| Mycoplasma synoviae(MS)mainly infects chickens and turkeys under natural conditions.MS mainly causes exudative synovial bursitis and tenosynovitis,also include the reducing of egg production in egg laying hens and the increase of carcass abandonment rate in broilers.MS has high transmission efficiency,which can be transmitted vertically as well as horizontally.In recent years,MS infection has been widely spread in China,resulting in high incidence and huge economic losses.At present,MS-H is the only commercial attenuated vaccine approved for use in China,but there are only a few breeding farms using it because of the highly price.Therefore,it is urgent to develop MS attenuated vaccine with low cost and consistent antigenicity with domestic epidemic strains.1 Pathogenicity of MS strains isolated from ChinaThis study based on 46 K-type,which was dominant genotype in China,MS strains isolated from different provinces in recent three years,some strains from different sources(Ningxia/2017-1,Ningxia/2019-3 and Shanghai/2017-2)were selected for systematic pathogenicity study.In this study,intra trachea inoculation and thoracic air sac inoculation were used respectively.When inoculated with 14-day-old SPF chickens,Ningxia/2017-1 caused 8/12 of air sacculitis,while this ratio increased to 11/12 and 13/13 in Ningxia/2019-3 and Shandong/2017-2 group,respectively.The severity of air sac lesions caused by Ningxia/2017-1 was significantly lower than that of Ningxia/2019-3 and Shandong/2017-2(p<0.05).The severity of air sac lesions caused by Shandong/2017-2 was slightly higher than that of Ningxia/2019-3,but no significant difference.Shandong/2017-2 caused 5/13 of foot pad lesions and 4/13 mortality,while Ningxia/2017-1 and Ningxia/2019-3 did not cause foot pads lesions and chicken death.The titer of Shandong/2017-2 was diluted gradiently and inoculated with 30-day-old SPF chickens to determine the minimum pathogenic dose of Shandong/2017-2.The results showed that Shandong/2017-2 can cause pathological change in all chickens when the titer was 5×105CCU/ml,while right thoracic air sac inoculation with 0.2ml each chicken.In this chapter,the pathological model of MS infection was established,and the scoring methods of air sac lesions and foot pad lesions are sorted out,which can provide reference for the follow-up experiments.When the 14 day old chicks were challenged by intra trachea inoculation,the clinical manifestations of the three challenged groups were significantly lighter than that by thoracic air sac inoculation,but the pathogenicity of the three strains was the same under the two attack methods.The virulence of Ningxia/2017-1 strain was the lowest,the incidence of air sac lesions was 20%after 14 day-post-inoculation,and there was no swelling of tarsal joint or foot pad.The virulence of Ningxia/2019-3 strain was in the middle,the incidence of air sac lesions was 30%after 14 day-post-inoculation,and there was no swelling of tarsal joint or foot pad.Shandong/2017-2 strain has the strongest virulence.The incidence of air sac lesions was 40%after 14 day-post-inoculation,and 5 cases of laryngeal hemorrhage occurred,including 3 cases with trachea hemorrhage;1 case with tarsal joint swelling,with purulent exudation in the swollen joint.The air sac lesion scores of Ningxia/2017-1 group was the lowest,but there was no statistical difference between the groups.The results show that Shandong/2017-2 showed the strongest pathogenicity and can be used as a standard virulent strain in subsequent tests.Ningxia/2017-1 is relatively weak in pathogenicity and its genotype is similar to the main domestic epidemic strains.Ningxia/2017-1 was selected as the parents strain for the development of MS attenuated strains.2 Cultivation of Temperature-Sensitive MS attenuated strainsThe ts+MS attenuated vaccine strain can colonize only in the upper respiratory tract of infected chicken,and cannot cause systemic infection.It can block the infection of MS field strains by competitively inhibition through "occupying effect" in the colonization site.Ningxia/2017-1 was continuously passaged and mutanted by three different concentrations of N-nitrogen-N'-methyl-N-nitrosoguanidine(N-nitro-N'-methyl-N-nitrosuguanidine(NTG))in this research.Sixty-six single colonies were obtained,as the progeny strains of Ningxia/2017-1.9 ts+strains were screened from these progeny strains.5-week-old SPF chickens were challenged with 9 ts+strains,respectively.The results showed that M2,M3,5-18 and 18-22 did not cause clinical symptoms or pathological changes after 4 weeks of inoculation,and the antibody titer at 2,3,and 4 weeks after vaccination were lower than those of the Ningxia/2017-1 challenge group.M10,M20,and 5-9 only caused slightly foot pads lesions and could not cause air sacculitis.5-9 and 5-18 could not cause air sacculitis when inoculated with 1-day-old chicks.M2,M3,M10 and 18-6 could cause slightly air sacculitis,but there was no significant difference between each other.The incidence of air sacculitis caused by M14 is 7/11,and the score of air sac lesions was significantly higher than other groups(p<0.05).M20 and 18-22 caused slightly higher air sac lesions than other groups,except M14 group,but there was no significant difference with any group including M14.7-day-old SPF chickens were inoculated with M2,5-9,and MS-H,and challenged with Shandong/2017-2 at 25 days after inoculation.5-9 can provide 30%protection,which increase to 50%in MS-H group,while only 10%in M2 group.The mean values of the air sac lesion scores of the 5-9 and MS-H groups were lower than the control group,but there was no significant difference.The air sac lesion scores of M2 group was significantly higher than that of MS-H group(p<0.05).Serum samples collected on days 7,14,21 and 25 after inoculation were all negative for MS,in M2,5-9 and MS-H groups,which indicated that the three strains failed to cause humoral immunoreaction within 25 days after inoculation.In general,in this study,Ningxia/2017-1,which was supposed to a lower virulence strain by animal experiments,was selected as the parents strain for development attenuated strains,and then different methods were used to induce mutations.Ts+ strains were screened,and 5-9 was selected as an attenuated strain that able to provide immune protection.In this study,the attenuated MS strain was prepared,which provides a potential vaccine strain for further development of live attenuated MS vaccine.In the follow-up work,the safety and efficacy of the attenuated strains can be systematically evaluated through more experiments.In order to develop a commercial live attenuated MS vaccine for clinical application. |
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