Establishment Of Diagnosis Method Of Mycoplasma Suis And Survey Of Infection

Eperythrozoonosis(EHS)is a severe zoonotic disease caused by Eperythrozoon,an organism that is parasitic on a variety of mammalian red blood cell surfaces without a cell wall structure.Eperythrozoon Suis is common in swines.The clinical manifestations are fever,cyanosis,hemorrhoids,jaundice and ecchymosis on skin.Eperythrozoon can cause the body’s immune system to decline,reproductive disorders of sow,and the serious may cause death.Because of the lack of standardized and effective diagnostic methods for Eperythrozoon,and the fact that the disease is often overlooked in the state of recessive infection,it is very necessary to establish an accurate and effective diagnosis method,to assemble a convenient diagnostic kit,and to find out the characteristics of clinical infection for the preventing and control Eperythrozoonosis in swines.This study will be carried out from the following three aspects.A pair of specific primers is designed to carry out the polymerase chain reaction(PCR)based on the reported MSG1 gene sequence of Eperythrozoon Suis in its conserved domain.The most suitable annealing temperature for PCR is 55 ℃,sequence the gel extraction products.The sequencing result is consistent with the prediction result and it is 1011 bp,which is in accordance with the homology of MSG1 gene sequence of Eperythrozoon published in Gen Bank.The specificity of the PCR diagnosis method established in this experiment is strong,and there is no cross-reaction with the blood of healthy swines,Toxoplasma gondii,Staphylococcus aureus,Escherichia coli,porcine pseudorabies,Polyporus umbellatus,porcine circovirus II.The minimum DNA concentration for positive pig blood samples is 20.0 fg/ul.The PCR diagnosis method has the characteristics of simple operation,high accuracy and high sensitivity,which provides a powerful tool for the diagnosis of Eperythrozoon Suis,the assembly of the kit and the investigation of the infection situation.According to the PCR diagnosis method established in this study,prepare PCR primers and PCR reaction solutions and assemble the PCR diagnostic kit for Eperythrozoon Suis byconstructing positive control and negative control.Carry out a test on the specificity,sensitivity,repeatability,stability,and shelf life of the kit.And using the assembled test kit,test the clinical positive sample to determine its accuracy.There is no cross reaction with the blood samples of healthy swine,Toxoplasma gondii,porcine parvovirus,porcine pseudorabies,porcine epidemic diarrhea,porcine Japanese encephalitis and the genome of porcine circovirus type II.The minimum detection concentration of the kit is 32.5 fg/ul.The repeatability is good.After 40 repeated freeze-thaw kits,the positive control amplification results still show bands.The shelf life of the kit is 6 months at-20°C.Using the kit to test the Eperythrozoon infection rate of 337 swine blood samples collected from parts of Hunan Province and detect the swine fever,blue ear,pseudorabies virus and Eperythrozoon Suis in 100 blood samples of growing and fattening swines.The results of investigation on the infection of Eperythrozoon in pigs in Xiangtan,Yiyang,Hengyang,Yongzhou and Chenzhou in Hunan Province show thatThe highest infection rate of Eperythrozoon was 69.2 in Chenzhou,61.5 in Yongzhou,58.4 in Hengyang,58.4 in Xiangtan and 55.3 in Yiyang.The infection rates of boars,sows,fattening pigs and piglets were 82.1,74.1,43.8 and 26.6 respectively.From this investigation,it can be seen that swines of different groups have Eperythrozoon infection,but the infection rate of Eperythrozoonosis is different.Among them,the infection rate of boar is 83.1%,that of sow is 74.1%,that of growing and fattening swines is 43.8% and that of piglet is 26.6%.Investigation of mixed infections: The individual infection rate is only 4.6%.The mixed infection rate with swine fever is 83.7%,that with blue ear is 88.3%,that with porcine pseudorabies is 51.1%,and the mixed infection rate of four diseases is 41.8%.Conclusion: this study established an accurate diagnostic method and assembled the diagnostic kit of Eperythrozoon porcine.The investigation of clinical infection found that the infection of Eperythrozoonosis was common,mainly in the form of mixed infection.The infection rate of boar was higher than that of sows and fattening pigs,and the infection rate of fattening pigs was higher than that of piglets.