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16 pages, 4095 KiB

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Loop-Mediated Isothermal Amplification for the Fast Detection of Bonamia ostreae and Bonamia exitiosa in Flat Oysters

by Irene Cano, Gareth Wood, David Stone, Mathilde Noyer, Lydie Canier and Isabelle Arzul

Pathogens 2024, 13(2), 132; https://doi.org/10.3390/pathogens13020132 - 30 Jan 2024

Cited by 1 | Viewed by 1629

Abstract

The haplosporidian parasites Bonamia ostreae (BO) and B. exitiosa (BE) are serious oyster pathogens. Two independent laboratories evaluated fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for rapidly detecting these parasites. Specific LAMP assays were designed on the BO actin-1 and BE actin genes. A further generic assay was conceived on a conserved region of the 18S gene to detect both Bonamia species. The optimal reaction temperature varied from 65 to 67 °C depending on the test and instrument. Melting temperatures were 89.8–90.2 °C, 87.0–87.6 °C, and 86.2–86.6 °C for each of the BO, BE, and generic assays. The analytical sensitivity of these assays was 50 copies/µL in a 30 min run. The BO and BE test sensitivity was ~1 log lower than a real-time PCR, while the generic test sensitivity was similar to the real-time PCR. Both the BO and BE assays were shown to be specific; however, the generic assay potentially cross-reacts with Haplosporidium costale. The performance of the LAMP assays evaluated on samples of known status detected positives within 7–20 min with a test accuracy of 100% for the BO and generic tests and a 95.8% accuracy for BE. The ease of use, rapidity and affordability of these tests allow for field deployment. Full article

(This article belongs to the Special Issue Aquatic Animal Pathogens: Epidemiology, Pathogenesis, Diagnosis, Preventive and Therapeutic Measures and Host–Pathogen Relationships)

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11 pages, 2261 KiB

Open AccessArticle

Bonamia exitiosa in European Flat Oyster (Ostrea edulis) on the Croatian Adriatic Coast from 2016 to 2020

by Dražen Oraić, Relja Beck, Željko Pavlinec, Ivana Giovanna Zupičić, Ljupka Maltar, Tihana Miškić, Žaklin Acinger-Rogić and Snježana Zrnčić

J. Mar. Sci. Eng. 2021, 9(9), 929; https://doi.org/10.3390/jmse9090929 - 27 Aug 2021

Cited by 3 | Viewed by 2926

Abstract

The annual production of European flat oysters (Ostrea edulis) in Croatia is about 50 to 65 tons, and it has a long tradition. All Croatian oyster farms are subjected to the national surveillance program aiming to detect the presence of Bonamia ostreae and Marteilia refringens according to the Council Directive 2006/88/EC. Within the surveillance program, the first findings of the parasite Bonamia spp. occurred in 2016 in two production areas in the north and south of the Eastern Adriatic coast. The repeated findings of the parasite were noted up to 2020 but also on two additional sites in the north. The parasite was detected by cytological analysis of stained heart smears, histological examination, and PCR. PCR positive samples were sequenced for SSU rDNA gene, and BLAST analysis confirmed infection with Bonamia exitiosa. Attempts to prove the Pacific oyster as a putative vector of the parasite failed. The infection prevalence from 2016 until 2020 ranged from 3.3 to 20% in different sites. No mortalities were reported from the infected sites, and it seemed that infection of flat oysters with B. exitiosa did not affect their health. The study has not shown the source and way of infection spread, which imposes the need for more comprehensive molecular and epidemiological studies. Full article

(This article belongs to the Special Issue Microbial Diseases of Marine Organisms)

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11 pages, 923 KiB

Open AccessArticle

Transcriptional Alteration of Gene Biomarkers in Hemocytes of Wild Ostrea edulis with Molecular Evidence of Infections with Bonamia spp. and/or Marteilia refringens Parasites

by Paolo Cocci, Alessandra Roncarati, Martina Capriotti, Gilberto Mosconi and Francesco Alessandro Palermo

Pathogens 2020, 9(5), 323; https://doi.org/10.3390/pathogens9050323 - 26 Apr 2020

Cited by 7 | Viewed by 3084

Abstract

The European flat Ostrea edulis is highly susceptible to intracellular parasitic infections, particularly bonamiosis and marteiliosis. The defensive response of oyster to both bonamiosis and marteiliosis is typically mediated by hemocytes, which play a pivotal role in immune system homeostasis. In the present study, we first used a DNA-based tool in order to rapidly and specifically detect the presence of parasites in oysters from natural banks in the middle Adriatic Sea. In a second step, we used qRT-PCR to analyze the mRNA levels of a set of genes (i.e., superoxide dismutase (SOD), glutathione S-transferase (GST), metallothionein (MT), heat shock protein (HSP) 70 and 90, inhibitor of apoptosis (IAP), fas ligand (FAS), galectin (GAL) and extracellular superoxide dismutase (Ec-SOD)) expressed by hemocytes of flat oysters infected by the parasites, present singularly or in combination, compared to hemocytes from non-infected specimens. The results indicate that the presence of parasite DNA may be associated to a general upregulation of host genes related to apoptosis, detoxification and oxidative stress protection, with the exception of Ec-SOD, whose trend to a downregulation might reflect a mechanism for parasite escape before internalization. Full article

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