Protective effects of Thai bentonite on aflatoxin B₁ contaminated in diet of tilapia fish

L Neeratanaphan¹ and B Tengjaroenkul

Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
btengjar@kku.ac.th
¹ Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand

Abstract

Efficacy of Thai sodium bentonite (TB) to ameliorate toxic effects, including growth, antibody titer, liver serum enzymes and histopathology of aflatoxin B₁ (AFB₁) in contaminated feed (CF) were investigated. The juvenile fish were fed with CF containing of <40 ppb and 10, 20 and 40 ppm AFB₁and CF containing of 10, 20 and 40 ppm AFB₁ mixed with 1% TB, respectively. Fish fed CF higher than 10 ppm significantly reduced body weight and antibody titer (p <0.05). Fish fed CF showed a significant increase aspartate amino transferase and alanine amino transferase (p<0.05). Histopathologically, gill of the fish fed 10 ppm AFB₁ and greater demonstrated ballooning lesion at the tip of gill lamellae. Liver of fish fed 10 ppm and greater demonstrated vascular congestion, sinusoidal enlargement, and white blood cell infiltration, whereas liver of the fish fed 20 and 40 ppm AFB₁ demonstrated irregular shape of nuclei, abnormal mitochondria, dissociation of endoplasmic reticulum and vacuolar degeneration. Fish fed all concentrations of the AFB₁ mixed with 1% TB demonstrated greater weight gain and vaccination titer, and lesser hepatic enzymes. TB can reduce aflatoxicosis in the tilapia, and help lower the risk of human consumption of fish as good protein food source.

Keywords: adsorbent, aquatic animal, growth, immunity, pathology, toxin

Introduction

Aflatoxins (AF) are a group of secondary metabolites produced by Aspergillus fungi, and usually found in high humid and hot climates.The toxin can be detected in feedstuffs such as fishmeal, soybean, corn, peanuts, wheat and rice, with mostly from South Asia followed by Southeast Asia, including Thailand (Schatzmayr and Streit 2013). AF, particularly AFB₁ is the most toxic form that can reduce productivity, and do harm to health of animals and human (Bryden 2012; Matejova et al 2017; Sahoo et al 2001; Schatzmayr and Streit 2013). Acute toxicities lead to relatively high morbidity and mortality, whereas chronic toxicities reduced growth performance, hematology, serum biochemistry, immunity, survival, and induced teratogeny, cytogenicities and carcinoma in a wide varieties of animal species, including aquatic creatures (Alinezhad et al 2011; Bbosa et al 2013; Gonzalez 2011).

To reduce aflatoxicosis in aquatic animals, one of prominent and acceptable approaches is an addition of binding substance to feedstuffs to inhibit the adsorption of the toxin in the gut. This is an economical and practical way to reduce AF in contaminated feed on an aquaculture scale. Several in vivo experiments showed that various toxin binders were to reduce effects of AF in animals (Avantaggiato et al 2005; Manafi 2011; Sadeghi et al 2012). Previous reports in vivo and in vitro studies demonstrated that the binders, including aluminosilicate, bentonite, activated charcoal and zeolite have shown considerable promise in preventing myxotoxicoses (Huebner et al 2004; Kossolova et al 2009; Santacroce et al 2008; Selim et al 2014; Wongtangtintan et al 2015). In Thailand, Thai bentonite (TB) clay from Lopburi province is capable to bind AFB₁ in vitro (Tengjaroenkul et al 2013). Adsorptions of zearalenone (ZEN) onto TB was greater than in mineral clays under different temperatures and pH levels. In addition, TB revealed its efficacy to anti-aflatoxicogenic effects in feedstuffs of Cherry Valley duck (Wongtangtintan et al 2016). However, in vivo report on efficacy of TB to reduce the toxic effects of mycotoxins in fish is limited, particularly on humoral immunity as well as cellular morphological changes. Thus, the purpose of this study was to determine efficacy of TB to ameliorate adverse effects of natural AFB ₁ on productive performance, immunity and pathology of the Nile tilapia.

Materials and methods

Aflatoxin

Contaminated corn contained AFB₁ as 264.67 ppm using HPLC (In-house method based on AOAC 2015) was extracted using acetonitrile, and then dissolved the toxin in 100% dimethyl sulfoxide solution. The AFB ₁ solution was later sprayed on fish commercial pellet feed (AF below 40 ppb) to achieve CF having 10, 20 and 40 ppm AFB₁ (Table 1).

