Transmission Routes and Experimental Infection Model of Inodosporus fujiokai (Microsporidia) in Fish

Ryuhei Kinami; Riku Kim; Sho Shirakashi; Kazuhiro Sugahara; Michitaka Yamamoto; Tetsuya Yanagida; Hiroshi Yokoyama

doi:10.3147/jsfp.60.61

Abstract

Inodosporus fujiokai is a lethal microsporidian that forms cysts in the muscle of salmonids. Fish are infected by ingesting the invertebrate host, the common prawn Palaemon paucidens. However, the precise transmission routes of I. fujiokai remain uncertain. Three trials were carried out to infect amago salmon Oncorhynchus masou ishikawae and rainbow trout O. mykiss through oral intubation or immersion using I. fujiokai spores from infected prawns. Both methods effectively induced I. fujiokai infections and high mortalities in challenged fish. Oncorhynchus masou ishikawae appeared more vulnerable to I. fujiokai infection, with up to 100% mortality. The fish cohabited with spore-intubated fish also exhibited high infection prevalences, suggesting that spores excreted from fish which consumed infected prawns cause waterborne transmission to other fish. Spore immersion at 2.6 × 10⁴ spores/mL and above led to 67% mortality and significant cyst formation in O. mykiss, while 2.6 × 10² spores/mL induced neither mortality nor cyst formation. Besides, fish fed with infected fish muscle tissue or cohabiting with infected fish did not show signs of illness or death, indicating that fish-to-fish transmission does not occur. This study provides significant insights into the transmission biology of I. fujiokai and establishes an experimental infection model for future research.

A new microsporidian disease affecting rainbow trout Oncorhynchus mykiss farmed in Shiga Prefecture, Japan, was first reported by Yamamoto et al. (2021). Subsequently, Yanagida et al. (2023) identified the causative organism as a new microsporidian species, Inodosporus fujiokai through molecular and morphological analyses. Infected fish exhibited white and/or red cysts in the trunk and heart muscles, leading to fatalities associated with hypoxic symptoms (Yamamoto et al., 2021). Histopathological examination of infected O. mykiss suggested that cyst formation in the heart induced myocarditis, impairing cardiac function and resulting in hypoxia (Yamamoto et al., 2023).

This microsporidian utilizes the common prawn, Palaemon paucidens, as its invertebrate host and produces morphologically distinct spores in both the prawn and fish hosts, with the spores in prawns possessing filamentous appendages that are absent in the fish host (Yanagida et al., 2023). While previous trials successfully reproduced the infection and resulting disease in two salmonids, O. mykiss and Biwa trout Oncorhynchus masou subsp. by feeding them infected P. paucidens collected from Lake Biwa, Shiga Prefecture, Japan, the possible transmission routes beyond the ingestion of infected prawns remain unexplored (Yamamoto et al., 2021, 2023).

In earlier experiments, infection prevalences of up to 100% were observed in the fish housed in tanks that received freshly captured wild P. paucidens from Lake Biwa (Yamamoto et al., 2021, 2023). Despite this, the infection prevalence among wild P. paucidens is low, estimated to be around 0.1% (Yanagida et al., 2023), suggesting that it is unlikely all fish become infected by consuming infected prawns alone. This discrepancy raises alternative transmission mechanisms that warrant further investigation.

The use of prawn-feeding for experimental infection has its limitations. Primarily, it does not allow for precise control of the amount of I. fujiokai spores ingested by each fish, resulting in non-quantifiable and inconsistent outcomes. Furthermore, prawns must be fed repeatedly to ensure infection, creating an extended infection window that makes it difficult to determine the exact onset of infection. Additionally, the prawn-feeding method may be unsuitable for comparing susceptibility across different fish species due to their varying appetites for prawns. To overcome these limitations, there is a need for a more controlled and quantitative experimental infection method. Therefore, this study aimed to investigate the potential transmission routes of I. fujiokai and establish a reliable, quantitative experimental infection method for future investigations.

