Effect of Biofilm of Aeromonas Hydrophila Oral Vaccine on Growth Performance and Histopathological Changes in Various Tissues of Striped Catfish, Pangasianodon Hypophthalmus (Sauvage 1878)

Aquaculture is one of the fastest growing food production sectors in the world including India. World fish production has reached at about 171 million tonnes in 2016, where aquaculture contributing 47 per cent of the total fisheries production (FAO, 2018). There has been a phenomenal shift from extensive to intensive culture of carps and catfishes in the last three decades. Striped catfish, Pangasianodon hypophthalmus has quickly become the third most cultured freshwater fish in India (Kumar et al., 2017). Intensive aquaculture offers an increased opportunity for spreading of infectious diseases at all stages of production. Pathogenic microorganisms (54.59% bacterial, 22.6% viruses, 3.1% mycotic) and parasitic agents (19.4%) are responsible for periodical disease outbreaks in aquaculture (Dhar et al., 2014). Among the bacterial pathogens P. hypophthalmus is most susceptible to the Aeromonas spp. and E. ictaluri causing motile aeromonas septicemia (MAS) and bacillary necrosis (BNP) respectively (Subagja et al., 1999; Ferguson et al., 2001). Fish immune systems are weak, compare to the higher vertebrates which is the major factor for predisposing them to innumerable disease outbreaks. Over the last few decades, vaccination has become the integral part of modern intensive aquaculture. Norwegian salmon industry is the best example of benefit of vaccinations (Sommerset et al., 2005). 
       
With respect to vaccine studies, the injection of antigens provides for better immune protection than does the immersion protocol (Tatner et al., 1986) and oral route (Ellis, 1988). However, injection vaccines are related to significant growth depression, internal adhesions and injection site melanization resulting in carcass downgrading (Evensen, 2003). Oral vaccines are an attractive alternative to overcome such constraints. Oral vaccination is ideal for economically vaccinating large number of animals with least stress. Unfortunately, free cells (FC) or planktonic bacterial oral vaccine have given poor or uneven performance in terms of protection, due to digestive hydrolysis and low pH of gut. To protect oral antigens from the gastric destruction several strategies were evaluated, such as microspheres, bioencapsulation.
       
Our laboratory has conceptualized and developed a novel bacterial biofilm. Biofilm (BF) of Aeromonas hydrophila, a fish pathogen developed on chitin flakes, inactivated and incorporated in fish feed (Fig 1). Bacterial biofilm is a structured community of bacterial cells enclosed in a self-produced polymeric matrix, glycocalyx. Within this glycocalyx, complex and differentiated associations can be formed, which facilitate nutrient uptake (Azad et al., 2000; Hall-Stoodley et al., 2004; Toutain et al., 2004).
 

       
In the earlier biofilm based research has restricted on the measurements of antibody titre and protection upon challenge (Azad et al., 1997, 1999; Nayak et al., 2004; Sharma et al., 2010; Siriyappagouder et al., 2014). Oral vaccines has great potential in Indian aquaculture.  However, so far, there has been no published information on growth performance and histopathology of fishes fed on biofilm A. hydrophila as oral vaccine. Histological changes provide a rapid method to detect effects of irritants, especially chronic ones, in various tissues and organs (Bernet et al., 1999, Wali et al., 2018). This paper describes the effects of biofilm of A. hydrophila oral vaccines on growth and histopathological features of striped catfish, P. hypophthalmus.

Bacterial isolate and maintenance of pure culture
 
A virulent A. hydrophila isolated from diseased gold fish was injected and re-isolated from common carp (Cyprinus carpio), using Rimler-Shotts (RS) selective medium for the pathogen (Himedia, Mumbai). One day old isolate grown in 1.5% (w/v) Tryptone Soya Broth (Himedia, Mumbai) was harvested by centrifugation at 10,000 rpm for 10 min and the cell pellet was re-suspended in sterile 1.5% TSB (w/v) with 15% glycerol. The cell suspension was aliquoted into 1.5 ml micro centrifuge tubes and stored at -20°C for further use.
 
