Toxicity Effects of Microplastics Individually and in Combination the Fish Pathogen Yersinia Ruckeri on the Rainbow Trout (Oncorhynchus Mykiss)

Exposure to xenobiotics such as Yersinia ruckeri can signicantly affect bacterial infections in sh. Microplastics (MPs) may predispose sh to infection and act as carriers in pathogen transmission. Therefore, this study is designed to evaluate MPs' effect on damage caused by exposure to Y. ruckeri in rainbow trout. In this study, blood biochemical parameters and hepatic oxidative biomarkers as clinical signs were measured in the sh co-exposed to Y. ruckeri (5 and 10% LD50) and MPs (500 and 1000 mg Kg-1) for 30 days. There were no signicant changes in the creatinine, triglyceride, cholesterol levels, and glutamic-pyruvic transaminase activity in the blood of sh infected with Y. ruckeri. In contrast, exposure to MPs had a signicant effect on most clinical parameters. The total protein, albumin, globulin, total immunoglobulins, High-density lipoprotein, low-density lipoprotein, cholesterol levels, and γ-glutamyltransferase activity decreased, whereas glucose, triglyceride, and creatinine levels, and glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, alkaline phosphatase, and lactate dehydrogenase activities increased in the plasma of sh after co-exposure to MPs and Y. ruckeri. Dietary MPs combined with a bacterial challenge decreased catalase activities, glutathione peroxidase, and total antioxidant levels. However, the superoxide dismutase activity and malondialdehyde contents in the hepatocytes increased in the hepatocyte of sh co-exposed to MPs and Y. ruckeri. In conclusion, this study showed that sh exposure to MPs and simultaneous challenge with Y. ruckeri could have a synergistic effect on clinical parameters. Y. ruckeri. This study revealed that sh exposure to MPs combined with Y. ruckeri could lead to a synergic effect on the triglyceride levels. The triglyceride levels were elevated in the serum of common carp, C. carpio and freshwater pond turtles, E. orbicularis after treatment with MPs (Banaee, et al., 2019; Banaee, et al., 2020b).


Introduction
Microplastics (MPs) are known as a newfound and unique pollutant in aquatic ecosystems . Along with the increase in plastic products' global production, the amount of garbage and plastic waste in the environment has also esclated (Iheanacho, et al., 2020;Zhang, et al., 2021). Destruction of plastic debris under the in uence of UV, physicochemical changes in the environment, and physical processes have caused most plastic compounds to become MPs and enter aquatic ecosystems through surface runoff (Frias & Roisin Nash, 2019). MPs may enter the sh body through the food chain or absorption by the gills. MPs accumulate in aquatic animals are transported through cells and spread throughout the body through the circulatory system or lymphatic (Wang, et al., 2020;Kim, et al., 2021). MPs often get into the digestive system but can also be transmitted to the liver or other vital organs (Jovanović, 2017). Therefore, the accumulation of MPs in sh's vital tissues can have toxic effects on growth and development, immune system function, oxidative stress, metabolism and energy budgeting, and various biomarkers (Qiao, et al., 2019a;Qiao, et al., 2019b;Bhagat, et al., 2020).
Prolonged exposure to MPs can lead to an in ammatory response in the gut, leading to metabolic disorders and diseases caused by imbalance and damage to the microbial ora of sh intestines (Kang, et al., 2021). Studies show that MPs in aquatic ecosystems can have detrimental effects on aquatic health (Brandts, et al., 2018;Ding, et al., 2018). Exposure of aquatic organisms to MPs can lead to oxidative stress, suppression of the immune system, changes in blood biochemical parameters, histopathological damage, and gene expression changes (Chen, et  Although MPs' effect on the pathogenicity of pathogens has not been studied extensively, there is a hypothesis that exposure of to MPs may suppress the immune system of sh and predispose them to pathogens.
Yersinia ruckeri is an opportunistic pathogenic gram-negative bacterium known to cause enteric redmouth disease. The spread of this bacterium among different commercial sh species can cause signi cant damage to the aquaculture industry. Y. ruckeri has been isolated from various species of sh in many parts of the world. Y. ruckeri is transmitted to sh via water, food, sediments, and aquatic animals. The Yersinia's infection is often transmitted through the faecal-oral route.
Y. ruckeri is an invasive microorganism that can induce infection via tissue destruction. The pathogen passes into the digestive system through contaminated feed and penetrates the intestine's epithelial cells. Y. ruckeri's infection may lead to mucosal ulcers and necrotic lesions in the digestive system of sh. The enterotoxin synthesised by Y. ruckeri may play a signi cant role in causing disease. After challenging sh with bacteria, Y. ruckeri can enter sh blood through gill lamella. The infection affects the intestine, liver, spleen, heart, and brain within 30 minutes to 3 days. Moreover, the bacteria can be detected in these tissues even up to 21 days after the initial challenge (Kumar, et al., 2015).
Since environmental conditions play a crucial role in the prevalence of opportunistic pathogens, the question is whether MPs can predispose sh to yersiniosis. Therefore, assessing the health of sh co-exposed to MPs and Y. ruckeri may help answer this question. Blood biochemical parameters are a good indicator for assessing the health of organs involved with pathogens and xenobiotics. Oxidative stress biomarkers are also an indicator of the cellular antioxidant defense system's power against free radicals due to xenobiotics and biological toxins' metabolism. Thus, this study was designed to study sh health based on changes in blood biochemical parameters and oxidative stress biomarkers. Rainbow trout is one of the commercial and farmed species in Iran that is very susceptible to Yersinia infection. In this study, the rainbow trout were used to investigate the clinical signs of the Y. ruckeri pathogenesis when facing MPs.

