| South American white-leg shrimp, Litopenaeus vannamei, is the major cultured shrimpin China, and very important to the ecolomy and food safety. However, diseases, such aswhite spot syndrome and vibriosis, are very harmful to L. vannamei, which limit healthy andsustainable development of culture industry of L. vannamei. Thus, it is very important andnecessary to select new L. vannamei line with disease resistance. In this paper, to exploresome immune indexes for selection and breeding of disease-resistant shrimp, thecharacteristics of disease term and the response styles of main immune factors of L. vannamei,after infection of pathogenic Vibrio harveyi, were investigated, as well as the relationshipbetween immune system and resistance of shrimp from which the key factors of immuneresponse of shrimp were found out. For this purpose, a pathogenic V. harveyi strain of NJ1was isolated and identified with API20E Microorganism Identification Test Kits,16S rRNAsequence analysis and phylogenetic tree analysis, and then a three rounds challenge procedurewas designed to select L. vannamei with different resistance to V. harveyi, and SPSS softwareand AMOS software were combined to analysis the disease term, immune response and keyimmune parameters of L. vannamei to V. harveyi infection. And results were found asfollows:1. Isolation and identification of pathogenicV. harveyi strain. Results of showed thatstrain NJ1had the typical biophysical and biochemical characteristics of Vibrio sp., such asgrowth on TCBS plate with green colony,0/129sensibility and producing of fluorescence.16S rRNA sequence analysis showed that strain NJ1identified to V. harveyi strains of LB4,persian1and S-16with an identity of99%, respectively. Phylogenetic tree analysis alsoshowed that strain NJ1belonged to the same clade with those three V. harveyi strains.Detection of virulence showed that strain NJ1could grow in the infected shrimp and madethem die with fluorescence. 2. Determination of resistance of L. vannamei families. In this paper, the96h medianlethal dose (LD50) of strain NJ1to25L. vannamei families was determined and used toexpress those shrimp families’ resistance to V. harveyi, respectively. And according to thoseLD50s, the25shrimp families were assigned to four shrimp groups: high susceptive, mediansusceptive, low susceptive and resistant shrimp. To investigate the disease term characteristicsof shrimp, these four groups were challenged with strain NJ1at different doses. When high,median, low susceptive shrimp and resistant shrimp were injected with strain NJ1at a dose of1.0×105-1.0×107cfu/shrimp, the time of death beginning was1.5h-5.2h,2.7h-5.5h,5.2h-12.2h and6.2h-23.5h post injection, the time of death ending was9.5h-31.3h,14.8h-27.7h,35.9h-42.5h and47.1h-47.5h, the stage of death peak was2-12h,4-24h,12-24hand24-48h in which the mortality was12.8%-42.1%,9.4%-44.5%,0%-26.1%and0%-16.4%, and the acute stage of death was2-12h,4-6h,4-24h and6-48h in which themortality rate was2.1%-21.4%,0%-22.3%,0%-4.9%and0%-1.9%per hour, respectively.Furthermore, resistant shrimp was more stable to infection of pathogen at different doses thannormal shrimp. With increasing of injection dose of V. harveyi NJ1from1.0×105to1.0×107cfu/shrimp, the time appearing death peak of high and median susceptive shrimpdecreased from6-12h and12-24h to2-4h and4-6h, respectively, but those times of lowsusceptive and resistant shrimp did not decreased which always appeared in12-24h and24-48h, respectively.3. Selection of V. harveyi-resistant L. vannamei. After three rounds selection, theresistance of shrimp was signigicnatly increased, the6h,12h,24h,48h and96h LD50ofNJ1to selected shrimp increased14.9(P <0.01),14.3(P <0.01),8.4(P <0.01),9.0(P <0.01) and9.4times (P <0.01), respectively. Furthermore, three L. vannamei groups withdifferent resistance were also obtained whose6h LD50was1.4±0.1×107,1.1±0.1×107and6.2±0.2×106cfu/shrimp, respectively. The resistance of shrimp before selection was8.8±0.4×105cfu/shrimp.4. The response characteristics of major immune factors of L. vannamei against infectionof V. harveyi. Resistant shrimp survived from the third round selection and normal shrimpwere challenged with strain NJ1at a dose of1.0×105cfu/shrimp, results shown that afterinjection, resistant and normal shrimp cleaned strain NJ1infecting hemolymph during3h and12h post injection, respectively. And the THC and PO activity, all significantly decreasedfrom30min and3h, and to the lowest level at3h and12h, then recovered after24h and48h, respectively. The SOD activity significantly decreased from30min and3h, and to thelowest level at3h and12h, then recovered after48h and72h, respectively. The hemocyaninconcentration significantly decreased from30min and3h, and to the lowest level at3h and 12h, then recovered after12h and48h, respectively. The bactericidal activity significantlydecreased from30min and3h, and to the lowest level at3h and12h, then recovered after12h and48h, respectively.5. Analysis of relationship between immune parameters and resistance of L. vannamei.In this paper, software of SPSS and AMOS were combined to research the relationshipbetween resistance and35immune parameters of shrimp, such as THC, HEM, and PRO, PO,SOA, NOS, GPX, CAT, SOD, BAC, LYS, ACP and AKP in hemolymph, hemocyte andhepatopancreas. Result showed that without infected by pathogen, there was a significantmultiple regression correlation between resistance and11immune parameters of shrimp, suchas THC, HEM, PO in hemolymph, hemocyte and hepatopancreas, PRO in hemocyte, AKP inhemolymph and hepatopancreas, and SOA, SOD and LYS in hepatopancreas, whosedetermination coefficient to resistance of shrimp was91.1%. All the factors in hepatopancreasamong those11immune parameters, such as PO, AKP, SOA, SOD and LYS, had negativerelationship with resistance of shrimp, PO and AKP in hemolymph had positive effect toresistance of shrimp, but negative in hepatopancreas.6. In conclusion, the time of death beginning and ending, the stage of death peak and theacute stage of death delayed, and the mortality in the stage of death peak and the mortalityrate in the acute stage of death decreased with increasing of the resistance of shrimp whensuffered infection of pathogen. The main immune factors of resistant and normal shrimpappeared a same response style, respectively, the level of factors decreased at first, and thenrecovered. The level of the activites of key immune factors in condition without infection ofpathogens could well predict the resistance of shrimp. And the same immune factor couldhave different predicting effcts to resistance of shrimp in different immune organs.In a word, in the present paper, some disease-term characteristics and immune responsecharacteristics of disease-resistant shrimp different from those of normal shrimp were found,and11immune parameters which could predict the resistance of shrimp effectively were alsoselected, indicating that the results of this paper could supply some valuable reference indexesfor selection and breeding of disease-resistant shrimp. |
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