Worms were harvested from the intestines of infected raccoons from Goleta, California, USA. Eggs were removed from adult female worms and allowed to embryonate in sterile saline at room temperature for 3 weeks.
To determine the thermal death point, 150 μL each of embryonated eggs, at a concentration of 100 eggs per μL were added to six 1-mL polypropylene tubes of sterile water. The 6 tubes were then added to a water bath at 35°C and allowed to sit for 10 min to equilibrate. Temperature of the water bath was increased at a rate of ≈5°C every 7 min, and 1 tube was removed at 5° increments from 37°C to 62°C. Eggs were then examined by light microscopy to determine viability as judged by larval motility. A minimum of 50 eggs were examined per assay, and larval motility was judged as +/–. The experiment was repeated using a more objective assessment of viability through examination of hatched larvae. Inactivation was measured by using a viability dye (methylene blue) exclusion method where uptake of dye indicates cell death and inactivation. After the eggs were removed from the heat, the mammilated layer was removed through exposure to undiluted household bleach (6% sodium hypochlorite) to promote subsequent emerging of larvae, then washed 5 times in 0.85% saline for 1 min at 600 × g
. Hatching was achieved by the glass bead method (6,7
). Hatched larvae were then removed and mixed 1:1 with a 1:10,000 dilution of methylene blue. Viable larvae remained motile and remained unstained (), whereas nonviable larvae absorbed the methylene blue stain () (8
Viable Baylisacaris procyonis larvae demonstrating intact membrane and impermeability to methylene blue. Original magnification ×40.
Hatched nonviable Baylisascaris procyonis larvae demonstrating uptake of methylene blue. Original magnification ×40.
The experiment was repeated by adding the heated water, in 5° increments from 37°C to 62°C, directly into the tube containing the eggs. Eggs were exposed to the water for <1 min. Eggs were then processed as described and examined by light microscopy. All experiments were replicated.
To determine the ability of eggs to withstand freezing temperatures, 150 μL each of embryonated eggs at a concentration of 100 eggs per μL were added to ten 1-mL polypropylene tubes of sterile water. The tubes of eggs were exposed to an environment of –15°C. Every 30 days, 1 tube was removed and thawed at room temperature. Viability was evaluated by using both motility and methylene dye exclusion as described above. After examination, tubes were refrozen. Each month, in addition to the evaluation of a fresh tube, tubes from the previous months were reevaluated to examine the effects of freeze–thaw. A total of 5 freeze–thaw cycles were evaluated.
To assess survival of eggs to total desiccation, 150 μL each of embryonated eggs, at a concentration of 100 eggs per μL, were added to ten 1-mL wells of sterile water in a microtiter plate. The plates were stored at room temperature and allowed to dehydrate to complete desiccation over a 1-week period. Monthly, 1 mL of sterile water was added and evaluated for eggs and assessed for viability. Assessments were made until complete inactivation occurred.
All larvae remained viable to 47°C; >90% of the larvae were viable at 52°C, and 50% were viable at 57°C. Larve were completely inactivated at 62°C (). When the heated water was added directly to the eggs, all larvae remained viable through 42°C, and most larvae were viable at 47°C and 52°C. Complete inactivation occurred at 57°C ().
Viability of Baylisacaris procyonis eggs in sterile water when exposed to 2 different heating conditions
Eggs survived complete desiccation for at least 6 months at room temperature. Total loss of viability was observed after 7 months of desiccation. Eggs frozen at –15°C for 6 months demonstrated no reduction in viability. Freeze–thaw, including exposure to 5 cycles, did not affect survival. Although we did not specifically design experiments to assess the effect of chlorine on inactivation of B. procyonis eggs, exposure to undiluted household bleach for 90 min to remove the mammillated layer did not affect viability.