Bacteria-induced egg hatching differs for Trichuris muris and Trichuris suis

Abstract

Background: Eggs of the porcine whipworm Trichuris suis are currently explored in human clinical trials as a treatment of immune-mediated diseases. In this context, only the infective, embryonated eggs, constitute the Active Pharmaceutical Ingredient (API). The rodent whipworm, Trichuris muris is commonly used as a laboratory model to study Trichuris biology. The embryonated eggs (containing a fully developed larva) are biologically active and will invade the large intestinal mucosa of the host. This study aims to assess the in vitro hatching of T. muris and T. suis eggs in various bacterial cultures as a measure for their biological activity.

Methods: Eggs of T. muris and T. suis were incubated with Escherichia coli strain (BL-21) at three concentrations in a slightly modified in vitro egg hatching assay previously developed for T. muris. Additionally, E. coli strains (M15, SG13009, PMC103, JM109, TUNER, DH5alpha, TOP10) and five Gram-positive bacteria (Enterococcus caccae, Streptococcus hyointestinalis, Lactobacillus amylovorus, L. murinus, and L. reuteri) were tested as a hatching stimulus for T. muris and T. suis eggs.

Results: Whereas T. muris eggs hatched, T. suis did not, even when exposed to different concentrations and strains of E. coli after 4 and 24-hour incubation. When incubated with Gram-positive bacteria, only T. muris eggs showed noticeable hatching after 20 h, although with high variability.

Conclusions: The observed difference in hatching of T. muris and T. suis eggs incubated with selected bacteria, indicate significant biological differences which may reflect specific adaptation to different host-specific gut microbiota.

Fig. 1

Trichuris muris eggs incubated with different Escherichia coli strains. Samples were incubated for 4 and 24 hours at 36 °C. The control group represents T. muris eggs incubated without the addition of E. coli. Arithmetic mean (bar) and standard deviation (error bars) for n = 3 per group

Fig. 2

Trichuris muris eggs incubated with Gram-positive bacteria in RPMI-1640 without antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. muris (Milli-Q or H₂SO₄ pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteri (n = 15), L. amylovorus (n = 15), and L. murinus (n = 10). Controls represent T. muris eggs incubated without the addition of bacteria (n = 25)

Fig. 3

Trichuris suis eggs incubated with Gram-positive bacteria in RPMI-1640 without antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. suis (Milli-Q or H₂SO₄ pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteri with T. suis in Milli-Q (n = 15), L. reuteri with T. suis in H₂SO₄ pH1 (n = 20), L. amylovorus (n = 15), and L. murinus (n = 10). Controls represent T. suis eggs without the addition of bacteria (n = 25)

Fig. 4

Trichuris muris eggs incubated with Gram-positive bacteria in RPMI-1640 with antimicrobials. Samples were incubated for 20 hours at 37 °C. Individual counts of T. muris (Milli-Q or H₂SO₄ pH1) and median for all experiments with Enterococcus caccae (n = 20), Streptococcus hyointestinalis (n = 10), Lactobacillus reuteriwith T. muris in Milli-Q (n = 20), Lactobacillus reuteri with T. muris in H₂SO₄ pH1 (n = 15), L. murinus (n = 10), and L. amylovorus (n = 15). Controls represent T. muris eggs without the addition of bacteria (n = 25)