A twelve-month extension of project 2001/621 is required to complete validation of the PCR primers for A. invadans and to continue testing the primers for A. astaci.
The primers for A. invadans (Epizootic Ulcerative Syndrome – EUS) are specific when tested on DNA from other Oomycete fungi, however need to be tested on tissue samples and wax-embedded tissue. The development of the fluorescent in-situ hybridization method (FISH) also needs to continue. Two isolates previously identified as A. invadans and used in research performed at other laboratories, do not amplify or produce a weak amplicon with the primers designed for A. invadans. This area needs investigation to determine the true identity of these two isolates.
The development and specificity of the primers for A. astaci (Crayfish Plague) needs to be completed. Sequence information for the chitinase and trypsin protease virulence genes of A. astaci were used to design a number of sets of primers including nested primers, which provide greatly increased sensitivity. However, product has not been amplified and we need to investigate this. We are confident with the DNA extraction technique, which can amplify fungal DNA from formalin-fixed crayfish using universal primers, therefore it is not known why the virulence genes cannot be amplified from the infected formalin-fixed crayfish tissue.
The primers will be assessed by trialing them at selected laboratories within Australia and overseas. Once this is completed the primers along with negative and positive controls can be developed into kits.
To ensure that reliable and robust diagnostic tests are developed for EUS and Crayfish Plague, this project needs to continue so that further research can be carried out to achieve these aims. Trialing the kits and assessing feedback from laboratories will greatly improve the uptake of these tests and also add to user confidence in these kits.
Crayfish plague and epizootic ulcerative syndrome are two fungal diseases that affect freshwater crayfish and freshwater finfish, respectively. Crayfish plague, which is caused by Aphanomyces astaci, is exotic to Australia, but is capable of causing massive stock losses of up to 100% which would be catastrophic should the disease occur in Australia. It is present in North America where the native freshwater crayfish are largely resistant to the disease and can act as carriers of the fungus. Crayfish plague was transferred to Europe in the 1870’s through infected stock and has wiped out many of the native freshwater crayfish in a number of countries. Epizootic ulcerative syndrome (EUS), caused by infection with Aphanomyces invadans, is endemic in many fish in river systems throughout Australia. The disease causes economic losses to the freshwater finfish aquaculture industry and affects local native stocks. Both diseases are almost impossible to eradicate.
Both diseases are difficult to diagnose, especially if laboratory personnel are not sufficiently skilled in recognising or differentiating the diseases. The culture and identification of both fungal species can be difficult and prolonged. Isolation and identification may be hampered by overgrowth from other fungi that contaminate the lesion or the isolation medium. Culture is tedious and may take up to 15 days before fungal growth is seen. It can then be difficult to identify the fungus species and often requires the culture being sent to a specialist laboratory for confirmation of identification. Examination of histological samples requires expertise and although the presence of hyphae of 7-10 µm in width is suggestive of Aphanomyces, it is not definitive.
Early detection and diagnosis of the disease is crucial to ensure rapid disease response and containment, particularly for crayfish plague, which is exotic to Australia and the Asian region. Therefore, there was a need to improve diagnostic capabilities of Australian laboratories in line with the Federal Budget Initiative “Building a National Approach to Animal and Plant Health” funding to improve disease diagnosis in aquaculture.
The research undertaken in this project sought to provide two diagnostic tests for each disease based on molecular (DNA) biological techniques, polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) using peptide-nucleic acid probes. In both techniques, identification is based on detection of a DNA sequence that is unique to the target organism. The uniqueness of the DNA sequence is demonstrated by testing specificity against other genetically similar organisms or those that may be found in the same ecological location. For detection and identification of A. invadans the tests were applied to fungal culture material, fresh tissue and formalin-fixed paraffin-embedded tissue. For A. astaci the tests were applied to formalin-fixed paraffin-embedded material.
Optimal extraction of DNA from samples is of prime importance for achieving an accurate and sensitive result. Two commercial methods were suitable; the DNAzol reagent (Invitrogen, Life Technologies) and the DNeasy mini plant tissue kit (Promega).
A PCR that was specific to A. invadans was developed. The PCR can be used on culture material and fresh fish tissue using primers AIF14 + AIR10. The test is specific and does not show cross-reaction with closely related oomycete fungi such as Saprolegnia species or fungal species that are likely to occur as plate contaminants on laboratory media, such as Aspergillus species and Penicillium species. The primers, which produce an amplicon of 554 base pairs (bp), were unable to amplify the DNA from paraffin-embedded tissue. This is most likely due to the effect of the formalin fixative on the DNA, which in the process of fixing the tissue cross-links amino acids groups. This cross-linking makes it difficult for primers that produce a large amplicon (greater than 500 bp) to amplify the DNA. A different fixative may be needed for these types of samples.
Four of the thirteen primers designed to amplify a sequence from the virulence genes (chitinase and trypsin proteinase) produced strongly staining amplicons. These were validated against two primer pairs published previously (Oidtmann et al., 2004, 2006). Primers 525f and 640 r published in 2004 were more sensitive and had the advantage that they detected DNA from formalin-fixed paraffin-embedded sections. However, they were not as specific as the primers developed in this project. Primers published in 2006 were specific but because they produced a large amplicon they were not tested on paraffin-embedded tissue. Further work is required before a specific and sensitive PCR for A. astaci can be recommended for diagnostic laboratories.
Peptide nucleic acid probes (PNA) for use in the fluorescent in situ hybridisation (FISH) method were developed for both A. invadans and A. astaci. The PNA-FISH for A. invadans was specific and successfully applied to fresh cultured hyphae. Tests on aged hyphae in samples such as formalin-fixed paraffin-embedded sections yielded a reduced fluorescent signal. The PNA-FISH for A. astaci could only be tested on paraffin-embedded sections and the same reduced fluorescent signal occurred with this species. Further work needs to be done to optimise the PNA-FISH for both fungal species using paraffin-embedded material.
Keywords: PCR, fluorescent in-situ hybridization, FISH, epizootic ulcerative syndrome (EUS), Crayfish plague, Aphanomyces astaci, Aphanomyces invadans.