Hematobiochemical Changes, Immunological Response and Immune Gene Expression in Nile Tilapia (Oreochrom niloticus) Experimentally Infected with Shewanella putrefaciens

Tilapia is considered as the second most farmed fish globally owing to its suitability for aquaculture and marketability (Wang and Lu, 2016). Tilapia is a candidate species for aquaculture in over more than 140 countries. Tilapia production in 2020 being 6,510,700 metric tonnes, which is predicted to increase to 7.3 million metric tonnes by 2030 (Singh, 2019). Nile tilapia (Oreochromis niloticus) contributed about 8.3% (4407.2 thousand tonnes) in 2020 in the total global aquaculture production (FAO, 2022). As in any other food production sector, diseases remain as the major challenge for aquaculture operations worldwide. It is reported that almost 50% production loss in tilapia farming occurs due to diseases (Assefa and Abunna, 2018). Currently there are more than 200 diseases identified in the cultured aquatic species, of which bacterial diseases are considered very important as they cause serious damage to the aquaculture operations and affect the livelihood of fish farmers. Shewanellosis is a bacterial disease of fish, which was first reported in 2002 in Poland (Kozinska and Pekala, 2004). The disease caused by Shewanella putrefaciens has emerged as an important bacterial pathogen in tilapia farming as it causes mortalities ranging from 30% to 80% and huge associated losses (Lu and Levin, 2010; Manal, 2017; Sood et al., 2020). S. putrefaciens is a gram-negative bacterial pathogen known to cause systemic infections with histopathological changes in cultured fishes viz., O. niloticus, Dicentrarchus labrax, Misgurnus anguillicaudatus and Carassius auratusauratus (Korun et al., 2009; Qin et al., 2014; Manal, 2017; Turgay et al., 2014). The study was conducted to understand the hematobiochemical changes, immunological response and immune gene expressions caused in O. niloticus during an experimental infection with S. putrefaciens.

S. putrefaciens isolate
 
S. putrefaciens isolate (4SK/SRLFDA/19) obtained from the naturally infected Tilapia sp. which was maintained at the repository of State Referral Lab for Aquatic Animal Health, Tamil Nadu Dr. J. Jayalalithaa Fisheries University (TNJFU) was used for experimental infection in this study.         

An isolate (MW341434) was grown in a nutrient broth at 28^oC for 18 h. The bacterial pellet was prepared by centrifugation (2000 g for 10 min) and suspended in phosphate-buffered saline (PBS). The stock culture with a concentration of 1×10⁹ CFU/ml was prepared based on the optical density (OD) values observed at 600nm in a spectrophotometer (Shimadzu, Japan). Serial dilutions were prepared from the stock culture (10⁸ to 10³ CFU/ml) to assess its pathogenicity based on the LD50 assay (Reed and Muench, 1938).
 
Collection and maintenance of fish samples
 
Apparently healthy O. niloticus (weight 25±5 g) were acclimatized in the wet lab facilities of the State Referral Lab for Aquatic Animal Health, TNJFU–Madhavaram campus for 3 weeks prior to the experiment with sufficient aeration, optimum water quality parameters and feeding ad libitum with 30% crude protein feed twice a day.
 
Experimental challenge
 
Experimental challenge with S. putrefaciens isolate (MW341434) was carried out in the fish of the treatment group by injecting 1.508×10⁶ CFU/ml, which was maintained in triplicates. Control fish received equal volumes of PBS. Observations on the moribund and dead fish were recorded in the experimental tanks daily for 4 weeks (Devi Kamilya and Abraham, 2012).
 
Hematobiochemical and immunological responses
 
Fish were anesthetized with MS-222 (20ppm) and blood samples (1 to 1.5 ml) were collected on days 0, 2, 4, 7, 10, 14, 20 and 27 post-infection to assess the hematological parameters viz, hemoglobin (Hb;g dl^-1), Lc (×10³µL^-1), erythrocyte (million/cumm^-1), hematocrit%, mean corpuscular volume (MCV;fl), mean corpuscular hemoglobin (MCH;pg), mean corpuscular hemoglobin concentration (MCHC;g dl^-1) and biochemical parameters viz., glucose (GLU;mg dl^-1), cholesterol (CHO;mg dl^-1), triglyceride (TG;mg dl^-1), respiratory burst activity (RBA), serum total protein (g dl^-1), albumin (g dl^-1) and globulin (g dl^-1). The erythrocyte (RBC) and leukocyte counts were analyzed by a Neubauer hematocytometer (Axiva Sichem Biotech, Delhi); Hemoglobin levels were determined by the cyanmethemoglobin method (Drabkin, 1946).
 
Hematocrit (Ht) and erythrocyte indices such as MCV, MCH and MCHC were calculated following the method of Nelson and Morris (1989) and Saravanan et al., (2011). The glucose concentration was assessed using a commercial kit (Sigma Diagnostics). Serum cholesterol and triglyceride concentrations were estimated following the method of Parekh and Jung (1970) and Rice (1970), respectively. RBA was estimated following the method of Stasiak and Baumann (1996). Serum total protein was determined based on the method of Reinhold (1953). Albumin and globulin levels were estimated by using a kit (Diatek, Kolkata).
 
