2025 Volume 60 Issue 3 Pages 145-156
Piscine intestinal coccidia, including Cryptosporidium spp., are significant pathogens in marine aquaculture, particularly affecting juvenile fish and leading to high morbidity and mortality. However, the pathological effects of coccidiosis in pearl gentian grouper (Epinephelus lanceolatus ♂ × E. fuscoguttatus ♀) have not been thoroughly studied. This research aimed to explore the histopathological changes and inflammatory responses linked to oxidative stress-induced tissue damage in juvenile pearl gentian groupers reared in marine cage farming systems. A total of 44 intestinal samples from juvenile groupers were classified as infected and non-infected based on the presence of Cryptosporidium. Nucleotide sequencing and phylogenetic analysis of the partial 18S rRNA gene identified the Cryptosporidium species detected in this study as C. bollandi. Histopathological analysis and immunofluorescence techniques were used to assess reactive oxygen species (ROS) production, DNA damage, and cellular apoptosis. The findings showed a significant increase in inflammatory cell infiltration, ROS production, DNA damage, and cellular apoptosis in infected fish compared to uninfected ones. These results indicate notable pathological alterations associated with C. bollandi infection in juvenile pearl gentian groupers. This research emphasizes the importance of understanding the effects of intestinal cryptosporidiosis on juvenile fish, as these pathological changes contribute to the health challenges that marine aquaculture currently faces.
The pearl gentian grouper is an aquacultural hybrid resulting from the crossbreeding of the female tiger grouper Epinephelus fuscogutattus and the male giant grouper E. lanceolatus (Chen et al., 2019). This hybrid species holds significant economic value in tropical and subtropical regions, particularly in China and Southeast Asian countries (Zhang et al., 2018). Although pearl gentian groupers exhibit advantageous traits such as rapid growth and strong disease resistance (Zhang, 2018), their culture is substantially hindered by various infectious pathogens, leading to considerable economic losses. Notably, diseases such as vibriosis (Yuan et al., 2025), pseudomoniasis (He et al., 2025), and infections caused by nervous necrosis virus (Jungi et al., 2022), have been identified as major threats. Nevertheless, studies have indicated that this hybrid species demonstrates resistance to certain parasitic protozoa, specifically Cryptocaryon irritans (Mo et al., 2021). However, research on infections caused by other pathogenic protozoa in pearl gentian groupers remains limited, highlighting the need for further investigation in this area.
Piscine intestinal coccidiosis represents a significant health challenge in both marine and freshwater aquaculture, contributing to substantial morbidity and mortality among cultured fish populations (Suyapoh et al., 2022b). This disease is primarily caused by apicomplexan parasites from the genera Eimeria, Goussia, Cryptosporidium, and Calyptospora (Dyková and Lom, 2006). Infection typically results in chronic and severe inflammatory responses, leading to epithelial damage, vascular congestion, focal necrosis, and sloughing of the intestinal epithelium (Suyapoh et al., 2024; Suyapoh et al., 2022b). The inflammatory process further triggers the production of reactive oxygen species (ROS), which induce DNA damage and promote cell death through apoptosis (Jantrakajorn et al., 2024). Despite the well-documented impact of coccidiosis in various fish species, information regarding its prevalence in pearl gentian groupers and the associated pathological effects remains limited, warranting further investigation.
To gain a deeper understanding of piscine intestinal coccidiosis in pearl gentian groupers, this study aimed to investigate its prevalence and assess its pathological impact. Specifically, a systematic and semi-quantitative approach was employed to evaluate the associated inflammatory responses, histopathological alterations, and oxidative damage within the intestinal system. By analyzing these pathological changes, the study sought to elucidate the potential pathogenic roles of the identified parasites in pearl gentian groupers.