Chemical materials
High-density polyethylene (PE100) powder (< 0.2 mm) was purchased from Eshragh Trading Co., Iran. All diagnostic kits used to measure biochemical parameters and antioxidant enzymes were obtained from Pars Azmun Co., and BiorexFars Co., Iran.

Fish
Juvenile rainbow trout were purchased from a private farm and transferred to the Aquatic Animal Health Division (School of Veterinary Medicine, Shiraz University, Iran). The sh were adapted to laboratory conditions (temperature: 15±2 °C, photoperiod cycle: 14 light/8 dark, dissolved oxygen: 8.1 ± 0.7 mg L -1 , pH: 7.3 ± 0.4, electrical conductivity: 693.83 ± 76 µS cm -1 , salinity: 0.3 ± 0.02 g L -1 ) for two weeks. The School of Veterinary Medicine's ethical committee con rmed this study's proposal and all the experimental procedures.

The median lethal dose (LD 50 ) of Yersinia ruckeri for rainbow trout
The Yersinia ruckeri (PTCC No: 1888) was cultured in Trypticase Soy Agar with 5% de brinated sheep blood at 25 °C for 48 h. Next, bacteria were washed twice using sterile phosphate-buffered saline (PBS). The bacterial concentration was adjusted to 10 10 CFU ml -1 using pour plate count method.
Rainbow trout were randomly distributed into eighteen tanks (9 trial groups in two replicates). Each tank contained fteen sh. Eight serial dilutions of Y. ruckeri suspension (10 3 to 10 10 cells ml -1 ), were added to each tank (10 L) for one hour. The sh in the control group were maintained in clean water. Clinical signs and the mortality rate was recorded following the challenge with bacteria for 7 days. During the LD 50 experiment, water was aerated and had the same conditions as the acclimation period. The dead sh were immediately removed from the tanks. Mortality due to bacterial infection was con rmed after sampling and bacterial culture in vitro. 7 days LD 50 (lethal dose affecting 50 percent of a population of animals) was determined by probit analysis.

