This study was conducted in accordance with internationally accepted guidelines for the care and use of animals in research and was approved by the Institutional Animal Care and Use Committee of Prince of Songkla University, Thailand (Approval No. MHESI 68014/1731). The study used moribund juvenile L. calcarifer submitted for diagnostic investigation during a natural disease outbreak; therefore, no animals were experimentally infected or intentionally sacrificed for research purposes. All procedures were performed in compliance with institutional and national ethical standards for animal welfare. Handling, transportation, and sampling of fish were conducted to minimize stress and suffering, following standard veterinary diagnostic protocols. Necropsy and tissue sampling were performed promptly after collection to ensure specimen integrity while adhering to humane practices. No endangered or protected species were involved in this study, and permission for sample collection was obtained from the aquaculture facility.

The study was conducted in January 2022 during a mortality outbreak at a commercial L. calcarifer nursery farm located in Samut Songkhram Province, central Thailand. Moribund juvenile fish were collected at the outbreak site and transported to the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, for diagnostic necropsy and histopathological evaluation.

This study was designed as a retrospective diagnostic case series investigating a natural outbreak of disease in juvenile L. calcarifer. Moribund fish collected during a mortality event were subjected to comprehensive pathological and molecular analyses to characterize concurrent infection with piscine intestinal coccidia and ISKNV. The study combined gross examination, histopathology, molecular detection, sequencing, and ultrastructural analysis to describe pathogen distribution, tissue tropism, and associated lesions. No experimental infection or controlled exposure trials were conducted, and all observations were based on naturally occurring infections.

Twenty moribund juvenile L. calcarifer, measuring 3–5 cm in total length and exhibiting lethargy followed by sudden death, were collected in January 2022 from nursery tanks at a commercial aquaculture facility in Samut Songkhram Province, central Thailand. The farm reported approximately 90% cumulative mortality during the outbreak period. Fish were reared in shaded ponds under routine aquaculture management conditions, with water salinity maintained at 30 ppt, temperature at 25°C, and pH at 8.2. They were fed a commercial pelleted diet (Profeed 902; Thai Union Feedmill Public Co. Ltd., Samut Sakhon, Thailand) containing 42% crude protein twice daily ad libitum. Moribund fish were submitted for diagnostic evaluation during an outbreak event, and no additional experimental sampling was performed. Specimens were placed in sterile containers within chilled, insulated boxes and transported to the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University. During transportation, water temperature was maintained at approximately 20–22°C. Necropsy and tissue fixation were performed promptly upon arrival, typically within 2 h of collection. Comprehensive gross and histopathological examinations were conducted. All major organs were inspected for macroscopic lesions. Representative samples of intracoelomic organs were fixed in 10% neutral-buffered formalin for 24 h, processed routinely, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E) according to standard histopathological procedures as described previously [36].

Evaluation of megalocytic cells: Evidence of ISKNV infection was initially evaluated using a Nikon ECLIPSE Ni-U advanced upright microscope equipped with a video-capture digital camera (Nikon, Tokyo, Japan). Diagnosis was based on the presence of characteristic lesions, including megalocytic cells, viral inclusion bodies, and basophilic hypertrophic cells in target organs such as the heart, kidney, gas gland, stomach, and other affected tissues [37].

Polymerase chain reaction (PCR) amplification and cloning of the MCP gene: Genomic DNA was extracted from the spleen and kidney tissues of L. calcarifer using the MasterPure™ Complete DNA & RNA Isolation Kit (Lucigen, Middleton, WI, USA) following the manufacturer’s instructions. DNA concentration and purity (A260/A280 ratio) were determined with a BioPhotometer D30 (Eppendorf AG, Hamburg, Germany). To confirm the presence of ISKNV, the complete ORF of the MCP gene was amplified by PCR using a T100™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). Each 50 μL reaction contained 1 U Phusion™ High-Fidelity DNA Polymerase (Thermo Fisher Scientific™, Waltham, MA, USA), 1× Phusion HF buffer, 200 μM dNTP mix, 250 nM of each primer, and 200 ng of template DNA. Full-length MCP (1362 bp) was amplified using primers derived from Huang et al. [38], and described by Subramaniam et al. [39], with minor 5′ extensions to improve amplification efficiency. The primer sequences were 5′-GGGGATGTCTGCAATACTCAGGTGC-3′ (forward) and 5′-GGGGTTACAGGATAGGGAAGCCTG-3′ (reverse).

