Molecular diagnostic methods for detection of Thelohania contejeani (Microsporidia), the causative agent of porcelain disease in crayfish

Abstract

Diagnosis of Thelohania contejeani in the crayfish Astacus astacus is currently based on observation of gross clinical signs -opaque appearance of the abdomen and whitish colouration of the musculature -and confirmed by microscopic examination of histological sections of muscle. We have developed 2 molecular diagnostic methods for sensitive and rapid detection of porcelain disease in its early stages: PCR and loop-mediated isothermal amplification (LAMP). The PCR test utilises a primer based on the T. contejeani small subunit ssu ribosomal RNA (ssu rRNA) gene and amplified parasite DNA with high specificity and a detection limit of 10 -5 dilution. The LAMP assay involves incubation of the target DNA with a set of 6 primers and Bst DNA polymerase for 60 min at 65°C in a water bath or heating block, followed by visualisation of the reaction products with the SYBR Green I stain; sensitivity of visual detection with SYBR Green I is equivalent to that with agarose gel electrophoresis. The LAMP assay can detect T. contejeani DNA to a dilution of 10 -7 . The LAMP assay is 100 times more sensitive than the PCR test and is the method we recommend as an alternative to traditional means of diagnosing T. contejeani. KEY WORDS: Thelohania contejeani · PCR · Loop-mediated isothermal amplification · Diagnosis · CrayfishResale or republication not permitted without written consent of the publisher

Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckerithe causative agent of enteric red mouth disease in fish

“…LAMP-based assays have been developed for numerous aquaculture animal pathogens, including white spot syndrome virus [ 30 ], yellow head virus [ 31 ], Edwardsiella tarda [ 32 ] and Nocardia seriolae [ 33 ], Tetracapsuloides bryosalmonae , Myxobolus cerebralis , Thelohania contejeani [ 34 - 36 ], Koi herpes virus (CyHV-3) and viral hemorrhagic septicaemia (VHS) [ 27 , 37 ]. The objective of this study was to develop and evaluate LAMP, as a simple, rapid and sensitive diagnostic tool for ERM disease.…”

Loop-mediated isothermal amplification (LAMP) based detection of bacteria: A Review

“…Li et al, 2009 Pseudomonas syringae pv. phaseolica 6.9x10 3 CFU/ml Hill et al, 2008 Escherichia coli 10 copies/reaction Salah et al, 2008 Renibacterium salmoninarum 1 pg Yamazaki et al, 2008 (a) Campylobacter jejuni 5.6 CFU/g Yamazaki et al, 2008 (b) V Boehme et al, 2007 Pulmonary tuberculosis 97.7% Kato et al, 2007 E. faecalis 10 µg/tube Aoi et al, 2006 Ammonia-oxidizing bacteri a 10 2 copies El-Matbouli et al, 2006 Thelohania contejeani 10 -5 Kamachi et al, 2006 Bordetella pertussis 10 fg/DNA tube Yeh et al, 2006 Flavobacterium columnare 30 pg/reaction tube Mukai et al, 2006 M. species 500 copies Kato et al, 2005 Clostridium difficile 50 ng-0.5pg Savan et al, 2005 Fish and shellfish pathogens 20 CFU Hara- Salmonella 2.2 CFU/test Saito et al, 2005 Mycoplasma pneumonia 2x10 2 copies Yeh et al, 2005 Edwardsiella ictaluri 20 CFU/ml El-Matbouli et al, 2005 Tetracapsuloides bryosalmonae 100 folds more sensitive Yoshida et al, 2005 Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola 1mg/tube(P.gingivalis),100fg/tube(T.forsythia),1m g/tube (T. denticola). Seki et al, 2005 S.. pneumonia 10 copies Maeda et al, 2005 Porphyromonas gingivalis 10 2 -10 6 cells Song et al, 2005 Shigella and enteroinvasive Escherichia coli 8 CFU/reaction Horisaka et al, 2004 Yersinia pseudotuberculosis 10 CFU Savan et al, 2004 Edwardsiellosis 3.8X10 2 CFU Enosawa et al, 2003 M. avium subsp.…”

Horizontal transmission of Thelohania contejeani in the endangered white-clawed (Austropotamobius pallipes) and the invasive signal crayfish (Pacifastacus leniusculus)

“…DNA was extracted from each sample using a phenol:chloroform method based on that of Kocher et al (1989). A nested PCR to amplify a portion of microsporidian small subunit ribosomal DNA was carried out using general microsporidian primers (all primers presented in 5′ -3′ orientation) V1f (CACCAGGTTGATTCT-GCCTGAC) and 1492r (GGTTACCTTGTTAC-GACTT) for the outer nest (Weiss et al 1994), and T. contejeani-specific primers F3 (AGCTAGTATG-TAGGGTAAGGGC) and B3 (ACTCTTGGAG-CTGGAATTACCG) for the inner nest (El-Matbouli et al 2006). See Table 1 for PCR protocols.…”

“…PCR protocols showing primers and expected fragment sizes, reagent quantities/concentrations, and PCR cycles for each reaction (Protocol for inner nest is that ofEl-Matbouli et al 2006. ) …”