Preparation of A. hydrophila biofilm cells
 
A. hydrophila biofilm was prepared according to Azad et al., (1997). Conical flask containing TSB (0.225% w/v) and chitin flakes (0.3% w/v) (Himedia, Mumbai) was autoclaved at 121°C for 15 min and cooled to room temperature. The medium was inoculated with A. hydrophila culture and agitated for 6 h daily in an incubator mechanical shaker (120 strokes/min) at room temperature. On the fourth day, the supernatant was decanted and the chitin flakes were washed thrice in the same flask with sterile phosphate buffer saline (PBS, pH 7.2) to remove free cells. Biofilm cells on chitin were then heat inactivated at 100°C for 50 min before incorporating in the feed.
 
Preparation of A. hydrophila free cells
 
A. hydrophila free cells were prepared according to Azad et al., (1997). TSB (1.5% w/v) was sterilized at 121°C for 15 min and cooled to room temperature. The medium was inoculated with 18 h old A. hydrophila culture and the flasks incubated for 18 h. The cells were harvested by centrifugation at 10,000 rpm for 10 min and further washed thrice using sterile PBS (pH 7.2). Finally the pellets were resuspended in PBS to 1010 cells/ml. The cells were heat inactivated at 90°C for 10 min before incorporating in the feed.
 
Incorporation of biofilm and free cells in feed
 
Feed ingredients, such as fish meal (24%), groundnut oil cake (24%), rice bran (17%), wheat flour (17%) tapioca flour (14%) were mixed together, cooked and cooled to room temperature. Cod liver oil (3%), vitamin and mineral mixture (1%) were added to the cooked ingredients separately followed by incorporation of heat inactivated biofilm cells on chitin (BF) and free cell (FC). Vaccine was incorporated in the feed according to Azad et al., (1999). Briefly, biofilm and free cells of A. hydrophila were grown to high density and quantified cells were incorporated at high density in feed, so as to achieve at the end 10¹⁰ cells/ fish/day. The feed paste was pelletized sun dried and dry pellets stored at 4°C in a refrigerator. A control diet (C) with above ingredients without A. hydrophila was prepared with sterile PBS (pH 7.2).
 
Fish stock maintenance
 
Healthy fry of P. hypophthalmus having mean weight (g) ranging from 0.524 ± 1.21 to 1.2 ± 1.13 and mean length (cm) 2.50 ± 0.94 to 4.50 ± 1.10 procured from Zonal Agricultural and Horticultural Research Station, Mudigere (13.1378°N 75.6060°E.), Chikkamagaluru, India. The fish was acclimatized in the Instructional Fish Farm, College of Fisheries, Mangalore and reared for 1 months to advanced fingerling. The fish were fed with dry pellets, prepared with feed ingredients such as fish meal 24%, groundnut oil cake 24%, rice bran 17%, wheat flour 17%, tapioca 14%, cod liver oil 3%, minerals and vitamins 1%.
 
Fish immunization
 
P. hypophthalmus (12.42± 0.14 g) were maintained in cemented rectangular tanks (950 l). Two treatments namely biofilm (BF) and free cell (FC) and one control (C) were setup with three replication in each. Each replicate tank was stocked with 30 fingerlings. Every alternate day, half of the tank water was siphoned off to remove the waste material to maintain better water quality. Fish were fed with biofilm cell incorporated feed at 1010/ fish/day and free cell feed at 1010/fish/day. Fish in the control was fed with control feed mixed with PBS (Phosphate Buffered Saline). The vaccinated feed was given at 4% of the body weight for 20 days and complete acceptance of feed was ensured each day. First 20 days fishes were vaccinated and control feed was given at 2.5% body weight for next 60 days. At the end of experiments, growth performance was evaluated and fishes were collected for histopathological analysis.  
 
Histopathology of the organs
 
Tissues for histopathological study, were aseptically collected. Healthy fishes were euthanized and tissue specimens of gills, liver and kidney were excised, rinsed in normal saline and fixed in 10% buffered formalin for 72 h.  After fixation, the tissues were dehydrated in an alcohol series of ascending concentration (70%, 80%, 90% and 100%, respectively), embedded in paraffin and sectioned at 5-6 μm. The tissue sections were stained with haematoxylin-eosin (H&E) and were examined by light microscope. All the histological procedures were followed as detailed by Bullock (1989). The tissues on the glass slides were analysed through the light microscope (Olympus, BX3-25ND25, Japan) to check for any signs of tissue damage.