PCR cycling conditions were as follows: initial denaturation at 98°C for 30 s; 35 cycles of 98°C for 10 s, 60°C for 15 s, and 72°C for 45 s; and a final extension at 72°C for 10 min. A no-template control and a positive control were included in each PCR run to monitor contamination and assay performance. The amplicons were separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized under ultraviolet illumination using a FireReader V10 Gel Documentation System (UVITEC Ltd., Cambridge, UK). A single band corresponding to the expected MCP ORF size (1,362 bp) was consistently observed. DNA fragments of the expected size were excised from the gel and purified using the Monarch® DNA Gel Extraction Kit (New England Biolabs, Ipswich, MA, USA). Purified products were cloned into the pJET1.2/blunt vector using the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and then transformed into Escherichia coli DH5α competent cells. Recombinant plasmids were recovered using the Monarch® Plasmid Miniprep Kit (New England Biolabs, Ipswich, MA, USA), and verification and bidirectional Sanger sequencing were performed by Macrogen Inc. (Seoul, Republic of Korea). Sequence alignment with reference ISKNV isolates in GenBank further verified amplification specificity.

The representative nucleotide sequence of the MCP gene was analyzed with BioEdit v7.2 and compared with available sequences in the GenBank database using BLASTn to verify nucleotide identity. Multiple sequence alignments and a phylogenetic tree of MCP gene sequences from ISKNV isolates across various fish hosts and countries, together with closely related iridoviruses, including red sea bream iridovirus, turbot reddish body iridovirus, and olive flounder iridovirus, were generated using maximum-likelihood analysis with the Kimura 2-parameter model [40]. Rate variation among sites was modeled using a gamma distribution [41], and node support was assessed using 1,000 bootstrap replicates in MEGA [42]. Sequence similarity of the MCP gene from the representative isolate was calculated against reference ISKNV sequences retrieved from GenBank using the p-distance model in MEGA software [42].

Morphological identification: Histological sections were examined under a Nikon ECLIPSE Ni-U upright microscope equipped with a DS-Fi3 digital camera and NIS-Elements imaging software (Nikon Corporation, Tokyo, Japan). Parasitic stages were identified according to previously published morphological criteria [43, 44]. For descriptive assessment, five non-overlapping high-power fields (×400) were examined in each of three regions of the small intestine (proximal, middle, and distal). Observations included evaluation of parasitic developmental stages and their localization within the intestinal epithelium, including epicellular, intracytoplasmic, and subepithelial positions. A parasitologist confirmed all morphological analyses. The coccidian infection rate was determined.

Morphometric observations using scanning electron microscopy (SEM): Representative intestinal tissues positive for coccidial infection, as confirmed by histopathology, and preserved in 10% neutral-buffered formalin, were selected for SEM. Preparation followed the protocol described previously [45]. Briefly, samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%), each step lasting 15–30 min, to ensure complete dehydration and prevent structural distortion during critical point drying with liquid CO₂ using an Autosamdri-931 critical point dryer (Tousimis Research Corporation, Rockville, MD, USA). Dried tissues were mounted on aluminum stubs with double-sided conductive carbon adhesive tape and coated with a 25 nm layer of gold using an SPI-Module sputter coater (SPI Supplies, West Chester, PA, USA) to prevent charging under the electron beam. Surface ultrastructural features of the coccidia were examined with a field-emission scanning electron microscope (Apreo FE-SEM; Thermo Fisher Scientific™, Eindhoven, Netherlands) operated in high-vacuum mode at 5–15 kV. Digital micrographs at multiple magnifications documented detailed coccidial morphology. Oocyst dimensions (length × width) were determined from calibrated digital micrographs using ImageJ software (National Institutes of Health, Bethesda, MD, USA). For each sample, 6–10 oocysts were measured, and values were expressed as mean ± standard deviation.

