In vitro culture of Echinococcus multilocularis producing protoscoleces and mouse infection with the cultured vesicles
© The Author(s). 2016
Received: 3 April 2016
Accepted: 7 July 2016
Published: 25 July 2016
Alveolar echinococcosis (AE) is a lethal zoonosis caused by the fox-tapeworm Echinococcus multilocularis. The disease is difficult to treat and an effective therapeutic drug is urgently needed. Reliable models are essential for drug development. In this study, we developed both in vitro and in vivo models of larval E. multilocularis.
The protoscoleces (PSC) of E. multilocularis from jirds were successfully cultured in a modified RPMI1640 based medium containing 25 % (v/v) fetal bovine serum (FBS). After 100 days of culture, PSC developed to larval vesicles (small unilocular cysts) and the fast growing vesicles produced PSC in brood capsules. In addition, mice were intraperitoneally injected with 30 cultured small vesicles and 100 % of the mice had resulting metacestode masses.
Larval protoscoleces and vesicles of E. multilocularis grow healthily in vitro in the RPMI1640 based medium containing 25 % FBS. Echinococcus multilocularis in vitro and in vivo models provide a valuable platform for investigating the biology of the parasite and screening effective therapeutic drugs against AE.
Alveolar echinococcosis (AE) is one of the most lethal helminthic infections in humans . The disease is caused by the fox-tapeworm Echinococcus multilocularis, which occurs in most areas of the northern hemisphere [2, 3]. Recent epidemiological studies showed that AE is highly endemic in Central Asia, including western China, Kazakhstan and Kyrgyzstan [4, 5]. AE is a chronic parasitic disease and can remain asymptomatic in a patient for up to 15 years . When an AE patient feels ill and requests to see a doctor, it is normally at a late stage. In this late stage, the parasite causes liver damage through its production of an infiltrating structure consisting of numerous small vesicles embedded in stroma of connective tissue, which makes surgical resection difficult and secondary re-infection often occurs . Normally, surgical removal, if applicable, is the only curative method for treating AE and patients have to take albendazole (ABZ) and/or mebendazole (MBZ) after surgical treatment for at least 2 years . Inoperable AE patients must undergo long term, often life-long, chemotherapy with ABZ and/or MBZ [9, 10]. Recent study has shown that these drugs are not parasiticidal in vivo against AE, and only inhibit E. multilocularis growth. This is the reason that AE patients have to take the drugs for a long time . In addition, the occurrence of side effects with these drugs has been often reported, leading to discontinuation of treatment or to progressive disease. An effective therapeutic drug for AE is urgently needed.
Metacestodes of E. multilocularis are fluid-filled grape-like vesicles. The asexually proliferating vesicles cause the disease and the vesicles have been used for drug screening [4, 12, 13]. A simple and stable method for culturing the metacestodes of the parasite will be beneficial not only for biological and physiological studies, but also for speeding up drug development against AE. Several previous studies have achieved successful cultivation of E. multilocularis metacestodes in vitro. The co-culture system contains host support or feeder cells for the parasite to be maintained in medium for the several months required to produce PSC. Without the feeder cells, the metacestodes can be only maintained for a few weeks [14–16].
In the present study, we cultured PSC of E. multilocularis in a modified medium without host feeder cells. In the culture system, PSC re-differentiate into vesicles which can be maintained for more than 100 days and the fast growing vesicles produced PSC in brood capsules.
Animal infections and parasites
Mongolian jirds (Meriones unguiculatus) were used for maintaining larval E. multilocularis in an animal house at the Veterinary Research Institute, Xinjiang Academy of Animal Science. Jirds were intraperitoneally (i.p.) infected with PSC of E. multilocularis collected from Yili, Xinjiang, China. Pathogen-free female BALB/c mice, aged 6–8 weeks, were purchased from Beijing Vital River Laboratory Animal Technology Company Limited, and raised in the animal facility of the First Affiliated Hospital of Xinjiang Medical University.
