Preparation And Immunoprotection Of Avian Leukosis Virus Subgroup B Inactivated Vaccine

Avian leukosis is one of the main diseases of causing poultry tumor, whose pathogen isavian leukosis virus (ALV) of α retrovirus genus. According to the viral envelopeglycoprotein antigenic structure, host range and mutual interference between different strainscultured in cells, ALV can be divided into10subgroups A to J, and subgroups A, B, C, D, Eand J exist in chickens. Subgroups A, B and J are the main exogenous ALV that cause chickentumors, but it is rare to hear tumors caused by subgroups C, D. Subgroup E is endogenousleukosis virus, which has low or no pathogenicity for chickens. In the last century before the80’s, avain leukosis virus subgroups A, B are the two viruses that caused chicken lymphoidleukemia and various tumors, but in the middle of the80’s, international large breedercompanies have basically eliminated the infection of ALV-A and ALV-B. In recent decades,China has never carried out any nationwide purification measures against ALV, exogenousALV infection most likely exist in chicken flocks in China, especially the local strains. Inrecent years, chicken/leukemia cases have encountered with many times, chicken leukosis hasbeen one of the important diseases that harm the poultry industy in our country. ZhuMei-zhen.ect isolated a strain of ALV-A from Shandong local strains, Zhao Dong-min.ectisolated a subgroup B avian leukosis virus from chickens of Chinese native breed luhua. Itshows that chicken in our country have presence of A, B subgroup avian leukosis virusinfection.To speed up the purification of avian leukosis virus subgroup B, the avian leukosis virusavian leukosis virus subgroup B SDAU09C2strain inactivated vaccine was successfullyprepared. We attempted from the approach of vaccination to reduce core breeder flock poisonrate, improve the level of specific antibody, thus protecting core breeder flock from avianleukosis virus infection, at the same time can provide maternal antibodies for the nextgeneration through eggs, protect chickens from early infection of ALV-B, accelerateeradication progress of avian leukosis virus subgroup B.To prepare the high quality inactivated vaccine, ensure that antigen content was must be, sowe firstly explore the optimal cells cultured conditions of subgroup B avian leukosis virusstrain SDAU09C2. ALV-B SDAU09C2strains inoculated the logarithmic growth phaseDF1cells in tissue culture plate12well, after2h incubation, changing the maintenance fluidof different serum concentrations (0.5%,1%,2%,3%), collected150ul supernatant incentrifugal tube daily, which used for p27detection, so as to determine the optimalconcentration of serum. The logarithmic growth phase cells of DF1in tissue culture plate24 well were vaccinated SDAU09C2, after2h incubation, changing the optimal serumconcentration maintenance fluid of different pH value (6.6~7.7), collected150ul supernatantin centrifugal tube daily, which used for p27detection, thereby determining the optimum pH.The logarithmic growth phase cells DF1were inoculated SDAU09C2, after2h incubation,changing DMEM culture medium (PH=6.8, containing0.5%of newborn bovine serum) for10days,150ul supernatant were collected in two1.5ml centrifugal tube daily,(one tube isused for p27antigen detection, anther tube used for TCID50determination). By continuousinspection, we found the S/P value is the highest when the ALV-B SDAU09C2strain culturedin cell supernatant whose serum concentration is0.5%, PH value is6.8, and the higher of S/Pvalues, the bigger of TCID50. A significant Pearson correlation was found between S/P valuesand TCID50.SDAU09C2was inoculated in20bottles of new growth monolayer CEF and pass3generations in the culture bottles, maintain with optimal conditions to for5days, collectALV-CEF. ALV-CEF were diluted to5ml with PBS, the0.2%volume ratio of formaldehyde,in the37℃incubator effected24hours, inactivated cells and MONTANIDE ISA775VG(adjuvant) volume ratio of2.6:7.4to full emulsify, prepared ALV-B cells inactivated vaccine.Multi-point injection of1ml (contain10millions ALV-CEF) inactivated vaccine to the9grandfather chickens, unvaccinated9adult progenitor chicken as control.4immunizedchickens and4control group chickens at the highest antibody levels of laying immunizedhens muscle multi-point injection of SDAU09C21ml (TCID50of104.6/0.1ml), collectanticoagulation and non-anticoagulant, cotton swabs weekly. The other5chickens wereimmunized four times and collected non-anticoagulant sanguis0.5ml weekly. The resultsshowed that the highest antibody level appear in the third week of the third immunization, S/Pvalues of ELISA detection in1.69~1.89, as well as antibody positive level could maintain fourweeks. Grandfather chickens after ALV-B injection neither immuned group nor control groupthe cotton swab p27was negative, and there was no viremia.Collected the eggs (include control group without antibodies) at the highest antibody levelsof laying hens for incubation. Incubated33chicks, in which there are17chicks hatched fromthe eggs of chickens having antibodies and16chicks hatched from chickens not havingantibodies. Chicks were divided into four group, the first group,13chicks hatched fromchickens having antibodies, abdominal injection of ALV-B at the age of1day, the secondgroup,4chicks with maternal antibodies, abdominal injection of PBS (the purpose is to testmaternal antibodies dynamics), the third group had13chicks hatched from the eggs ofchickens not having antibodies, abdominal injection of ALV-B at the age of1day, the fourth group had3chicks hatched from chickens not having antibodies, abdominal injection of PBS,as control group. Collected sanguis and cotton swabs weekly for tested. We can see from theresult that variation of maternal antibody, maternal antibodies could last about1week.1-day-old chicks injected ALV-B presented severe immune tolerance, only one chick withmaternal antibodies produced low-level antibody response in14-weeks-old.Group withoutmaternal antibodies never appear antibody positive because of immune tolerance, controlgroup don’t appear antibody any more. Maternal antibody group12chicks after ALV-Binjection at1-day-old, only4chicks in9th～10th week appeared cloaca swab P27transientpositive, last2to3weeks at most, and the S/P values were not high (compared withexcluding maternal antibody group), excluding maternal antibody group9chicks afterALV-B injection at1-day-old, appeared cotton swab p27positive in2th～6th weeks, lastabout8to9weeks, S/P values significantly higher than the positive cut off value. Chicks hadno maternal antibodies, began appearing viremia positive after ALV-B injection4weeks, andthen the proportion gradually increased, all9chickens positive in eighth weeks. Includingmaternal antibody group after ALV-B injection, only4/12chicks appeared detectable viremia,the earliest viremia being detected in7th weeks, and viremia is a cross, lasts4weeks at most,while the other8chicks was not appeared detectable viremia in the continuous observation of14weeks. From the results above we can draw the following conclusions: The vaccine canmake whole laying grandfather chicken produce higher levels of specific antibody, andprotected chicks pass the yolk antibody with maternal antibodies, can resist ALV-B earlyinfection, can effectively control the development of avian leukosis virus subgroup B,application in the core can accelerate the breeder flocks of subgroup B avian leukosis viruspurification process.