A cross-sectional study was conducted on juvenile pearl gentian groupers. Initially, approximately 10,000 juveniles, aged 1 to 1.5 months, were raised in a 500 m2 shaded cement pond at a stocking density of approximately 20 fish/m2. The pond was filled with seawater sourced directly from the sea, which was filtered and treated with chlorine at a concentration of 40 ppm prior to use. The fish were reared in the pond until they reached an average length of 6–8 cm at around two months of age. During this period, size-sorting was performed to promote uniform growth and reduce cannibalism-related mortality. Subsequently, the graded fish were transferred to sea cages for grow-out. Each cage measured 1 × 1 × 1.5 m and held fish at a stocking density of approximately 300 fish/m3. The cages were floated in the open sea, where water parameters included a salinity of 35 ppt, a temperature of 25°C, and a pH of 8.2. Fish were fed ad libitum twice daily with a commercial pelleted diet containing 42% crude protein (Uni-President). Two weeks after transfer to the sea cages in Satun Province, Thailand (January 2024), the fish exhibited clinical signs of non-specific illness, including anorexia, lethargy, and generalized body darkening. At this stage, 44 juvenile fish were randomly sampled alive, without bias toward clinically affected or unaffected individuals, for health assessment.
The sampled fish were transported over a two-hour period to the Department of Veterinary Science, Faculty of Veterinary Science, Prince of Songkla University, in well-aerated, temperature-controlled seawater to minimize handling stress. Clinical and postmortem examinations, as well as parasitological and bacteriological assessments were performed to determine the cause of illness. Intestinal samples were collected for both molecular and histopathological analyses. The anterior portion of the intestine was aseptically collected and stored in sterile microtubes for subsequent molecular analysis, while mid-to-posterior sections were fixed in 10% buffered formalin for histopathological examination. The presence of piscine intestinal coccidia was determined by identifying developmental stages within the intestinal epithelium through histological assessment and confirming infection by polymerase chain reaction (PCR). All 44 fish, including both infected and non-infected with piscine intestinal coccidia, were subjected to histopathological and immunofluorescence analyses to evaluate the associated inflammatory responses, characterize histopathological changes, and assess the production of free radicals in infected intestinal tissues. All animal management and experimental protocols were approved by the Institutional Animal Care and Use Committee at Prince of Songkla University (MHESI 68014/1457).
Assessment of clinical signs and postmortem examinationsClinical assessments of live fish including observations of swimming behavior, respiratory patterns, body condition, body surface abnormalities, and external organs, were conducted during the holding period in the laboratory prior to necropsy. All fish were maintained in aerated seawater tanks under controlled conditions (temperature: 26–28°C; pH: 6.5–8; salinity: ~20 ppt) for 8–12 hours before sample collection. Prior to postmortem examination, fish were euthanized by immersion in a solution of 100 mg/L Aquanes® (Better Pharma), which contains eugenol as the active ingredient, and maintained in the solution for a minimum of 20 min following the cessation of opercular movement. All fish were measured for total body length and body weight, followed by dissection to evaluate gross lesions in internal organs.
Bacteriological examinationBacteriological culture was performed as part of the routine diagnostic protocol. Sterile swabs were used to collect samples from the anterior kidney, spleen, and liver of all 44 sampled fish. The swabs were inoculated onto Tryptic Soy Agar (TSA; DifcoTM, Becton, Dickinson and Company) supplemented with 5% sheep blood, and Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar (DifcoTM). The cultures were incubated at 30°C for 24–48 h. In cases where a single bacterial colony was observed, it was subcultured onto TSA supplemented with 1.5% NaCl and subsequently identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, employing a Bruker Microflex MALDI Biotyper 2.0 system (Bruker Daltonics), as previously described by Assis et al. (2017). Due to the absence of distinct and severe clinical lesions, viral detection tests were not performed.
Intestinal parasite detection and evaluationIntestinal parasites were evaluated through gross examination, histological analysis, and conventional polymerase chain reaction (PCR) techniques. Intestinal helminths were primarily detected by gross examination, whereas the detection and assessment of coccidia were performed using histological examination and conventional PCR methods. For histological analysis, mid-to-posterior tissue samples (~3–6 cm) were fixed in 10% neutral buffered formalin for 18–24 hours, processed routinely, embedded in paraffin, sectioned at 4–5 μm, and stained with hematoxylin and eosin (H&E). Coccidian developmental stages and associated lesions were examined using a Nikon ECLIPSE Ni-U upright microscope equipped with a digital camera for image capture. The identification of all parasitic stages was based on methods from previous studies (Lovy and Friend, 2015). Quantification involved counting meronts, gamonts, and unsporulated oocysts across five non-overlapping fields under a light microscope. The prevalence and intensity of coccidial infection were calculated following the methodology outlined by Suyapoh et al. (2022a).
