Culture Of Trichinella Spiralis Muscle Larvae In Vitro

Trichinellosis is a serious food-borne parasitic zoonosis, which is caused by the nematode of Trichinella spiralis. It is acquired by ingesting the raw or insufficiently cooked meat containing Trichinella larvae. Typically, the patient has fever, eyelid and facial edema, myalgia, allergic rash and eosinophilia, even die of complication caused by Trichinella infection. 10 thousands of people were infected by Trichinella spp. during 1995-1997 according to the reports of International Commission on Trichinellosis. At present, the unsuccessful establishment of culture system of Trichinella larvae in vitro inhibits studies on molecular mechanisms of parasite invasion, worm explusion from host, development of medicine and vaccines against Trichinella. So it is very important to do some researches on culture of Trichinella muscle larvae in vitro.In this study, Trichinella muscle larvae were cultured in various liquid media in vitro. The molting time and rates of molting worms at different times were observed, and lengths of muscle larvae which han't molted were measured; liquid medium(complete medium+T84 monolayers), semisolid medium (complete medium + agarose, complete medium + agarose + T84 monolayers) were used to observe the ecdysis of larvae under different conditions; semisolid medium (complete medium + agarose + Caco-2 monolayers) was used to observe the ecdysis of differently treated larvae, and two cell strains were used to observe the impact of monolayers on larvae ecdysis.Materials and methods1. Trichinella spp., experimental animals and cell lines T. spiralis Henan isolate, male Kunming mice aged 6 weeks, male Sprague Dawley (SD) rats aged 6 weeks, T84 and Caco-2 cell lines were used. The muscle larvae were obtained from Kunming mice experimentally infected with T. spiralis by artificial digestion method.2. Preparation of rat gut contents and mouse bile①After the rat( from which feed had been withheld for 8 hours )was sacrificed, intestinal contents were prepared by rinsing the intestine with 5 ml saline containing antibiotics. By centrifugation at 1 200 g for 10 minutes, debris was removed. The supernatant was diluted 1:2 in saline and frozen at -20℃. Before being used in experiments, the intestinal contents were diluted 1:10 in saline.②After the mouse (from which feed had been withheld for 8 hours) was sacrificed, gallbladder was completely removed. Bile was obtained and frozen at -20℃. Before being used, bile was diluted 1:20 in saline.3. Culture of T. spiralis muscle larvae in various liquid media The muscle larvae collected were cultured in 0.9% saline, PBS, RPMI-1640 medium and RPMI-1640 medium containing 5% fetal bovine serum (FBS) respectively. From 12 hours post inoculation (hpi) on, larvae were taken out in each 2 hours and observed under inverted microscope to study the time that larvae molted. A few of larvae were taken out every 24 hours and suspended in saline, then larvae that molted were counted. About 15 larvae that hadn't molted were photographed and length measured.4. Culture of T. spiralis muscle larvae in various semisolid media The muscle larvae collected were incubated with gut contents or bile. Following treatment, the larvae were cultured in 3 culture systems, including complete medium (DMEM/F12 medium containing 10% FBS) + T84 monolayers, semisolid medium (complete medium+1.75%agarose), semisolid medium + T84 monolayers for 24 hours . then L1 larvae (non-ecdysis) and L2～L4 larvae (ecdysis) were counted under inverted microscope, respectively.5. Observing the impact of activators on larvae ecdysis Muscle larvae treated with 0.9% saline, PBS, gut contents of rat or bile of mouse were suspended in semisolid medium (DMEM medium containing 10% FBS+1.75%agarose). The mixture of larval suspensions and media was overlaid on Caco-2 monolayers. After 24 hours, L1 larvae and L2～L4 larvae were counted under inverted microscope, respectively.6. Observing the impact of intestinal epithelia on larvae ecdysis The impact of larvae ecdysis in T84 and Caco-2 monolayers was observed in semisolid medium, separately. Results1. Molting time