β-glucan alleviates the immunosuppressive effects of oxytetracycline on the non-specific immune responses and resistance against Vibrio alginolyticus infection in Epinephelus fuscoguttatus × Epinephelus lanceolatus hybrids

Highlights

• Oxytetracycline (OTC) suppresses phagocytosis and O₂⁻ production in head kidney cells.

• β-glucan can alleviate the immunosuppressive effects mediated by OTC in vitro.

• Residual OTC levels were detected in the muscle tissue of fish fed with OTC and β-glucan.

• Dietary β-glucan in combination with OTC improves the parameters of innate immunity.

• A combination of β-glucan and OTC provides better protection against bacterial infection than either alone.

Abstract

This study was conducted to examine the combinatory effects of β-glucan and oxytetracycline (OTC) on hybrid giant tiger groupers (Epinephelus fuscoguttatus × Epinephelus lanceolatus). In vitro tests, OTC significantly reduced superoxide anion production and phagocytic activity in primary head kidney leukocytes. However, this suppressive effect was alleviated by co-treatment with β-glucan. Subsequently, feeding trials were performed to investigate the potential immunomodulatory effects of dietary β-glucan alone or in combination with OTC on groupers. A total of 210 healthy groupers (368.00 ± 51.03 g) were divided into six groups. Group 1 was the control group, group 2 (BG) received 5 g β-glucan per kg feed weight, groups 3–5 received 5 g/kg β-glucan in combination with 10, 30, or 50 mg OTC/kg fish weight/day (groups M1, M2, and M3, respectively), and group 6 (O) received 50 mg OTC/kg fish weight/day. Fish were sampled to determine the innate immunity parameters and residual OTC levels in the muscle tissue during a 28-day feeding regimen. Residual OTC levels were considerably higher in groups M3 and O compared with the other groups, and peaked on day 14. This was followed by a slight decrease on day 28, despite a continuous supply of OTC. Notably, fish fed with OTC alone had significantly decreased phagocytic rates and superoxide anion production observed in head kidney leukocytes, as well as poorer protection against Vibrio alginolyticus infection. These immunosuppressive effects were not observed in the fish fed with β-glucan in combination with a lower dose of OTC (group M2). Thus, these data suggest that the combination of dietary β-glucan and OTC exerts synergistic immunostimulating effects that protect groupers from bacterial infection.

Introduction

Aquaculture is one of the key food-producing sectors, providing valuable source of protein to meet the growing demand worldwide [1]. Groupers are ray-finned bony fish distributed in tropical and subtropical waters with high market value and consumer demand [2]. In 2014, grouper production reached approximately 139 kilotonnes, valued at 654 million USD [3]. The hybrid giant tiger grouper, a crossbreed between tiger grouper (Epinephelus fuscoguttatus) and giant grouper (Epinephelus lanceolatus), is a popular choice for culture in Asian countries owing to its high tolerance to a wide range of rearing conditions, fast growth, and better feeding performance [4]. However, health management continues to be of serious concern as high stock density often results in crowding stress for aquatic animals, leading to disease outbreaks and substantial economic losses [5].

Vibriosis is the dominant disease affecting groupers, with several members being identified as pathogenic, such as Vibrio alginolyticus [6], V. parahaemolyticus [7], V. harveyi [8], V. vulnificus [9], and V. carchariae [10]. Currently, there is no commercially available vaccine for the protection of groupers from V. infection in Taiwan. Thus, a broad range of substances, including antibiotics, pesticides, immunostimulants, minerals, hormones, and vitamins, are utilized in daily practice to prevent or control diseases in grouper cultures [11]. β-1-3/1–6 glucans are important components of the outer cell walls of many medicinal mushrooms and fungi [12] and are recognized by pattern recognition receptors, which subsequently activate the expression of proinflammatory genes [13]. Many studies have demonstrated that β-glucan is an effective and promising immunostimulant for enhancing animal immunity and improving disease control in aquaculture [14].

Antibiotics and chemotherapeutic agents are often used for the prevention and control of diseases [15]. However, incorrect and excessive use of antibiotics may cause negative effects on aquatic animals and ultimately on human consumers, as well as increase the risk of raising antibiotic-resistant bacteria in the environment [16]. In addition, several antimicrobial agents are used as prophylactics and growth promoters, and these uses far outweigh their use as therapeutics [17]. Regulations on the use of antibiotics vary between different countries. For example, the use of antibiotics as a prophylactic measure has been prohibited in Europe since 2001; nevertheless, such regulations are lacking in many developing countries, which contribute 90% of aquaculture production worldwide [18].

Oxytetracycline (OTC) is a member of the tetracycline antibiotic family and is the first-choice antibiotic for nearly all systemic bacterial fish diseases, such as vibriosis, flavobacteriosis, furunculosis, yersiniosis, and columnaris [19]. Typically, OTC is administered via the oral route; however, it may be administered through an intravenous injection or bath [19]. OTC is cost effective, legally available, and is the most commonly used drug in aquaculture [20,21]. Nevertheless, several side effects, such as nephrotoxicity, growth retardation, immunosuppression, and drug residues in fish tissues have been linked to the overuse of antibiotics in fish [22].

To our knowledge, there has been no research carried out to examine the immunomodulatory effects of β-glucan, OTC, or their combination on the immune responses of hybrid giant tiger groupers. In the present study, OTC residues were assayed in muscle tissue from fish fed with various experimental diets. In addition, the effects of OTC with or without β-glucan on respiratory burst activity and phagocytosis of grouper head kidney leukocytes were examined in vitro and in vivo. Furthermore, we assessed the protective effects of dietary OTC and/or β-glucan as prophylactic agents against V. alginolyticus infection.