| Yak is an ancient and rare breed of cattle originating from China,which can adapt to the plateau environment.After long-term natural selection and artificial domestication,the yak has the advantages of cold tolerance,resistance to hypoxia,good climbing and omnivorous diet,as well as the ability to use the alpine pasture resources that other cattle species cannot digest,in addition,it integrates the performance of many excellent products of livestock,corresponding to the praise as "boat on the plateau" and "all-purpose livestock".Since 2012,the majority of the Yaks in Tibet have been often found to have infectious disease with respiratory symptoms,seriously threatening the development of the local cattle industry.Mycoplasmal Bovis(M.Bovis)is one of the most important pathogens causing Bovine Respiratory Disease Complex(BRDC),which can cause pneumonia,arthritis,mastitis,otitis media,keratoconjunctivitis,abortion and even death,so as to a serious threat to the cattle industry.In order to understand the epidemic situation and control of the disease,the isolation and identification of mycoplasma yaks in Tibet were carried out,and the related biological characteristics of the isolates were studied.The main studies were as follows:1.Isolation and identification of Mycoplasma Bovis from Yaks in TibetIn this study,the nasal mucus of Yak with respiratory symptoms was collected from Yaks in Tibet with positive antibody to Mycoplasma Bovis,and the isolation and identification of Mycoplasma Bovis were carried out.The 10 isolates,named from T 1 to T 10,was characterized by colony morphology observation,PCR amplification,sequence analysis of specific genes,growth curve determination,antimicrobial susceptibility test and biochemical test.The results showed that the colony morphology was typically fried egg-like,and M.bovis had high homology with PG45 and other M.bovis domestic isolates.The growth curve showed that the lag phase was 24 hours,then entered logarithmic phase,and stationary phase was after 42 hours,and the decline phase was after 84 hours.The isolated strains showed the different resistance for drugs.The isolated strains did not ferment glucose and lactose,did not hydrolyze gelatin and arginine,and did not decompose urea and mannitol,but the cholesterol test was positive.2.The resistance mechanism of Mycoplasma Bovis isolated strains from Yaks in Tibet to Fluoroquinolones antibioticsThree predominant strains were carried on the resistance mechanism of M.bovis isolated from Yaks in Tibet to Fluoroquinolones antibiotics.Three Fluoroquinolone antibiotics Enrofloxacin,Norfloxacin and Ciprofloxacin were selected as the experimental drugs,and the microdilution method was used to conduct drug sensitivity test,in vitro drug-resistant strain induction test,stability drug resistance test and cross drug resistance test on 10 strains of M.bovis from Yaks in Tibet,and taking fluoroquinolones sensitive strains,drug-resistant strains and highly resistant strains induced in vitro as the research objects,the target gene(gyr A,gyr B,par C,par e)mutation analysis of the Quinolone Resistance Determining Regions(QRDR)and the preliminary confirmation of the drug active efflux system were performed.The results showed that M.bovis from Yaks in Tibet were resistant to Ciprofloxacin,Norfloxacin and Enrofloxacin.Through in vitro induction,9 strains of M.bovis were found to be resistant to one fluoroquinolone antibiotic and cross-resistant to the other two fluoroquinolones.The sensitive strain had insignificant amino acid(Asp84)mutation in target gene par C,and the resistant strain had single amino acid(Ser 83 Phe/Tyr)or(Ser 80 Ile/Arg and Ser81 Phe)mutation in target gene gyr A or par C,and highly drug-resistant strains induced in vitro mainly had amino acid mutations(Ser 83 Phe and Ser 80 Ile)in the target genes gyr A and par C,while no mutations in the relevant amino acids were detected in the target genes gyr B and par E.The analysis of active efflux system showed that M.bovis from Yaks in Tibet did not express active efflux system based on Fluoroquinolone antibiotics as a substrate.The above mentioned results indicated that M.bovis from Yaks in Tibet were resistant to the three Fluoroquinolone antibiotics in various degrees.When M.bovis were kept continuously under the pressure of the drug,drug-resistant strains were prone to develop,and the amino acid at the gyr A or par C site of the drug target gene was prone to mutations.Moreover,if the amino acids of the two gene sites are mutated simultaneously,highly resistant strains will be produced.3.Clinical pathogenicity test of isolated Mycoplasma Bovis strains from Yaks in TibetFurthermore,another study(T 6,T 8 and T 10)was selected to carry out chicken embryo infection test and rabbit clinical