Author: marina02

Aspects of the histopathology of the stomach of Clarias gariepinus (Burchell, 1822) infected by Procamallanus (Procamallanus) pseudolaeviconchus Moravec & Van As, 2015.

African sharptooth catfish is a preferred aquaculture species. They are omnivorous and host many parasite species. The current study reports on the impact of Procamallanus (Procamallanus) pseudolaeviconchus Moravec & Van As, 2015 on the stomach of Clarias gariepinus (Burchell, 1822) and identifies the wound inflicting structures. Fishwere collected from Lake Heritage (Crocodile River), and Vaal River below Vaal Dam. Infected and uninfected stomach tissue as well as unattached parasites were fixed in 10% neutral buffered formalin for histological assessment or 70% alcohol for scanning electron microscope respectively. Specimens were embedded in TAAB epoxy resin and stained with Periodic-Acid Schiff or haematoxylin and eosin. No P. (P.) pseudolaeviconchus were found in the fish from the Vaal River therefore these fish were used as control.In Lake Heritage prevalence was 80% and mean intensity 2.5. The buccal capsule is large, lacks teeth, and has a round opening that cannot be closed and bears unlobed peribuccal flanges. The oesophagus is muscular. The parasites attach to the columnar epithelial lining of the mucosa and perforates it at the interface with the buccal capsule while the surrounding epithelium remains intact. The columnar epithelial cells and connective tissue are sucked into the buccal capsule, their shape is altered, and cells are sloughed off. The underlying gastric glands show signs of trauma. The impact is localized to the attachment site. There’s no inflammation, haemorrhage or cellular infiltration at the attachment site. Host tissue is present in the intestinum of the parasites. It is deduced that the nematode uses the peribuccal flanges, solid sturdy buccal capsule, and the vacuum created by the muscular oesophagus to attach and ingest host tissue. These structures create a suction cup which enables the parasites to ingest the host’s tissue into the buccal capsule and eventually into the intestine.It is concluded that the low intensity, localised trauma and absence of an immune response indicate limited impact on the host. The life cycle has not been studied but probably involves one or more intermediate host/s (copepods and/or smaller fish species) which can be excluded in fish culture. Therefore, the parasite poses a limited threat to aquaculture.

1. MATEA, KENNETH, UNIVERSITY OF JOHANNESNURG, Presenter
2. Avenant-Oldewage, Annemariè, UNIVERSITY OF JOHANNESBURG, Author

Aspects of the histopathology of the rectum of Clarias gariepinus infected with the camallanid parasite Paracamallanus cyathopharynx (Baylis,1923)

Various studies have reported on Paracamallanus cyathopharynx (Baylis,1923) morphology in various river systems all over the African continent. The data on the impact of the parasite on the host is incomplete and includes mixed species infections. Therefore, this study focuses on aspects of the histopathology of the rectum of Clarias gariepinus (Burchell, 1822) infected with P. cyathopharynx in South Africa. Twenty fish were collected from Lake Heritage (10 fish) (Crocodile River) and Vaal River below the Vaal Dam (10 fish). Infected and uninfected tissue as well as unattached parasite were collected. Specimens and tissues were fixed in 10% neutral buffered formalin or 70% alcohol, for histological assessment and scanning electron microscopy respectively. Samples for histological assessment were embedded in TAAB epoxy resin and sections were stained with haematoxylin and eosin. Prevalence and mean intensity in Lake Heritage (Crocodile River) were 80% and 3.3 and in the Vaal River 60% and 2.5 respectively. Macroscopically, attached parasites have a red tinge. The buccal capsule consists of two parts separated by an isthmus and the anterior part has longitudinal ridges dorsally and ventrally but lacks teeth. The buccal capsule has a slit like opening that can be closed. Externally, a pair of tridents can be seen as bumps on the lateral side of the parasite. The parasite attaches to the columnar epithelial lining of the rectum and apply negative pressure exerted by the muscular oesophagus, to suck the host’s epithelial cells and connective tissue into the buccal capsule. The longitudinal ridges create imprints on the host tissue. Furthermore, the shape of the columnar cells sucked into the buccal capsule is altered. The surrounding epithelium and the submucosa remain intact. None of the specimens sectioned contained blood in the buccal capsule or intestine. There is no inflammation, haemorrhage, or cellular infiltration at the attachment site. There is also no statistical difference between the number of goblet cells in infected and uninfected tissue. It is concluded that contrary to previous reports P. cyathopharynx does not infect the stomach but instead the rectum of C. gariepinus. The low intensity and limited impact indicate that the parasite does not pose threat to its host and will probably not impact on catfish aquaculture. The origin of the red coloration in the parasites remains unsolved.

