Mona Saleh

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

BackgroundEnteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium Yersinia ruckeri. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the Pathogen using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel.ResultsA loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of Y. ruckeri the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the yruI/yruR gene, which encodes Y. ruckeri quorum sensing system, in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with HphI restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected Y. ruckeri not only in pure bacterial culture but also in tissue homogenates of infected fish.ConclusionThe ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of Y. ruckeri in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.

Detection of cyprinid herpesvirus type 3 in goldfish cohabiting with CyHV-3-infected koi carp (Cyprinus carpio koi).

has confirmed its place in the family Herpesviridae (Akoi and others 2007). The virus has had an impact on common carp that are raised for food fish production in Europe, Israel, Japan and Indonesia (Hedrick and others 2000, Perelberg and others 2003, Sano and others 2004, Hutoran and others 2005, Sunarto and others 2005).Outbreaks of

Recent progress in applications of nanoparticles in fish medicine: A review.

UNLABELLED Nanotechnology has become an extensive field of research due to the unique properties of nanoparticles, which enable novel applications. Nanoparticles have found their way into many applications in the field of medicine, including diagnostics, vaccination, drug and gene delivery. In this review, we focused on the antimicrobial effects of nanoparticles, with particular emphasis on the problem of antibiotic resistant bacteria in fisheries. The use of nanoparticle-based vaccines against many viral pathogens is a developing field in fish medicine research. Nanoparticles have gained much interest as a specific and sensitive tool for diagnosis of bacterial, fungal and viral diseases in aquaculture. Nevertheless our review also highlights the many applications of nanotechnology that are still to be explored in fish medicine. FROM THE CLINICAL EDITOR Advance in nanotechnology has enabled the development of nanomedicine, with many ideas being used in clinical diagnosis and therapy. In this review article, the authors described the current use of nanotechnology in fish medicine. The knowledge would also impart important information for our daily living.

Antibody-coated gold nanoparticles immunoassay for direct detection of Aeromonas salmonicida in fish tissues.

Aeromonas salmonicida is the causative agent of furunculosis, a disease that affects both salmonid and non-salmonid fish. Detection of A. salmonicida can be labour intensive and time consuming because of the difficulties in distinguishing the bacterium from other species given the wide variety of existing biochemical profiles and the slow growth characteristics which allow other organisms to overgrow the A. salmonicida. Herein, we report the development of a specific immunoassay using gold-conjugated polyclonal antibodies for the rapid detection of A. salmonicida in fish tissues. Monodispersible 13-nm gold nanoparticles were coated with polyclonal antibodies specific to A. salmonicida. Reddish purple agglutination of gold particles indicated the presence of A. salmonicida in samples. Positive reactions were detected visually with the naked eye. No agglutination was observed when A. salmonicida antibody-coated gold nanoparticles were tested with other common bacterial fish pathogens, thereby verifying the specificity of the assay. The assay could detect A. salmonicida in fish tissues down to 1 × 10(4)  CFU mL(-1) , and results were obtained within 45 min. The antibody-coated gold nanoparticles were stable for at least 2 months at 4 ° C. The immunoassay using antibody-coated gold nanoparticles represents a promising tool for the rapid and specific detection of A. salmonicida in fish tissues.

Fate of Tetracapsuloides bryosalmonae (Myxozoa) after infection of brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss

Tetracapsuloides bryosalmonae (Myxozoa) is the causative agent of proliferative kidney disease in salmonids. We assessed differences in intensity of T. bryosalmonae infection between brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss from the clinical phase of infection onwards. Specific pathogen-free fish were exposed to T. bryosalmonae spores under controlled laboratory conditions and sampled at 6, 8, 10, 12, 14, and 17 wk post exposure (wpe), and the transmission of T. bryosalmonae from infected fish to the bryozoan Fredericella sultana was observed. Parasite load was determined in fish kidneys by quantitative real-time PCR (qRT-PCR), and parasite stages were detected in kidney, liver, and spleen tissues at different time points by immunohistochemistry. T. bryosalmonae was successfully transmitted from infected brown trout to F. sultana colonies but not from infected rainbow trout. Body length and weight of infected brown trout did not differ significantly from control brown trout during all time points, while length and weight of infected rainbow trout differed significantly compared to controls from 10 to 17 wpe. qRT-PCR revealed that parasite load was significantly higher in kidneys of brown trout compared with rainbow trout. Immunohistochemistry showed high numbers of intra-luminal stages (sporogonic stages) in kidneys of brown trout with low numbers of pre-sporogonic stages. Sporogonic stages were not seen in kidneys of rainbow trout; only high numbers of pre-sporogonic stages were detected. Numbers of pre-sporogonic stages were low in the spleen and liver of brown trout but high in rainbow trout. These data confirmed that there are differences in the development and infection progress of T. bryosalmonae between brown trout and rainbow trout.

Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease

A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Renibacterium salmoninarum in 1 h without thermal cycling. A fragment of R. salmoninarum p57 gene was amplified at 63 degrees C in the presence of Bst polymerase and a specially designed primer mixture. The specificity of the BKD-LAMP assay was demonstrated by the absence of any cross reaction with other bacterial strains, followed by restriction digestion of the amplified products. Detections of BKD-LAMP amplicons by visual inspection, agrose gel electrophoresis, and real-time monitoring using a turbidimeter were equivalently sensitive. The BKD-LAMP assay has the sensitivity of the nested PCR method, and 10 times the sensitivity of one-round PCR assay. The lower detection limit of BKD-LAMP and nested PCR is 1 pg genomic R. salmoninarum DNA, compared to 10 pg genomic R. salmoninarum DNA for one-round PCR assay. In comparison to other available diagnostic methods, the BKD-LAMP assay is rapid, simple, sensitive, specific, and cost effective with a high potential for field application.