Intracoelomic organs, including the heart, kidney, gas gland, stomach, bulbus arteriosus, and intestine, were fixed in 10% neutral-buffered formalin for 24 h. After fixation, tissues were dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin wax according to routine histological procedures. Paraffin blocks were sectioned at 5 μm thickness using a rotary microtome, mounted on glass slides, and stained with H&E following previously described protocols [36]. Histological changes were evaluated by veterinary pathologists with attention to the distribution and severity of inflammatory cell infiltration, tissue degeneration, necrosis, vascular alterations, and the presence of characteristic ISKNV-associated megalocytic cells and coccidian developmental stages. The presence, extent, and pattern of these lesions were recorded to assess the impact of dual infection on organ pathology and to relate microscopic findings to the observed high mortality in juvenile L. calcarifer.

All quantitative data are presented as descriptive statistics. Oocyst measurements obtained from SEM were expressed as mean ± standard deviation. No inferential statistical analyses were performed, as the study was designed as a retrospective diagnostic case series without predefined comparative groups or hypothesis-driven testing. Data processing and calculation of descriptive statistics were performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).

In January 2022, farms in central Thailand reported a severe mortality event affecting early juvenile L. calcarifer, with cumulative losses estimated at approximately 90% of the stock. The fish originated from broodstock maintained in closed earthen ponds. Fertilized eggs were transferred to hatching tanks supplied with brackish water, and the resulting larvae were reared in nursery tanks until the juvenile stage. During rearing, the fish became lethargic and died suddenly without exhibiting specific external signs of disease. Farmers routinely used prophylactic antibiotics to prevent bacterial infections and enhance productivity; however, mortalities persisted, suggesting that bacteria were not the primary cause. These observations indicated that other pathogens, such as parasites or viruses, were likely responsible for the outbreak.

Fish carcasses were placed in sterile containers within large, chilled plastic buckets and transported to the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, for necropsy. Each specimen was weighed, measured, and examined for gross pathological changes. Affected juvenile L. calcarifer typically displayed grayish-black body discoloration and marked emaciation. No obvious external lesions such as necrosis, lepidorthosis, cloudy eyes, hemorrhage, or ulcers were detected. Congestion of intracoelomic organs, particularly the intestine, liver, kidney, and spleen, was consistently observed. Based on these external and internal findings, bacterial disease was considered unlikely, suggesting that viral or parasitic pathogens were the primary causes of mortality.

To assess possible parasitic involvement in the mortality event, gastrointestinal tissues, including the stomach, pyloric ceca, and intestine, were examined microscopically for the presence of piscine intestinal coccidia. All 20 fish (100%) were positive for coccidial infection. Histological identification was based on morphological characteristics of the parasite, which occupied epicellular, intracellular, and subepithelial locations within the mucosa and submucosa (Figure 1a). Three major developmental stages were observed: meronts, gamonts, and oocysts.

Oocysts were typically oval and frequently aggregated within intracellular or subepithelial positions of the pyloric cecum and intestine. Most were unsporulated and surrounded by a thin oocyst wall. Gamonts, which develop from merozoites, differentiate into macrogamonts and microgamonts. Macrogamonts were irregular to oval, with prominent central nuclei and distinct eosinophilic peripheral bodies, whereas microgamonts were spherical to oval and contained multiple peripheral dark basophilic nuclei. Both gamont types were most often located at epicellular positions, admixed with abundant mucus and cellular debris. Some macrogamonts were embedded in the intestinal epithelium and aggregated into small clusters.

Because caps or micropyles of oocysts are not discernible by light microscopy, SEM was employed to characterize surface ultrastructure. SEM revealed rough-surfaced oocysts lacking both a polar cap and a micropyle (Figures 1b–d). The mean oocyst size was 5.18 ± 1.20 μm in length and 3.38 ± 0.59 μm in width (n = 6), with observed ranges of 3.8–6.9 μm in length and 2.6–4.1 μm in width. These dimensions fall within the reported size range of small- to medium-sized piscine intestinal coccidia described in previous studies, although definitive genus-level assignment cannot be established based solely on morphology.