of T. spiralis muscle larvae Following inoculation in RPMI-1640 medium, PBS, RPMI-1640 medium containing 5% FBS and 0.9% saline at 37°C in 5% CO₂, time of larvae molting was 15 h, 19 h, 23 h and 37 h, respectively.2. Observing activity of T. spiralis muscle larvae and rates of molting When being inoculated in media, larvae moved actively , doing stretching and curling movements. After cultured in 0.9% saline and PBS for 3 days, the motility of larvae was decreased seriously. Most larvae died on 5 days post incubation (dpi) in 0.9% saline and PBS. However, some larvae still moved on 21 dpi in RPMI-1640 medium containing 5% FBS. Only a single cuticle was formed when larvae were cultured in 0.9% saline and PBS. Among 5 378 larvae observed, the number of larvae with 1-4 cuticles was 3386 (62.96%), 1377(25.60%), 71(1.32%) and 14(0.26%) respectively. Larvae formed 1-2 cuticles in RPMI-1640 medium containing 5% FBS, and the rates of larvae with 1 and 2 cuticles were 66.78% (3699/5539) and 0.45% (25/5539) respectively.3. Observing lengths of larvae cultured in liquid media The lengths (mm) of larvae cultured in RPMI-1640 medium during 1-21 dpi were 0.881±0.024, 0.865±0.079, 0.905±0.068, 0.843±0.029, 0.900±0.083, 0.826±0.082, 0.835±0.117, 0.792±0.205, 0.854±0.115, 0.913±0.178, 0.751±0.214, 0.834±0.140, 0.882±0.169, 0.953±0.133, 0.811±0.168, 0.827±0.152, 0.788±0.147, 0.755±0.161, 0.786±0.187, 0.667±0.071 and 0.733±0.070 respectively. The length decreased along with the increase of days post inoculation (F= 54.639, P<0.01). When cultured in RPMI-1640 containing 5% FBS, the lengths (mm) of the larvae were 0.884±0.041, 0.862±0.054, 0.877±0.043, 0.868±0.031, 0.912±0.067, 0.852±0.043, 0.802±0.066, 0.939±0.094, 0.871±0.080, 0.808±0.115, 0.779±0.075, 0.741±0.092, 0.811±0.092, 0.766±0.131, 0.867±0.235, 0.767±0.091, 0.770±0.093, 0.785±0.075, 0.747±0.046, 0.790±0.038 and 0.772±0.036 respectively. The length also decreased along with the increase of days post inoculation (F= 26.928, P<0.01). Length difference between RPMI-1640 medium and RPMI-1640 medium containing 5% FBS was not statistically significant (t=0.138, P>0.05).4. Observing the condition for ecdysis of larvae cultured in vitro Muscle larvae activated by rat gut contents were inoculated to semisolid medium + T84 monolayers, moved through T84 monolayer and left serpentine trails. Complete cuticles after ecdysis of larvae were not observed in complete medium + T84 monolayers. The proportions of L2-L4 larvae in semisolid medium + T84 monolayers and semisolid medium were 40% and 4.67% respectively. The number of L2-L4 larvae in semisolid + T84 monolayers is obviously higher than that in semisolid medium only (x²=11.590, P<0.01).5. Observing the impact of activators on larvae ecdysis When muscle larvae activated by rat intestinal contents incubated 24 hpi, the proportion of L2-L4 larvae was 50%, which was the highest. Following with larvae activated by mouse bile which proportion of 2-4 phase larva was 34.78%, and larva activated by RPMI-1640 medium with 8.70% as a proportion of L2-L4 larvae. However, no complete cuticles after ecdysis of larvae were found in saline-treated larvae after inoculation. The numbers of L2-L4 larvae activated by rat gut contents and mouse bile before inoculation were higher than that activated by RPMI-1640 medium after inoculation (x₁²=5.978, P<0.05; x₂²=4.600, P<0.05).The activation of muscle larvae by rat gut contents and mouse bile was similar. (x²=0.836, P>0.05).6. Observing the impact of intestinal epithelia on larvae ecdysis Muscle larvae activated could invade the monolayers. The proportions of L2-L4 larvae cultivated in T84 Caco-2 and monolayers were 40% and 50% respectively. There was no statistical significance between them (x²=0.546, P>0.05).Conclusion1. T. spiralis larvae had the highest rate of molting when cultivated in RPMI-1640medium, and had best activity in RPMI-1640 medium containing 5% FBS.2. Development of muscle larvae in semisolid media + T84/Caco-2 monolayers is better, more larvae develop to L2-L4 larvae than in other media.3. The treatments of gut contents and bile on muscle larvae are beneficial to the development.