Isolation,Identification,Epidemiological Analysis,Prevention And Control Technology Study Of Infectious Synovitis In Chinese Native Breed Chickens

In 2010,a disease characterized as infectious synovitis that occurred in native-type chickens in Guangdong and Guangxi province.The disease gradually spread to 16 provinces across the country,leading to considerable losses to the breeding industry of China.In an effort to systematically understanting the pathogen and epidemiology of this disease,and explore effective prevention and control measure,this study was designed to aim at determining and chacterizing the pathogen,understanding its epidemiology,explored an prevention and control system intergrating vaccines and drug treatment.The results were as follow:1.Pathogen identification and epidemiological analysis of infectious synovitis in native breed chickens.A total of 18063 synovial fluid sercreted swabs,1688 serum samples and 215 tissue samples from sick chicken legs were collected from native chicken breeding farms or broiler farms in 16 provinces of China.All samples above were experimentaly detected and analyzed.Our results showed that the Mycoplasma synoviae(M.synoviae)positive rate of swabs was 96.31%,and it was 69.57%-95.16%for the serum collected from diseased chickens.In total,there were 110 M.synoviae strains isolated and inoculated into SPF chickens,and the results indicated that all infected chickens exhibited typical clinical symptoms.Our data suggested that the disease with infectious synovitis prevalent in multi-aged breeder farms was caused by M.synoviae infection.M.synoviae is an important pathogenic agent that can infect chickens or turkeys,resulting in acute or chronic infectious synovitis.We demonstrated that the epidemiology of M.synoviae infection in Chinese native breed chickens were characterized as follow:(1)The M.synoviae infection was universal in multi-aged native breeders farms,and the egg transmission ratio was high.The averaged positive rates of M.synoviae antibody in 8-29 week-old chickens were 7%-97%and interestingly,it reached 100%in 30-week-old chickens or older.The average proportion of M.synoviae infection in 7-to 9-day-old embryos was 16.14%,and the highest was 36.8%.(2)After infected with M.synoviae,slow antibody response,periodically excreting bacteria and strong horizontal transmission was observed in the chicken.We found that M.synoviae maternal antibody and M.synoviae could be simultaneously detected in chickens.10 percent of the chickens at 1 day-old was confirmed infection with M.synoviae,while antibody respone was firstly observed at 50 days-old and positive antibody ratio reached 100%at 100 days-old.10-70 days of age,the chicken with the positive ratio of M.synoviae antigen ranged from 10%to 80%,which tended to be increased periodically.(3)This was an pandemic of M.synoviae in China,and susceptible of native bred chickens.Our epidemiologic analysis indicated that M.synoviae were widely prevalent in 16 provinces,as a total of 9773 chicken flocks with more than 10 million chickens were affected by this disease.Over 10 native breeds were susceptible for M.synoviae.(4)The antibody level of breeders could affect the ability of M.synoviae's horizontal transmission and propagation in the offspring chickens.The analysis of three native breeds chickens found that the silky layers hold the lowest antibody level and the embryo eggs carried the highest infection ratio,while the earliest antibody respone and the highest positive antibody ratio was observed in those offspring chicken.Interestingly,the highest antibody level of layers and the infection rate of embryo eggs were close to that of silky were observe in Qingyuanma,but the slowest antibody respone and the lowest antibody positive ratio was observed in those offspring chicken.2.Phylogenetic and pathogenicity analysis of M.synoviae isolatesThe vlhA gene sequence fragments(nt 76 to 421)of M.synoviae isolates were compared with the reference sequences released in Genbank.The results indicated that the new isolates were classified into a new group which was tentatively termed as subgroup K.Two M.synoviae strains isolated from Muscovy duck and MS-H were identified as the E subtype.Significant difference was detected in the virulence of M.synoviae isolates in subgroup K,but no regularity among the isolates in the sub-branches.Consecutively passaging of M.synoviae in vitro resulted in mutations at two regions,aa 161-240 and 240-400 of the VlhA protein.Intriguingly,a mutation of 130-aa deletion at aa position 70-200 was observed in the strain QZ-ZZX after 70 passages in vitro,leading to lower virulence.These result indicated that the aa residues at position 70-200 in the VlhA protein was likely closely related to the pathogenicity of M.synoviae.3.Develepment and immune effect evaluation of M.synoviae vaccineOur study development and evaluation of M.synoviae inactivated vaccines illustrated that intramuscular injection was the optimal vaccination route for the inactivated vaccine.We confirmed that,HN03 strain could be served as ideal vaccine candidate,and good vaccination effect could be obtained by immunizing chickens at age of 20 and 41 days with a dose of 0.3 mL/chicken/time.We also indicated that the SPF chickens and native chickens immunized with the inactivated vaccine resulted in different degree of protection against M.synoviae infection.The rate of positive antibody titer was greater than 60%and the mean of antibody titer was greater than 5000 in the immunized chickens,and the antibody lasted for 10 weeks.Furthermiore,boosting immunization increased rapidly the level of antibody of the immunized chickens.Chicken carrying M.synoviae maternal antibody could be effectively protected against M.synoviae experimental infection,whereas SPF chickens carried maternal antibody showed lower protection.The result indicated that the M.synoviae inactivated vaccines could offer effective protection against M.synoviae infected,nevertheless it could not completely eliminate the of M.synoviae.Our results indicated that the chickens inoculated with 0.2 � 104 EID50 infectious bronchial virus strain(IBV-GX)and 107 CCU M.synoviae strain(QZ-ZZX)were comfortable for MS-H vaccine evaluation.The native chickens immunized with the MS-H vaccine were 100%protected from experimental infection with M.synoviea.The evaluation of MS-H vaccine used in field on more than 1.24 million native broilers demonstrated significantly decreased morbidity,comparing with the control group.Overal,our results suggested that MS-H could offer effective,but not complete,protection for native chickens from M.synoviea infection.4.Evaluation effect of the M.synoviae medication in native breed chickensThe M.synoviae stains isolated from 2010 to 2015 showed stably susceptibility with tyvancin,tylosin,chlortetracycline,doxycycline,spectinomycin,tiamulin,but significant resistance to oxytetracycline and norfloxacin in vitro.We found that chickens fed with aureomycin(150 ppm)after experimental infection of M.synoviae were not protected,compared with the control group in vivo.Chickens fed with aureomycin(150 ppm)before experimental infection of M.synoviae were protected,as showing delayed syndrome and reduced incidence of the disease,however,the drug has different effect on different strains.Breeding hens periodically fed with chlortetracycline(400 ppm)and tyvancin(50 ppm)showed significant difference in the level of antibody against M.synoviae compared with the control group.It was indicated that chickens received drug at earlier time were protected better.In was indicated,however,medication using drug could not completely eliminate the consequence of M.synoviae infection.In sum,our results indicated that medication with drug could alleviate M.synoviae infection,and thus it might be useful for M.synoviae prevention.5.Evaluation effect of comprehensive prevention and control measure for M.synoviae infection.Based on the above results,we have established a strategic system for the prevention and control of M.synoviae infection,which employing in 16 provinces since the end of 2013.The results showed that clinical cases of M.synoviae infection declined rapidly from a peak of 4500 flocks in 2013 to less than 1000 flocks in 2015.The monthly incidence within group of more than 1%was reduced from the highest number of 300 in 2013 to less than 50 in 2015.Our results indicated that through carrying out the prevention and control system we established,the number of disease caused by M.synoviae decreased by 80%from 2013 to 2015,and the prevalence of M.synoviae infection was effectively control.