The Experimental challenge trials
Two hundred and seventy rainbow trout (15±2.5 g) were randomly distributed in twenty-seven berglass tanks (100 L) into nine separate experimental groups that included group I or control group (without MPs and Y. ruckeri), group II (5% LD50 of Y. ruckeri), group III (10% LD50 of Y. ruckeri), group IV (500 mg Kg -1 of MPs), group V (1000 mg Kg -1 of MPs), group VI (5% LD50 of Y. ruckeri & 500 mg Kg -1 of MPs), group VII (5% LD50 of Y. ruckeri & 1000 mg Kg -1 of MPs), group VIII (10% LD50 of Y. ruckeri & 500 mg Kg -1 of MPs), and group IX (10% LD50 of Y. ruckeri & 1000 mg Kg -1 of MPs) in triplicate following completely randomized design (CRD). Fish were continuously exposed to the MPs for 30 days and challenged with Y. ruckeri for one hour on the 1 st , 8 th , 15 th and 22 nd day of the experiment. Every 24 h the water was renewed (100%) and fresh suspension of MPs were prepared and added to water to maintain the nominal dose. In the experimental periods, rainbow trout were fed two times daily with commercial feed. One day before sampling, sh feeding was stopped. At the end of the challenge period, the sh were anaesthetized using clove powder (150 mg L -1 ). The blood was sampled from the venous stem vein using a 2.5 mL syringe impregnated with an anticoagulant. The blood samples were centrifuged (6000 g, at 4º C for 15 min), and plasma fractions were collected and stored at -25º C until the biochemical analyses.
After the sacri ce, the sh were dissected, then the liver was quickly dissected and homogenized on ice in 100 mM cold potassium phosphate buffer (Sigma-Aldrich, Germany) pH 7.0, with 2 mM EDTA (Riedel-Haën, Germany). Tissue homogenates were centrifuged at 12,000 g-force for 15 minutes at 4 °C and the supernatants were collected and maintained at -80 °C until further analysis.

Blood biochemical analysis
Glutamic-oxaloacetic transaminase (SGOT), glutamic-pyruvic transaminase (SGPT), alkaline phosphatase (ALP), γglutamyltransferase (GGT), and lactate dehydrogenase (LDH) activities, glucose, total cholesterol, triglyceride, high density lipoprotein (HDL), low density lipoprotein (LDL), creatinine, total protein and albumin levels in plasma were evaluated based on the standard methods provided in the diagnostic kit protocol. The globulin levels were also computed based on the following formula:

Globulins = Total protein -Albumin
Total immunoglobulin (Ig) was estimated using polyethylene glycol solution according to the procedure described by Banaee et al. . All blood biochemical parameters were measured utilizing a UV-visible spectrophotometer (Unico, 2100) and standard biochemical reagents obtained from Pars-Azemun Co, Tehran, Iran.

Oxidative biomarker analysis
Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in the hepatocytes were assessed using oxidative biomarker kits purchased from the Biorexfars Co., Iran. The total antioxidant capacity was estimated with the ferric reducing ability of plasma (FRAP) technique using TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine) as a substrate (Benzie and Strain, 1996). Per-oxidative damage in the hepatocytes was evaluated in terms of malondialdehyde (MDA) production by the method presented by Placer and others (Placer et al., 1996), using thiobarbituric as substrate acid and monitored at 532 nm. Catalase (CAT) activity was measured spectrophotometrically in tissue homogenates by using a hydrogen peroxide solution (30 mM) as a substrate and monitored at 450 nm (Góth, 1991).

Assessment of synergism and antagonism
In the present study, the following mathematical models were used to predict the interaction of MPs and Y. ruckeri on each other. According to Banaee et al. (2020), the synergism rate is calculated only when the predicted effect is higher than the observed one.
1. Predicted effect of the endpoints of rainbow trout exposed to either MPs or ruckeri 2. Observed effect of the endpoints of rainbow trout exposed to both ruckeri and MPs 3. The synergism effect

Data analysis
The Shapiro-Wilk test calculated the normality of all data. Then, changes among the different variables of rainbow trout exposed to Y. ruckeri combined with MPs were analyzed using two-way analysis of variance (two-way ANOVA). A Tukey test was used to compare differences in each parameter between control and experimental groups. Statistical analyses of all data were carried out using Graph Pad Prism 8.0.2. A signi cant difference was assessed at P < 0.05 levels, and results were shown as Mean ± Standard deviation.