For PCR identification, genomic DNA was extracted from one gram of intestine from each fish using the PureLinkTM Genomic DNA Kit (Invitrogen). Briefly, the samples were homogenized with a tissue grinder and digested with proteinase K. DNA was then isolated with a DNA extraction buffer and precipitated with ethanol according to the manufacturer’s instructions. The DNA concentration was measured using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Inc.). The nested PCR assay was conducted to amplify 18S rRNA gene of Cryptosporidium spp., following the protocol according to Certad et al. (2015). The external primer pair used was 5′-GACATATCWTTYAAGTTTCTGACC-3′ and 5′-CTGAAGGAGTAAGGAACAACC-3′, while the internal primer pair consisted of 5′-CCTATCAGCTTTMGACGGTAGG-3′ and 5′-TCTAAGAATTTCACCTCTGACTG-3′, producing a ~588 bp product. The PCR conditions included an initial denaturation at 95°C for 2 min, followed by 40 amplification cycles consisting of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 1 min. The PCR products were analyzed via electrophoresis on a 1.5% agarose gel (Invitrogen,) and visualized under UV light using an E-Box VXII transilluminator (Vilber).
18S rRNA Sequencing and phylogenetic analysis of CryptosporidiumSix representative PCR-positive products were purified using the GenepHlowTM Gel/PCR kit (Geneaid Biotech Ltd.) and submitted for outsourcing to a commercial sequencing service (ATGC Co., Ltd., Thailand). Nucleotide sequences were analyzed using BioEdit v.7.2 and were cross-referenced with available sequences in the GenBank database using the BLASTN tool provided by the National Centre for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/BLAST) to determine their nucleotide identity.
Our nucleotide sequences for 18S rRNA gene were multiple aligned with additional Cryptosporidium sequences published in GenBank, covering isolates from fish and other hosts species, using the Clustal W algorithm of the MEGA 11 software (Saitou and Nei, 1987). A phylogenetic tree was constructed using the Maximum Likelihood (ML) method and Hasegawa-Kishino-Yano model (Hasegawa et al., 1985) including estimates of invariant sites (I) and gamma distribution. The tree was resampled by 1,000 bootstrap replicates to evaluate the reliability of the groups (Tamura et al., 2021).
Semi-quantitative histopathological studyHistopathological evaluation was conducted to assess intestinal inflammation and lesions, including inflammatory cell infiltration, intestinal necrosis, congestion, and epithelial desquamation. Intestinal inflammation was quantified by semi-quantitative grading the inflammatory cells infiltrating the intestinal tissue, with leukocyte identification criteria based on Clauss et al. (2008). Grading was conducted across five non-overlapping microscopic fields per intestinal section, specifically within the mucosal and submucosal layers, using a light microscope. For histopathological lesions, semi-quantitative grading was applied based on previous related studies (Suyapoh et al., 2022b; Suyapoh et al., 2021). The severity of lesions was scored numerically as 0, 1, 2, and 3, corresponding to “absent,” “mild,” “moderate,” and “severe,” respectively. The grading was based on the proportion of the affected area relative to the unaffected tissue under 40× magnification, with the following criteria: absent = no lesion or ≤1% lesion development; mild = 2–25%; moderate = 26–50%; severe = >50%. Five non-overlapping fields were evaluated per fish, and the mean was calculated to represent the severity of lesions for each fish.