Materials and Methods

Experimental animals

All fish used in this study were bred at two hatcheries. They were raised from eggs and fed formulated pellet diets until use. Amago salmon Oncorhynchus masou ishikawae was produced and reared in stream water at the Shingu Station of Aquaculture Research Institute at Kindai University (Shingu ARIKU) in Wakayama Prefecture, while O. mykiss and O. masou subsp. were produced and reared in well water at the Samegai Trout Farm of the Shiga Prefectural Fisheries Experiment Station (Shiga FES). Both hatcheries have no prior records of microsporidian infection. Ten fish from the same production batches used for each trial were tested by the PCR described below to confirm the absence of I. fujiokai infection prior to the experiment.

Spores of I. fujiokai used for infection experiments were obtained from naturally infected wild P. paucidens captured in Lake Biwa. Prawn individuals exhibiting an opaque appearance, characteristic of microsporidian infection (Yanagida et al., 2023), were selectively captured with a dipnet and kept alive until the infection trials.

Preparation of I. fujiokai spore suspension

The spore suspension of I. fujiokai was prepared by processing opaque prawns, presumed to be infected. These prawns were decapitated, and their carapace was removed to collect the tail muscle. A wet mount of a minute piece of the muscle tissue was examined under a light microscope to confirm microsporidian infection. If spores with characteristic filamentous appendages were detected, the prawn was tentatively determined to be infected with I. fujiokai, and this was later confirmed by the multiplex PCR (Yanagida et al., 2023). The infected tail muscles were pooled in a tube and homogenized with a plastic pestle with approximately 1–2 times the volume of Dulbecco’s phosphate-buffered saline (DPBS) (−) to prepare the spore suspension. This suspension was kept on ice until used for the experimental infection.

Spore concentration was calculated later from the freeze-thawed spore suspension subsample. Spore suspensions were appropriately diluted in DPBS (−) when necessary, and images were captured on a hemocytometer using a digital microscope (VHX-7000, Keyence). From these images, the number of sporophorous vesicles, a parasite-formed membrane, was counted using ImageJ software (Abramoff et al., 2004). Spore density was estimated by calculating the average number of sporophorous vesicles in five 0.1 mm³ areas and multiplying it by 8, representing the typical number of spores in a vesicle (Yanagida et al., 2023).

Experimental infections with spore intubation or immersion

A total of four infection trials (Trials 1 to 4) were conducted (summarized in Table 1). Trials 1 and 2 were performed concurrently at different locations to investigate the possibility of waterborne transmission in two salmonid species. In Trial 1, conducted at the Shingu ARIKU, O. masou ishikawae (approx. 5 g in body weight) were infected with I. fujiokai via either oral intubation or immersion in the spore suspension.

Table 1. Experimental conditions, mortality and Inodosporus fujiokai infection in Oncorhynchus spp. in infection trials

