Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci.

BACKGROUND: The oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes. RESULTS: Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences. CONCLUSION: The simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods. Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.

Toxicity assessment of wastewaters, river waters, and sediments in Austria using cost-effective microbiotests.

The toxicity and chemical quality of surface water and sediment in the River Traun in Austria were studied because of recurrent fish mortality in some alpine rivers over the last few years. The analyses were carried out on samples collected during winter and summer upstream and downstream of two municipal wastewater treatment plants (WWTPs) and on effluents taken at the points of discharge of these two plants. Toxicity tests were performed on 20 samples of surface water, effluent, and sediment pore water. The test battery was composed of microbiotests with protozoans (Protoxkit F), microalgae (Algaltoxkit F), crustaceans (Daphtoxkit F magna and Thamnotoxkit F), and a higher plant (seed germination and root elongation assay on cress). Direct contact tests were performed on whole sediment with crustaceans (Ostracodtoxkit F). The physical-chemical characteristics of the surface water, effluent, and sediment pore water samples analyzed were conductivity, total hardness, pH, O(2), BOD(5), TOC, DOC, AOX, NH(4), NH(3), NO(2), PO(4)--P, Cd, Pb, Cu, and Zn. The toxicity data were expressed as percentage mortality or percentage inhibition, depending on the effect criterion of the respective assay. None of the water samples collected upstream and downstream of the WWTPs showed any significant (short-term) toxicity in either winter or in summer, but the effluents of the first municipal wastewater treatment plant were toxic to some of the test biota. All the sediment pore water samples induced serious inhibition of root growth of cress, and several pore waters were toxic to other test biota as well, particularly at the outlets of the WWTPs. The toxic character of some sediments was confirmed by direct contact tests with the ostracod crustacean. The chemical analyses did not reveal particularly high concentrations of any chemical that is very toxic. As a result no direct causal relationship could be established between the detected toxic effects and the chemical composition of the surface waters or sediment pore waters. The outcome of this preliminary study again highlights the need to complement chemical analyses with toxicity tests to determine the toxic hazard to aquatic environments that may be threatened by contamination. Furthermore, the investigations also confirmed the need to apply a battery of tests for an ecologically meaningful evaluation of the hazards of waters, sediments, and wastewaters. Finally, the results of the 360 bioassays performed show that culture-independent microbiotests are practical and reliable tools for low-cost toxicity monitoring of aquatic environments.