Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fish. In this study, a multienzyme isothermal rapid amplification (MIRA) assay targeting a conserved region of the IHNV N gene was established for rapid detection. The performance of the assay was evaluated using IHNV RNA standards, and further compared with digital PCR (dPCR) using 8 IHNV isolates, 12 cell culture supernatants, and 67 tissue samples from rainbow trout suspected of IHNV infection. The MIRA assay produced detectable signals within approximately 20 min. A detection rate of 100% was achieved at 10 2 copies μl -1 using RNA standards. The assay successfully detected all 8 IHNV isolates and showed no cross-reaction with non-target fish viruses. For cell culture supernatants and tissue samples, the MIRA assay showed a high level of agreement with dPCR results. These findings indicate that the MIRA assay may serve as a rapid and practical method for IHNV detection, particularly for on-site screening and preliminary diagnosis in resource-limited settings.
Enteromyxum leei is a significant myxosporean parasite associated with emaciation syndrome in cultured fish, particularly olive flounder Paralichthys olivaceus in Korea, that can lead to substantial economic losses. Accurate quantification of parasite load is crucial for monitoring disease progression and assessing infection intensity. While 18S rDNA-targeted quantitative PCR (qPCR) has been widely used, the inherent copy-number plasticity of ribosomal arrays across different developmental stages limits its precision for absolute quantification, often introducing unpredictable signal inflation. In this study, we developed and validated a novel qPCR assay targeting the minicollagen ( ncol- 2 ) gene, a putative low-copy structural gene expected to maintain a relatively stable genomic ratio per nucleus. The ncol-2 qPCR assay demonstrated high linearity (R 2 = 0.999) and an amplification efficiency of 95.4% over a range of 10 2 to 10 7 copies μl -1 , with a robust limit of detection (LoD 95 ) of 8.57 copies μl -1 . The assay showed high specificity with no cross-reactivity against other myxosporean parasites and exhibited acceptable repeatability across different real-time PCR platforms. Comparative analysis using 100 clinical samples revealed a strong correlation (r = 0.978) between the ncol-2 and 18S rDNA assays. Notably, copy numbers estimated by the ncol-2 assay were, on average, 29.68-fold lower than those from the 18S rDNA assay, with a marked sample-to-sample fluctuation (SD ± 13.8). This disparity demonstrates that ncol-2 effectively reduces copy-number variability associated with ribosomal gene duplication, providing a more biologically accurate and standardized molecular tool for improved quantification of E. leei in both diagnostic and epidemiological research.
Mud crab reovirus (MCRV) has emerged as a major challenge in mud crab farming, often leading to sudden mortality and severe economic losses. This study documents an outbreak of MCRV in a Scylla olivacea fattening facility at Bideipur, Odisha, India. Crabs in poor health condition were collected for analysis. The affected animals were sluggish, showed a grey and unhealthy hepatopancreas with autotomy. Histological examination revealed basophilic inclusion bodies together with necrotic changes in the hepatopancreas, gills, and muscle. Transmission electron microscopy confirmed the presence of non-enveloped, icosahedral viral particles arranged in crystalline arrays predominantly within the cytoplasm, together with nuclear alterations including chromatin condensation and nucleolar displacement. Reverse transcription (RT)-PCR produced a distinct amplification at ~433 bp, verifying MCRV infection. Experimental challenges using oral exposure, waterborne transmission, and cohabitation all resulted in complete mortality, demonstrating both the virulence of the virus and its ease of transmission. This study provides the first evidence of MCRV infecting S. olivacea in India. This outbreak underlines the need for consistent health screening, the use of virus-free seed, and strict biosecurity protocols if mud crab aquaculture is to remain sustainable.
Tilapia (genus Oreochromis ) contribute significantly to food security and aquaculture development in the Department of San Martín, an administrative region in the Peruvian Amazon. During the final quarter of 2024, an investigation was carried out following reports of infectious disease outbreaks associated with unusually high mortality in farmed tilapia located in the provinces of Mariscal Cáceres and Bellavista. A total of 153 samples were analysed, including 120 broodstock, 24 fry, and 9 juvenile specimens. PCR testing detected Streptococcus agalactiae in at least 1 juvenile sample from each of Farms A and B, representing the first confirmed cases of S. agalactiae associated with the outbreaks. In the hatchery, microbiological analysis of broodstock and fry samples yielded 6 bacterial isolates compatible with Streptococcus spp., which were subsequently confirmed as S. agalactiae by PCR. At Farm C, S. agalactiae was confirmed through PCR, and 3 bacterial isolates (P01, P02, and P03) were recovered and comprehensively characterized. Molecular analyses confirmed all isolates as S. agalactiae . Further characterization identified serotype Ia using conventional PCR and sequence type ST7 through multilocus sequence typing (MLST). Three representative isolates obtained from diseased juvenile tilapia were further characterized by whole-genome sequencing and MLST analysis, confirming their classification as S. agalactiae serotype Ia sequence type ST7. Clinical signs observed during the outbreaks included erratic swimming, lethargy, exophthalmos, fin erosion, and hepatic alterations, consistent with streptococcosis. This study represents the first molecular characterization of S. agalactiae serotype Ia ST7 associated with streptococcosis outbreaks in farmed tilapia in Peru.