1. MATEA, KENNETH, UNIVERSITY OF JOHANNESNURG, Presenter
2. Avenant-Oldewage, Annemariè, UNIVERSITY OF JOHANNESBURG, Author

Completing the life cycle of Myxobolus tihanyensis (Cnidaria, Myxozoa) by molecular evidence

Myxozoans represent a diverse group of parasitic cnidarians that display a complex life cycle alternating between two different spore types in two different hosts: the myxospore type infecting a vertebrate host and the actinospore type infecting an invertebrate host.

In the present study, a total of 1,248 oligochaetes collected at Keszthely, from Lake Balaton in Hungary, were placed into wells of microtiter plates. Each plate was examined daily to detect the production of actinospores. Three specimens were identified as Tubifex sp. with sequencing and were detected to release actinospores, which were morphologically classified as aurantiactinomyxon type actinospores. The mature actinospores have three stout, semicircular, curved downwards, leaf-like valvular appendices attached to a spore body, three protruding polar capsules at the apex of the spore, and a sporoplasm which displays numerous secondary cells. Actinospore’s body length was 19.03 ± 1.26 (17.30−21.0) µm, and width was 17.79 ± 11.37 (15.18−19.66) µm. Caudal valvular appendices’ length was 22.28 ± 1.41 (20.24−24.92) µm, and width was 8.01 ± 1.05 (6.61−10.78) µm at the base.

Molecular analysis based on the 18S rDNA sequences showed ≥99.9% similarity to the available sequences of the parasite Myxobolus tihanyensis, which infects the muscle adjacent to fins and vertebrae of the European perch (Perca fluviatilis), documented at Tihany from Lake Balaton in Hungary. In order to strengthen this finding, the 28S rDNA gene was amplified from both aurantiactinomyxon type actinospores and myxospores of M. tihanyensis. The genetic similarity between the two spore types was found to be ≥99.2%. Phylogenetic reconstruction based on the 28S rDNA, the aurantiactinomyxon type actinospores and myxospores of M. tihanyensis sequences were clustered into a monophyletic clade.

The genetic similarity and the phylogenetic analysis provide together compelling evidence that the aurantiactinomyxon type actinospores and myxospores of M. tihanyensis belong to the same species.

This study is the second report about a Myxobolus species with an aurantiactinomyxon type actinospore; and it is the first to employ 28S rDNA to elucidate the life cycle of a myxozoan parasite.

Acknowledgements: This study was funded by the Stipendium Hungaricum Program. Graciela Colunga-Ramírez is supported by SECIHTI, Mexico (grant number: CVU 769732).

1. BOGLÁRKA, SELLYEI, HUN-REN VETERINARY MEDICAL RESEARCH INSTITUTE, Presenter
2. GRACIELA, COLUNGA-RAMÍREZ, HUN-REN VETERINARY MEDICAL RESEARCH INSTITUTE, Author
3. CSABA, SZÉKELY, HUN-REN VETERINARY MEDICAL RESEARCH INSTITUTE, Author
4. GÁBOR, CECH, HUN-REN VETERINARY MEDICAL RESEARCH INSTITUTE, Author

Antiparasitic efficacy of glutaraldehyde against Ichthyobodo salmonis and Trichodina spp. in chum salmon (Oncorhynchus keta) and barfin flounder (Verasper moseri), with a toxicological assessment on host species

Introduction: Ichthyobodo spp. and Trichodina spp. cause significant parasitic diseases in aquaculture, particularly affecting salmonid and flounder fishes. Since the ban on formalin use in many countries, effective alternatives for managing these parasites have been limited. This study investigated the antiparasitic efficacy of glutaraldehyde (GA) against I. salmonis and Trichodina spp. in chum salmon (Oncorhynchus keta) and barfin flounder (Verasper moseri).

