Sacbrood virus (SBV) infects larvae of the honeybee (Apis mellifera), resulting in failure to pupate and death. Until now, identification of viruses in honeybee infections has been based on traditional methods such as electron microscopy, immunodiffusion, and enzyme-linked immunosorbent assay. Culture cannot be used because no honeybee cell lines are available. These techniques are low in sensitivity and specificity. However, the complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a reverse transcription-PCR (RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. RT-PCR was used to target five different areas of the SBV genome using infected honeybees and larvae originating from geographically distinct regions. The RT-PCR assay proved to be a rapid, specific, and sensitive diagnostic tool for the direct detection of SBV nucleic acid in samples of infected honeybees and brood regardless of geographic origin. The amplification products were sequenced, and phylogenetic analysis suggested the existence of at least three distinct genotypes of SBV.
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.
BackgroundThe 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.ResultsTwo 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.ConclusionThe 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 m...
Red mark syndrome (RMS) or cold water strawberry disease (CWSD) is a non-lethal skin disease of rainbow trout Oncorhynchus mykiss that is of high economic importance in the UK. The disease is temperature-dependent, with up to 60% morbidity at water temperatures below 15°C. Although CWSD is horizontally transmissible, the aetiology is still unknown. Here we describe the first cases of RMS on the European mainland in the alpine regions of Switzerland and Austria. In Switzerland, morbidity remained around 1% after the first outbreak, whereas in Austria no further cases were diagnosed.KEY WORDS: Red mark syndrome · Coldwater strawberry disease · Oncorhynchus mykiss · Austria · Switzerland · Water temperature Resale or republication not permitted without written consent of the publisherDis Aquat Org 88: 65-68, 2009 al. 2008). It is notable that Lloyd et al. (2008) also reported no statistical association between SD lesions and F. psychrophilum based on PCR assays, but they did find a strong association between SD lesions and a Rickettsia-like organism. Verner-Jeffreys et al. (2008) proposed that RMS is caused by 1 or more infectious agents with a long latency (> 500 day-degrees).RMS was first reported in 2003 in Scotland. RMS seemed to spread in the UK, as it was subsequently diagnosed in England and Wales (Verner-Jeffreys et al. 2008). Outbreaks outside the UK have not been reported yet. Here we describe the first likely cases of RMS on the European mainland, in 2 Swiss and 1 Austrian trout farm. CASE STUDIES AND DISCUSSIONThe 2 affected Swiss farms exclusively raise rainbow trout. Despite belonging to the same owner, they are completely separated. Farm A consists of a hatchery and a grow-out facility, which are located at 2 different sites with separate water supplies. The farm produces 250 t of trout yr spring water with a temperature of 5 to 14°C. Eggs are supplied by brood stock kept in the grow-out facility. Additionally, each year 2 batches of eggs are imported from different farms in the European Union, but not from the UK. In the hatchery, fingerlings up to 50 g are reared in separate troughs and concrete raceway systems with gravel bottoms. Older animals are moved into the grow-out facility that is supplied by spring water at 250 l s -1 with water temperature of 4 to 14°C. Trout are reared in separate tanks and concrete raceway systems.Farm B produces 100 t of rainbow trout yr -1 hatched on site in recirculation units supplied with tap water. Afterwards, fish are reared in separated flow-through troughs and tanks and then moved to independent recirculation systems. The farm is supplied by 80 to 110 l s -1 spring water flowing in an open channel for about 1 km that is inhabited by wild fish. The water is not disinfected before entering the fish farm. Water temperature ranges from 5 to 15°C. In 2007, rainbow trout from Farm A were once transported to Farm B.Water parameters of both farms were checked regularly and showed no unusual variation. Rainbow trout from both farms regularly tested negative...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.