Trials location	Date Water temp.	Fish species Mean body weight	Infection methods	Challenge doses and experimental period	Cumulative Mortality*¹	Cysts positive*¹	Detection rate cysts+PCR*¹
Trial 1	6/2–7/2, 2021	O. masou ishikawae	Intubation	3.3 × 10⁸ spores/fish	94.7% (18/19) ^a	63.2% (12/19) ^a	100% (19/19) ^a
Shingu ARIKU	15.0–17.3°C	5.30 ± 0.57 g	Int. cohabited		100% (5/5) ^a	100% (5/5) ^a	100% (5/5) ^a
			Immersion	1.8 × 10⁶ spores/mL	100% (19/19) ^a	36.8% (7/19) ^a	89.5% (17/19) ^a
			Imm. cohabited		0.0% (0/5) ^b	100% (5/5) ^a	100% (5/5) ^a
Trial 2	6/9–7/6, 2021	O. mykiss	Intubation	6.9 × 10⁸ spores/fish	73.0% (27/37) ^a	94.6% (35/37) ^a	100% (37/37) ^a
Shiga FES	17.0–18.1°C	4.83 ± 1.06 g	Immersion	1.4 × 10⁶ spores/mL	70.0% (14/20) ^a	80.0% (16/20) ^a	90.0% (18/20) ^a
			Control		27.8% (10/36) ^b	0.0% (0/36) ^b	0.0% (0/36) ^b
Trial 3	6/9–7/6, 2021	O. mykiss	Immersion	2.6 × 10⁶ spores/mL	66.6% (10/15) ^a	73.3% (11/15) ^a	100% (15/15) ^a
Shiga FES	17.3–18.1°C	5.14 ± 1.14 g	Immersion	2.6 × 10⁴ spores/mL	66.6% (10/15) ^a	80.0% (12/15) ^a	100% (15/15) ^a
			Immersion	2.6 × 10² spores/mL	0.0% (0/15) ^b	0.0% (0/15) ^b	100% (15/15) ^a
Trial 4	7/30–9/6, 2020	O. mykiss (ca. 300 g)	Fed infected muscle*²	1.2 g muscle × 10 d*⁴	0.0% (0/30) ^a	0.0% (0/30) ^a	0.0% (0/30) ^a
Shiga FES	18.0–18.3°C	O. masou. subsp. (ca. 90 g)	Fed infected muscle*²	0.9 g muscle × 7 d*⁴	0.0% (0/10) ^a	0.0% (0/10) ^a	0.0% (0/10) ^a
		O. mykiss (ca. 300 g)	Cohabitation*³	34 d	0.0% (0/20) ^a	0.0% (0/20) ^a	0.0% (0/20) ^a
		O. masou. subsp. (ca. 90 g)	Cohabitation*³	38 d	0.0% (0/19) ^a	0.0% (0/19) ^a	0.0% (0/19) ^a

*¹ Different letters indicate significant difference within each trial (p < 0.05).

*² Feeding muscle of heavily infected O. mykiss with high density of I. fujiokai cysts (spores).

*³ Cohabitation with fish that have already developed I. fujiokai cysts within the trunk muscle.

*⁴ Calculated by dividing the average amount of muscle tissue from infected fish administered to the tank by the number of fish in the tank.

For intubation infection, 20 fish were anaesthetized with 2-phenoxyethanol, individually weighed, and marked by clipping the adipose fin. Each fish was orally intubated with 0.1 mL of spore suspension (3.3 × 10⁸ spores) directly into the stomach using a feeding needle. The fish were then transferred to a recovery tank, observed to ensure no regurgitation, and subsequently placed in a 100 L tank.

For spore immersion, 20 fish were anaesthetized, weighed, and marked as described above. They were collectively immersed in a plastic container with 5 L of well water containing spores whose concentration of 1.8 × 10⁶ spores/mL for 4 h. The immersion tank was aerated and maintained at a constant water temperature (15–16°C) using a water bath. After immersion, the fish were transferred to another 100 L tank. One fish from both intubation and immersion groups was lost during or shortly after the infection, reducing the group size to 19.

To determine if I. fujiokai could transmit from fish that have been intubated with or immersed in spore suspension to other fish, five naïve fish were added to each tank (width 40 cm × length 60 cm × height 30 cm, water depth 15 cm) the day after the infection trials. All fish were reared for 30 days in a water exchange system using well water, with the water being completely changed several times a day, and were fed pellet feed daily. Dead or moribund fish were sampled, stored individually in plastic bags, and frozen. At the end of the experiment, all remaining fish were sampled and kept frozen for further examination.

In Trial 2, similar infection methods were applied to O. mykiss (approx. 5 g) at Shiga FES. For oral intubation, 37 fish were orally administered a spore suspension (6.9 × 10⁸/fish) as described above. For spore immersion, 20 fish were immersed in 15 L of well water containing spores (1.4 × 10⁶ spores/mL). After the challenge, the fish from each group were housed in separate 65 L tanks. Additionally, 36 untreated fish were kept in a control group tank. All fish were reared for approximately one month in flow-through well water and regular feeding. Samples were collected as described in Trial 1.

Trial 3, conducted at Shiga FES, aimed to determine the spore concentration required for infection by immersion. Forty-five O. mykiss were equally divided into three containers, each with 15 L of water. Spore suspensions pre-diluted to either 0-, 100- or 10,000- fold with DPBS (−). were added to each container, resulting in immersion concentrations of 2.6 × 10⁶, 2.6 × 10⁴ or 2.6 × 10² spores/mL, respectively. After immersing for 4 h, fish were reared in separate tanks and sampled similarly to the previous trials.