Methodology: Infected fish were exposed to GA concentrations ranging from 28ppm to 44ppm for 15, 30, and 60 min, with 93ppm formaldehyde (FA) serving as a positive control, while rearing water was used as a negative control. At 24 h post-initiation (hpi), fish mortality was recorded, and parasites were preserved and counted under a microscope. For acute toxicity tests, fish were exposed to GA for 1 h at concentrations ranging from 98ppm to 3,897ppm for chum salmon, and 35ppm to 866ppm for flounder. FA was tested under the same 1 h exposure condition, at concentrations ranging from 93ppm to 2,327ppm for chum salmon and 93ppm to 2,270ppm for flounder. Fish mortality was recorded at 24 hpi and every 24 h thereafter up to 96 hours.

Results: Treatment with GA achieved I. salmonis clearance rates exceeding 90%, comparable to those observed in the FA group across all tested concentrations. GA was also effective against T. cf. truttae in chum salmon, with most GA bath treatments achieving over 90% clearance. Fish mortality ranging from 11% to 25% was observed only in the longest exposure groups (60 min treatment).

Similarly, both GA and FA demonstrated antiparasitic activity against T. cf. jadranica in barfin flounder, with parasite eradication rates exceeding 99%. Notably, GA at a concentration of 28ppm achieved 99.7% clearance within a 30-min bath, with no mortality recorded in any treatment groups.

Acute toxicity assessments showed 96 h LC₅₀ values of 659ppm (FA) and 205ppm (GA) for salmon fry, and 352ppm (FA) and 184ppm (GA) for barfin flounder.

Conclusions: Our findings indicate that GA shows antiparasitic activity against I. salmonis and T. cf. truttae in chum salmon and T. cf. jadranica in flounder, without evident toxicity to the host fish at the concentrations used for parasite eradication. These results highlight the potential of GA as a chemotherapeutic agent for controlling these parasites in aquaculture.

This study was supported by the Ministry of Agriculture, Forestry and Fisheries of Japan and JSPS KAKENHI (Grant number 23K05381).

1. SAKALA, MASAUTSO EMMAUEL, HOKKAIDO UNIVERSITY, Presenter
2. YAMAMOTO, KANTARO, HOKKAIDO UNIVERSITY, Author
3. AMENO, MAKOTO, HOKKAIDO UNIVERSITY, Author
4. YAMAGUCHI, KEITO, HOKKAIDO UNIVERSITY, Author
5. TIPPAYAKRAISRI, KARN, HOKKAIDO UNIVERSITY, Author
6. NITTA, MASATO, NANSEI FIELD STATION, FISHERIES TECHNOLOGY INSTITUTE, JAPAN FISHERIES RESEARCH AND EDUCATION AGENCY, Author
7. OSEKO, NORIHISA, NANSEI FIELD STATION, FISHERIES TECHNOLOGY INSTITUTE, JAPAN FISHERIES RESEARCH AND EDUCATION AGENCY, Author
8. NAGATA, JUN, HOKKAIDO UNIVERSITY, Author
9. KASAI, HISAE, HOKKAIDO UNIVERSITY, Author

Histopathological changes caused by parasitic copepods in marine fish from Tunisian coasts

Parasitic copepods are of the most common ectoparasites on marine fish. Many species of copepods have long been recognized to have the potential to affect the growth, fecundity and survival of their hosts. Parasitic copepods feed on host mucous, tissues, and blood, and cause direct harm to their hosts through attachment mechanisms and feeding activities. The aim of this study was to determine the pathological changes induced by parasitic copepods on economically important fish Sardina pilchardus, Merluccius merluccius and Scorpaena notata from Tunisian waters.

