Clinical Case Analysis Of Malassezia Otitis Externa In Cat,and Eukaryotic Expression Of Malassezia Globosa Lipase SMG1

Clinically,Malassezia otitis externa is commonly diagnosed in pet cats with a high incidence and recurrence rate,distressing a large number of cat owners.Malassezia is lipid-dependent,which secretes lipases that degrade lipids on the host skin surface to provide nutrients for its growth,and Malassezia lipases are pathogenic.In this study,133 cats of otitis externa were collected from the Teaching Animal Hospital of Yangtze University in Jing Zhou and the United Animal Hospital of Wuhan for diagnosis and treatment analysis,and these cases information were statistically analyzed.A Malassezia globosa strain was isolated from one case of Malassezia otitis externa and its lipase activity was investigated under different culture conditions.The SMG1 gene from Malassezia globosa was modified and made to express in eukaryotes by using the Pichia pastoris expression system.The following main research results are achieved:(1)95% of the cases with Malassezia otitis externa were cured by medicine application after removing the large amount of fat accumulated inside the ear canal.The recurrence rate was 16%.According to the etiology analysis results,the otitis externa was caused by ear mite infection in 36.1% of the cases and Malassezia infection in 32.3% of the cases.In cats with Malassezia otitis externa,there were 65.1% males and 34.9% females.41.8% of the cats were aged under 6 months,and the prevalence decreased with the increase of age.In all the infected cats,British Shorthair cats(13.3%)showed the highest incidence,while Felis catus(4.7%)had the lowest incidence.(2)The ear canal secretions collected from clinical cases of Malassezia otitis externa were stained,isolated and purified.Malassezia globosa was identified by PCR,and then cultured under different conditions.Its lipase activity was measured to be 0.01624 U/mL at 34℃,0.02450 U/mL at 35℃ and 0.02425 U/mL at 39℃.The optimum culture temperature of Malassezia was close to the skin temperature of the cat ear canal.The lipase activity of Malassezia in media containing 2%,4%,6% and 8%olive oil was 0.01624 U/mL,0.02062 U/mL,0.02363 U/mL and 0.02625 U/mL,respectively.The oil concentration was positively correlated with Malassezia lipase activity.(3)By optimizing the codon of Malassezia lipase SMG1,CAI was improved to 0.95,and the cloning vector of SMG1 was successfully constructed.After plasmid extraction and enzyme digestion,the target gene was acquired,which was then further constructed into a recombinant plasmid p GAPZαA-SMG1 and transformed into Pichia pastoris X33.The results of SDS-PAGE gel electrophoresis analysis preceded by methanol-induced expression showed that the protein product had a size of 35 k D and its lipase activity was 0.023 U/mL.The activity of the recombinant lipase SMG1 was the highest at 30℃(0.027 U/mL).The lipase activity increased with the rising temperature in the range of 20℃ to 30℃,but decreased as the temperature ascended from 30℃ to 45℃.In conclusion,the disease presents with a relatively clear gender bias,and more frequently occurs in cats of a certain age range.The cure rate of Malazzia otitis externa can be improved by the administration of the correct ear medication and proper nursing care.Malassezia secretes lipases that have high activity in the skin temperature range of infected cats,and lipases promote the growth of Malassezia.The Pichia pastoris expression system successfully expresses and secretes the recombinant lipase SMG1,which lays a foundation for the pathogenesis study of Malassezia otitis externa in cats,the preparation of lipase inhibitors and the development of rapid detection technology.