Quantitative real-time PCR (qRT-PCR)
 
Total RNA was extracted from the muscle and kidney tissues collected aseptically from fish using RNA iso-plus kit (Takara Bio Inc. Japan) following the manufacturer’s instructions. The first-strand (cDNA) complementary DNA was created from 2 mg of total RNA by following the manufacturer’s instructions for the first-strand cDNA synthesis reagent (Thermo Scientific, USA). To study the quantitative real-time (qRT-PCR), a b-actin transcript was used as an internal control. A C1000 Touch thermal cycler with a CFX96 Real-time PCR was used to conduct the qRT-PCR (Bio-Rad, USA). A specific set of primers was used to analyze gene expression (Table 1). 20 ng of cDNA template, 10 M of two primers (forward and reverse), 1x SYBR Green PCR Master Mix Kit (Takara Bio Inc. Japan), and 20 μl of nuclease-free water were used to perform RT-PCR. A relative expression level of the gene was displayed as 2^-ΔΔCt and calculated using the qRT-PCR cycle threshold (Ct) values (Livak and Schmittgen, 2001).
 

 
Statistical analysis
 
One-way analysis of variance and Tukey’s test were conducted for multiple comparisons to study the variation among the groups on various days post-infection (p<0.05). The data were statistically analyzed by SPSS version 20.0, software (SPSS Inc.) for Windows.

Behavioral changes viz., swimming near the surface of water, lethargy, hemorrhages on the body, fin rot, and mortality were observed in O. niloticus experimentally challenged with S. putrifaciens 5 to 6 days post-infection. Cyprinus carpio, Oncorhynchus mykiss (Pezkala et al., 2015), O. niloticus (Manal, 2017) and Seabass (Korun, 2009) infected with S. putrefaciens exhibited similar behavioral changes.
       
O. niloticus experimentally infected with S. putrefaciens showed significantly (p<0.05) lowered hematocrit and hemoglobin values compared to the controls from day 2 to day 4 post-challenge, respectively. Yu et al., (2010) observed a significant reduction of erythrocytes in peripheral blood, hemoglobin level, and hematocrit percentage, which are indices of anemia. Aeromonas hydrophila infection in spotted snakehead,Channa punctatus (Podeti and Benarjee, 2017), Aeromonas and Streptococcus sp. infections in cichlid fish also caused a reduction in Hb and RBC levels (Rehulka, 2002; McNulty et al., 2003).
       
Albumin and globulin levels were also lower than control. Decreased albumin level has been observed in Labeo rohita infected with columnaris disease (Tiwari, 2014). The leucocyte counts (p<0.05) were significantly higher in the infected O. niloticus than in the control fish from day 4 post-infection. Harikrishnan et al., (2010) observed an increased leucocyte count in the gold fish infected with A. hydrophila.
       
A significant (p<0.05) increase in the MCV and MCH levels was observed in the infected O. niloticus than in control fish from day 7 post-infection. However, MCHC values (p<0.05) significantly reduced in the infected O. niloticus compared to uninfected control fish from day 10 post-infection (Table 2). Similar to our results, Ranzani-Paiva, et al., (2004) reported that there was a decrement in the RBC indicator MCHC in O. aureus challenged by Corynebacterium sp. In addition, Haniffa and Mydeen (2010) showed that catfish (Silurus asotus) revealed a decreased MCHC during A.hydrophila infection.
 

       
RBA and serum total protein levels showed significantly lowered values (p<0.05) in the infected O. niloticus than the control fish from day 2 post-infection (Table 3). A decreasing RBA trend was observed after bacterial infection in Rohu challenged with E.tarda (Mohanty and Sahoo, 2010). The reduction in TP has also been reported in various fish infected with pathogenic bacteria viz., Campylobacter cryaerophila, Arcobacter halophilus, Renibacterium sp., Flavobacterium sp., Streptococcus sp., Aeromonas sp. and Vibrio sp. (Harbell et al., 1979; Moyner, 1993; Aydin et al., 2000; Rehulka 2002). Decreased serum total protein was observed in the present study following the observations made by Evenberg et al., (1986) in Cyprinus carpio challenged with Aeromonas salmonicida and Labeo rohita infected with Flexibacter columnaris (Tiwari and Pandey, 2014). Glucose levels increased significantly (p<0.05) whereas cholesterol and triglyceride levels decreased in the infected fish (Fig 1-3). Similar increase in blood glucose levels has been reported by Evans et al., (2006) following exposure of O.niloticus to unionized ammonia. Decreased cholesterol levels were recorded in brook trout post infection with A. salmonicida and Atlantic salmon infected with Vibrio sp.
 

 

 

 

 
Quantitative real-time PCR (qRT-PCR)
 
Intraperitoneal injection of S. putrefaciens induced a significant increase in the expression of all genes after infection in the samples examined (Fig 3). When compared to the control (1.01±0.1) significantly up-regulated TLR4 (3.64±0.1), GST (4.9±0.3), IL6 (4.41±0.1), HSP70 (3.64±0.1) and HSP90 (3.80±0.1) expression were observed after infection (Fig 4). Similarly, the expression levels of various immune-related genes, namely TLR4, GST, IL6, HSP70, HSP90 in both the spleen and head kidney of mandarin fish (Wang et al., 2016). Similarly A. hydrophila infected L. rohita showed reduced gene expression of HSP70 and HSP90 (Das et al., 2016).
 

To the best of our knowledge, this is the first study to document the hematobiochemical, immune response and immune gene expression of Nile tilapia against S. putrefaciens infection. It may be tremendously helpful to the fish farming and large-scale aquaculture industry for diagnostic purpose.

The authors acknowledge the grants and facilities offered by Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, Tamil Nadu, India.

The authors declare that they have no conflict of interest.