pathogenicity test to study the clinical pathogenicity of M.bovis from Yaks in Tibet.The results showed that the embryos were delayed in development and free of blood vessels in different extents,and even died after infection.The results of necropsy identified that the embryos in the control group developed well without any pathological changes,and the dead embryos in the infected group showed different extents of hyperemia and hemorrhage on the surface of the dead embryos,and the undead embryos were smaller than the control group.However,on the day of infection,the body temperature rose slightly,and the body temperature reached its peak on the 5th day,which was about 2 to 2.5 °C above normal body temperature,then the body temperature slowly dropped,finally on the 10 th day,the body temperature basically returned to normal.With the increase of body temperature,animals accompanied by depression and slow responses,decreased water and food intake,and had slower weight gain,especially in the later stages,had white mucus secretion and slight respiratory sounds in the nasal cavity,but once the body temperature returned to normal,the above symptoms disappeared.The pathological autopsy showed that there were varying degrees of effusion in the thoracic cavity,lung enlargement and hemorrhage,lung tissue hyperplasia,hard touch texture,white nodules on the surface,pulmonary mucosa shedding,and no changes in other organs.Through histopathological observation,it was seen that there were varying degrees of hemorrhage in the alveolar cavity,the collapsed alveolar cavity,and there were a large number of exfoliated alveolar epithelial cells and lymphocyte cells with a large number of inflammatory cells infiltrating,increased macrophages and edema fluid,serum oozing,alveolar wall thickening.At the same time,the lungs were collected for isolation and identification of M.bovis.Not only was PCR identification as positive,but also the M.bovis was isolated,which indicated that the above symptoms and pathological changes were caused by M.bovis,and T 6 was the most pathogenic.4.Differential analysis of micro RNA in rabbits infected with isolated Mycoplasma Bovis from Yaks in TibetIn this study,the difference of Mirna expression in lung tissue of rabbits infected with mycoplasma Yaks in Tibet were studied.The results showed that the experimental group and the Control Group had 687 Mirna,and the experimental group had 57 different Mirna,there were 163 up-and 188 down-regulated Mirnas in the control group,of which49 up-and 69 down-regulated Mirnas were significantly different,34 up-and 42down-regulated Mirnas showed significant differences,while 80 up-and 77down-regulated mirnas showed no significant differences.In GO enrichment and Kegg enrichment,the most significant concentrations were protein binding pathway,Pathways in cancer and TNF signaling pathway We identified the highest frequency of Mirna in HSA(Homo Sapiens,human)and the second highest in Bta(Bos Taurus,bovine).This study provides a theoretical basis for studying the pathogenesis of mycoplasma infection at Mirna level.5.Differential analysis of lnc RNA and circ RNA in rabbits infected with Mycoplasmas Bovis and the construction of ce RNA networkIn this study,the differences in the expression of m RNA,lnc RNA and circ RNA in the lung tissues of rabbits infected with mycoplasma Yaks in Tibet were studied.A total of 683 m RNA expression differences,of which 366 m RNA expression was up-regulated,317 m RNA expression was down-regulated;a total of 844 lnc RNA expression differences,of which 416 lnc RNA expression was up-regulated and 428 lnc RNA expression was down-regulated;a total of 317 circ RNA expression differences,of which231 circ RNA expression were up-regulated,and the expression of 86 circ RNA was down-regulated.Finally,based on the construction of target miRNA-m RNA,we finished miRNA-lnc RNA,miRNA-circ RNA,ce RNA(lnc RNA,circ RNA)-miRNA-m RNA network,it has laid a solid foundation for the further development of the functions and mechanisms of lnc RNA and circ RNA.To sum up,the respiratory infectious diseases frequently occurred in Yaks of Tibet herds and were mainly caused by Mycoplasma Bovis,which was mainly transmitted through the respiratory tract,digestive tract and reproductive tract.The sensitive antibiotics should be used for therapy in clinic treatment,and there is no effective vaccine for the prevention of the disease;only quarantine can be used to continuously eliminate positive cattle to reduce the number of pathogens in the external environment,so as to achieve the purpose of prevention and control.The results of this study provide a scientific basis for the treatment,prevention and control of BRDC caused by M.Bovis in Tibet. |
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