German cockroach
Four lines of German cockroach (WATn, WATp, NIIDn, and NIIDp) were used in the study. WATn is originally a pinworm-uninfected WATARIDA strain, and is abbreviated as WATn by adding “n” which means pinworm-negative. WATp is a cockroach strain that artificially infected the WATARIDA strain with B. blattae, and is designated as WATp by adding “p”, which means pinworm- positive. WATn cockroaches were artificially infected with B. blattae by exposing them to feces of infected cockroaches for 6 months in the breeding environment. The prevalence of B. blattae infection was 0% (0/40) in the WATn line and 83% (20/24) in the WATp line.
NIIDp is naturally pinworm-infected NIID strain, with "p" meaning pinworm-positive. The NIIDn strain was artificially generated by treating pinworm-infected NIIDp line with an anthelmintic (2000 ppm pyruvinium pamoate solution) for 16 days, and the negative meaning "n" was added to make NIIDn. The prevalence of B. blattae was 93% (40/43) for the NIIDp line and 6.7% (1/15) for the NIIDn line.
Adult German cockroaches 3 to 30 days old for the WATp and WATn lines and 24 to 50 days old for the NIIDp and NIIDn lines were used in the study.
Cockroach breeding environment
A total of 10 to 15 adult German cockroaches were bred in a plastic cage (size: 175 × 140 × 105 mm) with a porous cover to allow air entry. Escape of cockroaches was prevented by applying a 50-mm wide layer of Vaseline (manufactured by Kanto Chemical Co., Inc.) to the upper surface of the inner wall of the container. A piece of folded paper (approx. 70 × 70 mm in size) was placed in the cage to provide a hiding place. Water and food (control area only) were also provided, and the cages were kept in a constant temperature environment (25 ± 1°C).
Comparison of survival rate
Pinworm-infected cockroaches (WATp & NIIDp), uninfected cockroaches (WATn), and anthelmintic-treated cockroaches (NIIDn) were bred separately in two groups, a normal feeding group and a non-feeding group. The number of surviving cockroaches was recorded at the end of each day. Feces excreted by the cockroaches during the test period were not removed until the end of the experiment.
Statistical analysis
For each group, the 50% survival days (the number of days until the survival rate reached 50%) was determined from the survival rate in the elapsed days. The 50% survival days was calculated according to the Kaplan-Meier method and the log-rank test was used to examine the significance of the differences. The analysis was performed in a high-level test using Statcel4 software (OMS Publishing, Tokyo, Japan) and a P value of ≤0.05 was considered significant.