Weight-dependent susceptibility of tilapia to tilapia lake virus infection

Animals and experimental designs

In total, 400 red tilapia hybrid (Oreochromis spp.) with an initial body weight of 1.0 ± 0.1 g were acquired from a tilapia hatchery with a TiLV-free status from Petchaburi province, Thailand. The fish were kept in the animal research facility of the Faculty of Veterinary Medicine at Kasetsart University, Bangkok, Thailand, in 400 L tanks at 28 °C with daily water exchanges up to 50%. After 1 week of acclimatization, five fish were randomly selected for screening for important pathogens, including TiLV by reverse transcription-quantitative PCR (RT-qPCR), bacterial isolation by anterior kidney sampling, and parasites by skin scraping and gill excision. Fish were fed with commercial feed three times per day until reaching an average size of 5 g, 25 g, and 65 g. The average age of 5 g, 25 g, and 65 g fish were 8, 12, and 16 weeks, respectively. At the same density (5 g/L), sixty fish from each weight group were equally divided into two 150 L tanks (for 5 g fish), three 150 L tanks (for 25 g fish), and six 150 L tanks (for 65 g fish). These tanks were dedicated to recording mortality. A further 30 fish of 5 g, 25 g, and 65 g were placed in additional 150 L tanks at the same density for sample collection. For each weight group, an additional 15 fish were used as the control group. The animal use protocol was approved by the Kasetsart University Institutional Animal Care and Use Committee (protocol number ACKU63-VET-011).

Virus propagation and challenge study

The TiLV strain VETKU-TV08 isolated from red hybrid tilapia collected in 2019 was used in the challenge study. The virus was propagated in the E-11 cell line, which is a clone of SSN-1 from snakehead fish (Ophicephalus striatus). The E-11 cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK). The E-11 cells were maintained in Leibovitz’s L-15 medium (Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 5% (vol/vol) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 2 mM L-glutamine. The cells were propagated at 25 °C without CO₂. The infected E-11 cells were harvested through centrifugation at 3,000×g for 10 min, and the supernatant was then collected and stored at −80 °C for later use. Before the IP injection challenge, the fish were sedated with a 1 mL/L eugenol (Better Pharma, Bangkok, Thailand) solution for 3 min. The fish were IP-challenged with 50 µL of TiLV at 10⁵ TCID₅₀/mL or the L-15 medium for the control group. During the experiment, the decision criteria to terminate fish included the appearance of severe clinical signs with two or more appearance including stop feeding for 3 consecutive days, severe erratic swimming, skin erosion, skin hemorrhage, scale protrusion, extensive abdominal swelling, and exophthalmia. For viral quantification, the liver tissues were collected from the control fish (n = 3) at day 0 of all weight groups and TiLV IP-challenge fish (n = 7−8) at 7, 14, and 22 days post-infection (dpi). The fish were euthanized using an overdose of eugenol solution (3 mL/L) for 5 min prior to fish necropsy and sample collection. The samples were placed in separate 1.5 mL microcentrifuge tubes, and stored at −20 °C for further RNA isolation.

For the cohabitation study, 10 fish of 5 g and 25 g were IP-injected with 50 µL of the TiLV strain VETKU-TV08 at 10⁵ TCID₅₀/mL and then placed in a 150 L glass tank containing 30 fish (cohabitation fish) of either 5 g or 25 g fish, giving a ratio of inducer to cohabitant of 1:3 (Liamnimitr et al., 2018). All IP-injected fish (inducer) were trimmed on the pelvic fin to differentiate them from cohabitating fish. Clinical signs of infection and cumulative mortality were observed and recorded until 28 days post challenge. At 7 and 14 dpi, three cohabitation fish from the 5 g and 25 g groups were randomly euthanized to collect liver samples and for processing for RNA isolation.

RNA extraction and cDNA synthesis

The total RNA was extracted from the liver using GENEzol™ reagent (Geneaid Biotech Ltd., New Taipei City, Taiwan) according to the manufacturer’s instructions. Briefly, 30 mg of liver samples were mixed and homogenized in 1 mL of GENEzol™ reagent using a hand-held pestle homogenizer. Thereafter, 200 μL of chloroform was mixed and incubated at room temperature for 3 min. The samples were then centrifuged at 4°C and 12,000×g for 15 min. The supernatant was transferred to a new microcentrifuge tube, mixed with 500 μL isopropanol, and incubated at −20 °C for 2 h. The samples were centrifuged at 4 °C and 12,000×g for 15 min to precipitate the RNA. After discarding the supernatant, the RNA pellet was washed with 75% ethanol and centrifuged at 4 °C and 10,000×g for 15 min. The RNA pellet was resuspended in 50 μL prewarmed RNase-free water (60 °C). The RNA quality and quantity were examined using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

For cDNA synthesis, a 20 µL mix reaction containing 4 μL of 5X RT buffer mix, 1 μL of primer mix, 1 μL of RT enzyme mix, 4 μL of nuclease-free water, and 10 μL total RNA template (1 μg) was prepared using a reverse transcription kit (Toyobo, Osaka, Japan). The reaction was incubated in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA) at 42 °C for 60 min, followed by 98 °C for 5 min.

Reverse transcription-quantitative polymerase chain reaction

The TiLV genomic RNA was measured using an SYBR-based reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay (Tattiyapong, Sirikanchana & Surachetpong, 2018). Briefly, the reaction was performed in a 10 μL reaction containing 4 μL of 400 ng cDNA, 5 μL of 2X iTaq™ universal SYBR supermix (Bio-Rad, Hercules, CA, USA), and 0.3 μL of forward and reverse primers. The final volume was adjusted to 10 μL using molecular water. The reactions were performed in a PCR thermal cycler, CFX96 Touch™ (Bio-Rad, Hercules, CA, USA). At the end of the qPCR reaction, the samples were processed for melting curve analysis at 65–95 °C with increments of 0.5 °C per 5 s. The TiLV viral concentration was extrapolated by comparing the Ct value of the tested samples to the standard curve generated from a 10-fold serial dilution of a plasmid-containing segment 3 of TiLV.