Trial 4 was additionally designed to investigate the potential for horizontal transmission of I. fujiokai between fish. Conducted at Shiga FES, the experiment employed two challenge methods; feeding infected trout muscle tissue and cohabitation with infected fish. The challenges used two susceptible species, O. mykiss and O. masou subsp. For the feeding challenge, O. mykiss were fed with chopped trunk muscle from heavily infected conspecifics, confirmed to harbor a high density of I. fujiokai cysts. A total of 23–70 g (1.2 ± 0.4 g/fish/d) of chopped trunk muscle tissue was given daily to 30 naïve O. mykiss (approx. 300 g) kept in a 1,000 L tank over 10 consecutive days (Table 1). In a parallel setup, O. masou subsp. (approx. 90 g) were fed minced infected muscle tissues that was mixed with formulated pellets to improve palatability, as they showed low preference for raw fish muscle. They were administered at 0.9 ± 0.2 g tissue per fish per day for over a seven-day period. During feeding, fish were closely observed to ensure consumption of the infected tissue. After the feeding period, all fish were maintained on pellet feeds, and sampled at 25 and 30 days post-feeding for O. mykiss and O. masou subsp., respectively, to assess I. fujiokai infection.

For the cohabitation challenge, naïve O. mykiss and O. masou subsp. (marked by fin clipping) were stocked in tanks (diameter 136 cm × depth 70 cm, water depth 45 cm) housing infected conspecifics that had been previously fed with I. fujiokai infected P. paucidens (Table 1). The naïve fish were added to the tank 16 or 20 days after the end of prawn feeding period, ensuring minimal prawn-derived spores remained, as the tank received flow-through water at a rate exceeding 10 water volumes per day. By the start of cohabitation, the previously infected fish had already developed I. fujiokai cysts in their trunk muscle, with infection prevalence confirmed to exceed 80%. Throughout the cohabitation period, only pellet feed was provided, and the cohabiting fish were sampled at 34 and 38 days after the initiation of cohabitation to evaluate the potential for horizontal transmission.

Examination of I. fujiokai infection

Each sampled fish was microscopically examined for microsporidian infection following the method described by Yamamoto et al. (2023). Briefly, defrosted fish samples were measured, filleted, and the skinned left-side trunk muscle was flattened between two glass plates for cyst detection. If cysts were observed under a stereo microscope, a tissue sample was further examined under a light microscope at higher magnifications to confirm the presence of microsporidian spores. Fish with confirmed cysts were classified as cyst-positive. The heart muscle was examined similarly, except in Trial 3 for which heart was not checked. If neither cysts nor spores were observed, the I. fujiokai-specific PCR targeting small subunit ribosomal DNA (SSU rDNA) was conducted for detecting subclinical infection. DNA was individually extracted from approximately 30 mg of homogenized trunk muscle using QIAamp DNA Mini Kit (QIAGEN) as per manufacturer’s instruction and eluted in 100 μL of Buffer AE.

Examination of the PCR method

In this study, a new I. fujiokai-specific PCR detection method was developed to reduce cost and labour compared to the previously established multiplex PCR method that distinguishes four microsporidian species in P. paucidens (Yamamoto et al., 2023). Species-specific forward primer (MspIf_F: 5′- GCC TAC ACT GTT AAA GAT AAA AT -3′), which is also used in the multiplex PCR (Yamamoto et al., 2023), and the newly designed species-specific reverse primer (MspIf_R: 5′- CAC TCA CTC GCT TTC A -3′) were used for the PCR. Reaction mixtures were assembled in 20 μL volume with; 0.5 units of TaKaRa Ex Taq HS (TaKaRa Bio Inc.), 1 × Ex Taq Buffer, 0.2 mM each of dNTPs, 15 pmol of each primer and 1 μL of template DNA. After initial denaturation at 95°C for 3 min, DNA was amplified by 35 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, followed by final extension of 72°C for 5 min. PCR amplicons were visualized after 1.5% agarose gel electrophoresis, and samples with the specific 588 bp band were judged as PCR-positive. The sensitivity of the PCR was estimated using a series of serial 10-fold dilutions of the plasmid containing the rRNA gene fragment of I. fujiokai as previously described (Yamamoto et al., 2023). The specificity of the PCR was verified using the genomic DNA extracted from the four microsporidians infecting P. paucidens in Lake Biwa, namely I. fujiokai, Microsporidium sp. CP-2022A, Microsporidium sp. CP-2022B, and Microsporidium sp. CP-2022C (Yanagida et al., 2023). For molecular confirmation, the same PCR was conducted on three cyst-harboring individuals from each trial.