Results And Discussion
Clinical signs such as haemorrhage on the body, especially at the base of the ns and anus, bleeding and in ammation in the intestine's distal parts, petechiae bleeding around the jaw and mouth, bleeding or pale gills were observed in the experimentally infected rainbow trout. We were able to isolate Y. ruckeri from the intestine, kidney, liver and gill of all recently dead and survied sh. A cumulative mortality rate of 20%, 60%, 70%, 80%, and 100% was reached in rainbow trout injected with Y. ruckeri isolate at doses of 10 6 , 10 7 , 10 8 , 10 9 , and 10 10 CFU/L, respectively and LD 50 value for Y. ruckeri was estimated to be 1.42×10 7 CFU L -1 .
Although, none of the sh used in the experiment died during co-exposure to MPs and Y. ruckeri, clinical signs of yersiniosis were observed in sh. The pathogenicity of Y. ruckeri and sh mortality may be attributed to virulence factors. The most well-known virulence factors are lipopolysaccharides, hemolysin, Yrp1 protease, ruberbactin, and extracellular metalloproteinase (Portnyagina, et al., 2021). Furthermore, Y. ruckeri can damage host cellular membranes through secreting nonspeci c pore-forming proteins (porins) (Portnyagina, et al., 2021).
Glucose is an important energy source in sh, especially in the brain, which gets most of its energy from carbohydrates. However, sh is known as a species with low glucose tolerance. Exposure of sh to MPs and Y. ruckeri led to elevated glucose levels ( Figure 1 & Table 1). Elevated glucose may be a physiological response to increased energy demand to combat biotoxins and MPs. Bao et al. (2020) showed that the liver's glucose metabolism gene altered after xenobiotic exposure. An increase in blood glucose may also increase the conversion rate of excess glucose to triglycerides and increase its level. . The highest levels of glucose were observed in the blood of sh exposed to 1000 mg Kg -1 MPs and challenged with 5% LD 50 Y. ruckeri. This result suggest that MPs and Y. ruckeri exert a synergistic effect by increasing the glucose levels.
Creatinine is the most critical metabolite produced by the muscles and excreted through the kidneys. The concentration of creatinine in the blood of sh exposed to 1000 mg Kg -1 MPs increased signi cantly compared to the control group.
However, there was no signi cant change in creatinine level in sh fed with 500 mg Kg -1 MPs (Figure 1 & Table 1). Exposure to MPs could impair the kidney and decrease its ability to secrete creatinine through reducing glomerular ltration. A study done by Hamed et al. (2019) showed that exposure of tilapia O. niloticus to MPs increased creatinine levels (Hamed, et al., 2019). No signi cant differences were found in the creatinine level in sh after being challenged with Y. ruckeri alone. However, creatinine concentration in sh co-exposed to both MPs and bacterial infections was signi cantly higher than the control group. This study showed that sh exposure to MPs challanged with Y. ruckeri could lead to a synergic effect on creatinine levels.
Results showed that bacterial infection of sh did not cause a signi cant change in triglyceride concentration. Fish exposure to MPs resulted in a meaningful increase in triglyceride concentration compared to the control group ( Figure 1 & Table 1). Simultaneous exposure of sh to MPs and challenge with Y. ruckeri signi cantly increased triglyceride concentrations. Bao et al. (2020) found that exposure of sh to xenobiotics could alter the expression of genes involved in lipid metabolism and adipogenesis (Bao, et al., 2020). Morover, increased metabolic expenditure after Y. ruckeri challenge and MPs exposure to maintain homeostasis and detoxi cation can lead to metabolite concentrations changes . The glycerol in triglycerides can be converted to glucose (Wen, et al., 2021). Therefore, an increase in triglycerides may indirectly increase the blood glucose of sh exposed to MPs and Y. ruckeri. This study revealed that sh exposure to MPs combined with Y. ruckeri could lead to a synergic effect on the triglyceride levels. The triglyceride levels were elevated in the serum of common carp, C. carpio and freshwater pond turtles, E. orbicularis after treatment with MPs There were no signi cant changes in the cholesterol levels in the blood of sh infected with Y. ruckeri. Treatment of