Over 20% of our samples tested positive for
Bd infection (). We detected no fungus in native bullfrogs from Ontario even though our sampling effort per pond was sufficient to detect 10% disease prevalence (
DiGiacomo & Koepsell 1986). However, other research has shown that
Bd has been present in bullfrogs from Eastern Canada since the 1960s (
Ouellet et al. 2005), suggesting that there is spatial heterogeneity in infection within this region. Samples from Japan exhibited no sign of
Bd infection. No reports of mass die-offs in Japan are published and amphibian declines are attributed to factors other than infectious disease (
Ota 2000). Asia continues to stand out as an area rich in amphibian species but where
Bd appears absent. Whether this truly indicates low prevalence or a lack of adequate screening for the pathogen remains to be seen, but it is notable that in one of our populations, sample sizes were large enough to have been able to detect a 10% prevalence of infection (
DiGiacomo & Koepsell 1986). In general, New World populations in our study exhibited high prevalence () and extremely high burdens (), the latter of which differed significantly among the two regions (
t0.05(2),20=2.086,
p=0.035). We do not know why this is, but reduced burden does not mean Europe is relatively
Bd-free.
Batrachochytrium dendrobatidis is known to occur in six European countries and in over 20 species of European amphibian (
Garner et al. 2005). Our study adds France to the list of European countries with amphibians that are known to be infected with
Bd.
| Table 2Genomic equivalents (GE) for all positive bullfrog samples in this study. (GE is corrected for a 1/10 dilution factor.) |
Histological sections of positive samples from Canada (Vancouver Island), South America, France, the UK and Italy contained typical signs of
Bd infection: hyperkeratosis, along with empty and zoospore-bearing zoosporangia embedded in the stratum corneum and stratum granulosum of bullfrog digits (). Sequences were generated from bullfrogs sampled in British Columbia, Canada (
n=3 Beaver Lake Ponds,
n=5 Maltby Lake) and France (
n=3 Loir et Cher). We scored 238 bases of the
ctsyn1 locus and detected the single homo/heterozygote polymorphism published by
Morehouse et al. (2003) located at position 117 of our sequences. Beaver Lake frogs included both homozygotes (2A, 1G), Maltby Lake samples included all three genotypes (1A, 3G, 1A/G) and Loir et Cher samples included both homozygotes (1A, 2G). Research by
Morehouse et al. (2003) previously described heterozygosity at the
ctsyn1 locus in 32 out of 35
Bd isolates, including three bullfrog-derived isolates. The authors concluded that excess heterozygosity at the
ctsyn1 and another locus was evidence for rare, ancestral recombination, and that the predominant mode of reproduction in this species is clonal (
Morehouse et al. 2003). The life history of
Bd certainly suggests that recombination should be rare, as no sexual structures have yet been identified, and fixed heterozygosity is accepted evidence for an absence of typical segregation at meiosis. However, our results provide no evidence that heterozygosity is fixed, as only one of our 11 samples was heterozygous, and we observed nearly equal numbers of each homozygote.
Bullfrog introductions have occurred throughout western North America (
Kats & Ferrer 2003). While the relationship between bullfrog introductions and the spread of
Bd has not been clearly established, studies of this area suggest
Bd may be playing a powerful role in species declines. The Boreal toad (
Bufo boreas) has tested positive for infection in the wild (
Annis et al. 2004), suffers increased mortality after experimental exposure to
Bd (
Blaustein et al. 2005) and a recent analysis shows that
Bd is the most likely agent of toad declines in the region (
Scherer et al. 2005). In contrast, Pacific tree frogs (
Pseudacris regilla) do not seem to be affected by bullfrog introduction (
Kupferberg 1997;
Govindarajulu 2004) and experimental
Bd exposure had no effect on
P. regilla (
Blaustein et al. 2005). We have detected infection in adult and apparently healthy adult
P. regilla (T. W. J. Garner & P. Govindaraju 2005, unpublished data).
It is likely that disease, predation and competition all influence native species after bullfrog introductions. Amphibian declines are frequently due to a complexity of causative agents (
Adams 1999;
Blaustein & Kiesecker 2002;
Relyea 2003;
Pearman & Garner 2005), and though
Bd alone can be responsible for rapid declines (
Bosch et al. 2001), predation and competition would exacerbate the process; experiments have shown significant interactions between predators and
Bd exposure (
Parris & Beaudoin 2004). Although introduced bullfrogs can be removed as a conservation measure,
Bd likely has a capacity to persist across a variety of environmental conditions (
Piotrowski et al. 2004). Predation and interspecific competition are therefore manageable, but removing
Bd from the environment has yet to be accomplished. Conservation plans for amphibian populations invaded by bullfrogs involve eliminating bullfrogs and the reintroduction of native species. These projects will likely yield unsatisfactory results until amphibian pathogens are also treated as invasive.
This article has been 
μl initial PCR product, 1.25