The growth performance of African catfish fed with probiotic and paraprobiotic B. cereus BR2 for 30 days of cultivation showed better results than the control group (Table 2). This result is in line with Hamid et al. (2021), that probiotic supplementation of Enterococcus hirae was able to increase the growth of Clarias gariepinus × Clarias macrocephalus hybrid catfish compared to the control. Similarly, paraprobiotic supplementation of heat-killed Lactobacillus plantarum L-137 was reported to provide better growth performance in bighead catfish Clarias macrocephalus (Hien et al. 2021). The increase in the growth rate of catfish in the treatment was due to the performance of probiotics and paraprobiotics which were able to increase feed utilisation to be more efficient as stated by the low feed conversion value in this study.
The efficiency of feed utilisation is supported by the activity of digestive enzymes of the fish. The digestive enzyme activities (amylase, protease, lipase) of the treatment groups were observed to be higher than the control (Fig. 2A, 2B and 2C, respectively). The high enzyme activity in the probiotic treatment came from its involvement as a living cell in the competitive exclusion of infectious pathogens. Probiotics produce inhibitory molecules, promote their colonisation and compete for adhesion sites, nutrients, chemicals, as well as energy sources that can interfere with pathogen growth or activity (Kuebutornye et al. 2020). The ability of probiotics to produce a number of extracellular amylase, protease and lipase enzymes in the gut of fish indicates that probiotics have a direct contribution to digestion (Ramesh et al. 2015; Ray et al. 2012). Yang et al. (2017) described the application of probiotic B. cereus strain G19 can affect microbial interactions and the number of generalists to improve the homeostasis of the gut microbiota of sea cucumber Apostichopus japonicus. Meanwhile, in contrast to probiotic treatments, paraprobiotics has its own mechanism in increasing the activity of catfish digestive enzymes. Paraprobiotic B. cereus BR2 is a crude para-probiotic material that was pre-heat-killed. According to Akter et al. (2020), structural components contained in paraprobiotics such as teichoic acids (TA) and lipoteichoic acids (LTA), cell homogenates, and peptidoglycans (PGN) can produce immunomodulating effects in fish regardless of viability, through various structural components of bacterial cells that secrete bioactive compounds when bacterial cells dissolve in the digestive system. Li and Tran (2022) added that paraprobiotics as inactive cells induce fish immune responses through interactions with gut associated lymphoid tissue (GALT). GALT contains mucosal immune cells such as lymphocytes, plasma cells, granulocytes, and macrophages that make mucosal tissue an active immune organ. The gut microbiota plays a central role in the development of the GALT (Dawood, 2021; Lauriano et al. 2023; Lee et al. 2021). This is in line with Wu et al. (2020), who reported that the presence of commercial paraprobiotic cells Rhodotorula minuta and Cetobacterium somerae (HWF™) in the digestive tract of hybrid sturgeon Acipenser baerii x Acipenser schrenckii can increase the abundance and diversification of gut microbiota that are beneficial in secreting digestive enzymes to facilitate digestion and nutrient absorption. Diversification of symbiotic microbes in the gut is considered very important in host metabolism because microbes have 100-fold more genes than the host and can synthesise a large number of enzymes (Zhu et al. 2010). Thus, the gut microbiota is considered as an auxiliary metabolic organ and participates in several processes such as amino acid, carbohydrate, energy, and lipid metabolism through the provision of fermentation end products (Portincasa et al. 2022). Relying on complex species-species interactions, the bacterial community maintains its stability in the intestine, and completes the metabolic functions of the system simultaneously (Yang et al. 2020).