The European pilchard Sardina pilchardus harbors the parasitic copepod Peroderma cylindricum (Pennellidae). The female of P. cylindricum excavates a deep gallery into the fish musculature and ultimately attach itself in the anterior kidney using its cephalothorax, equipped with a branched system of rhizoids. The invasion of rhizoids into the kidney disrupts renal tissue, leading to the disappearance of renal tubules. Histological sections reveal that the kidney has lost its cellular architecture, with its central region predominantly occupied by the rhizoids of the parasite. Furthermore, hemorrhagic zones are observed within the kidney.

The European hake, Merluccius merluccius, is frequently parasitized by Lernaeocera lusci (Pennellidae). The female attaches its head to the branchial gill arch, causing multiple forms of damage : moderate to severe alterations of the secondary gill filaments in contact with the parasite, and structural disruption of the gill arch, which is deeply affected by the anchoring apparatus of the copepod. The alterations identified in gill tissues included foci of inflammation and hyperplasia of the epithelium.

The small red Scorpaena notata is infected by females of Strabax monstrosus (Chondracanthidae). One to two females was found infecting the upper corner of the gill chamber with posterior extremity protruding into the gill cavity and the anterior part of the body was deeply embedded into the host tissue, while the head and neck were inserted into the cardiac region. Histopathological examination revealed that the penetration of anterior part and neck of Strabax monstrosus into the gill tissues of the fish leads to necrosis of these tissues. However, different degrees of histopathological damages were observed, after the attachment of the parasite in the cardiac region, mainly a compression of the aorta, necrosis of the arterial bulb, and inflammatory fibrosis in adjacent tissue.

1. BAHRI, SIHEM, UNIVERSITY OF TUNIS EL MANAR, FACULTY OF SCIENCES OF TUNIS, Presenter

Parasitic diversity of the endemic Gobiidae species, Zosterisessor ophiocephalus (Pallas, 1814) along the Tunisian coast (Mediterranean Sea)

Zosterisessor ophiocephalus, an endemic goby species from the Mediterranean Sea, represents a valuable indicator of marine ecosystem health and local environmental conditions. This study provides, for the first time, a comprehensive assessment of the parasitic diversity of this species along the Tunisian coast. A total of 221 specimens of Z. ophiocephalus were collected from five marine localities: Ghar El Melah lagoon, Lake of Tunis, Sfax, Zarzis, and the Kerkennah Islands.

Microscopic identification of parasites revealed five species of metazoan parasites belonging to three taxonomic groups: isopods, acanthocephalans, and nematodes. Among the isopods (ectoparasites), we collected Gnathia sp. larvae in the buccal cavity and beneath the pectoral fins. Three species of acanthocephalans were collected from the fish intestine including Acanthocephaloides irregularis (Amin et al., 2011), A. propinquus (Dujardin, 1845), and Telosentis exiguus (von Linstow, 1901). These endoparasites were also reported in Gobius niger (Kvach & Sasal, 2010), however, only A. propinquus has been observed in Zosterisessor ophiocephalus from the Black Sea (Kvach, 2006). The nematode Hysterothylacium reliquens (Norris & Overstreet, 1975) was collected from the intestine and liver of the grass goby for the first time. Thus, Z. ophiocephalus is considered a new host for this parasite. Morphological study confirmed the distinctive features of each acanthocephalan species, and revealed morphological variations particularly in hook size and proboscis shape, that may be related to host species and geographical locality.