Weight gain (g/fish)
 
Biofilms are gathering of microbial cells that is immutable associated with a surface  material and enclosed in a matrix of primarily polysacchride material. Biofilm organism in natural environment are tiny and highly nutritious. Biofilm act as a bioactive compunds and dietary stimulants which can enhance growth perforance of fishes. Several research conducted employing natural substrate based biofilm, revealed that higher biomass compare to control at the end of the experiments. Ramesh et al., (1999) have reported better growth of Rohu (Labeo rohita) fed on biofilm, settled on the substrates. Similar results found in shrimp (Thompson et al., 2002), scampi (Abdullah et al., 2012).
       
In the present study, biofilm oral vaccines enhanced the growth of pangasius catfish (Fig 2). From the initiation of the trial to 15 d, no significant difference (P>0.05) was evident in mean weight gain (g/fish) in all the experimental diets. From 30^th day onwards, till end of the trial, the mean weight gain (g/fish) was significantly higher (P<0.05) in BF treated group that of FC and control group. The mean weight (g/fish) recorded after 80 days of feeding trial in BF treatment was 59.51 g, followed by 46.93 g in FC and 47.26 g in control. The average weight of fish in BF group was higer by 26.80, 25.92% than FC and control group respectively. Similar result was reported that in the presence of sugarcane bagasse, paddy straw and dried Eichhornea for settelement of biofilm, was higher the growth of rohu by 47.5, 29.1 and 17.6%, respectively than the control (Ramesh et al., 1999). Significant improvements of growth performance were noted in silver pomfret, Pampus argenteus diets supplemented with Bacillus subtilis, Lactobacillus plantarum and Clostridium butyricum (Gao et al., 2017).
 

       
There is limited knowledge about the effects of oral vaccines on the growth of P. hypophthalmus, in particular with biofilm of A. hydrophila (Azad et al., 1999, Nayak et al., 2004; Sharma et al., 2010; Siriyappagouder et al., 2014). This research demonstrates for the first time for growth-related effects in striped catfish, P. hypophthalmus, following the oral vaccination with the biofilm and free cell of A. hydrophila antigen. Biofilms are group or micro-organisms in which microbes produced an extracellular polymeric substances (EPS) such as proteins (<1-2%) including enzymes, DNA (<1%), polysaccharides (1-2%) and RNA (<1%) and in addition to these components, water (up to 97%) is the major part of biofilm which is responsible for the flow of nutrients inside biofilm matrix (Jamal et al., 2015). The elevated growth in BF fed group fishes might be due to the extra protein substances generated by the biofilm of A. hydrophila. We assuming, glycocalyx matrix might proectcd the A. hydrophila from the gastric digestion  and fecilated to the hind gut enterocytes in a form which can be taken up by these cells and translocated in an immunogenic form to hind gut and provide the probiotic effect. Kahieshesfandiari et al., (2019) and Azad et al., (2000) convincingly validated the availability of greater quantities of antigen and its prolonged retention and protection as demonstrated by GALT and MAb based immunofluorescence respectively. Studies revealed that Bacillus subtiliis act as probiotics since they have promoted for growth (Hoa et al., 2000). According to Balcázaret_al(2006) probiotic microorganisms are able to aggregate in the gastrointestinal tract when administered over a long period of time could generated their multiple benefits. The increased growth performance reported Gao et al., (2017) in silver pomfret fed with probiotics could be due to better utilization of the diets through enhancement of digestive enzyme activity.
 
Histopathological changes in the gill tissue

Gills are responsible for regulating the exchange of salt and water and play a major role in the excretion of the nitrogenous waste products. Histopathological changes in the gill tissues in various treatments have shown in Fig 3. In biofilm fed fishes showed normal architechture of the gill. Similar findings of striped catfish P. hypophthalmus fed with dietary nucleotides has been reported (Pournori et al., 2017). Nouh et al., (2009) has mentioned no remarkable pathological alterations were recognized in groups treated with single or mixed probiotic. Feeding with FC and control feed resulted several histopathological abnormalities in gill tissues, such as filamentous clubbing, cellular necrosis, blood cells, vasodialation, congestion, hypertrophy. Sharaf and Tag (2011), indicated that feeding of Cyprinus carpio with the higher dose of humic acid caused more potent destructive effect in the gill, liver and kidney tissues.
 