Assessment of oxidative damageThe effect of coccidia infection on host was evaluated by assessing oxidative damage through immunofluorescence (IF). This method was used to detect the expression of reactive oxygen species (ROS) using the 4-HNE marker, DNA damage using the 8-oxodG marker, and cellular apoptosis using the Caspase-3 marker. Paraffin-embedded intestinal sections from all fish were processed for staining. Briefly, 4 μm sections of intestinal tissue were pre-treated at 60°C for 15 min, followed by deparaffinization in xylene and rehydration through a series of ethanol solutions. Antigen retrieval was carried out using citrate buffer (pH 6.0) in a high-pressure cooker for 10 min. Non-specific binding was blocked by incubating the sections in 1% normal horse serum in PBS for 45 min at room temperature (Tangkawattana et al., 2023). To assess the production of an individual marker, the sections were incubated overnight at 4°C with a different primary and secondary antibodies following protocol from (Jantrakajorn et al., 2024). Briefly, the individual sections were incubated overnight at 4°C with a different primary antibodies, including, monoclonal mouse anti-4 Hydroxynonenal antibody (ab48506, Abcam) at a dilution of 1:50, polyclonal goat anti-8 Hydroxydeoxyguanosine antibody (AB5830, Sigma-Aldrich) at a dilution of 1:200, and polyclonal rabbit anti-caspase 3 antibody (SAB5700196, Sigma-Aldrich, USA) at a dilution of 1:200. After washing with PBS, sections were incubated for 1 h at room temperature in the dark with a different secondary antibody raised in different species: DyLight-488 Donkey anti-mouse IgG (H+L) Antibody (Invitrogen), Alexa FluorTM 555 rabbit anti-goat IgG (H+L) Antibody (Invitrogen), and CFTM 555 Goat anti-rabbit IgG (H+L) Antibody (Sigma-Aldrich), both in 1% BSA. Sections were then mounted with ProLongTM Gold Antifade Mountant containing DNA Stain DAPI (Thermo Fisher Scientific, USA). Fluorescence visualization was conducted using a fluorescence microscope. Immunofluorescence procedures were standardized using previously validated polyclonal antibodies, with consistent commercial batches and a comprehensive control system (positive, negative, true positive, and true negative controls) to ensure antibody specificity and minimize background fluorescence.
Quantitative image analysis of immunofluorescence stainsPositive expression for 4-HNE and 8-oxodG was observed in the cytoplasm and nucleus, respectively, while Caspase-3 positivity was seen in both the cytoplasm and nucleus of intestinal tissue. For the single-marker tests, color intensity was measured in 10 non-overlapping fields of view at ×20 magnification (VDO capture digital camera, ECLIPSE Ni-U, Nikon) using ImageJ software (Schneider et al., 2012).
Statistical analysisAll statistical analyses were performed using SPSS version 23.0 (SPSS Inc.). Normality of the data was assessed using the Shapiro–Wilk test. Group comparisons were conducted using the Mann–Whitney U test, and p-values < 0.05 were considered statistically significant. Spearman’s rank correlation coefficient (Spearman’s r) was used to assess the relationships between fish size and various condition factors associated with intestinal coccidia. Prior to applying Spearman’s correlation, linearity and bivariate normal distribution were initially evaluated. The correlation coefficient (r) and corresponding p-values were calculated, with p < 0.05 considered statistically significant.
A total of 44 juvenile pearl gentian groupers, aged 60 days, with an average length of 9.0 ± 1.0 cm and an average body width of 2.6 ± 0.3 cm, were examined. Some individuals exhibited mild emaciation, lethargy, and sluggish swimming with delayed opercular movement. Postmortem examination of these fish revealed a scaphoid (sunken) abdomen, and slight darkening of the skin pigmentation was observed in some cases. No erosions or hemorrhagic lesions were detected on the fins or skin. Internal gross lesions were observed in clinically affected fish, with pale livers and distended gall bladders noted in 16 out of 44 individuals. Additionally, 16 out of 44 fish exhibited dilated, fluid-filled intestines, although no evidence of intestinal edema was detected. Intestinal hemorrhage and hyperemia were not prominently observed. In contrast, 27 out of 44 fish that did not exhibit clinical signs showed no remarkable gross lesions. The intestinal organs in these fish were within normal limits in terms of location, size, color, and consistency.
Parasitological and bacteriological examinations of all specimens revealed no evidence of co-infection or concurrent infection with pathogens other than coccidia infection.
Detection and identification of intestinal coccidia in pearl gentian groupersTo assess the coccidial infection status, intestinal samples from juvenile pearl gentian groupers were examined using both PCR and histological techniques. Of the 44 fish assessed, 22 individuals (50.0%) tested positive for coccidia by both methods, while the remaining 22 were negative. Coccidia, in their unsporulated oocyst, were found exclusively in the intestine and were located at various sites within the supra-epithelial regions (epicellular location) (Fig. 1). The average length (mean ± SD) of the unsporulated oocyst stage was 3.2 ± 0.3 μm, and the average width was 2.5 ± 0.1 μm. Quantitative analysis of the coccidia in the intestinal sites revealed that the parasites were most prevalent in the luminal surface, with an infection count of 5.3 ± 2.6 oocysts/high power field (HPF). The intensity of infection was assessed by quantifying the number of coccidia per infected area. An average infection intensity was 217.3 ± 95.3 coccidia/mm2. The morphological characteristics, including the spherical to ovoid shape and basophilic staining with H&E were consistent with those of Cryptosporidium spp.
