Detection Of Virulence Genes And Pathogenicity Of Streptococcus Pneumoniae And Staphylococcus Aureus From Mink

Mink is one of the important economic animals and has developed into an important industry.The largest province of mink farming is Shandong Province.But with the development of intensive and large-scale mariculture,epidemic disease seriously hinders the healthy development of mink breeding industry and causes great economic losses.Hemorrhagic pneumonia is one of the most important infectious diseases in mink.In recent years,our laboratory has carried out epidemiological investigation on mink hemorrhagic pneumonia.The results show that besides Pseudomonas aeruginosa,Klebsiella spp.,Pasteurella pasteurella and other pathogenic bacteria,there should be other pathogeny.To this end,this study is aimed at the prevalence and pathogenicity of Streptococcus pneumoniae(S.pneumoniae)and Staphylococcus aureus(S.aureus)in mink.To clarify the role of both in mink hemorrhagic pneumonia.During 2017 to 2018,120 samples of mink pneumonia were taken from mink farms in Zhucheng,Shandong province.Two strains of S.pneumoniae(Sp-1,Sp-2)and three strains of S.aureus(Sa-6,Sa-16,Sa-24)were isolated.Nine virulence genes of S.pneumoniae and 17 virulence genes of S.aureus were detected and analyzed by molecular cloning and sequencing.The results showed that ply,nan A,lyt A,psp A,psa A and rrg A genes were present in both isolates,and pav A,hys A virulence gene was also found in Sp-1 isolates,while iga gene was negative in both isolates.Among the 3 isolates of S.aureus,Sa-16 and Sa-24 isolates were methicillin-resistant Staphylococcus aureus(MRSA),Sa-6 isolates were methicillin-sensitive Staphylococcus aureus(MSSA),in which MSSA carried pvl gene,and three strains of Staphylococcus aureus all carried coa,nuc and clf A genes.Among them,Sa-6 and Sa-24 strains carried hla,hlb,hld and hlg hemolysin genes,and Fnbp B virulence genes at the same time.Enterotoxin is one of the most important pathogenic factors in food poisoning cases.In this study,only Sa-6 isolates were positive for enterotoxin seb and seg gene,while Epidermal exfoliative toxin gene eta,etb and tsst-1 were not detected.The drug resistance of 5 strains of bacteria was detected by disk diffusion method,and21 kinds of antibiotics were used in 9 categories.In this study,Sp-1 and Sp-2 isolates were completely resistant to roxithromycin and were sensitive to piperacillin,cefotaxime,ceftazidime,cefoxitin,gentamicin and amikacin.Two strains of MRSA were more sensitive to piperacillin,cefotaxime,ceftazidime,cefoxitin,gentamicin and amikacin.MRSA strains were more resistant to commonly used clinical antibiotics and were completely resistant to penicillins(ampicillin,penicillin)and sulfonamines(compound Xinomin).Only some quinolones(ciprofloxacin,norfloxacin),cephalosporins(ceftazidime,cefoxitin)showed a good bacteriostatic effect on Sa-16 and Sa-24 isolates.Sa-6 isolates were completely resistant to compound Xinomin,quinolones and nitrofurans.Glycopeptide and most cephalosporins showed good bacteriostatic effect on MSSA isolates.According to the results of virulence gene detection,the pathogenicity of S.pneumoniae strain Sp-1 and S.aureus isolate Sa-6 were studied,respectively.In the infection model of mice,the mice were challenged by intraperitoneal inoculation,combined with the observation of clinical symptoms and the statistics of onset time and mortality of mice.The results showed that,the pathogenicity of Sa-6 isolates to mice was stronger than that of Sp-1 isolates.And then,return to experimental animals,Sp-1 isolate and Sa-6 isolate were inoculated intraperitoneally,intramuscularly and intranasally,respectively,to infect mink in three different ways.Combined with histopathological changes,and the recovery rate of target bacteria in different organs of infected mink,the pathogenicity of the isolates to mink was determined.At the same time,the difference of pathogenicity caused by S.pneumoniae and S.aureus was compared.The results showed that the S.pneumoniae isolates were more likely to infect minks through nasal cavity and then descend from nasopharynx to respiratory tract,causing lung infection.Staphylococcus aureus isolates are easy to infect mink through damaged skin,causing pyogenic infection of mink,cellulitis and so on.This study shows that the S.pneumoniae and S.aureus exist in mink population and can cause mink pneumonia.These results enrich the data of mink hemorrhagic pneumonia,and have important significance in public health and production practice.