| Leeches have more than 100 different biologically active substances,these active substances have a variety of medicinal effects.Hirudotherapy can promote blood circulation by the blood-sucking function of leeches.At the same time,the blood silted up in the tissues can be removed through the active substances with anticoagulant function released by leeches in the process of blood sucking,so as to increase the perfusion flow of tissues,this kind of auxiliary medical method is often used in various fields in modern medicine.However,the infection caused by leech bacteria during the hirudotherapy is still a major concern at present,there is no clearly clean breeding technology for medical leeches at home and abroad,there has been no guideline for preventing bacterial infection of hirudotherapy.Poecilobdella manillensis is a kind of leech which is widely used in our country and many Southeast Asian countries,In order to reduce the risk of bacterial infection of Poecilobdella manillensis during hirudotherapy,this thesis has studied the artificial breeding and disinfection treatment technology of medicinal Poecilobdella manillensis.The aim is to minimize the pathogenic bacteria carried on the body surface and digestive tract of Poecilobdella manillensis by artificial breeding and disinfection treatment technology.At the same time,antibiotic sensitivity of the bateria in the digestive tract of Poecilobdella manillensis was detected.This study has a guiding significance for the clean breeding of medicinal leeches and the prevention of bacterial infection caused by hirudotherapy in clinic.Methods:1.Preparation of artificial feedThe experiment was carried out by preparing 9 groups of nitrogen feeds(the crude protein content of the feed is 21%,which was close to the protein content of mammalian blood)with different proportions of plasma protein and blood cell protein(1:0,6:1,4:1,2:1,1:1,1:2,1:4,1:6 and 0:1),the fresh blood was used as the control,and the feeding was carried out for 31 days to explore the effect of different protein ratio on the growth of Poecilobdella manillensis.The artificial feed with different protein ratio was evaluated from three aspects of Poecilobdella manillensis:survival rate,weight gain rate and specific growth rate,and a better formula of artificial feed formula without pathogenic microorganism was determined to replace traditional blood feeding.2.Detection of the digestive tract flora of Poecilobdella manillensis during the fasting periodHigh-throughput sequencing technology and bioinformatics software analysis were used to investigate the effect of different fasting times(7 days,45 days,120 days)on the flora in the digestive tract of Poecilobdella manillensis.3.The disinfection method of the body surface of Poecilobdella manillensisThe semi-hydrostatic biological toxicity test method was used to study the acute toxicity of benzalkonium bromide,potassium permanganate and povidone-iodine to Poecilobdella manillensis.by detecting the total number of bacteria on the surface of Poecilobdella manillensis treated with different concentrations of disinfectants and testing the sucking ability of the Poecilobdella manillensis after treatment,to determine the body surface disinfectant and disinfection method before the use of the medicinal Poecilobdella manillensis4.Isolation,identification and antibiotic sensitivity of bacteria in the digestive tract of Poecilobdella manillensisThe traditional aerobic culture method was used to isolate and purify the bacteria in the digestive tract of Poecilobdella manillensis,morphological observation,biochemical identification and 16S rRNA gene sequence analysis were carried out on the isolates,the sensitivity of the isolates to 11 commonly used antibacterial drugs was detected by Kirby-Bauer disc agar diffusion test.Results:1.The artificial feed formula for medicinal Poecilobdella manillensis for hirudotherapyWhen the crude protein content in the feed is 21%,the different ratios of plasma protein and hemoglobin have a significant influence on the weight gain rate and specific growth rate of Poecilobdella manillensis,but no influence on the survival rate.The groups of adding the compound protein powder feed(6:1,4:1,2:1,1:1,1:2,1:4 and 1:6)and the groups of adding a single protein powder feed(0:1,1:0),both significantly improve the weight gain rate and specific growth rate of Poecilobdella manillensis(p<0.05).Compared with adding a single protein powder feed group(0:1,1:0),adding a compound protein powder feed group(6:1,4:1,2:1,1:1,1:2,1:4 and 1:6)can significantly increase the weight gain rate and specific growth rate of Poecilobdella manillensis(p<0.05).When the ratio of plasma protein to hemoglobin in the feed is 1:4,the weight gain rate and specific growth rate of Poecilobdella manillensis reached the maximum,and the difference is not significant compared with the control group for blood-fed(p>0.05),the results showed that the survival rate,weight gain rate and specific growth rate of the feed with this protein ratio(i.e.4.66%of plasma protein powder and 22.19%of blood cell protein powder)were comparable to blood fed.Therefore,4.66%plasma protein powder,22.19%blood