Phylogenetic analysis of 18S rRNA gene confirmed the presence of Cryptosporidium bollandi (Fig. 2). Nucleotide sequences of the partial 18S rDNA gene (552–561 bp) from Cryptosporidium isolates in this study (isolates. PSU01–PSU06) were deposited in GenBank under the accession numbers PV624682–PV624687. BLASTN analysis revealed that all sequences were 100% identical to each other and showed 100% identity with C. bollandi (PP294650–PP294652), previously isolated from Asian seabass Lates calcarifer in Thailand. Phylogenetic analysis demonstrated two major clades: one comprising piscine Cryptosporidium species and the other including sequences from terrestrial animals and human. The study isolates clustered within the piscine clade alongside C. bollandi from Asian seabass and spotted scat Scatophagus argus in Thailand, as well as Cryptosporidium sp. from angelfish Pterophyllum scalare in Australia, with strong bootstrap support (97%). In contrast, Cryptosporidium piscine genotype 5 (KR610344), C. molnari, C. abrahamseni, and other species from freshwater fish formed separate branches, indicating genetic divergence from the isolates in this study.
Infected fish exhibited a mean total length of 8.1 ± 0.6 cm and a mean body width of 2.3 ± 0.05 cm. In contrast, non-infected fish had a significantly greater mean length of 9.6 ± 0.6 cm and a mean body width of 2.7 ± 0.3 cm. The average body weight of infected fish was 4.8 ± 0.6 g, whereas non-infected individuals had a significantly higher mean weight of 9.8 ± 0.8 g. These differences in morphometric parameters between infected and non-infected groups were statistically significant (Mann–Whitney U test, p < 0.05), indicating signs of cachexia.
The average condition factor (K), calculated as body weight divided by the cube of body length, of Cryptosporidium-infected fish was significantly lower (K = 0.9 ± 0.1) than that of uninfected fish (K = 1.1 ± 0.2) (Mann–Whitney U test, p < 0.05). The condition factor showed a significant negative correlation with vascular congestion (Spearman’s r, r = –0.4, p = 0.02), indicating that increased congestion was associated with reduced body condition. Other pathological features, including submucosal inflammation (r = –0.2, p = 0.24), mucosal inflammation (r = –0.2, p = 0.33), and goblet cell hyperplasia (r = –0.2, p = 0.32), also showed negative correlations with condition factor, though these were not statistically significant (Table 1).
| Parameters | Spearman’s r | p value | Interpretation |
|---|---|---|---|
| Congestion | –0.4 | 0.02 | Moderate negative correlation, statistical significance |
| Submucosal inflammatory cell infiltration | –0.2 | 0.24 | Weak negative correlation, no statistical significance |
| Mucosal inflammatory cell infiltration | –0.2 | 0.33 | Weak negative correlation, no statistical significance |
| Goblet cell hyperplasia | –0.2 | 0.32 | Weak negative correlation, no statistical significance |
| 4-HNE | +0.2 | 0.33 | Weak positive correlation, no statistical significance |
| 8-oxodG | +0.1 | 0.59 | Weak positive correlation, no statistical significance |
| Caspase-3 | –0.3 | 0.08 | Moderate negative correlation, no statistical significance |
The relationship between condition factor and cellular-level responses was also investigated using immunofluorescence markers. A negative correlation was observed between condition factor and Caspase-3 expression (r = –0.3, p = 0.08), suggesting a trend toward increased apoptotic activity in fish with lower body condition, although this finding did not reach statistical significance. In contrast, 4-HNE (a marker of lipid peroxidation) and 8-oxodG (a marker of oxidative DNA damage) demonstrated weak positive correlations with condition factor (r = 0.2 and r = 0.1, respectively), without statistical significance (Table 1). These findings indicate that fish infected with C. bollandi have significantly lower condition factors, reflecting compromised body condition. The condition factor is also associated with the severity of intestinal lesions and trends in apoptotic responses at the cellular level.