ISKNV infection was further investigated in juvenile L. calcarifer. Histopathological identification was based on the presence of megalocytic or basophilic hypertrophic cells containing inclusion bodies scattered throughout the parenchyma of various coelomic organs. Microscopic examination revealed numerous hypertrophic cells in the heart, kidney, bulbus arteriosus, submucosal vessels, and gas gland (Figures 2a–f). These cells exhibited a high nucleus-to-cytoplasm ratio, a centrally located prominent nucleolus, enlarged nuclei with peripherally distributed chromatin, and distinct intracytoplasmic basophilic inclusions.

Consistently, the stomach and intestinal tissues already affected by coccidiosis exhibited marked lymphocytic infiltration with numerous megalocytic cells (Figure 2f). In contrast, no significant lesions were detected in the spleen, liver, gills, or brain.

To confirm the viral etiology, molecular detection targeting the ORF of the MCP gene was performed. PCR amplification of kidney and spleen samples produced an approximately 1.4 kb fragment (Figure 3). The amplicons were cloned into a vector, transformed into E. coli, and sequenced. Sequence analysis confirmed the presence of a 1,362-nucleotide MCP ORF, thereby verifying ISKNV infection.

All juvenile L. calcarifer samples tested positive for ISKNV (Figure 3). Molecular identification was conducted by PCR amplification and sequencing of the MCP gene. Pairwise identity matrix analysis of MCP gene sequences revealed that isolate CU-1 was 100% identical to ON743043 (China), AF370008 (China), AB666337 (Malaysia), PV101205 (Singapore), and PQ351746 (Thailand). The next closest match was MZ152127 (India), sharing 99.70% similarity with CU-1 (Table 1). The representative nucleotide sequence obtained in this study has been deposited in GenBank under accession number PX442651.

Phylogenetic analysis of MCP gene sequences demonstrated that ISKNV, red sea bream iridovirus, turbot reddish body iridovirus, and olive flounder iridovirus each form distinct, well-supported monophyletic clades, consistent with their classification as separate but closely related megalocytiviruses. The majority of ISKNV isolates clustered into a strongly supported monophyletic group (99% bootstrap). Within this group, bootstrap support for internal subclades varied; however, the CU-1 isolate in this study clustered with a broad range of hosts, including humpback grouper (Malaysia), albino rainbow shark (USA), mandarin fish (China), hybrid grouper (Indonesia), Banggai cardinalfish (USA), dwarf gourami (Singapore), tilapia (Brazil), and L. calcarifer (Thailand and Indonesia), with strong support (99% bootstrap), confirming its identity as ISKNV. Notably, this isolate was clearly distinct from ISKNV previously reported in L. calcarifer from Vietnam (KY440040) and in marble sleepy goby from China (HM067835) (Figure 4).

To evaluate the pathological impact of concurrent piscine intestinal coccidia and ISKNV infection, detailed histopathological examinations were performed on all submitted fish. Overall, ISKNV infection was characterized by hypertrophic (megalocytic) cells accompanied by variable degrees of inflammation and necrosis. Minimal lymphocytic infiltration was observed around hypertrophied cells in organs not affected by coccidia, including the heart, kidney, bulbus arteriosus, and gas gland (Figures 5a–c).

In contrast, tissues exhibiting dual infection, notably the stomach and intestine, showed more extensive inflammatory lesions where both pathogens were present (Figures 5a–c), which were associated with moderate to severe inflammation, mucosal injury, and cellular necrosis.

