Comparison Of Collection Methods Of Trichinella Spiralis At Different Developmental Stages And The Distribution Of Migratory Larvae

Trichinella spiralis is one of the most widely distributed zoonotic parasites,it was found in the muscle tissues of all kinds of terrestrial vertebrates except Antarctica,Trichinellosis can be caused when people or other animals ate raw meat infected with Trichinella spiralis.In order to promote immune related research and provide reliable insights into host pathogen interactions,the current research on Trichinella spiralis focuses on identifying important genes in its life cycle.However,the basis of discussing these problems is inseparable from the body of Trichinella spiralis at each developmental stage.The requirement for collection of Trichinella spiralis should not be limited to quantity,and the maintenance of normal life activities of Trichinella spiralis is also the basis for all kinds of experiments.Therefore,this experiment compared several collection methods of Trichinella spiralis at different stages,and summarized the most suitable collection methods of Trichinella spiralis muscle larvae,adults and newborn larvae.At the same time,fluorescence quantitative PCR was used to investigate the distribution of the migrating larvae in mice on different infection days,which provided a new idea for further study on the migration path of the migrating larvae of Trichinella spiralis.This experiment based on the existing methods of collecting Trichinella spiralis in different periods,combined with the experience of collecting Trichinella spiralis in our laboratory.At the time when collected muscle larvae,two kinds of collecting liquid and three kinds of collecting methods were selected : Baermann’s standing method,Baermann’s mixing round method and precipitation centrifugation method.The viability and infectivity of the collected worms were analyzed by counting the number of dead worms,in vitro infection test,small intestine perfusion test and reinfection mice test,The results showed that : the type of collecting fluid had no significant effect on the number of collected worms(P>0.05),however,the number of dead worms collected by Baermann’s standing method was significantly higher than that by Baermann’s mixing round method(P<0.05)when used physiological saline;after 24 hours,the number of dead larvae collected by precipitation centrifugation method in water was significantly higher than that collected by Baermann’s standing method(P<0.01).In vitro infection test,the activated muscle larvae were mixed with semi-solid medium and added to the cell monolayer,after 5 hours of continuous observation,the results showed that the muscle larvae collected by precipitation centrifugation in water group had the weakest infection ability,and the invasion rate was only11.17% after 5 hours,the invasion ability of muscle larvae obtained by physiological saline Baermann’s standing method was the strongest,reaching 24.5% after 5 hours;the activated muscle larvae were incubated with sterile Tyrode in the intestinal bag at 37℃for 2 hours in small intestine perfusion test,the ability of muscle larvae collected by Baermann’s mixing round method and Baermann’s standing method to invade intestinal mucosa was the strongest in water group,and there was a significant difference between precipitation centrifugation and the other two methods(P<0.05);the muscle larvae collected by different methods were used to infect the mice again,then collected the muscle larvae after 35 days,LPG of muscle larvae in water group that collected by precipitation centrifugation was significantly lower than that in physiological saline group(P<0.05).Based on the above results,using physiological saline as the collecting solution and using Baermann’s mixing round method to collect can not only meet the requirement of collecting quantity,but also ensure the infectivity of the insect.If only the number of muscle larvae is needed,the precipitation centrifugation method can be used,and there is no special requirement for the collected liquid.The study showed that the differences in the number and fecundity of the adults collected after scraping the intestinal contents with glass slide and flushing the intestinal contents with physiological saline,as a result,63.5% of the adults were collected by scraping the intestinal contents with glass slide,and the impurities in the collected liquid were less,also there was no significant effect on the fecundity of the female(P>0.05).Therefore,it can be widely used in the experiments which need to collect a large number of adults.Based on this method,different collection devices are used,including screen passing equipment and Behrman equipment,although the two apparatus could remove most of the impurities,the recovery rate of adults was significantly different from that of conventional equipment(P<0.0001).In the experiment of collecting newborn larvae,the adult and newborn larvae were separated by 200 mesh screen,and the filtrate containing newborn larvae was collected by natural precipitation method and centrifugation method respectively.The collection effect was studied by comparing the residual number of newborn larvae in supernatant fluid and the number of muscle larvae recovered by tail intravenous.The results showed that the natural precipitation method took a long time,while the centrifugation method could collect a large number of larvae in a short time,but after the newborn larvae were inoculated into mice through the tail vein,the infectivity of the larvae collected by the natural precipitation method was significantly stronger than that by the centrifugation method(P<0.001).In the experiment of studying the distribution of migratory larvae,the newborn larvae stage specific gene T668 was selected to locate the newborn larvae in mice,after verifying the specificity of T668,the standard curve of fluorescence quantitative PCR for detecting T668 gene in newborn larvae by SYBR green dye method was successfully drawn,the linear equation is Y=﹣ 3.673X+ 68.784.The number of copies of T668 gene in each tissue and organ was calculated according to the equation,which indirectly indicated the content of newborn larvae in various parts of the body.The results showed that the first organ of larva was lung,and it was also found in psoas muscle,masseter muscle and hind leg muscle,on the 8th day,the larvae could be detected in many muscle tissues,and then could not be detected.It was speculated that the larvae had developed into muscle larvae,but the larvae could be detected in the heart until the 15 th day,and the number decreased on the 15 th day,but it could still be detected in the liver and kidney.The results of this experiment roughly reflected the distribution of migrating larvae in various tissues and organs of mice infected with muscle larvae over time,which laid a solid foundation for further depicting the migration path of newborn larvae in vivo.