A novel gold nanoparticles-based assay for rapid detection of Melissococcus plutonius, the causative agent of European foulbrood

European foulbrood (EFB) is a severe bacterial brood disease of honey bees (Apis mellifera) caused by Melissococcus plutonius. Diagnosis of EFB in the field is based on visual inspection of brood-combs and detection of diseased larvae. However, symptoms of EFB may be easily obscured by other diseases or abnormalities in the brood, making definitive diagnosis difficult. Hence, confirmatory laboratory assays, such as PCR and real-time PCR, are used to verify the presence of M plutonius in suspected colonies. While these methods are accurate and specific, they are time consuming and labour intensive. Herein, we report development of a label-free colorimetric nanodiagnostic method for direct detection of unamplified M plutonius DNA using unmodified gold nanoparticles. Under appropriate conditions, the DNA probes hybridised with their complementary target sequences in the sample DNA, which resulted in aggregation of the gold nanoparticles and a concomitant red to blue colour change, which was observed visually. The assay could detect as few as 25 copies of the M plutonius cell wall-associated protease gene within 20 minutes. The assay results were in 100 per cent concordance with real-time PCR-positive and PCR-negative samples. Our study demonstrated that the gold nanoparticles-based assay is a specific and sensitive tool for rapid detection of M plutonius.

Direct detection of unamplified spring viraemia of carp virus RNA using unmodified gold nanoparticles.

Spring viraemia of carp (SVC) is a viral disease that mainly affects carp Cyprinus carpio and other cyprinid fish, causing severe economic losses. Rapid detection and identification of spring viraemia of carp virus (SVCV) is crucial for effective disease management. Recent advances in nanoscience are having a significant impact on many scientific fields, especially biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection. Single- and double-stranded oligonucleotides can be adsorbed on gold nanoparticles (AuNPs) in colloidal solution under certain conditions. We exploited this phenomenon to develop a specific hybridization assay for direct detection of SVCV-RNA without prior amplification. The result of the hybridization process could be detected visually within 1 min when the colour of the reaction mixture changed from red to blue (positive reaction) or remains red (negative). The lower detection limit of the assay was estimated to be 10-3 TCID50 ml-1 SVCV-RNA, and it has the feasibility to detect the target virus-RNA in clinical specimens without previous amplification. In order to obtain an indication of the assays performance on clinical samples we compared the optimized assay with nested RT-PCR in detection of SVCV-RNA in infected fish samples. The concordance of the 2 methods was defined as 100% when compared to nested RT-PCR positive and negative samples. The SVC-AuNPs assay requires only 15 min, eliminates the need for thermal cycling or detection instruments and is a specific and rapid tool for detection of SVCV-RNA directly from clinical samples.

In vitro antimicrosporidial activity of gold nanoparticles against Heterosporis saurida

BackgroundWorldwide, there is a need to expand the number of drugs available to treat parasitic infections in aquaculture. One of the new materials being tested is metal nanoparticles, which have unique chemical and physical characteristics owing to their extremely small size and high surface area to volume ratio. We examined the effectiveness of gold nanoparticles against the microsporidian parasite Heterosporis saurida, which causes severe economic losses in lizard fish, Saurida undosquamis aquaculture.ResultsWe synthesized gold nanoparticles by chemical reduction of tetrachloroauric acid as a metal precursor. We assessed the antimicrosporidial efficacy of the nanoparticles against H. saurida using an in vitro screening approach, which we had developed previously using the eel kidney cell line EK-1. The number of H. saurida spores produced in EK-1 cells was reduced in a proportional manner to the dosage of gold nanoparticles administered. A cell metabolic activity test (MTT) indicated that the gold nanoparticles did not appear to be toxic to the host cells.ConclusionsGold nanoparticles can act as an effective antimicrosporidial agent and hold promise to reduce disease in lizardfish aquaculture. Metal nanoparticles should be considered as an alternate choice for development of new antimicrosporidial drugs to combat disease problems in aquaculture.

Rapid detection of Cyprinid herpesvirus-3 (CyHV-3) using a gold nanoparticle-based hybridization assay

Cyprinid herpesvirus-3 (CyHV-3) is a highly infectious pathogen that causes fatal disease in common and koi carp Cyprinus carpio L. CyHV-3 detection is usually based on virus propagation or amplification of the viral DNA using the PCR or LAMP techniques. However, due to the limited susceptibility of cells used for propagation, it is not always possible to successfully isolate CyHV-3 even from tissue samples that have high virus titres. All previously described detection methods including PCR-based assays are time consuming, laborious and require specialized equipment. To overcome these limitations, gold nanoparticles (AuNPs) have been explored for direct and sensitive detection of DNA. In this study, a label-free colorimetric nanodiagnostic method for direct detection of unamplified CyHV-3 DNA using gold nanoparticles is introduced. Under appropriate conditions, DNA probes hybridize with their complementary target sequences in the sample DNA, which results in aggregation of the gold nanoparticles and a concomitant colour change from red to blue, whereas test samples with non complementary DNA sequences remain red. In this study, gold nanoparticles were used to develop and evaluate a specific and sensitive hybridization assay for direct and rapid detection of the highly infectious pathogen termed Cyprinid herpesvirus-3.