Full length articleOptimized dexamethasone immunosuppression enables Echinococcus multilocularis liver establishment after oral egg inoculation in a rat model
Graphical abstract
Introduction
Oral inoculation with Echinococcus multilocularis eggs, termed primary infection, is the natural route of infection in the intermediate or accidental host. After intestinal oncosphere invasion and venous migration, the parasite mainly establishes and develops in the liver, causing a disease referred to as alveolar echinococcosis (AE) (Stojkovic et al., 2014), which might be lethal if left untreated. Immunocompetent humans are considered rather resistant to infection after ingestion of eggs (Vuitton, 2003, Gottstein et al., 2015). However, in patients with impaired immunological status due to co-infections e.g., with HIV (Sailer et al., 1997) or due to drug-based immunosuppression (Chauchet et al., 2014), the progression of AE development may considerably be increased (Vuitton et al., 2015).
Experimentally, secondary larval E. multilocularis infections comprising parenteral parasite inoculation have been used for decades. Thus, parasite isolates are maintained for research and diagnostics by serial propagation of metacestode material by intraperitoneal inoculation (Romig and Bilger, 1999). Although many AE animal models bypass the gastrointestinal exposure during oncosphere invasion including defensive local host immune regulations and responses, secondary AE infection models are widely used since parenteral application of metacestode material allows for the most rapid and potent development of AE (Asanuma et al., 2006, Küster et al., 2013, Song et al., 2014). Experiments with AE animal models extensively contributed to the current knowledge on host-parasite interaction such as host immune responses and parasite evasion strategies (reviewed by Gottstein et al., 2015, Gottstein et al., 2017), parasite development (Ohbayashi, 1960, Ohbayashi et al., 1971, Eckert et al., 1983, Mehlhorn et al., 1983) as well as treatment opportunities (Eckert, 1986; reviewed by Hemphill et al., 2014). However, since secondary echinococcosis in rodents considerably varies with regard to parasite growth and development characteristics, obtained results may be difficult to compare or even extrapolate to the natural situation after oral ingestion of parasite eggs. To overcome these limitations, primary infection models were propagated in several studies (Siles-Lucas et al., 2003, Deplazes et al., 2005, Matsumoto et al., 2010, Woolsey et al., 2016, Armua-Fernandez et al., 2016). Depending on host species and strains, metacestode growth and protoscolex formation (fertility) may be delayed, poor or absent (Matsumoto et al., 2010). A major obstacle of these experiments is the fact that parasite eggs are difficult to obtain, either after necropsy of wild foxes in endemic areas (most cost-effective option) or by experimental infections of foxes, dogs or raccoon dogs (Kapel et al., 2006; reviewed by Matsumoto and Yagi, 2008).
Comparable to the situation in humans, the genus Rattus is considered highly resistant to oral E. multilocularis egg exposure and does not contribute to the maintenance of the general life cycle in nature. In a highly endemic region such as Hokkaido, Japan, only two natural AE cases in Rattus norvegicus were described (Okamoto et al., 1992, Iwaki et al., 1993). Under experimental conditions, no hepatic AE could be induced in Wistar or T-cell deficient nude rats after oral egg inoculation (Webster and Cameron, 1961; Iwaki et al., 1995, Armua-Fernandez et al., 2016). In contrast, intrahepatic or intraperitoneal inoculations of metacestode or oncosphere material led to local AE development in rats (Yamashita et al., 2013, Armua-Fernandez et al., 2016). These observations suggest that resistance to oncosphere invasion or AE development most probably involves different mechanisms.
Glucocorticoids such as dexamethasone (DXM) are potent immunosuppressive and anti-inflammatory agents which induce multiple effects on immune cell counts and functions (reviewed by Coutinho and Chapman, 2011). After immunosuppression of Wistar rats with DXM, the barrier-mediating resistance was broken, leading to the assumption that immune-derived mechanisms or the intestinal endothelial or villous structure are responsible for the resistance of rats against E. multilocularis oncosphere invasion. Since recent DXM dosage schemes of Wistar rats led to the exclusion of several animals from the experiment due to weight losses >20% (Armua-Fernandez et al., 2016), the aim of this study was to improve the rat model by an optimized treatment regime, causing less animal weight losses but allowing hepatic AE development in the liver after oral egg inoculation. As different strains of mice are well known to vary in susceptibility to oral egg inoculation, as documented by differences in the number of hepatic AE lesions (Matsumoto et al., 2010), a second rat strain named F344/DuCrl was orally inoculated with eggs without immunosuppressive treatment.
Section snippets
Parasite eggs
Echinococcus multilocularis eggs were obtained from naturally infected foxes during the regular Swiss hunting season in spring 2016. After harvesting adult parasites from the small intestines of foxes, Echinococcus eggs were collected after squashing and filtering the worms through 41 μm followed by 21 μm meshes (Lanz-Anliker AG, Switzerland) before being kept at 4 °C in PBS with 100 IU penicillin, 100 μg streptomycin (Life Technologies, Switzerland) for 11 weeks until use. Eggs resistant to
Animals
No rat had to be excluded from the experiments due to unexpected clinical signs or weight losses >20%. The rats of group R3 showed weight losses after DXM treatments (day -13 and -9) to a maximum of 20%, and weights continuously increased 5 days after the last treatment until necropsy. Rats of groups R4 and R5 revealed weight losses after a single DXM treatment up to a maximum of 17%, and weight gain was observed from day 5 (group R4) and day 14 (group R5) after treatment. Weight losses and
Discussion
Laboratory rats are considered resistant to oral inoculation with E. multilocularis eggs. However, dexamethasone (DXM), but not methotrexate treatment, allowed intestinal oncosphere invasion and subsequent settlement in the liver following progressive metacestode growth in naturally resistant Wistar rats, and it was hypothesized that this was due to an extensive impairment on various immunological patterns or intestinal structural changes (Armua-Fernandez et al., 2016). In that study, DMX
Financial support
This research was financed by funds of the Institute of Parasitology, University of Zurich.
Competing interest
The authors declare that they have no competing interests.
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