Establishment of a secondary infection laboratory model of Echinococcus shiquicus metacestode using BALB/c mice and Mongolian jirds (Meriones unguiculatus)

Abstract

Echinococcus shiquicus is peculiar to the Qinghai–Tibet plateau of China. Research on this parasite has mainly focused on epidemiological surveys and life cycle studies. So far, limited laboratory studies have been reported. Here, experimental infection of E. shiquicus metacestode in BALB/c mice and Mongolian jirds (Meriones unguiculatus) was carried out to establish alternative laboratory animal models. Intraperitoneal inoculation of metacestode material containing protoscoleces (PSCs) obtained from infected plateau pikas were conducted on BALB/c mice. Furthermore, metacestode material without PSCs deriving from infected BALB/c mice was intraperitoneally inoculated to Mongolian jirds. Experimental animals were dissected for macroscopic and histopathological examination. The growth of cysts in BALB/c mice was infiltrative, and they invaded the murine entire body. Most of the metacestode cysts were multicystic, but a few were unilocular. The cysts contained sterile vesicles, which had no PSCs. The metacestode materials were able to successfully infect new mice. In the jirds model, E. shiquicus cysts were typically formed freely in the peritoneal cavity; the majority of these cysts were free while a small portion adhered loosely to nearby organs. The proportion of fertile cysts was high, and contained many PSCs. The PSCs produced in Mongolian jirds also successfully infected new ones, which confirms that jirds can serve as an alternative experimental intermediate host. In conclusion, a laboratory animal infection was successfully established for E. shiquicus using BALB/c mice and Mongolian jirds. These results provide new models for the in-depth study of Echinococcus metacestode survival strategy, host interactions and immune escape mechanism.

Keywords: BALB/c mouse; Echinococcus shiquicus; Mongolian jird; Protoscolex; model.

Graphical abstract

Figure 1.

Collection and identification of PSCs of Echinococcus shiquicus in plateau pikas. Macroscopical finding in the infected plateau pikas showed the cystic lesions (arrowhead) were all found in the lungs, including single cyst (a), cysts in small groups (b) or cysts in dense aggregations (c), metacestode materials collected in the dish (d), scale bar: 5 mm. Light microscopic of the fertile cysts (e), methylene blue-staining image of PSCs (f) demonstrated that those with no absorbed dye were considered potentially viable (white arrow) and otherwise, they were recorded as dead (yellow arrow). H&E staining of metacestode revealed brood capsules within the cysts (black arrow), and green arrow indicates the germinal layers in the metacestode tissue, scale bar: 100 μm (g). PAS staining of metacestode revealed the laminated layers (blue arrow) (h). Agarose gel electrophoretogram displaying PCR amplified cox1 fragments from germinal layer of the cysts. M: Molecular marker 100–2000 bp; Lane 1: test sample; Lane 2, positive control (DNA of E. shiquicus); Lane 3, negative control (i).

Figure 2.

BALB/c mice anatomy after the intraperitoneal injection of Echinococcus shiquicus PSCs after 1 (a–e), 3 (f–j) and 6 (k–o) months. After 1 month p.i., cysts lesions (arrowhead) attached to mesentery (a). The contents of the cysts have no PSCs and vesicles (b). After 3 month p.i., cystic lesions infiltrated multiple organs in the abdominal cavity (f). Collected cyst contents were observed to contain a lot of micro-vesicles (red arrow) under light microscopy (g). After 6 month p.i., cystic lesions almost occupied the entire abdominal cavity and organs were severely extruded (k), and micro-vesicles could be observed in the cyst contents (l). H&E staining indicating that metacestode tissue contained sterile cysts (c, h and m). Green arrow indicates the germinal layers of the larvae. Images ‘d’, ‘i’ and ‘n’ are the higher magnification images of the boxed areas in ‘c’, ‘h’ and ‘m’, respectively. PAS staining indicating the laminated layers (blue arrow) of images ‘e’, ‘j’ and ‘o’. Scale bar: 100 μm.

Figure 3.

Metacestode tissues of Echinococcus shiquicus in the BALB/c mice (a–d) and Mongolian jirds (e–h) 6 month post injection. Black arrow showing cysts observed under light microscope without PSC in mice (a, b), and fertile cysts with PSCs (red arrow) in Mongolian jirds (e, f). Purple arrow showing budding capsule observed under light microscope without PSC in mice (b), and with PSCs in Mongolian jirds (f); Green arrow in the H&E staining showing the germinal layer of the larvae (c, g), and black arrow showing brood capsules with PSCs (red arrow) in Mongolian jirds (g). Blue arrow in the PAS staining showing laminated layer (d, h). Scale bar: 100 μm.

Figure 4.

Agarose gel electrophoretogram showing PCR amplified cox1 fragments from cysts of BALB/c mice and Mongolian jirds. M: Molecular marker 100–2000 bp; Lanes 1–3, test samples at 1-, 3- and 6-month's post-injection, respectively (BALB/c mice); Lane 4, positive control (Echinococcus shiquicus); Lane 5, negative control; Lanes 6–8, test samples at 2-, 6- and 12-month's post-injection, respectively (Mongolian jirds), Lane 9, positive control (E. shiquicus); Lane10, negative control.

Figure 5.

Mongolian jirds anatomy after the intraperitoneal injection of Echinococcus shiquicus metacestode materials after 2 (a–f), 6 (g–l) and 12 (m–r) months. After 2 month p.i., cystic lesions (arrowhead) attached to mesentery or free in abdominal cavity (a, b). The contents of metacestode materials had no PSCs and brood capsules (c). H&E staining showed the metacestode had germinal layers (green arrow) (d, e). Blue arrow in the PAS staining showing laminated layer (f). After 6 month p.i., multichambered cystic lesions adhered gently to mesentery but did not infiltrate other organs (g, h). Lots of PSCs (red arrow) in collected cysts contents (i). H&E staining of metacestode revealed fertile cysts within brood capsules (black arrow) (j). Enlarged view of the brood capsule in ‘j’ showed the PSCs in the cysts. Green arrow in the HE staining indicates the germinal layers of the larvae (j, p), and blue arrow in the PAS staining showing laminated layer (l, r). After 12 month p.i., multichambered cystic lesions occupy almost the entire abdominal cavity but did not infiltrate other organs (m, n). H&E staining of metacestode revealed more fertile cysts. Image ‘e’, ‘k’ and ‘q’ is the higher magnification images of the boxed areas in ‘d’, ‘j’ and ‘p’, respectively. Scale bar: 100 μm.