In the stomach, inflammatory cell infiltration extended through the mucosa, submucosa, and muscularis. The infiltrate was composed primarily of mononuclear cells (macrophages and lymphocytes), with granulocytes present in lesser numbers. Inflammation typically surrounds viral lesions, which are marked by basophilic hypertrophic cells. Moderate to large numbers of inflammatory cells were concentrated in the mucosa and submucosa, particularly around the vascular endothelium, in perivascular areas, and around epithelial cells. Fewer macrophages and lymphocytes were found in the muscular layer and gut wall, whereas granulocytes were more abundant in the mucosal and submucosal zones. Gastric pathology included necrosis, vascular congestion and hemorrhage, edema, degeneration of the muscular layer, and mucosal and submucosal fibrosis (Figures 5a and b). Necrosis, characterized by nuclear and cell membrane disruption, was associated with moderate-to-severe inflammation. Marked congestion or hyperemia, caused by dilation of blood vessels with erythrocyte accumulation, was common in areas with active leukocytic infiltration. In severe cases, inflammation extended into the muscular layer and the gastric wall, resulting in myocyte degeneration and muscle edema. Chronic lesions were accompanied by mucosal and submucosal fibrosis.

Inflammation in the intestine was more severe than in the stomach. The infiltrate consisted predominantly of macrophages and lymphocytes, with occasional granulocytes in the lamina propria. Lesions initially appeared around the submucosa, where both coccidia and ISKNV were present (Figure 5c), and extended into the epithelium and muscular layers. Severe inflammatory changes correlated with epithelial desquamation, villous denudation, congestion, nuclear centralization within enterocytes, and widespread tissue necrosis.

These findings demonstrate concurrent infection of piscine intestinal coccidia and ISKNV-associated with marked gastrointestinal pathology, characterized by extensive mucosal damage in juvenile L. calcarifer.

Single infections with piscine intestinal coccidia, apicomplexan protozoa that parasitize the gastrointestinal tract, represent a significant health concern for juvenile fish in Southeast Asia and are frequently associated with diverse intestinal pathologies [2, 10, 46]. Most coccidian species display strict host specificity and are typically reported as single infections in various fish species [12, 47, 48]. Although mixed infections have been described, such as coccidia–bacteria [49], coccidia–helminth [25], and coccidia–coccidia combinations including mixed Goussia spp. [44], mixed Cryptosporidium spp. [50], and mixed Eimeria spp. [51], co-infection of coccidia with major pathogenic viruses has not been described previously.

The present study provides the first evidence of concomitant infection of piscine intestinal coccidia with a pathogenic virus, ISKNV, in juvenile L. calcarifer. We documented the general infection status, performed phylogenetic analysis, and demonstrated concurrent infection associated with pronounced gastrointestinal lesions observed on histopathological examination. A high proportion of dual infections was observed in the intestinal tract, consistent with previous reports of coccidial [10, 14, 44] and viral infections [9]. The marked susceptibility of juvenile fish suggests an immature or compromised immune system, which reduces host defenses against both protozoal and viral pathogens [52, 53].

Piscine intestinal coccidia were identified in the intestinal tract of L. calcarifer through light microscopic examination and SEM-based morphological assessment. The measured oocyst dimensions were 5.18 ± 1.20 μm in length and 3.38 ± 0.59 μm in width (range 3.8–6.9 × 2.6–4.1 μm). These measurements are inconsistent with Cryptosporidium spp., which are typically considerably smaller [25, 48]. Based on size, the observed parasites are more compatible with members of the larger-bodied coccidian genera reported in fish, including Eimeria spp., Goussia spp., Epieimeria, Calyptospora, and Crystallospora, as described in previous studies [44, 54, 55]. Considering the anatomical location of infection (intestinal epithelium), epicellular development, and the predominantly oval rather than spherical oocyst morphology, the findings are less consistent with Calyptospora and Crystallospora spp., which are generally described as spherical and have distinct morphological characteristics [56].

Among the remaining candidates, the morphometric profile and site of infection are most compatible with Eimeria-like or Goussia-like piscine intestinal coccidia [54, 55, 57]. However, due to overlapping morphological characteristics among piscine coccidian genera, definitive genus- or species-level identification cannot be established without molecular confirmation, and this taxonomic limitation should be considered when interpreting the findings. Infection with piscine intestinal coccidia, including Goussia spp. and Eimeria spp., has been reported to disrupt intestinal homeostasis [44], alter the gut microbiota, and increase susceptibility to secondary pathogens in fish, potentially predisposing affected individuals to disease or mortality [16, 17]. Previous reports from Thai aquatic environments have documented Eimeria spp. and Cryptosporidium spp. in various fish hosts [24, 25, 48], highlighting the diversity of piscine intestinal coccidia in the region. These findings suggest that the diversity and distribution of piscine intestinal coccidia in Thailand may be broader than currently recognized and remain incompletely characterized.

