| Ichthyophthirius multifiliis is a highly pathogenic protozoan parasite,which can cause Ichthyophthiriasis of freshwater cultured fish and cause great economic losses to freshwater aquaculture.A large number of I.multifiliis samples are needed to study the pathogenic mechanism,drug development and prevention and control measures of I.multifiliis.Obtaining I.multifiliis samples directly from diseased fish is limited in time and region,and it is difficult to guarantee the continuity of research.Therefore,it is necessary to carry out the research on the culture of I.multifiliis and obtain a large number of I.multifiliis.This paper carried out the study of in vivo and in vitro culture of I.multifiliis,and preliminarily explored the in vivo and in vitro culture methods and conditions of I.multifiliis,the main results are as follows:1.To determine the conditions for the in vitro incubation and the in vivo culture of I.multifiliis.And optimization of the in vivo culture system of I.multifiliis:By comparing the effects of different physicochemical factors on the hatching rate,incubation time and the number of theronts released by one tomont,the following results were obtained:(1)The suitable incubation temperature of tomont was 25℃.The hatching rate of tomont was greater than 90%;the shortest incubation time was 13.37±0.70 h and the maximum number of theronts released were 1,433.40±353.32 at 25℃.(2)The suitable salinity of tomont was 0.The highest hatching rate was 95.83±0.072%;the shortest incubation time was 17.76±1.73 h and the largest number of theronts released were 404.77±96.47.(3)The tolerance of tomont to p H,nitrite and ammonia nitrogen was high.When p H 5-9 or nitrite concentration<5 mg/L or ammonia nitrogen concentration<6 mg/L,the hatching rate could reach more than 90%.And there was no significant effect on the incubation time of tomont and the number of released theronts.The optimal infection conditions were determined by comparing the survival rate of goldfish and the influence of different infectious doses on the development of I.multifiliis in the tail fin of goldfish.When the infectious dose is less than 10,000 theronts/fish,the survival rate of goldfish can reach more than 90%.Compared with the infection intensity in the caudal fin of goldfish,it was found that the infection intensity was the highest when the infectious dose was 10,000,on the first day of infection which was 2.51±0.30trophonts/mm~2.Therefore,10,000 theronts/fish was determined as the optimal infectious dose for in vivo culture system.At this dose,the development time of trophont on the tail fin of goldfish was 4 to 5 days,and the size of trophont increased from55.13±13.31×46.57±12.28μm to 436.49±50.22×343.65±57.19μm.2.The effects of different medium,cell aggregate,host tissue homogenate or serum on the survival and development of theront was compared,and the preliminary study on the in vitro culture of I.multifiliis was carried out.(1)The optimal culture medium for in vitro culture was composed of 44.5%aerated water,44.5%M199 culture medium,10%fetal bovine serum,and 1%penicillin-streptomycin.The longest culture time for theront was 6 d.(2)Part of theront in the optimal medium with the additionepithelioma papulosum cyprini(EPC)or fathead monnow(FHM)cell line aggregate could be transformed into trophont.I.multifiliis died within 3 days.The trophont size of EPC cell aggregate at day 0 and 1 were 30.02±0.19and 29.93±1.62μm.The trophont size of FHM cell aggregate at 0 and 2 d were30.90±5.21 and 31.92±1.89μm.There was no significant change in the size of trophont in the cell aggregate.And there was no significant difference between the groups.(3)The effects of host tissue homogenate and serum on survival rate and development were compared.It was found that I.multifiliis in each experimental group with tissue homogenate and serum died within 3 days;There was no significant change in the size of trophont in each group.(4)To compare the effects of three different models(Theronts and cells were mixed in the upper layer of agarose medium;Mixed theronts,cells and agarose medium;Theronts between agarose medium and cells)on survival and development of I.multifiliis.The results showed that after the addition of EPC or FHM cells,I.multifiliis died within 4 days in the three models.Part of theronts can be transformed into trophonts.And the trophont size tended to increase,but there was no significant difference among the models.After adding EPC cells,the trophont size in three models was 29.51±11.96,28.79±11.95 and 30.41±13.26μm,on the first day of culture.On the third day,the trophont size was 37.40±3.99,39.51±8.51 and 45.14±10.92μm,respectively.A similar phenomenon was observed in different model groups supplemented with FHM cells. |
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