Total gut bacterial abundance constantly increased during rearing (Fig. 2A), and was directly proportional to probiotic B. cereus BR2 count, which also showed an increase (Fig. 2B). This indicates the direct involvement of probiotics and indirect involvement of paraprobiotics in increasing the number of bacteria in the host gut. Similar results were reported by Yuhana et al. (2022), an increase in probiotic Bacillus sp. NP5 RfR given in the feed treatment of vaname shrimp Litopenaeus vanamei increased the number of bacteria in the gastrointestinal tract. The presence of probiotic B. cereus BR2 in the gut in this study is an indicator of viability, that the probiotic can still live in the gut of catfish during the growth period and is able to survive the digestive process. In line with Opiyo et al. (2019), probiotic supplementation of Saccharomyces cerevisiae and Bacillus subtilis can increase the total bacterial gut of Oreochromis niloticus tilapia. According to Ringø et al. (2016), the increase in beneficial microbes in the gut is an indication of the positive role of probiotics in improving the host's gut microbial balance by replacing harmful bacteria with beneficial bacteria. The production of antimicrobial compounds by beneficial microbiota in the gut has a major influence on the blood profile which ultimately leads to improved physiology and growth (Verschuere et al. 2000). These improvements can further enhance not only growth but also the immune system of the fish, resulting in better health status and production.
Total E. tarda ETS1.1 in catfish kidney and liver organs showed the highest abundance on day 1 and gradually decreased until day 14 post-infection (Fig. 3A and 3B). The presence of E. tarda ETS1.1 in both catfish organs is due to the ability of E. tarda ETS1.1 to survive and replicate in phagocytes, develop in the vascular stream and cause systemic infection in the host (Park et al. 2012). E. tarda ETS1.1 septicaemia induces systemic immunosuppression through lymphocyte apoptosis, which suppresses systemic immune responses during the early stages of septicaemia (Pirarat et al. 2006). The faster decrease in the number of E. tarda ETS1.1 n the treatment group compared to the control was due to the competition and suppression of pathogenic E. tarda ETS1.1 cells by probiotic and paraprobiotic B. cereus BR2. Through the capacity of probiotics to produce bacteriostatic or bactericidal substances against pathogenic microbes, such as lysozyme, proteases, siderophores, hydrogen peroxide or bacteriocins, probiotics consume nutrients available in the host body, so competition for nutrients is one of the mechanisms of probiotics to inhibit pathogen colonisation (Chen et al. 2019). B. cereus has been reported to be able to produce indole acetic acid (IAA), ACC-deaminase and siderophores to support its growth (Zhou et al. 2021). Siderophore secretion by B. cereus species produces 2 products, namely bacillibactin (BB) and petrobactin (PB) which function for iron acquisition through membrane-associated substrate binding proteins (SBPs) (Zawadzka et al. 2009).
Cytokines play an important role in the immune system through their specific receptors that bind to cell membranes, providing induction of cascade enhancement, stimulation, or suppression of cytokine-regulated genes (Mokhtar et al. 2023). Interleukin 1β (IL-1β) is a pro-inflammatory cytokine that is involved as a major mediator in the control of fish immunity to overcome stressful conditions and pathogenic infections (Tsarouchas et al. 2018). IL-1β stimulates immune responses by activating lymphocytes or by inducing the release of other cytokines capable of triggering macrophages, granulocytes, lymphocytes and NK (natural killer) cells (Secombes et al. 2015). Administration of probiotics and paraprobiotics during 30 days of rearing showed upregulation of the IL-1β gene in catfish kidneys (Fig. 5A), indicating an increase in pro-inflammatory factors to fight infection that is thought to occur by invaders or other environmental stressors. This is in line with Villamil et al. (2014) reported that Oreochromis niloticus tilapia fed with probiotic Lactobacillus acidophilus showed higher expression of IL-1β, and that Lactobacillus acidophilus showed higher expression of IL-1β. Jang et al. (2021) by feeding the vegetative paraprobiotic Bacillus sp. to olive flounder Paralichthys olivaceus has significantly upregulated the cytokine IL-1β. After the challenge test with E. tarda, there was an acute upregulation of the IL-1β gene in DPI 1, indicating inflammation and a high immune status in catfish to avoid or resist the spread of E. tarda. Similar results were stated by Mohanty and Sahoo (2010) stated that Indian major carp Labeo rohita challenged with E. tarda showed significant induction of immune response and expression of several immune-related genes, including IL-1β, TNF-α, inducible nitric oxide synthase (iNOS), complement component C3, β2-microglobulin, CXCa, lysozyme type C and type G. Several days post-challenge, there was a downregulation of IL-1β in DPI 3 and DPI 7. The changes in catfish behaviour that occur during the challenge are thought to have an impact on feeding and consumption behaviour which affects the suppression of digestive enzyme activity in the digestive tract. According to Magouz et al. (2020), when available nutrients in the gut are reduced, local gut immunity decreases, and general body immunity is suppressed. Under these conditions, the fish body cannot resist the influence of pathogens, invaders, and environmental conditions, thus indicating a downregulation of the IL-1β gene.