ISKNV was detected in diseased juvenile L. calcarifer by both histopathological and molecular methods. Megalocytic cells containing basophilic inclusions were systemically distributed across multiple coelomic organs, consistent with previous descriptions in L. calcarifer aquaculture [26, 30]. Phylogenetic analysis of the MCP gene revealed that the L. calcarifer ISKNV isolates obtained in this study clustered firmly within the established ISKNV lineage. This placement confirms their phylogenetic identity and evolutionary relatedness to previously reported isolates (genotype I ISKNV) from Asia, particularly those from Thailand, Malaysia, and Indonesia [26, 58].

Notably, this cluster was clearly distinct from genotype II ISKNV, which has been reported from Vietnam [27]. ISKNV has been widely reported in L. calcarifer and is recognized as a major viral pathogen in marine aquaculture. Previous studies have documented co-infections involving ISKNV and other viral pathogens, such as red-spotted grouper nervous necrosis virus, associated with increased mortality in juvenile L. calcarifer [33]. However, to our knowledge, concurrent infection of ISKNV with piscine intestinal coccidia has not been documented in this host species.

This study documents a previously unreported concurrent infection of piscine intestinal coccidia and ISKNV-associated with mass mortality in juvenile L. calcarifer in Thailand. Histopathological examination revealed classical systemic ISKNV-associated megalocytic changes in multiple visceral organs, accompanied by extensive necrotizing enteritis localized to the intestine, the primary site of coccidian infection. Extra-intestinal changes, including mild to moderate gastric perivasculitis, myocarditis, aerocystitis, and tubulointerstitial nephritis, were comparatively less severe.

In previously described ISKNV infections in L. calcarifer, systemic dissemination with prominent megalocytic cells in the spleen and kidney represents the dominant pathological pattern, whereas severe necrotizing enteritis is not consistently emphasized as a major feature [30]. In contrast, isolated intestinal coccidiosis is typically characterized by epithelial disruption and localized mucosal inflammation without systemic viral-type pathology [10, 24, 59, 60]. The combined distribution of systemic viral lesions and pronounced intestinal necrosis observed in the present study, therefore, differs from the typical presentation of either condition alone and supports the interpretation of concurrent infection.

Previous studies have shown that intestinal coccidia preferentially infect specific intestinal segments in juvenile L. calcarifer, particularly the anterior portion, where localized tissue damage and inflammatory responses may compromise mucosal integrity [10]. Experimental and observational studies in fish have also suggested that piscine intestinal coccidia can disrupt intestinal homeostasis, alter the gut microbial community [16], and induce oxidative stress responses, including the production of reactive oxygen species leading to epithelial damage and apoptosis [61]. Such alterations may be associated with reduced host resilience and increased susceptibility to secondary infections.

In the present study, however, no immune gene expression analysis, cytokine profiling, microbiome assessment, or experimental challenge trials were performed. Therefore, any proposed interactions between piscine intestinal coccidia and ISKNV remain speculative and should be interpreted with caution. In addition to these limitations, this investigation was conducted as a retrospective outbreak-based study. Quantitative lesion scoring, statistical comparisons, quantitative viral load assessment (including real-time PCR), and parasitic intensity measurements were not performed. Only moribund fish from a single outbreak were examined, and no healthy or single-pathogen control groups were available, limiting causal interpretation.

In addition, molecular characterization of piscine intestinal coccidia and bacteriological examination were not undertaken. Accordingly, the findings should be interpreted as descriptive evidence of concurrent infection associated with marked gastrointestinal pathology rather than proof of direct pathogen interaction or causation. Further controlled experimental studies are warranted, including challenge trials in juvenile L. calcarifer using molecularly characterized ISKNV isolates and intestinal coccidia, with sequential or simultaneous infection designs to assess lesion progression, pathogen load, and host immune responses.