Statistical analysis

Mortality comparisons between groups were analyzed using a Kaplan-Meier survival analysis with a log-rank test, followed by post-hoc pairwise comparisons. The proportions of individuals testing positive for cysts and PCR (detection rates) were compared between groups using a Chi-square test. All statistical analyses were performed using JMP version 11 (SAS Institute), with a significance level set at 0.05.

Results

Effectiveness of the PCR method

The I. fujiokai species-specific PCR conducted in this study yielded an amplicon of the expected size only when the genomic DNA of I. fujiokai was used among the four microsporidians tested. Sensitivity test using the serially diluted plasmid revealed the lowest limit of detection was 10 copies (Fig. 1), which is more sensitive than that of the multiplex PCR (100 copies) previously reported (Yamamoto et al., 2023).

Fig. 1. Sensitivity of the Inodosporus fujiokai species-specific PCR. Plasmids containing rRNA gene of I. fujiokai were diluted ten-fold from 10⁵ to 10⁰ copies/μL. M: 100 bp DNA ladder marker. N: Negative control with distilled water as the template.

Trial 1: Transmission route and infection method to amago salmon

In Trial 1, mass mortality in O. masou ishikawae occurred around 20 days post-challenge (dpc) in both the spore-immersion and intubation groups (Fig. 2). The majority of mortalities occurred within a short time frame of just a few days, resulting in cumulative mortality rates of 100% in the immersion group and 94.7% in the intubation group (Table 1). Although mortality in the spore-immersed fish began 9 days earlier, at 10 dpc, compared to the intubated group, there was no statistically significant difference in mortality rates between the two groups. Approximately 4 days after the peak mortality at 20 dpc in the challenged groups, the fish cohabited with the spore-intubated group also began to die, with all five fish succumbing within 3 days (Fig. 2). In contrast, no mortality was observed in the fish cohabited with the spore-immersed group (Table 1, log-rank test, p < 0.001). The dead O. masou ishikawae did not display typical signs of hypoxia, such as open mouth and opercula, but exhibited hypodynamia, including loss of appetite and lethargy, often resting at the bottom of the tank before death.

Fig. 2. Trial 1 Kaplan-Meier survival curves after infection challenge of Inodosporus fujiokai spores from Palaemon paucidens to Oncorhynchus masou ishikawae through spore intubation (black solid line), spore immersion (gray solid line), cohabitation with spore-intubated fish (black broken line), or cohabitation with spore-immersed fish (gray broken line).

Microsporidian cysts were detected in 36.8% of the spore-immersed and 63.2% of spore-intubated O. masou ishikawae, with no statistically significant difference between the two groups. All cyst-harboring individuals were identified after 19 dpc, and no detectable cysts were found in the small number of fish that died earlier. Some of these fish died from jumping out of the tank, while the cause of death in others remain unknown. Among the 24 fish with cysts in the trunk muscle, noticeable cysts in the heart were observed in only one individual. PCR testing performed on fish without detectable cysts confirmed the presence of the I. fujiokai gene in all fish from the spore-intubation and intubation-cohabited groups, resulting in a 100% overall detection rate (Table 1). Two individuals from the spore-immersion group that died early in the trial tested PCR-negative, with the cause of death unknown, resulting in an overall detection rate of 89.5% for this group. All five fish cohabited with the spore-immersed group showed neither cysts, mortality nor abnormalities but were PCR-positive, indicating 100% infection prevalence (Table 1). No statistically significant difference was found in the overall I. fujiokai detection rate, as determined by microscopy and PCR, between the groups. No microsporidian infections were detected in the untreated fish from the same production batch, and no other pathogens and signs of other infectious diseases were observed during the trial.