The evaluation of quantitative parasitism parameters revealed significantly higher parasite diversity and abundance in southeastern localities (Sfax, Zarzis, and Kerkennah) compared to northeastern sites (Ghar El Melah and Lake of Tunis). Among the identified species, H. reliquens was particularly frequent in the south, with prevalence rates of 54.28% in Sfax, 24.07% in Kerkennah, and 6.66% in Zarzis, while it was absent in the north. A. propinquus was dominant in Zarzis (100%), but rare in Kerkennah (9.25%) and Ghar El Melah (3.89%). Telosentis exiguus was found in both areas but with moderate to low prevalence: 12% in the Northern Lake of Tunis, 6.49% in Ghar El Melah lagoon, and 3.33% in Zarzis. Finally, A. irregularis was detected only in Zarzis, with a prevalence of 23.33%. These results reflect heterogeneity in parasites distribution which is probably influenced by host features (size, age, density, feeding habits…) and by the quality of their environment.

1. SLAMA, WIDED, UNIVERSITY OF TUNIS EL MANAR, FACULTY OF SCIENCES OF TUNIS, Author
2. BAHRI, SIHEM, University of Tunis El Manar , Faculty of Sciences of Tunis, Presenter
3. BAHRI, LILIA, UNIVERSITY OF TUNIS EL MANAR, FACULTY OF SCIENCES OF TUNIS, Author

Gill histopathology associated with Lernanthropus kroyeri and Diplectanum aequans infestation in European seabass (Dicentrarchus labrax)

Gill-associated parasites are a significant concern in marine aquaculture, particularly in intensively farmed species such as European seabass (Dicentrarchus labrax). Lernanthropus kroyeri, a parasitic copepod commonly found on the gills of marine fish, poses significant threats to host health and aquaculture productivity. Diplectanum aequans is a monogenean ectoparasite belonging to the family Diplectanidae, primarily known to infest the gills of marine teleost fish, particularly species within the Moronidae family such as European seabass. This study examines the histopathological effects of two prevalent ectoparasites, L. kroyeri and D. aequans, on the gill tissues of infested specimens. This study was conducted seasonally and involved a total of 100 European seabass (Dicentrarchus labrax) specimens sampled from a marine cage aquaculture facility. Sampling was conducted on a seasonal basis from 2024 to 2025, and gill tissues were thoroughly evaluated using macroscopic, microscopic, and histopathological methods to determine the presence and impact of parasitic infestations. The prevalence and mean density values of each parasite species were determined. Copepod and monogenean species determination were done according to published identification keys. For histopathological examinations, gills were excised, fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 µm, and stained using hematoxylin and eosin (H&E) for microscopic analysis. The most common parasite in European seabass was L. kroyeri, with the highest prevalence in summer (53.5%); however, D. aequans reached peak prevalence (73%) in autumn. Lernanthropus kroyeri specimens were primarily located on the proximal branchial lamellae, while D. aequans was distributed across the secondary lamellae. Histological analysis revealed common lesions, including epithelial hyperplasia, lamellar fusion, sub-epithelial edema, mucus cell proliferation, and localized necrosis. Complete degeneration of secondary lamellae and structural breakdown of primary filaments were observed, indicating substantial respiratory compromise. Additionally, the presence of hemorrhagic lesions and excessive mucus secretion indicates a disruption of the protective mucosal barrier, increasing susceptibility to secondary infections. In conclusion, these parasites exacerbate gill damage together and impair gas exchange. These findings highlight the pathological burden imposed by ectoparasitic infestations and underscore the importance of regular health monitoring and integrated parasite control in European seabass aquaculture.