cell protein powder,0.9%sodium chloride,0.21%L-arginine salt,0.05%VC and 71.99%distilled water were used to make the formula of no pathogenic microorganism artificial feed for Poecilobdella manillensis.It can be used as an artificial compound feed instead of traditional blood,which can not only reduce the intake of pathogenic microorganisms,but also ensure the growth and development of Poecilobdella manillensis.2.The effect of fasting on the digestive tract flora of Poecilobdella manillensisThe results of a diversity analysis showed that there were significant differences in species richness and species diversity among different fasting groups(fasted for 7 days,for 45 days,for 120 days after feeding).At the group of fasting for 45 days,the richness and diversity of the digestive tract flora of Poecilobdella manillensis were the lowest.The results of PCoA analysis and ANOSIM analysis showed that there was a significant difference in the overall structure of the digestive tract of Poecilobdella manillensis(P<0.05).At the phlum level,Proteobacteria,Bacteroidetes and Firmicutes were the dominant bacteria in digestive tract(crop and intestine)of Poecilobdella manillensis in the group of fasting for 7 days,45 days and 120 days.The total number of bacteria in the three gates accounted for more than 91%.The relative abundance of Proteus was the lowest in the group of fasting 45 days while Bacteroides was the highest,the relative abundance of Firmicutes decreased with the increase of fasting time and only 0.22%in the group of fasted 120 days.At the genus level,the dominant bacteria of crop in digestive tract of Poecilobdella manillensis in the group of fasted 7 days(F7d)were Aeromonas and Mucinivorans,and the dominant bacteria in intestine were Mucinivorans,uncultured bacterium_f_Elsteraceae and Aeromonas;The dominant bacteria in crop and intestine were Bacteroides in the group of fasted 45 days(F45d),and Elstera and uncultured_f_Elsteraceae in the crop and intestine in the group of fasted 120 days(F120d).The relative abundance of Aeromonas in crop and intestine decreased with the increase of fasting time(crop:F7d(38.23%)>F45d(0.57%)>F120d(0.04%);intestine:F7d(11.92%)>F45d(0.7%)>F120d(0.02%));Mucinivorans also showed a decreasing trend with the increase of fasting time(crop:F7d(20.41%)>F45d(3.94%)>F120d(0.20%);intestine:F7d(20.81%)>F45d(1.6%)>F120d(0.07%)).However,the relative abundance of Bacteroides was the highest at 45 days of fasting.After fasting for 45 days,the relative abundance of Bacteroides decreased gradually(crop:F7d(0.07%)<F45d(60.11%)>F120d(0.48%);intestine:F7d(0.18%)<F45d(64.73%)>F120d(0.02%)).Three kinds of bacteria in digestive tract decreased gradually after fasting for 45 days,and almost reached the undetectable level after fasting for 120 days.Therefore,at the level of phlum and genus,fasting can change the composition and overall structure of dominant bacteria in digestive tract of Poecilobdella manillensis,after fasting for 45 days,the abundance of common pathogens in digestive tract of Poecilobdella manillensis decreased significantly.3.Disinfection of the body surface of Poecilobdella manillensisThe LC50 of benzalkonium bromide,povidone iodine and potassium permanganate were 29.53mg/L,14.37mg/L and 5.54mg/L at 24h;at 48h,they were 24.54mg/L,10.95mg/L,4.36mg/L;at 72h,they were 13.11mg/L,8.68mg/L,3.29 mg/L;at 96h,they were 10.17mg/L,6.34mg/L,2.75mg/L respectively,and the safe concentrations(SC)were 5.08mg/L,1.91mg/L,0.81mg/L.Therefore,the acute toxicity of the three disinfectants to Poecilobdella manillensis were as follows:potassium permanganate>povidone iodine>benzalkonium bromide.The bacteria on the body surface could be reduced to the undetectable level by disinfecting the body surface of Poecilobdella manillensis with 100 mg/L benzalkonium bromide solution for 5 minutes,and the sucking ability of Poecilobdella manillensis would not be damaged.4.Isolation,identification and the antibiotic sensitivity of bacteria in digestive tract of Poecilobdella manillensis84 strains of bacteria were isolated from the digestive tract of Poecilobdella manillensis,including 41 Aeromonas(22 Aeromonas hydrophilia,19 Aeromonas veronii),19 Morgannell morganii,15 Chryseobacterium indogenes and 9 Acinetobacter calcoaceticus.Aeromonas and Morganella were highly sensitive to the third generation of cephalosporins(cefotaxime,ceftazidime,ceftriaxone),and Chryseobacterium indogenes and Acinetobacter calcoaceticus were highly sensitive to fluoroquinolones(levofloxacin,ciprofloxacin).The isolates were resistant to other antimicrobial agents to varying degrees.Conclusion:The clean breeding of Poecilobdella manillensis which was used for hirudotherapy,was fed with artificial feed instead of blood,fasted for more than 45 days,and disinfected with benzalkonium bromide on the body surface,which could significantly reduce the pathogenic bacteria of Poecilobdella manillensis.In clinic,the third generation cephalosporins(cefotaxime,ceftazidime,ceftriaxone)and fluoronorone drugs(levofloxacin,ciprofloxacin)are the first choice antibiotic to prevent bacterial infection during hirudotherapy caused by Poecilobdella manillensis. |
