Effects of the insecticide chlorpyrifos, on hatching, mortality and morphology of Duttaphrynus melanostictus embryos.

In an attempt to assess the effects of chlorpyrifos [O,O-diethyl O-(3,5,6-trichloropyridin-2-yl) phosphorothioate], the second largest selling insecticide in India, studies were made with reference to some non-target organisms. The present study was undertaken to evaluate the effects in the embryos of Duttaphrynus melanostictus caused by the commercial formulations of chlorpyrifos (Tricel, chlorpyrifos, 20% EC). The LC50 value for Duttaphrynus melanostictus embryos after 48 h (h) of treatment with chlorpyrifos was found to be 57.525 ppm. The mortality of the embryo was significantly affected by different concentrations of chlorpyrifos when compared with the control groups. An increase in concentration of chlorpyrifos resulted in the simultaneous decrease of the hatching percentage and an increase in the morphological abnormalities such as compression of the embryo, reduced body size and curling of tail.

A novel cathelicidin from Bufo bufo gargarizans Cantor showed specific activity to its habitat bacteria.

Toad Bufo bufo gargarizans Cantor is still used in China as traditional Chinese medicine. However, present investigations on its skin secretions were mainly focused on the bufadienolides, the proteins/peptides contained in the secretions are largely unknown. A cDNA encoding a novel cathelicidin termed BG-CATH was identified by analysis of the toad skin transcriptome. The BG-CATH precursor was predicted to have 2 possible cleavage sites following dibasic cleavage signals at its C-terminal, which will generate two mature peptides, BG-CATH37 and BG-CATH(5-37). Phylogenetic analysis suggests that amphibian cathelicidins might evolve from common ancestors. The two predicted mature cathelicidins from B. bufo gargarizans were synthesized and both of them showed weak antimicrobial activities against human pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus (MIC ≥ 200 µg/mL). However, BG-CATH37 and BG-CATH(5-37) had strong antimicrobial activities against aquatic bacteria of Vibrio splendidus, Streptococcus iniae and Aeromorus hydrophila, which were common microorganisms in the habitat of B. bufo gargarizans (MIC 3.125-40 µg/mL). BG-CATH37 and BG-CATH(5-37) showed no hemolytic activity even at high concentrations (400 µg/mL). CD spectra analysis suggested that structure rigidity of BG-CATH37 and BG-CATH(5-37) might play an important role to regulate their biological activities. Selective antimicrobial activity against habitat microorganisms might reflect the adaptation of amphibians to their living environments.

Migrating common toads (Bufo bufo) in rural temperate regions: reservoirs of Salmonella?

Salmonella infections in amphibians are supposedly highly prevalent. Migrating common amphibian species in cultivated areas such as common toads (Bufo bufo) may thus promote spread and zoonotic transfer of Salmonella to humans, both indirectly by crop and livestock contamination and by direct contact. Between February and April 2011, the intestinal content of 1,740 samples of road-killed migrating common toads in five Flemish provinces of Belgium was examined for the presence of Salmonella using bacterial culture and PCR. All the samples were negative. These results suggest that the role of migrating common toads in maintaining the infection cycle of Salmonella in northern European temperate regions is negligible.

Proteomic and phenotypic profiling of the amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked to virulence.

Population genetics of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) show that isolates are highly related and globally homogenous, data that are consistent with the recent epidemic spread of a previously endemic organism. Highly related isolates are predicted to be functionally similar due to low levels of heritable genetic diversity. To test this hypothesis, we took a global panel of Bd isolates and measured (i) the genetic relatedness among isolates, (ii) proteomic profiles of isolates, (iii) the susceptibility of isolates to the antifungal drug caspofungin, (iv) the variation among isolates in growth and phenotypic characteristics, and (v) the virulence of isolates against the European common toad Bufo bufo. Our results show (i) genotypic differentiation among isolates, (ii) proteomic differentiation among isolates, (iii) no significant differences in susceptibility to caspofungin, (iv) differentiation in growth and phenotypic/morphological characters, and (v) differential virulence in B. bufo. Specifically, our data show that Bd isolates can be profiled by their genotypic and proteomic characteristics, as well as by the size of their sporangia. Bd genotypic and phenotypic distance matrices are significantly correlated, showing that less-related isolates are more biologically unique. Mass spectrometry has identified a set of candidate genes associated with inter-isolate variation. Our data show that, despite its rapid global emergence, Bd isolates are not identical and differ in several important characters that are linked to virulence. We argue that future studies need to clarify the mechanism(s) and rate at which Bd is evolving, and the impact that such variation has on the host-pathogen dynamic.

A case of pulmonary cryptococcosis in a free-living toad (Bufo bufo).

Pulmonary cryptococcosis was observed in a free-living adult female common toad (Bufo bufo) that was killed by a vehicle. Both lungs had various eosinophilic, monomorphic, and spherical to elliptical organisms identified as Cryptoccocus spp. The yeasts were demonstrated by Grocott's silver method and the periodic acid-Schiff reaction and the capsule was positive for mucin with a mucicarmine stain. The agent was confirmed by immunohistochemistry, using the monoclonal antibody anti-Cryptococcus neoformans, and by a polymerase chain reaction-based method using a C. neoformans-specific primer. This report, to the best of our knowledge, represents the first case of cryptococcosis in a common toad.

Persistence of the emerging pathogen Batrachochytrium dendrobatidis outside the amphibian host greatly increases the probability of host extinction.

Pathogens do not normally drive their hosts to extinction; however, Batrachochytrium dendrobatidis, which causes amphibian chytridiomycosis, has been able to do so. Theory predicts that extinction can be caused by long-lived or saprobic free-living stages. The hypothesis that such a stage occurs in B. dendrobatidis is supported by the recent discovery of an apparently encysted form of the pathogen. To investigate the effect of a free-living stage of B. dendrobatidis on host population dynamics, a mathematical model was developed to describe the introduction of chytridiomycosis into a breeding population of Bufo bufo, parametrized from laboratory infection and transmission experiments. The model predicted that the longer that B. dendrobatidis was able to persist in water, either due to an increased zoospore lifespan or saprobic reproduction, the more likely it was that it could cause local B. bufo extinction (defined as decrease below a threshold level). Establishment of endemic B. dendrobatidis infection in B. bufo, with severe host population depression, was also possible, in agreement with field observations. Although this model is able to predict clear trends, more precise predictions will only be possible when the life history of B. dendrobatidis, including free-living stages of the life cycle, is better understood.