Trial 2: Transmission route and infection method to rainbow trout

In Trial 2, using O. mykiss, both the spore-intubation and immersion groups exhibited similar mortality patterns (Fig. 3). Fish began dying around 14 dpc, and their mortalities continued for almost two weeks, resulting in cumulative mortality rates of 73.0% and 70.0%, respectively (Table 1). Some of the dead fish showed signs of hypoxia, although the exact number of individuals displaying these symptoms was not determined. In this trial, 27.8% of untreated control group also died, though these fish did not exhibit hypoxic symptoms. Numerous multinucleate spherical bodies were observed in the trunk muscle and heart tissues of the dead control fish and PCR analysis of muscle tissue from one such individual tested positive for Ichthyophonus (Whipps et al., 2006), suggesting that their deaths were likely due to ichthyophonosis. Ichthyophonus infections were detected in all groups; however, the mortality rates of the two treatment groups were significantly higher than that of control (log-rank test, p = 0.0011, Fig. 3).

Fig. 3. Trial 2 Kaplan-Meier survival curves after infection challenge of Inodosporus fujiokai spores from Palaemon paucidens to Oncorhynchus mykiss through spore intubation (black line), or spore immersion (gray line), and untreated control (broken line).

Among the 27 dead individuals from the spore-intubation group, heavy cysts formation in the trunk muscle (dozens of cysts per microscopic field) was observed for 26 individuals (96.3%). Cysts were also detected in 9 out of the 10 surviving fish, resulting in a total cyst detection rate of 94.6% (35/37) (Table 1). The two individuals without detectable cysts in the trunk muscle were PCR-positive, bringing the overall detection rate to 100%. Examination of cysts in the heart tissues was challenging due to small heart size and tissue degradation, but microsporidian cysts were confirmed in 6 individuals out of 63 examined.

In the spore-immersion group, 13 out of 16 dead fish and 3 out of 4 surviving fish harbored cysts, resulting in a total cyst detection rate of 80.0% (16/20). There was no significant difference in cyst detection rate between the spore-intubation and spore-immersion groups. Two individuals in the spore-immersion group tested negative by both microscopy and PCR, resulting in an overall detection rate of 90.0%. Cysts in the heart were confirmed in 3 individuals. No microsporidian infections were detected in the control fish.

Trial 3: Dose of the spore immersion method

In Trial 3, which compared three doses of spore immersion for O. mykiss, 66.6% cumulative mortality was observed in both the high-dose (2.6 × 10⁶ spores/mL) and mid-dose (2.6 × 10⁴ spores/mL) groups, with similar mortality patterns (Fig. 4). In contrast, no fish died in the low-dose group (2.6 × 10² spores/mL). Furthermore, none of the fish in the low-dose group exhibited cysts, while over 70% of the fish in the high-dose and mid-dose groups had detectable cysts (Table 1). Despite the absence of cysts, the I. fujiokai gene was detected in the trunk muscle of all 15 fish in the low-dose group, resulting in an overall detection rate of 100% across all groups (Table 1).

Fig. 4. Trial 3 Kaplan-Meier survival curves after immersion of Inodosporus fujiokai spores from Palaemon paucidens to Oncorhynchus mykiss at the concentrations of 2.6 × 10² (broken line), 2.6 × 10⁴ (grey line), or 2.6 × 10⁶ (black line).

Trial 4: Probability of fish-to-fish transmission

In Trial 4, I. fujiokai infection was not detected in any of the fish that were fed infected muscle tissue or those cohabited with infected conspecifics (Table 1). Moreover, there was no mortality observed among the challenged fish across both experimental setups, indicating the absence of I. fujiokai related disease under the tested conditions.