The analysis of the relationship between infection intensity, histopathological alterations, and cellular-level responses revealed that only submucosal inflammation demonstrated a moderate and statistically significant positive correlation with infection intensity (Spearman’s r = 0.6, p = 0.01). This finding indicates that fish with higher C. bollandi burdens tend to exhibit more pronounced submucosal inflammatory responses. In contrast, other pathological and immunofluorescence markers showed only weak or negligible correlations, none of which reached statistical significance (Table 2).
| Parameters | Spearman’s r | p value | Interpretation |
|---|---|---|---|
| Congestion | 0.02 | 0.93 | Weak positive correlation, no statistical significance. |
| Submucosal inflammatory cell infiltration | 0.6 | 0.01 | Moderate positive correlation, statistical significance. |
| Mucosal inflammatory cell infiltration | 0.4 | 0.06 | Moderate positive correlation, no statistical significance. |
| Goblet cell hyperplasia | –0.2 | 0.51 | Weak negative correlation, no statistical significance. |
| 4-HNE | 0.1 | 0.66 | Weak positive correlation, no statistical significance. |
| 8-oxodG | –0.03 | 0.90 | Negligible negative correlation, no statistical significance. |
| Caspase-3 | 0.3 | 0.19 | Weak positive correlation, no statistical significance. |
To determine whether piscine intestinal coccidia induce significant inflammation in the gut of pearl gentian groupers, we examined the infiltration of inflammatory cells in the intestines of both infected and uninfected fish. Infected fish exhibited predominant infiltration of mononuclear cells, particularly lymphocytes, which were localized to the submucosa (Fig. 3Ai–Aii) and intraepithelial layers (Fig. 3Aiii–Aiv), without extending into the deeper intestinal layers. In some infected fish, infiltration of polymorphonuclear cells was also observed. The Cryptosporidium-infected group demonstrated significantly higher grades of submucosal inflammation (1.8 ± 0.8, n = 22) and intraepithelial inflammation (0.8 ± 1.0, n = 22) compared to the uninfected group, which exhibited grades of 0.8 ± 0.2 (n = 22) and 0.2 ± 0.5 (n = 22), respectively (Fig. 3B). Mild to moderate active congestion or hyperemia, indicated by dilation of blood vessels, was observed in the inflammatory regions (Fig. 3Avii–Aviii). The infected group showed a significantly higher degree of congestion (1.0 ± 0.2) compared to the uninfected group (0.2 ± 0.6) (Fig. 3B). Additionally, goblet cell hyperplasia was predominantly observed in the infected group (1.8 ± 0.3), while the non-infected group exhibited a lower degree of hyperplasia (1.0 ± 1.3) (Fig. 3Av–Avi & 3B). The differences between the groups were statistically significant (Mann–Whitney U test, p < 0.001). However, there was no evidence of extensive inflammation, epithelial desquamation, or intestinal necrosis in both groups.
Immunofluorescence staining using 4-HNE, 8-oxodG, and Caspase-3 markers was performed on the intestines of all 44 fish to study the effects of C. bollandi infection-induced inflammation on ROS production, DNA damage, and cellular apoptosis in enterocytes and peripheral structures. Cells positive for 4-HNE exhibited cytoplasmic staining, while 8-oxodG-positive cells displayed nuclear staining, and Caspase-3-positive cells displayed both intranuclear and cytoplasmic staining. In infected fish, 4-HNE expression was first detected in the epithelium, particularly in areas with epicellular (supra-epithelium) coccidia. Increased expression of 4-HNE was observed in regions with higher levels of inflammatory cell infiltration. The areas of intense 4-HNE expression also prominently co-expressed 8-oxodG (Fig. 4A). Similarly, elevated Caspase-3 expression was found in regions with increased 4-HNE expression (Fig. 4B). Both markers were detected in areas with infiltration of various inflammatory cells.