1. TURGAY, EMRE, Dept. of Aquaculture and Fish Diseases, Faculty of Aquatic Sciences, Istanbul University, Presenter
2. YARDIMCI, REMZİYE EDA, Dept. of Aquaculture and Fish Diseases, Faculty of Aquatic Sciences, Istanbul University, Author
3. ŞUAYİP, HAKAN, Institute of Graduate Studies in Sciences, Istanbul University, Author
4. KARATAŞ, SÜHEYLA, Dept. of Aquaculture and Fish Diseases, Faculty of Aquatic Sciences, Istanbul University, Author
5. STEINUM, TERJE MARKEN, Dept. of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Author

Distribution of Anisakis species from Atlantic horse mackerel (Trachurus trachurus) in the Portuguese coast: an update

Introduction:

Third-stage larvae of the genus Anisakis (Nematoda: Anisakidae) parasitize the viscera and muscles of fish and have been associated with human anisakiasis due to the consumption of raw or undercooked infected fishery products. The traditionally high fish intake and the recent introduction of exotic eating habits (such as sushi, sashimi and ceviche) have raised the risk of this zoonosis in Portugal. It has been suggested that Anisakis species’ distribution may vary depending on factors such as geographical location and water temperature. Therefore, the present study aims to examine the distribution of Anisakis species in the viscera and muscles of fishery products collected from six localities in Portugal at two times of the year. Additionally, it will provide updated epidemiological data and insights on food safety.

Methodology:

Anisakis larvae were collected from Atlantic horse mackerel (Trachurus trachurus) captured in the winter and in the summer of 2024, after individual dissection and separate digestion of the viscera and the muscles of the hosts using pepsin solution. Molecular identification of some of the larvae was then performed through the amplification of the ITS region followed by Restriction Fragment Length Polymorphism (PCR-RFLP) using Hinf I and Hha I enzymes.

Results:

Preliminary results indicate Anisakis pegreffii and A. simplex sensu stricto (s.s.) as the most frequent species detected in the Portuguese coast, apart from hybrid genotypes between these two sibling species. Data provided shows a variation in the proportion of each of the parasitic species identified according to geographical locality (north vs south), biotope in the host (muscle vs viscera) and time of the year (winter vs summer).

Conclusions:

This study highlights Portugal as a crucial region where A. simplex (s.s.), prevalent in colder Atlantic waters, and A. pegreffii, frequently associated with warmer Mediterranean environments, converge. These findings deepen our understanding of Anisakis spp. and underscore the potential effects of global warming on their distribution and potentially on public health.

1. Gomes, Tiago, Faculty of Veterinary Medicine, Lusófona University; I-MVET; Veterinary and Animal Research Centre (CECAV), Presenter
2. Mendes, Beatriz, Faculty of Veterinary Medicine, Lusófona University, Author
3. Medeiros, Monalisa, Faculty of Veterinary Medicine, Lusófona University, Author
4. Abegão, Leonor, Faculty of Veterinary Medicine, Lusófona University, Author
5. Barbosa, Ricardo, Faculty of Veterinary Medicine, Lusófona University, Author
6. Moita, Tomás, Faculty of Veterinary Medicine, Lusófona University, Author
7. Almeida, Laura, Faculty of Veterinary Medicine, Lusófona University, Author
8. Mira, Inês, Faculty of Veterinary Medicine, Lusófona University; University of Évora, Author
9. Ramos, Sónia, Faculty of Veterinary Medicine, Lusófona University; I-MVET; Veterinary and Animal Research Centre (CECAV), Author
10. Munhoz, Ana, Faculty of Veterinary Medicine, Lusófona University; I-MVET; Veterinary and Animal Research Centre (CECAV), Author

Emergence of serotype III in Miamiensis avidus: molecular identification and virulence in olive flounder

Introduction: Miamiensis avidus is a highly pathogenic ciliate responsible for scuticociliatosis in olive flounder (Paralichthys olivaceus) in Korea and Japan. Current vaccines targeting serotypes I and II have shown limited effectiveness, probably due to the presence of uncharacterized serotypes.

Methodology: In this study, the BB19 strain was isolated from diseased olive flounder and identified as M. avidus via phylogenetic analysis of the cytochrome c oxidase subunit 1 (cox1) gene. Serotype-specific PCR, agglutination assays, indirect ELISA, and western blot analyses were performed to determine serotype and antigenic properties. Virulence was assessed through intraperitoneal challenge in olive flounder at different ciliate concentrations.