Antigen Processing and Presentation (APP) is the process by which antigenic proteins are processed into peptides. Peptides are loaded and transferred to the cell surface on Major Histocompatibility Complex (MHC) proteins (Galluzzi et al. 2017). Histocompatibility molecules are glycoprotein receptors that play an important role in exogenous and endogenous antigens (Goldsby et al. 2003). MHC class II is only expressed on professional Antigen-Presenting Cells (pAPCs), which mainly inhabit lymphoid organs including the kidney head, spleen, or thymus, and are capable of triggering T lymphocytes into CD8+ T cytotoxic lymphocytes or CD4+ T helper lymphocytes. pAPCs express MHC class II molecules that generally engulf pathogens, and display peptides processed from exogenous antigens via the lysosomal pathway (Johnstone and Chaves-Pozo, 2022). pAPCs in some fish species are B cells, dendritic cells (DCs), monocytes or macrophages, granulocytes, and erythrocytes (Iliev et al. 2013).
Dietary supplementation with probiotics and paraprobiotics for 30 days of rearing showed upregulation of the MHC II gene in catfish kidneys (Fig. 5B). This is different from the results of Muñoz-Atienza et al. (2014) who reported that the administration of probiotic lactic acid bacteria (LAB) by immersion to turbot fish Scophthalmus maximus L. did not stimulate the transcription of MHC I and MHC II genes. After the challenge test with E. tarda, there was an upregulation of MHC II genes in DPI 1 with peak expression regulation at DPI 7. According to Deng et al. (2020), gene expression in the MHC II antigen presentation pathway shows different patterns in response to different challenges, including bacteria, viruses, and LPS. The upregulation of MHC II shows faster viral stimulation (24 hours) than bacterial stimulation (72 hours). This suggests catfish are infected and MHC is executing immune response mechanisms in the recognition of non-self antigens. In line with Ni et al. (2014), the expression of MHC I and MHC II can be influenced by host susceptibility to infection or disease. Similar findings were stated by Chaves-Pozo et al. (2004), intraperitoneal injection of intact Vibrio anguillarum in gilthead seabream Sparus aurata L. induced an increase in the number of acidophilic granulocytes in the kidney head that constitutively expressed MHC class II mRNA and produced IL-1β post bacterial infection.
The survival rate of catfish after the challenge test with E. tarda was observed for 14 days. The highest survival rate was obtained in the probiotic and paraprobiotic B. cereus BR2 (1010 CFU mL− 1) treatments (Fig. 6). In line with the research of Nayak et al. (2007), the administration of probiotic B. subtilis in Indian major carp Labeo rohita, and Lee et al. (2013) by administering probiotic L. pentosus PL11 to Japanese eel Anguilla japonica showed significant survival rate against E. tarda infection. Similar results were found in the application of paraprobiotics reported by Barui et al. (2023) where feed treatment supplemented with paraprobiotic formalin-killed B. amyloliquefaciens significantly (p < 0.05) increased the survival of Indian major carp Labeo rohita against A. hydrophila infection.