Discussion

The findings from Trial 4 indicate that the horizontal transmission of I. fujiokai between fish does not occur. This aligns with observations for Kabatana takedai, a close congener of I. fujiokai, and Microsporidium seriolae, the causative agent of beko disease, where attempts to induce infection via immersion, intramuscular injection, and oral administration of fresh spores from infected fish were unsuccessful (Fujiyama et al., 2002; Yokoyama et al., 2011). For both K. takedai and M. seriolae, a non-fish host has not yet been identified, and their experiments rely on natural infections via exposing fish in endemic waters (Awakura, 1974; Miyajima et al., 2007; Yanagi et al., 2021). In contrast, the results of Trial 1 to Trial 3 in this study clearly demonstrated that I. fujiokai derived from P. paucidens can be transmitted to fish via waterborne routes, in addition to transmission through ingestion of infected prawns, as previously reported (Yamamoto et al., 2021). The high infection prevalence and associated mortality among fish cohabiting with the spore-intubated group was suspected to be due to the prawn-derived spores excreted from the intubated fish, although the presence of the spores in feces or other sources was not confirmed in this study. This waterborne transmission may also explain the previous observations. There was a discrepancy between the low infection prevalence in wild P. paucidens and the high infection prevalences in prawn-fed fish (Yanagida et al., 2023), and control fish placed in the half section of a concrete pond divided by a mesh partition—without direct access to infected prawns—also became infected (Yamamoto et al., 2021). The present study provides a following hypothesis: prawn-fed fish, acting as Trojan horses, excreted spores through feces, likely contaminated the water and spreading the infection throughout the ponds.

Waterborne transmission may represent the primary route of I. fujiokai infection. Spores of I. fujiokai in the prawn host are characterized by a short anterior appendage and three long, tape-like appendages at the posterior end (Yanagida et al., 2023). These appendages are absent in spores developed in fish hosts. The long appendages likely enhance buoyancy, facilitating spore dispersal in water, whether released from fish that ingested infected prawns or from decaying prawn carcasses. Although the exact portal of spore entry into fish remains unclear, these appendages may also assist spore in attaching to fish tissues, such as gill filaments. The distinct morphological features of the spores, combined with the findings from this study, provide valuable insights into the transmission biology of I. fujiokai.

This waterborne transmission route has important implications for disease control in fish farms. A previous study recommended preventing I. fujiokai infections by avoiding the use of wild prawns, particularly P. paucidens, as feed or by freezing them before feeding (Yamamoto et al., 2021). Some salmonid fish farms in Shiga Prefecture keep live P. paucidens for selling as fishing bait. Farmers should be aware that drainage water from tanks holding these prawns could serve as a source of infection. Additionally, water from streams or lakes where infected prawns reside poses a risk if introduced into farm systems. However, spore density is crucial factor in causing disease, including cysts formation and mortality. In Trial 3, exposure to a concentration of 2.6 × 10² spores/mL for 4 h did not induce mortality or cyst formation, in contrast to the 66.6% cyst detection rate and over 70% mortality observed at concentrations of 2.6 × 10⁴ spores/mL and higher. This indicates that there is a threshold spore density necessary for effective waterborne transmission, and the minor spore contamination in water may not induce the disease. This could also explain the subclinical infections observed in the fish cohabited with the spore-immersed group in Trial 1, where cysts and mortality were absent, but PCR tests were positive. It is likely that a relatively small number of spores detached from the surface of the spore-immersed fish dispersed in the tank, leading to low-level, asymptotic infections.

This study also demonstrated that O. masou ishikawae is highly susceptible to I. fujiokai, making it the third Oncorhynchus species known to be prone to infection, alongside O. mykiss and O. masou subsp. However, differences in susceptibility and pathological responses to I. fujiokai were observed between salmonid species. A previous study reported that O. mykiss was more susceptible, exhibiting greater mortality compared to O. masou subsp. (Yamamoto et al., 2023). In contrast, the present study found that O. masou ishikawae was particularly vulnerable, experiencing acute mass mortality resulting in cumulative mortality of up to 100% (Trial 1), whereas O. mykiss showed mortality rates of up to 73.3% over a prolonged period (Trials 2 and 3). Considering that some mortality in O. mykiss in Trial 2 was likely due to ichthyophonosis, the difference in susceptibility between the two species may be even more pronounced.