Animal, diet and experimental design

A total of 420 juvenile tilapia fish were weighed approximately 24.3 g, and randomly allocated into 7 treatments for 8 weeks toxin binder feeding trial. CFs were prepared as follow: 1) commercial pellet feed (crude protein 32%; 400 kcal gross energy/100g feed) with AFB₁ contamination lower than International standard allowance (40 ppb) as the control, 2) CF₁₀ with AFB₁ = 10 ppm, 3) CF ₁₀ + 1% TB, 4) CF₂₀ with AFB₁ = 20 ppm, 5) CF₂₀ + 1%TB, 6) CF₄₀ with AFB₁ = 40 ppm, and 7) CF₄₀ + 1%TB. Each treatment had three replications with 20 fish per net cages (size 1x1 m.) in concrete pond. Water quality, including pH, dissolved oxygen, alkalinity and ammonia were measured weekly and maintained to be suitable for aquaculture throughout the experiment. This experiment was approved by the Animal Ethics Committee, Khon Kaen University, Thailand.

Survival and productive performance

The fish were daily recorded for morbidity and mortality. At the end of 8 weeks of the experiment, the fish were weighed individually after 12 h of starvation.

Vaccine and antibody measurement

Streptococcus agalactiae was isolated from fish reared in floating basket in Che river, Khon Kaen province, Thailand. The bacteria were identified and cultured in brain heart infusion (BHI) broth for 24 h, killed by 3% neutral buffer formalin for 12 h, washed in sterilized normal saline 3 times and streaked on BHI agar plate to reconfirm as no bacterial growth on the plate as being killed. Then, the killed streptococcus was adjusted its concentration to 10⁸ CFU/mL using spectrophotometer (Hach DR300, Oriel Corporation, USA) at wavelength 610 nm, and calculated from linear equation of the standard curve, before vaccination.

Humoral antibody titer test, all fish were injected twice in a two-week interval with 10⁸ CFU/mL formalin-killed streptococcal vaccines, 0.1 mL per 100 g of fish weight. After 3 weeks post vaccination, blood from caudal vessels of each of the 6 fish samples was collected, the serum was obtained and kept -20°C upon immunity measurement using direct agglutination test. The titer test was demonstrated as agglutinated (positive) or precipitated (negative) (Toranzo et al 1987).

Liver enzyme test

At the end of experiment, sera from caudal vessels of 6 fish from each treatment were collected to determine levels of aspartate amino transferase (AST) and alanine amino transferase (ALT) using titrated-biochemical reaction by Automate Analyzer (ROCHE/Hitachi Cobas C501, ROCHE/Hitachi, Japan).

Histopathology

The fish were daily recorded for clinical signs. Upon termination, gill and liver of the 6 randomly sampling fish were excised, fixed in 10% neutral buffered formalin, dehydrated in series grades of absolute ethanol, embedded in paraffin wax, sectioned using microtome, stained with Hematoxylin and Eosin (H&E) and examined under a light microscope.

Ultrastructurally, fish liver was washed in 0.1 M phosphate buffer, fixed in 3% Modified Karnorvsky for 48 h, washed in 0.1 M phosphate buffer, fixed in 1% osmium tetroxide for 1 h, washed in 0.1 M phosphate buffer, immersed in propylene oxide and eppon, embedded in capsule with eppon, then sectioned using ultra-microtome, stained with uranylacetae and lead citrate, and examined under transmission electron microscope (JEOL100, JEOL, Japan).

Data analyses

Weight, antibody titer and liver enzyme levels were statistically analyzed using Proc. GLM (SAS 2004). The differences of means were compared using Duncan’s multiple range test (DMRT). Statements of statistical significance were based on p<0.05. Histopathological data were reported descriptively.

Results

Productive performance, survival and clinical signs

Dissolved oxygen, pH, temperature and total ammonium concentration of water were 5.7±0.34 mg/L, 6.9-7.1, 25.5°C-30.5°C and 0.01 ppm, respectively.

Average final weight decreased as the concentration of AFB₁ increased. Fish weight in the control treatment had the highest 102.50 g, when the fish fed the toxin 40 ppm showed the lowest weight gain 53.67 g. Weight of the fish fed 10 ppm AF as well as 10 ppm AF mixed with 1% TB were significantly different from the control treatment (p<0.05). Weight gain of the fish fed 10 ppm toxin was significantly different from 20 ppm AF mixed with 1% TB (Table 1). Weights of fish fed 20 ppm AF and higher were significantly reduced when compared to the fish fed 10 ppm toxin and lower (p<0.05). There were not significantly different on survival of all treatment of juvenile fish fed toxin 10 to 40 ppm.

Fish fed AFB₁ for 8 weeks demonstrated several toxicological signs, including lethargy, and reduced appetite starting from day 14 of the experiment, later that they showed signs of darkened skin, highly mucous skin with hemorrhage, slough off scale and pale liver.

Liver enzymes

Levels of AST and ALT were elevated as dose-dependent with AFB₁ concentrations (Table 1). Both enzymes were highest in 40 ppm AFB ₁ treatment, and they were not significantly different from 40 ppm AFB₁ mixed with TB treatment (p>0.05). The enzyme levels were lowest in the control treatment.