 
Histopathological changes in the liver tissue
 
Histopathological changes in the liver tissues in various treatments have shown in Fig 4. Liver tissue of biofilm fed group was natural and healthy with regular liver cells. This might be due to the capability of biofilm of A. hydrophila to reduce the effect of stressors. Proper nutrition is extremely important for maintaining sound health status of fish. Fish feeding on probiotics manipulate microbiota in the gastrointestinal tract, which produce exoenzymes that can increase nutrient digestibility and promote better health conditions (Zhang et al., 2010). Babiñska et al., (2005) reported no negative impact of Lactobacillus acidophilus on the morphology of the liver and gastrointestinal tract when given in piglets feed. In control and free cell treatments, vacuolization in the tissues, fibrosis of hepatocyte cells, dilated sinusoids, vacuolization of cytoplasm, cellular necrosis of hepatocytes, increase in the number of Kupffer cells, pyknotic nuclei were observed. The histopathological abnormalities observed in control and free cell vaccine either for nutrition imbalance or environmental irritants or presence of ubiquitous bacterial pathogen, A. hydrophila that are present in the same water environment.
 

 
Histopathological changes in kidney tissue
 
The kidneys are paired elongated structures placed above the alimentary canal close to the vertebral column. Histopathological changes in the kidney tissues of the fish in different treatments have shown in Fig 5. In the biofilm fed group, kidney was perfectly healthy consisting of glomeruli, tubules, hematopoietic tissue and a variable number of visible melanocytes. The fingerlings of L. rohita fingerlings fed with 10⁶ CFU/g L. rhamnosus probiotic feed showed intact structure of gills, liver and kidney (Gobinath, 2014). Fishes in the  free cells and control group showed histopathological alterations such destruction of Bowman’s space, inflammatory exudate, glomerular necrosis, tubular necrosis, infiltration of leukocyte cells were detected. Most common alterations found in the kidney of fishes exposed to water contamination are tubule degeneration and changes in the corpuscle, such as dilation of capillaries in the glomerulus and reduction of Bowman´s space (Takashima and Hibiya, 1995). Our results are in accordance with Moneim et al., (2019) reported disruption of kidney tissues in wild captured Nile tilapia, Oreochromis niloticus.
 

       
The induction of a local or systemic immune response after oral immunization is dependent on uptake of antigens from the gut lumen. Histological results revealed that biofilm fed fishes showed healthy structure of gill, liver and kidney tissues compare to free cell and control group. As there was no previous results we could assume that biofilm based oral vaccine might induced in the integumentary immune response in the gill, by providing the protection from ubiquitous bacterial pathogen, A. hydrophila that are present in the same water environment. Moreover, normal structural morphology of the biofilm fed group might be due to the probiotic effect. Furthermore, biofilm might reduce the stress and improved the health status of P. hypophthalmus fingerlings. Azad et al., (2000) found that quantity of BF antigen in the lumen of the gut, spleen and kidney were significantly higher than that of free cell with high retention time. It’s because BF cells are encased in the glycocalyx material which renders protection against digestive hydrolysis.

Oral vaccines are ideal for aquaculture to vaccinate millions of fish of different size with least stress and cost. Till now, no known vaccines are being commercialized or marketed in India despite the fact that market attractiveness is very high. Against this, biofilm oral vaccine could be the ideal vaccine for Indian aquaculture. Biofilm fed fishes were revealed healthy status of gill, liver and kidney tissues compare to free cell and control group. It can be concluded that feeding with biofilm of Aeromonas hydrophila oral vaccines can provide the better growth and histological results in this experiment. Further studies should be carried out in field conditions to ascertain the benefit of biofilm based oral vaccine on growth and histopathology. 

First two authors gratefully acknowledge the funding received towards their Ph.D. from the Indian Council of Agricultural Research for awarded of Netaji Subhas- ICAR International fellowship 2015-16.