sh with MPs alone and MPs combined with Y. ruckeri resulted in a signi cant reduction in cholesterol concentration. This study showed that sh co-exposure to MPs and Y. ruckeri could lead to a suppressive effect on the cholesterol levels ( Figure 1 & Table 1). Changes in the concentration of cholesterol in sh's blood showed an inverse trend of triglyceride concentration, which may be related to its different biological role. Cholesterol is not known as the primary energy source but is involved in the biosynthesis of biological membranes, steroid and corticosteroid hormones and bile acids (Wen, et al., 2021). Therefore, cholesterol depletion may be attributed to impaired cholesterol biosynthesis in the liver or its absorption in the intestine, increased biosynthesis rates of corticosteroid hormones, and bile acids. Porins and lipopolysaccharides play an essential role in increasing cytokinin biosynthesis in hepatocytes and macrophages (Portnyagina, et al., 2021). Hence, the liver tries to excrete cytokines by enhancing bile acids' biosynthesis to maintain biochemical homeostasis (Li, et al., 2006). A signi cant decrease in cholesterol levels was observed in the serum of African cat sh, Clarias gariepinus exposed to MPs .
High-density lipoprotein concentrations reduced signi cantly after sh exposure to Y. ruckeri and MPs alone and in combination. A signi cant decline was detected in low-density lipoprotein concentrations in the plasma of sh after coexposure to MPs and Y. ruckeri. Decreased LDL and HDL may be due to lower blood cholesterol levels ( Figure 1 & Table 1).
Total protein, globulins, and total immunoglobulins content in the plasma decreased signi cantly following sh exposure to Y. ruckeri and MPs alone and in combination. The globulin and total immunoglobulin levels in sh exposed to both MPs and bacteria were considerably lower than the other experimental groups. Co-exposure to MPs and bacteria resulted in a decrease in albumin in the plasma of sh ( Figure 2 & Table 1).
Decreased total protein may be due to increased protein catabolic rate to counteract the toxicity of MPs and bacterial biotoxins. Morover, MPs may inhibit the absorption of essential amino acids and reduce food digestibility in the aquatic digestive system. Decreases in globulin, albumin, and immunoglobulin levels also re ect a decrease in total plasma protein. Damage to the intestinal epithelium of sh after challenge with Y. ruckeri may also prevent the absorption of essential amino acids. Furthermore, Fernandez et al. (2003) found that Yrp1 protease plays a critical role in the biodegradation of a wide variety of matrix and muscle proteins (Fernandez, et al., 2003). Therefore, the increased biodegradation rate of plasma proteins could decrease total proteins, albumin, and globulin. Total proteins, albumin, and globulin levels decreased in the serum of freshwater pond turtles, E. orbicularis exposed to MPs (Banaee, et al., 2020b).   Table 2). Damage to hepatocytes may lead to increased SGOT, SGPT, ALP and LDH activities. The results showed that SGPT activity signi cantly increased in sh exposed to 1000 mg Kg -1 MPs ( Figure 3 & Table 2). Furthermore, combined exposure to MPs and bacteria increased SGPT activity in sh plasma. The change in SGOT, SGPT, LDH, and ALP activities can be attributed to the hepatotoxicity effect of bacterial lipopolysaccharide (Beheshti, et al., 2021). Also, porins can signi cantly affect cellular biochemical homeostasis by disrupting the selective permeability of cell membranes (Portnyagina, et al., 2021). ALP is a transmembrane metalloenzyme involved in protein phosphorylation, cell growth, apoptosis, and cell migration (Derikvandy, et al., 2020).