Despite the high mortality in O. masou ishikawae, no hypoxic symptoms were observed, and cysts were rarely detected in the heart. In contrast, histological analysis of infected O. mykiss showed that myocarditis, associated with cyst formation in heart muscle tissue, induced hypoxia, contributing to mortality (Yamamoto et al., 2023). The absence of hypoxic symptoms and heart cysts in O. masou ishikawae suggests that a different pathological mechanism may underlie the mortality in this species. Furthermore, the relatively high occurrence of PCR-positive O. masou ishikawae individuals without detectable cysts in the muscle may suggest differences in pathogen development between host species. However, it is important to note that the two salmonid species were not directly compared in this study. The spore-immersion method used here proved to be an effective and quantitative experimental infection model. This method now enables direct comparison of susceptibility and pathology across various fish species. Incorporating this experimental method with histopathological, immunological, and other studies will pave the way for future research to better understand microsporidian diseases in aquaculture. Together, these findings provide a solid foundation for advancing our understanding of I. fujiokai infections and improving disease management in aquaculture.

References

Abramoff, M. D., P. J. Magalhaes and S. J. Ram (2004): Image Processing with ImageJ. Biophotonics Int., 11, 36–42.
Awakura, T. (1974): Studies on the microsporidian infection in salmonid fishes. Sci. Rep. Hokkaido Fish Hatchery, 29, 1–95. (In Japanese with English summary)
Fujiyama, I., S. Urawa, H. Yokoyama and K. Ogawa (2002): Investigation of the transmission stage of the microsporidian Kabatana takedai in salmonids. Bull. Natl. Salmon Resources Center, 5, 1–6.
Miyajima, S., S. Urawa, H. Yokoyama and K. Ogawa (2007): Comparison of susceptibility to Kabatana takedai (Microspora) among salmonid fishes. Fish Pathol., 42, 149–157.
Whipps, C. M., T. Burton, V. G. Watral, S. St-Hilaire and M. L. Kent (2006): Assessing the accuracy of a polymerase chain reaction test for Ichthyophonus hoferi in Yukon River Chinook salmon Oncorhynchus tshawytscha. Dis. Aquat. Organ., 30, 141–147.
Yamamoto, M., S. Shirakashi, N. Asai, T. Yanagida, K. Sugahara and H. Yokoyama (2021): A new microsporidian disease of rainbow trout Oncorhynchus mykiss farmed in Shiga Prefecture, Japan, and possible involvement of common prawn Palaemon paucidens for transmission of the pathogen. Fish Pathol., 56, 130–139.
Yamamoto, M., K. Sugahara, N. Asai, T. Yanagida, H. Yokoyama and S. Shirakashi (2023): Pathology of a microsporidian Inodosporus fujiokai in two Onchorynchus spp. Fish Pathol., 58, 60–70.
Yanagi, S., J. Satoh, T. Mekata, T. Sakai, H. Kawakami, S. Harakawa, S. Shirakashi, S. Hamano, K. Fukudome, K. Wada, M. Yamasaki, T. Nishioka, C. Nakayasu and K. Mori (2021): Chemical treatment of beko disease in Seriola fishes: Laboratory trials of commercially available fishery and veterinary medicines. Fish Pathol., 56, 62–70.
Yanagida, T., N. Asai, M. Yamamoto, K. Sugahara, T. Fujiwara, S. Shirakashi and H. Yokoyama (2023): Molecular and morphological description of a novel microsporidian Inodosporus fujiokai n. sp. infecting both salmonid fish and freshwater prawns. Parasitology, 150, 1–14.
Yokoyama, H., D. Ayado, J. Miyahara, K. Matsukura, I. Takami, F. Yokoyama and K. Ogawa (2011): Infection dynamics of Microsporidium seriolae (Microspora) causing the beko disease of Seriola spp. Fish Pathol., 46, 51–58.

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