Quantitative analysis revealed pronounced cytoplasmic expression of 4-HNE in the intestinal epithelium of both Cryptosporidium-infected and uninfected fish (Fig. 4C). The median intensity of 4-HNE expression was significantly higher in the infected group (4.3 ± 5.0) compared to the uninfected group (1.4 ± 4.0). Specifically, infection with C. bollandi. in juvenile fish led to a significant increase in 4-HNE expression (Mann–Whitney U test, p < 0.05), while minimal 4-HNE expression was observed in uninfected fish. Similarly, 8-oxodG expression was significantly upregulated only in the infected group (0.1 ± 0.3). The average intensity of nuclear staining for 8-oxodG was notably higher in the infected group compared to the uninfected group (Mann–Whitney U test, p < 0.001) (Fig. 3C). Furthermore, Caspase-3 expression levels were significantly higher in the Cryptosporidium-infected group (3.8 ± 1.8) compared to the uninfected group (0.002 ± 0.003) (Mann–Whitney U test, p < 0.001) (Fig. 3C). Infection resulted in a significant increase in Caspase-3 expression intensity in the infected fish (Mann–Whitney U test, p < 0.001) (Fig. 4C). No Caspase-3 immunofluorescence was observed in the uninfected fish.
Through morphological assessment by histology based, and PCR-based detection, we identified the presence of the piscine intestinal coccidia, C. bollandi., in the intestinal tracts of 22 juvenile pearl gentian groupers obtained from a marine cage farm. This protozoan has been recognized as an emerging aquatic pathogen, previously reported in various fish species, including black ghost knife fish Apteronotus albifrons, neon tetra Paracheirodon innesi, oscar fish Astronotus ocellatus, red-eye tetra Moenkhausia sanctaefilomenae, and Asian seabass L. calcarifer (Bolland et al., 2020; Jantrakajorn et al., 2024). Our findings indicate a moderate prevalence of C. bollandi infection (50%) in pearl gentian groupers. This prevalence is comparable to that reported in black ghost knifefish at 50% (Bolland et al., 2020), but lower than the rates observed in 60- and 90-day-old juvenile Asian seabass, which reached 65.9% (Jantrakajorn et al., 2024), and in red-eye tetra reported at 62.5% (Bolland et al., 2020). Conversely, the prevalence in our study is higher than that documented in oscar fish at 37.5% and neon tetra at 6.1% (Bolland et al., 2020). These findings suggest that C. bollandi infection is relatively common in pearl gentian groupers. However, the absence of wet mount examination of stool samples—which could facilitate identification of characteristic morphological structures and more accurate assessment of parasite burden—represents a limitation of this study.
Phylogenetic analysis revealed a clear genetic distinction between piscine-derived Cryptosporidium isolates and those from terrestrial vertebrate hosts, consistent with previous study (Couso-Pérez et al., 2022). All isolates in this study exhibited 100% genetic identity to C. bollandi, previously identified in Asian seabass and spotted scat —two economic fish species in Southern Thailand— and clustered within a distinct clade, separate from other piscine-derived Cryptosporidium species. These findings suggest that C. bollandi has a broad host range in Thailand. This is in line with a previous report indicating that parasitic infections, such as monogeneans, myxosporeans, and copepods, can infect multiple fish species within saltwater cage systems (Jahangiri et al., 2022). The common practice of culturing different fish species within shared marine cage environments may facilitate cross-species transmission of C. bollandi, particularly under high-density conditions that increase stress and susceptibility to infection (Sitjà-Bobadilla et al., 2005). To the best of our knowledge, this is the first report of C. bollandi infection in juvenile pearl gentian grouper.