Histopathology

For the histopathological analysis, two control and three TiLV IP-challenge fish were collected from each weight group at 7, 14, and 22 dpi. Tissues, including the liver, spleen, and intestine, were removed and placed in 10% (vol/vol) neutral buffered formalin. At 24 h, the tissues were transferred to 70% ethanol. The samples were then processed using a standard histopathology protocol in which they were embedded in the paraffin block, sectioned at 5 µM thick, and stained with hematoxylin and eosin (H&E). Thereafter, the tissue slides were scanned using VS120^® Virtual Microscopy Slide Scanning (Olympus, Tokyo, Japan) and examined and graded under an Olympus OlyVIA Ver.3.1 program (Olympus, Tokyo, Japan).

Statistical analysis

The difference in the cumulative mortality, and mean TiLV concentration in the IP-challenge and cohabitation experiments, pathological scores from each weight groups and different time points were determined using GraphPad Prism software version 5.0 (GraphPad, San Diego, CA, USA). Significant differences were assessed using one-way ANOVA with Tukey’s multiple comparisons test or nonparametric Mann–Whitney test (Gibson-Corley, Olivier & Meyerholz, 2013). A p-value less than 0.05 was considered significant.

Susceptibility of small fish to TiLV infection

After TiLV infection, an earlier onset of clinical signs, including lethargy, anorexia, schooling cessation, and lying on the floor of the tank, was observed in the small fish (5 g) at 2 dpi, while these clinical signs started at 4 dpi in the medium (25 g) and large fish (65 g). More severe clinical signs and gross lesions, including exophthalmos, skin hemorrhage, scale protrusion, anemia, fin erosion, and ascites, were exhibited in all weight groups after 4 dpi (Fig. S1). At the end of the experiment, the mean cumulative mortality and standard error of the mean (SE) of the small, medium, and large red hybrid tilapia was 85% (±1.67), 55% (±2.89), and 51.67% (±7.49), respectively, with significantly higher mortality in the 5 g fish than the other groups (p < 0.05; Fig. 1). The small fish had first mortality at 3 dpi, while this occurred in the medium and large fish at 7 dpi. Notably, the mortality of the small fish continued until 16 dpi, while the mortality of the medium and large fish stopped at 13 and 14 dpi, respectively. No mortality or signs of TiLV infection were recorded in the control fish in any weight group at any time during the study.

Amount of TiLV in small fish

At 7 dpi, the mean TiLV genomic RNA concentration in the liver of the 5 g, 25 g, and 65 g fish was 7.4 log₁₀, 7.1 log₁₀, and 6.7 log₁₀ TiLV copy/µg total RNA, respectively (Fig. 2). In all weight groups, the mean viral load at 14 dpi was significantly lower at 5.7 log₁₀, 2.7 log₁₀, and 3.1 log₁₀ TiLV copy/µg total RNA in the 5 g, 25 g, and 65 g fish, respectively. No difference in the mean viral load was found at 22 dpi (3.4 log₁₀, 3.2 log₁₀, and 3.0 log₁₀ TiLV copy/µg total RNA in the 5 g, 25 g, and 65 g fish, respectively). From the three time points, the 5 g fish showed more TiLV genomic RNA in the liver than the 25 g fish (p < 0.001) and 65 g fish (p < 0.01) at 14 dpi (Fig. 2). No TiLV genomic RNA was detected in any of the weight groups at 0 dpi before the TiLV challenge.

Histopathological scores of the organs in the different weight groups

Histopathological changes in the liver, spleen, and intestine of all the weight groups were scored according to the scoring system described in Table 1 and Fig. 3. The scores were given after examining three fish per weight group per time point. The three categories of scores were normal (0), mild (1), moderate (2), and severe (3) (Fig. S2). At 7 dpi, all the weight groups obtained severe pathological scores in the liver, spleen, and intestine. At 14 dpi, lower pathological scores were given for the liver, spleen, and intestine of the 25 g and 65 g fish than the 5 g fish. In particular, most of the 5 g fish had moderate or severe pathological scores in these organs compared to the 25 g and 65 g fish (Table 2). Low histopathological scores were obtained in all weight groups at 22 dpi (data not shown) and no pathological changes in the control unchallenged fish.

Weight susceptibility to TiLV in the cohabitation challenge

The effect of weight on TiLV susceptibility was further tested in the cohabitation challenge study. As shown in Fig. 4, the cumulative mortality of the inducer 5 g and 25 g fish were 85% and 60%, respectively, while the mortality in the cohabitation 5 g and 25 g fish was 38% and 23%, respectively. Both the inducer 5 g and 25 g fish started showing clinical signs of TiLV infection on days 3–4, with the first mortality observed on 7 dpi. Notably, the cohabitation 5 g and 25 g fish had delayed clinical signs and mortality onset, which started on 9–11 dpi. Interestingly, while the mortality in the 5 g fish ceased at 20–24 dpi, the mortality of the 25 g fish stopped earlier, at 15–18 dpi (Fig. 4). Further analysis of the TiLV concentrations in the liver of the cohabitation 5 g fish at 7 and 14 days showed the viral load between 3.50 log₁₀ and 5.64 log₁₀ TiLV copy/µg total RNA. The mean viral load in cohabitation 25 g fish ranged between 2.86 log₁₀ and 5.71 log₁₀ TiLV copy/µg total RNA.