Collection of protoscoleces and in vitro cultivation
Mongolian jirds were infected with PSC of E. multilocularis for 4–8 months, and those with a heavy infection were sacrificed with CO2 anaesthesia. Metacestode masses were collected from the peritoneal cavity of the infected jirds under aseptic conditions and then washed with sterile phosphate-buffered saline (PBS). After removal of possible host tissues, the masses were minced into small tissue pieces. The parasite tissues were then passed through a sterilized sieve (100 mesh sieve) by grounding with a glass pestle. The passed through parasite tissues containing PSC were washed five times with PBS containing 100 U/ml penicillin and 100 μg/ml streptomycin (PBS-PS) by placing the tube on the bench for 5 min to allow the PSC to sediment at room temperature. The sedimented PSC were digested with 1 % (w/v) pepsin (Sigma-Aldrich, Louis, MO, USA) at pH 2.0 (adjusted with 2 M HCl) in Hank's Buffer for 20 min at 37 °C. After the digestion procedure, the PSC were washed five more times with PBS-PS to remove debris by natural sedimentation for 5 min at room temperature. The viability of PSC was determined by staining with 0.1 % methylene blue, with dead PSC staining blue.
Only samples with ≥ 95 % viability were subsequently cultured in RPMI 1640 medium (Gibco, Auckland, New Zealand) containing 25 % (v/v) FBS (Gibco), 0.45 % (w/v) yeast extract, 0.4 % (w/v) of glucose, 100 U/ml penicillin and 100 μg/ml streptomycin in a culture flask at 37 °C in the presence of 5 % CO2. The medium was changed every 3 days. Each culture was monitored weekly under an optical microscope to check the growth status of the larval vesicles.
BALB/c mice were each i.p. transplanted with 30 small (250–300 μm in diameter) cultured vesicles of E. multilocularis. After 6 months, parasitic masses were collected from the mice and histopathological examination was performed by hematoxylin and eosin staining.
Results and discussion
A comparison of different conditions used for culturing E. multilocularis in vitro
Hemphill et al. 
Jura et al. 
Spiliotis et al. 
Initial parasite material
Small tissue blocks/ vesicles
Homogenized parasite tissue
Adding feeder cells
Stopped at day 100
At least 2 months
Stopped at day 56
More than 100 days
We have developed a simple and practical in vitro culture method for generating vesicles from PSC of E. multilocularis which in turn produced PSC in vitro in the absence of host feeder cells. This successful development of vesicles in a host feeder cell-free cultivation system represents a useful model for investigating the biology of E. multilocularis and for screening drugs for treatment of alveolar echinococcosis. Furthermore, we have developed a method for the secondary infection of E. multilocularis by simply transferring the cultured vesicles into the peritoneal cavity of mice, which provides a valuable animal model for investigating parasite development and elucidating host-parasite interactions.
AE, alveolar echinococcosis; ABZ, albendazole; BC, brood capsule; cox1, cytochrome c oxidase subunit 1; E-PSC, evaginated PSC; FBS, fetal bovine serum; H, hooks; i.p, intraperitoneally; I-PSC, invaginated PSC; LL, laminated layer; MBZ, mebendazole; PSC, protoscoleces; PBS, phosphate-buffered saline; Pre-BC, pre-brood capsule; S-PSC, “swollen” PSC; SU, suckers
We thank Mr Tao Jiang and Mr Chun Zhang for their support for maintaining experimental rodent animals and E. multilocularis parasite.
The work was supported by the grants from the National Natural Science Foundation of China (31260272, U1303222).
Availability of data and material
Data supporting the conclusions of this article are included within the article.
WH and ZW-B conceived and designed the experiments; WH and ZL performed the experiments; GB-P, ZZ-Z participated in animal model and sample collection; WH wrote the manuscript; ZW-B, LJ and DPMcM critically revised the manuscript. All authors have read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was conducted in accordance with the Chinese Laboratory Animal Administration Act (1988) and the study protocol was approved by the Ethical Committee of the First Affiliated Hospital of Xinjiang Medical University (Approval No IACUC-20120625003).
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