Results and Conclusions: BB19 was classified as cox1 genotype IV and identified as serotype III based on PCR results. Agglutination and ELISA confirmed that BB19 reacted exclusively with anti-serotype III sera (Mie0301 and BB19), without cross-reactivity to other serotypes. Western blotting revealed a unique 34 kDa antigenic protein in BB19, distinguishing it from the 30 kDa and 38 kDa proteins observed in other serotypes. In challenge tests, BB19 induced 65% cumulative mortality at a dose of 1 × 10⁵ cells/fish, similar to other virulent strains, confirming its high pathogenicity. This study reports the first isolation and characterization of serotype III of M. avidus in Korea. The distinct antigenicity and high virulence of the BB19 strain indicate that existing vaccines may not confer cross-protection. These findings highlight the need for continuous surveillance of emerging serotypes and support the development of multivalent vaccines for effective control of scuticociliatosis in olive flounder aquaculture.

1. JUNG, SUNG-JU, COONNAM NATIONAL UNIVERSITY, Presenter
2. KIM, JIN-YOUNG, CHONNAM NATIONAL UNIVERSITY, Author
3. KIM, JEONG-HOON, CHONNAM NATIONAL UNIVERSITY, Author

Non-Invasive Monitoring of Miamiensis avidus in Seawater Using a Real-Time PCR Targeting the cox1 Gene

Introduction: Miamiensis avidus (syn. Philasterides dicentrarchi) causes significant mortality and economic loss in olive flounder (Paralichthys olivaceus) aquaculture. Effective disease control requires sensitive detection tools for early diagnosis and monitoring. This study aimed to develop a sensitive and specific real-time PCR assay targeting the cytochrome c oxidase subunit I (cox1) gene of M. avidus for detection and quantification in seawater.

Methodology: Primers were designed using Primer3Plus and Primer-BLAST to amplify the 159–274 bp region of the cox1 gene, covering all known genotypes (1–4). A standard curve was generated by plotting Ct values against 10-fold serial dilutions of a plasmid containing the cox1 target sequence. The 95% limit of detection (LoD95%) was calculated using probit regression. The number of cox1 gene copies per M. avidus cell was estimated using counted and diluted single cells. Specificity was validated against five M. avidus strains (genotypes 1–4), Uronema elegans, and Cryptocaryon irritans. Sensitivity was determined by detecting the minimum number of M. avidus cells in 1 L of seawater. Reproducibility was tested through ten replicate experiments. The assay was applied to fertilized eggs, juvenile flounder (30, 60, 90 days old), and associated seawater to compare invasive and non-invasive detection. Environmental monitoring included inflow and effluent seawater from tanks with diseased, recovered, and healthy fish.

Results and Conclusions: The real-time PCR assay showed a strong linear correlation between Ct values and log plasmid copy number (R² = 0.9985) with an amplification efficiency of 96.26%. The LoD95% was 35.16 copies, and the average cox1 gene copy number per cell was estimated at 6017 ± 2794. The assay was specific to M. avidus and showed no cross-reactivity with C. irritans or U. elegans. It reliably detected as few as two M. avidus cells per liter of seawater. Non-invasive seawater sampling demonstrated greater sensitivity than tissue-based detection. High parasite densities (up to 57.81 cells/10 L) were observed in tanks with clinically infected fish. Sporadic detection was noted in both inflow and effluent seawater. The developed real-time PCR assay is a sensitive, specific, and non-invasive tool for monitoring M. avidus in aquaculture environments. Its application can significantly enhance early detection and disease management strategies in olive flounder farming.

1. JUNG, SUNG-JU, COONNAM NATIONAL UNIVERSITY, Author
2. KIM, HYEON-KYEONG, CHONNAM NATIONAL UNIVERSITY, Author
3. KWON, YOUNG-HYEOK, CHONNAM NATIONAL UNIVERSITY, Author
4. KIM, JIN-YOUNG, CHONNAM NATIONAL UNIVERSITY, Presenter