The clinical outcomes observed in juvenile pearl gentian groupers—mild emaciation, darkened skin pigmentation, lethargy, sluggish swimming, and delayed opercular movement—were noted prior to confirming C. bollandi infection. These manifestations are common nonspecific signs of coccidiosis in fish (Pasnik et al., 2005). However, our findings support a link between infection and reduced body condition. Infected fish had significantly lower condition factors (K = 0.9) compared to uninfected fish (K = 1.1), and infection intensity showed a significant positive correlation with submucosal inflammation (r = 0.6, p = 0.01). These results suggest that C. bollandi infection may contribute to the observed clinical outcomes, particularly emaciation and reduced vitality. Previous reports in other fish species infected with piscine intestinal coccidia have described more severe clinical signs, including erosions and hemorrhagic lesions on the skin and gills, often associated with concomitant bacterial infections (Suyapoh et al., 2024). The observed changes in nonspecific clinical signs suggest that Cryptosporidium infection can impact the systemic health condition of juvenile fish (Suyapoh et al., 2024). Numerous studies in other animal species have further demonstrated that coccidial infections can impair growth performance, leading to reduced bone growth rates, inferior meat quality, diminished egg production and quality, and overall impaired growth and development (Chen et al., 2025). However, the potential relationship between Cryptosporidium infection and growth performance in fish remains unclear. Necropsy evaluation revealed internal gross lesions, including pale livers, distended gall bladders, and dilated, fluid-filled intestines. These lesions were consistent with findings from previous studies (Suyapoh et al., 2024). Histopathological examination revealed the moderate presence of C. bollandi in an epicellular location within the intestinal mucosa of juvenile pearl gentian grouper, accompanied by varying degrees of tissue damage. Mild to moderate intestinal inflammation was observed, localized to the mucosal and submucosal layers, without evidence of extension into the deeper muscular layers of the intestine. It is well established that lymphocyte infiltration into the submucosa is a hallmark of cryptosporidiosis throughout the course of infection (Sitjà-Bobadilla et al., 2016), followed by granulocyte and lymphocyte infiltration within intracellular compartments (Sitjà-Bobadilla et al., 2016). This infiltration pattern contributes to persistent or chronic inflammation, exacerbating pathological changes in the gut (Suyapoh et al., 2024; Suyapoh et al., 2022b). The inflammatory lesions observed in fish in this study were consistent with those reported in other fish species affected by Cryptosporidium infections (Suyapoh et al., 2024; Thongrin et al., 2025). However, the severity of pathological changes appeared to be lower in pearl gentian grouper compared to other fish species. In addition to inflammation, intestinal congestion was a commonly observed lesion, a feature frequently reported in coccidiosis-affected fish (Lovy and Friend, 2015). Furthermore, goblet cell hyperplasia was evident in infected fish, which is consistent with the pathogenesis of piscine intestinal coccidial infections (Thongrin et al., 2025). This phenomenon may be linked to the role of mucin in protozoan disease pathogenesis, particularly in facilitating adhesion and colonization of the parasite at the site of entry (Martínez-Ocaña et al., 2020).
The pathological effects of C. bollandi infection is closely associated with inflammatory responses, particularly through the significant production of ROS, which can exacerbate tissue damage via DNA damage and cellular apoptosis (Jantrakajorn et al., 2024). In the present study, we observed an increased production of ROS in infected fish, correlating with inflammatory activation. This response is consistent with previous findings indicating that Cryptosporidium infection stimulates the generation of both reactive nitrogen species (RNS) and ROS (Jantrakajorn et al., 2024; Suyapoh et al., 2024). During persistent inflammation, excessive free radical production is primarily attributed to the activity of peroxidase enzymes and teleost granulocytes (Reite and Evensen, 2006). Additionally, intestinal dysbiosis induced by infections with other coccidian parasites, such as Eimeria spp., has been shown to enhance host-bacterial pathogen interactions, further contributing to the overproduction of ROS in teleost fish (Lu et al., 2021). This is another source of the overproduction of ROS in teleost (Sayyaf Dezfuli et al., 2023). The increased oxidative stress associated with Cryptosporidium infection has been linked to significant DNA damage and elevated cellular apoptosis, a pathogenic mechanism that has been previously reported in other fish species (Jantrakajorn et al., 2024). Overall, our findings provide significant insights into the pathological impact of piscine intestinal cryptosporidiosis by elucidating intestinal lesions and the underlying mechanisms linking inflammation to DNA damage and cellular apoptosis through ROS overproduction in juvenile pearl gentian grouper. Our findings contribute to a deeper understanding of piscine cryptosporidiosis. Given the economic importance of pearl gentian grouper, further research is essential to develop effective mitigation strategies, including improved management practices and targeted therapeutic interventions, to minimize the impact of C. bollandi infection in aquaculture systems.
This research was supported by Faculty of Veterinary Science, Prince of Songkla University (VET6704157S).
The authors declare that there is no conflict of interest.
Conceptualization, WS, PS, NK; Methodology, WS, PS, NK, AY, KN, WM, and KS; Validation, WS; Formal Analysis, WS, PS, NK; Investigation, WS; Resources, WS; Writing – Original Draft Preparation, WS; Writing – Review & Editing, WS, PS, NK, SJ; Supervision, WS. All authors have read and agreed to the submitted version of the manuscript.