Antibody titer

Positive antibody titer test demonstrated as agglutinated as shown in Table 1 and Figure 1. Fish in the control treatment fed pellet feed containing <40 ppb AFB₁ showed the highest antibody titer, while the average titers were significantly and relatively lowered when fed the toxin, particularly of the greater concentrations. However, the fish fed AFB₁ mixed with TB increased immunity as compared with the fish fed CF without TB. The fish fed 40 ppm AFB₁ had significantly decreased titer levels when compared with the control (p<0.05).

Figure 1. Immune response using direct agglutination technique of vaccinated tilapia sera afterfed AFB1 and AFB1 mixed with TB

Histopathology

Gill of the fish fed 10 ppm AFB₁ and greater demonstrated ballooning lesion at the tip of gill lamellae (Figure 2). Liver of fish fed 10 ppm AFB₁ and higher presented vascular congestion, sinusoidal enlargement and hepatocellular swelling, while of CF containing 20 and 40 ppm AFB₁ the liver demonstrated irregular shape and pyknosis of the nuclei, white blood cell infiltration and vacuolar degeneration (Figure 3). Moreover, the fish fed AFB₁ mixed with TB reduced tissue lesions as compared with the fish fed CF without TB.

Ultramicroscopic results demonstrated pathological structures in liver cells (Figure 4), especially the fish fed 10 ppm AFB₁ and greater. Cell membrane and organelles of the fish fed 20 and 40 ppm were demonstrated degenerative changes as compared with the fish in the control and the 10 ppm treatment. In degenerative cells, enlarged and irregular shaped nuclei and loosen nuclear chromatin at the central region as well as the condensed chromatin adhered to the nuclear membrane were observed. Abnormally arranged and less stained cristae were presented predominantly in the mitochondria, particularly when fish fed 10 ppm AFB₁ and greater. Endoplasmic reticulum was arranged as disorient direction in 10 ppm treatment and greater, and showed differently from those in the control. Liver of TB with 10 ppm AFB₁ fish showed lesser cellular changes than those without TB (Figure 4).

Figure 2. Gill lamellae in control treatment(A); Fish in AFB1 mixed with TBtreatment (B); Fish in AFB1 treatment (C); Ballooning lesion at the tip of the lamellae (BL) x 40, H&E

Figure 3. Liver of fish in control treatment (A); Fish in AFB1 treatment (B); Fish in 10 ppm AFB1 mixed with TB treatment (C); Fish in 40 ppm AFB1 mixed with TB treatment (D); Blood congestion (BC); Pancreas (P); Sinusoidal enlargement (SE); Vacuole (V); White blood cell (W) x 40, H&E

Figure 4. Electron micrographs of liver cells in control treatment (A); 10, 20 and 40 ppm aflatoxin B1treatments (B-D, respectively); 10, 20 and 40 ppm aflatoxin B1 mixed with 1% Thai bentonite treatments (E-G, respectively) in the Nile tilapia; Cell membrane (CM); Mitochondria (M); Nuclear membrane (NM); Nucleolus (NCL); Rough endoplasmic reticulum (ER); Bar 2,000 nm

Discussion

Productive performance, survival and clinical signs

Juvenile tilapia fed 10 ppm AFB₁ and greater reduced growth performance as the AFB₁ concentration increased (p <0.05). In fingerlings, Tuan et al (2002) demonstrated that the toxin 2.5 ppm reduced growth performance and feed utilization. Royes and Yanong(2002) revealed that 10 ppm toxin reduced growth in fingerlings up to 90% of 8 weeks feeding trial. Deng et al (2010) reported the hybrid tilapia fed 245 ppb AF and higher concentrations reduced their growth after a long exposure. Sherif and Mahfouz (2015) demonstrated a significant decrease on growth performance of tilapia fed 20-100 ppm AF for 12 weeks. Liver is an important organ involving several body functions, including protein, carbohydrate and lipid metabolisms, andalso a major target for AF epoxide metabolites to induce damages to DNA, RNA and proteins (Coppock et al2012). These cytotoxicities could affect growth, immunity and other cellular functions as presented in this study as previous reports related to AF in aquatic creatures.

Productive performance of the fish exposed to AF was generally varied, and closely related to fish size, weight, exposure time and AF concentration as well as fish species (Deng et al 2010). Younger and smaller fish tend to get more sensitive to the toxin than the aged and larger fish (Matejova 2017; Sahoo et al2001; Santacroce et al2008).