Therefore, any damage to the cell membrane can lead to a change in ALP activity in the blood (Banaee, et  LDH plays a vital role in converting lactate to pyruvate, NAD + to NADH and vice versa (Soleimany, et al., 2016). LDH is an indicator of hypoxia and mitochondrial oxidation function. Also, an increase in extracellular LDH can indicate cell death or necrosis (Maes, et al., 2015). Increased LDH activity was detected in the C. carpio and S. aequifasciatus exposed to MPs combined with Cd (Wen, et al., 2018;. GGT is an anchorage enzyme in the cell membrane that plays a unique role in the gamma-glutamyl cycle in the biosynthesis and biodegradation of glutathione and detoxi cation from xenobiotics (Hatami, et al., 2019). Compared with the reference group, GGT activity decreased in the plasma of sh exposed to MPs and Y. ruckeri individually and concurrently ( Figure 3 & Table 2). Changes in the catalytic domain of the GGT can lead to its inactivation. GGT is often degraded after inactivation, and its metabolites are excreted in the bile (Fornaciari, et al., 2014). The relationship between decreased GGT activity in the blood and increased biliary excretion of its metabolites has also been demonstrated (Li & Chiang, 2014). Decreased GGT activity in serum of freshwater pond turtles (E. orbicularis) exposed to MPs may indicate a reduction in detoxi cation capacity in hepatocytes (Banaee, et al., 2020b). Damage to the intestinal epithelium after bacterial challenge and exposure to MPs may lead to gastrointestinal disorders.
Therefore, a reduction in GGT activity can be attributed to the disturbance in the absorption of minerals and vitamins.
A signi cant decrease was observed in CAT activity in the hepatocyte of sh after co-exposure to MPs and Y. ruckeri. The result shows that MPs combined with Y. ruckeri inhibited CAT ( Figure 4 & Table 2). CAT plays a vital role in the breakdown of hydrogen peroxide (Banaee, et al., 2013). Therefore, inhibition of CAT activity may lead to an increase in H  Table 2). GPx is an enzyme involved in GSH-related antioxidant defense mechanisms that can neutralize organic peroxides (ROOH) and hydroperoxides (Banaee, et al., 2013). Therefore, decreased GPx activity can lead to oxidative stress in sh exposed to MPs and Y. ruckeri. The GPx activity signi cantly decreased in D. rerio after exposure to MPs (Umamaheswari, et al., 2020). Signi cant activation of SOD occurred after sh exposure to MPs and/or Y. ruckeri ( Figure 4 & Table 2). SOD is the only antioxidant enzyme that neutralizes the superoxide anion by transforming this ROS to oxygen and hydrogen peroxide (Banaee, et al., 2013). Increased SOD activity may be a physiological response to enhanced superoxide anions in cells. SOD activity signi cantly increased in the liver of gold sh, Carassius auratus, and common carp, C. carpio after exposure to MPs (Xia, et al., 2020; Yang, et al., 2020).
Exposure to MPs and/or Y. ruckeri resulted in a signi cant decrease in total antioxidant levels in sh's hepatocyte. MDA content in the hepatocytes of sh exposed to 500 mg Kg -1 MPs and 5% LD 50 Y. ruckeri alone did not show any signi cant changes to the control group. However, MDA content was increased considerably in the hepatocytes of sh exposed to   (Guijarro, et al., 2018) found that inoculation of trout renal macrophages with Y. ruckeri in the in vitro condition can induce reactive oxygen species production. Thus, changes in antioxidant enzyme activities and cellular antioxidant capacity may have been due to ROS increase by macrophages of sh after challenging with Y. ruckeri. Morover, bacterial metalloproteinases may activate mechanisms involved in producing reactive oxygen species that could lead to oxidative stress (Dasgupta, et al., 2010).

Conclusion
This study showed that sh exposure to doses of 5 and 10% LD 50 of Y. ruckeri did not signi cantly change some clinical parameters. However, simultaneous exposure to MPs and Y. ruckeri resulted in changes in all clinical parameters. These results indicated that MPs have been able to have a synergistic effect on Y. ruckeri pathogenesis.
Declarations Figure 1 Changes of glucose, creatinine, cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein levels in the blood of rainbow trout after exposure to MPs and Y. ruckeri. Results are illustrated as mean ± SD. Signi cant differences between groups were identi ed by alphabetical characters (P < 0.05).

Figure 2
Changes of total protein, albumin, glubolin, and total immunoglubolins levels in the blood of rainbow trout after exposure to MPs and Y. ruckeri. Results are illustrated as mean ± SD. Signi cant differences between groups were identi ed by alphabetical characters (P < 0.05).

Figure 3
Changes of SGOT, SGPT, ALP, LDH, and GGT activities in the blood of rainbow trout after exposure to MPs and Y. ruckeri.
Results are illustrated as mean ± SD. Signi cant differences between groups were identi ed by alphabetical characters (P < 0.05).