the NADC30 strain which is reported to have spread throughout China. The objective of the present study was to evaluate the cross-protective efficacy of the live vaccine TJM-F92 in young pigs against challenge with a NADC30-like strain, HN201605. Twenty-five PRRSV-and antibody-free pigs were randomly divided into the following five groups: Vac/ChA, Unvac/ChA, Vac/ChB, Unvac/ChB and the mock.The pigs in groups Vac/ChA and Vac/ChB were inoculated intramuscularly with 1 mL TJM-F92(10^(5.0) TCID_(50)/mL). At28 days post vaccination(0 days post challenge), groups Vac/ChA and Unvac/ChA were inoculated intranasally with 10^(4.5) TCID_(50)/mL PRRSV strain TJ F3(2 mL/pig), while groups Vac/ChB and Unvac/Ch B were inoculated, using the same route, with the same dose of the NADC30-like strain HN201605 F3. Protective effects of the PRRSV strain were observed in all pigs in the Vac/ChA and Vac/ChB groups. Neither high fever nor signs of clinical disease were observed through the experiment in these groups, whereas pigs in Unvac/ChA group exhibited serious clinical symptoms, pathological lesions,and weight loss. In Unvac/ChB group, pigs developed milder clinical symptoms, which demonstrated that the NADC30-like strain HN201605 had moderate pathogenicity. The results suggest that the MLV vaccine strain TJM-F92 is an effective and safe vaccine candidate for use in China.