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Amphibians are suffering unprecedented global declines. A leading cause is the infectious disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis. Chytridiomycosis is a skin disease which disrupts transport of essential ions leading to death. Soluble factors produced by B. dendrobatidis impair amphibian and mammalian lymphocytes in vitro, but previous studies have not shown the effects of these inhibitory factors in vivo. To demonstrate in vivo inhibition of immunity by B. dendrobatidis, a modified delayed-type-hypersensitivity (DTH) protocol was developed to induce innate and adaptive inflammatory swelling in the feet of Xenopus laevis by injection of killed bacteria or phytohemagglutinin (PHA). Compared to previous protocols for PHA injection in amphibians, this method induced up to 20-fold greater inflammatory swelling. Using this new protocol, we measured DTH responses induced by killed bacteria or PHA in the presence of B. dendrobatidis supernatants. Swelling induced by single injection of PHA or killed bacteria was not significantly affected by B. dendrobatidis supernatants. However, swelling caused by a secondary injection of PHA, was significantly reduced by B. dendrobatidis supernatants. As previously described in vitro, factors from B. dendrobatidis appear to inhibit lymphocyte-mediated inflammatory swelling but not swelling caused by an inducer of innate leukocytes. This suggests that B. dendrobatidis is capable of inhibiting lymphocytes in a localized response to prevent adaptive immune responses in the skin. The modified protocol used to induce inflammatory swelling in the present study may be more effective than previous methods to investigate amphibian immune competence, particularly in nonmodel species.
FIG 1 Experimental design for injection of feet with inducers of inflammatory swelling. (A) X. laevis feet were injected intramuscularly in the middle of the foot on the plantar side (arrow). Measurements of the foot thickness (bracket) and width (perpendicular to the thickness) were recorded with a caliper. (B) Schematic of foot injections. Foot injections occurred on day 0 with either APBS, PHA alone, killed E. coli alone, PHA and B. dendrobatidis supernatant (Bd Sup), or killed E. coli and B. dendrobatidis supernatant. Intraperitoneal (i.p.) injection with PHA only occurred for defined experiments in which frogs were primed with PHA. The time points at which feet were measured are indicated (+).
FIG 2 Intramuscular injection of phytohemagglutinin (PHA) into the foot of X. laevis induces inflammatory swelling after 24 h (A, B, and E) and 48 h (C, D, and F). PHA injections induced significantly greater swelling than buffer (APBS) controls (*, P < 0.01; **, P < 0.001 [paired Student's t test; alpha set to 0.017 for multiple tests]). The data show the mean (± the SEM) increase in actual size (A and C) or percent increase (B and D) in foot size compared to each foot's measurement before injection from both feet of six frogs. Representative photographs of individuals at 24 h (E) or 48 h (F) after injection of APBS in the leftfoot and PHA into the rightfoot (pictures show ventral side of frogs).
FIG 3 Intramuscular injection of killed E. coli into the foot of X. laevis induces inflammatory swelling after 24 h. E. coli injections induced significantly greater swelling than buffer (APBS) controls (*, P < 0.01; **, P < 0.001 [paired Student's t test; alpha set to 0.017 for multiple tests]). The data show the mean (± the SEM) increase in actual size (A) or percent increase (B) in foot size compared to each foot's measurement before injection from both feet of six frogs. (C) Representative photograph of an individual 24 h after injection of APBS in the rightfoot and of killed E. coli into the leftfoot (the picture shows the ventral side of the frog).
FIG 4 B. dendrobatidis infection does not affect the number of leukocytes present in the spleen (splenocytes) of X. laevis. (A) Splenocyte number positively correlates with size in X. laevis. A significant correlation exists (P = 0.03) between the total body mass of individuals, and the total number of splenic leukocytes. n = 75, R = 0.25; the best fit line is shown. (B) No significant relationship exists between the infection load and relative number of splenocytes (relative to body mass) by regression analysis (all: P = 0.64; infected: P = 0.99). Bd Load, B. dendrobatidis load. (C) There is also no significant difference in the mean number of splenocytes (± the SEM) between individuals that were or were not infected with B. dendrobatidis by two-tailed Student's t test (P = 0.37). The number of splenocytes was normalized (B and C) to the size of each individual by dividing the total splenocytes by the mass in mg of the frog. The Bd load was determined by quantitative PCR using zoospore equivalent standards and log transformed. n = 50 for individuals that were not infected; n = 11 for infected individuals. The mean infection load (± the SEM) for infected individuals was 17.8 ± 0.9 zoospore equivalents.
FIG 5 B. dendrobatidis supernatant (Bd Sup) does not impair inflammatory swelling induced by a single PHA injection (frogs were not primed with PHA). X. laevis feet were injected with PHA alone or with PHA and Bd Sup. Feet were measured before injection and at 24 h (A and B) and 48 h (C and D) after injection. The data show the mean (± the SEM) increase in actual size (A, C) or percent increase (B, D) in foot size compared to each foot's measurement before injection from both feet of six frogs. Swelling was not significantly different between treatments (paired Student's t test).
FIG 6 B. dendrobatidis supernatant (Bd Sup) reduces inflammatory swelling induced by a second PHA injection. X. laevis feet were injected with PHA alone or with PHA and Bd Sup 7 days after priming with intraperitoneal injection of PHA. Feet were measured before injection and at 24 h (A, B, and E) and 48 h (C, D, and F) after injection. The data show the mean (± the SEM) increase in actual size (A and C) or percent increase (B and D) in foot size compared to each foot's measurement before injection from both feet of 12 frogs. Swelling was only significantly different between treatments in feet 48 h after injection. n.s., P > 0.017; *, P < 0.01; **, P < 0.001 (paired Student's t test; alpha set to 0.017 for multiple tests). Representative photographs of an individual at 24 h (E) and 48 h (F) after the injection of PHA and Bd Sup in the leftfoot and PHA alone into the rightfoot (the pictures show the ventral side of the frog).
FIG 7 B. dendrobatidis supernatant (Bd Sup) does not impair inflammatory swelling induced by killed E. coli. X. laevis feet were injected with either killed E. coli alone or E. coli with Bd Sup. Feet were measured before injection and 24 h after injection. The data show the mean (± the SEM) increase in actual size (A) or percent increase (B) in foot size compared to each foot's measurement before injection from both feet of 12 frogs. Swelling was not significantly different between treatments (paired Student's t test).
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