| Bovine mastitis is one of the most familiar diseases in the dairy farming industry and it can cause financial losses to countries around the world grievously.The disease causes the quality and yield of milk produced by diseased animals to decrease,and the pathological changes of breast tissue.In addition,once such dairy products enter the market,it will also affect the health of consumers.Subclinical mastitis has high incidence without obvious symptoms and it can cause serious damage.Pathogenic microorganisms is one of the important reasons of causing mastitis in animals.According to statistics,its variety can currently reach more than 150 species,and research shows that new pathogens are constantly appearing.Such as Aerococcus viridans that can cause lobster sepsis and serious human diseases.Aerococcus viridans is easily confused with streptococcus in isolation and identification,and the research data such as pathogenicity and drug resistance are not perfect.This study collected 193 subclinical mastitis samples after CMT detection from some large-scale dairy farms in the Guanzhong area of Shaanxi Province;separating and identificating Aerococcus viridans by Gram stain microscopy,biochemical tests,molecular biological tests,etc;determining the microstructure by scanning electron microscopy and transmission electron microscopy;further sensitivity analysis of 13 antimicrobial agents against isolates by K-B diffusion method;pathogenicity of clinical isolates was tested in vivo in animals,these provide basic information for the isolation and identification of Aerococcus viridans from bovine and the prevention and treatment of mastitis.The results are as follows:1.Ten suspected Aerococcus viridans were isolated from 193 milk samples.Clinical isolates are either two-linked or four-linked Gram-positive cocci under the microscope.The smooth gray-white colonies isolated on the blood agar plates were with α-hemolytic and1~3mm in diameter.Scanning electron microscopy showed that the surface of the uneven cells had strip-like protrusions.Under transmission electron microscopy,Gram-positive bacteria can be observed with thicker cell walls and structures with obvious nucleoid as typical features.The PCR reaction products of clinical isolates were sequenced and compared in GenBank and construct evolutionary tree.The results showed that the 10 suspected strains were all Aerococcus viridans2.Each strain is sensitive to ciprofloxacin,ofloxacin,vancomycin,ceftiofur,ampicillin,enrofloxacin,cefoxitin and penicillin.The resistance to compound sulfamethoxazole is very serious,and the drug resistance rate is as high as 100%.And there are different degrees of resistance to streptomycin,gentamicin,tetracycline and erythromycin.3.The results of the mouse pathogenicity test showed that the mice inoculated with the clinical isolates had normal appetite and mental status,and no mice died during the test;after necropsy,there was no significant change in the appearance of tissues,organs such as heart,liver,spleen,lung and kidney;the culture medium coated with the organ did not grow a suspected colony;the paraffin sections and HE staining were performed on the heart,liver,spleen,lung,kidney,ovary,brain and cerebellum of the sacrificed mice.No obvious histopathological changes were observed under the microscope.And this indicates that the isolated milk-derived Aerococcus viridans is not pathogenic to the mice which have normal immune function.The above results indicate that the morphology,colony morphology and hemolysis of Aerococcus viridans are similar to those of streptococcus.Therefore,preliminary identification cannot be performed correctly by ordinary epigenetic observation.At present,the multi-drug resistance of Aerococcus viridans in some parts of Shaanxi is not as serious as Staphylococcus aureus.However,the resistance to cotrimoxazole suggests that Aerococcus viridans can also have a high rate of resistance.Therefore,it is very necessary to regularly replace antibiotics for disease prevention and control.The pathogenicity of Aerococcus viridans clinical isolates in parts of the experiment is weak.No clinical death occurred in the mice inoculated with the strain,and no obvious pathological changes were observed in the main organs after the necropsy. |