AFB₁ concentrations from 10 to 40 ppm was not induce any mortality in this study. Tuan et al(2002) demonstrated that 10 ppm AF did not cause mortality in experimental fish. However, Deng et al(2010) showed that tilapia fed different AF levels up to 1.641 ppm for 20 weeks revealed significantly different on mortality among AF treated fish. Santacroce et al(2008) mentioned that rainbow trout fed AFB₁ 0.5-1 ppm increased mortality 50%. Mahfouz and Sherif(2015) reported that the Nile tilapia fed 200 ppb AF for 12 weeks increased mortality to nearly 4%, and Cagauan et al(2004) found that tilapia fed AF upto 115.34 ppb for 120 days had mortality 67%. In contrast, young tilapia, El-Banna et al (1992) demonstrated that fish consumed 0.2 ppm toxin for 10 weeks induced mortality 16.7%. Tuan et al (2002) demonstrated that tilapia fingerlings exposed to 100 ppm toxin for 6 weeks showed mortality rate 55%. Lethal sensitivity to the toxin was also reported as species dependent; for example, rainbow trout 50 g and catfish 35 g fed AFB₁ concentrations of 0.5-1.0 and 11.5 ppm, respectively, showed the death rate 50% (Lethal dose 50%). In this study, it has shown that tilapia was relatively resistant as compared the lethal concentrations reported in other fish species, this may be due to the tilapia has a great physical and biological systems involving biochemical and detoxification processes.

Reports on subacuteaflatoxicosis signs in animals, in general, are cachexia, yellowed mucous skins or membranes, darkened scales, reduction of growth, immunosuppression, liver lesions, kidney damages and premature death (Sahoo et al 2001; Santacroce et al 2008; Zaki et al2008). Most aflatoxicosis symptoms revealed in this study were in accordance with the previous reports, however, darken skin and slough skin were more prominent, this may be related to tissue hypoxia and toxic stress from the toxin on the fish tissues and organs.

Liver enzymes

Fish fed CF showed a significant increase in liver enzymes; AST and ALT ( pp<0.05) as compared with the control. The results are in accordance with other research studies in tilapia. Deng et al(2010) reported that both enzymes in hybrid tilapia (O.Niloticusand O.aureus) were elevated when the young fish fed 0.085-1.641 ppm AF for a long exposure time (20 weeks). Mahfouz and Sheriff (2015) revealed that sera AST and ALT of the Nile tilapia fed 0.1 ppm AFB₁ for 12 weeks were increased and significantly different from the control and the fish fed 0.02 ppm AFB ₁. In this study, the enzymes of the fish fed AF with TB were relatively lower. This indicates that 1%TB can prevent AFB₁ toxic effect to liver and other organs in tilapia. It has known that absorbed AF through intestinal tract can react to microsome in liver cytosol, and produce epoxide substances that bind covalently and induce damages to cell membrane (Coppock et al 2012). The membrane damages increase cell membrane permeability, and allow the moving out of AST and ALT from liver cytosol into the blood stream, and make them to be detected the increments in blood sera.

Antibody titer

Antibody titer test demonstrated as agglutinated and precipitated as shown in Table 1 and Figure 1. Fish in the control treatment fed control diet showed the highest antibody titer, and the average titers were significantly and relatively lowered in fish sera fed toxin, particularly of the high AFB₁₁ concentrations. However, the fish fed AFB ₁ mixed with TB significantly increased immunity as compared with the fish fed CF without TB. The fish fed 10 ppm AFB₁ and greater significantly decreased antibody titers when compared with the control (p<0.05). Additionally TB can recover antibody titer approximately 1.38-2.0 times compared between 2 treatments as T2 and T3, T4 and T5, as well as T6 and T7.

Sahoo and Mukherjee (2001) revealed that AF reduced fish immunity by reduction of neutrophile phagocytic activities, serum protection against Aeromonas hydrophilla challenge test. Mahfouz and Sherif (2015) reported that hybrid tilapia fed AFB₁ showed an increase bacterial susceptibility, particularly 100 ppb AF. The lower immunity titer in AF treatment in this study as well as previous studies, occurred probably because AF deters protein metabolism in liver, the AF primarily target organ, and directly affect production of protein globulin levels as the indicator of vaccination titer.

Histopathology

This study demonstrated that AF caused skin lesions and morphological changes in gill, spleen and liver tissues, particularly when the fish fed 20 ppm AFB₁₁ and higher. Gill showed ballooning feature at tip of the lamellae. This similar lesion was occurred when it exposed to pyrethroid insecticide (Vidhya and Nair 2013), heavy metals (Koca et al 2005), detergents (Ogundiran et al 2009) and contaminant stressors (Marina et al 2007).

Hepatic cells exposed AF showed the pyknotic nuclei, condensed nuclear chromatin adhere to the nuclear membrane, discoloration or less staining of the cytoplasm and vacuolar degeneration. As the toxin concentration increased, the liver tissue lesions of light and electron microscopy were increased. These results indicated that the liver histological lesions of aflatoxicoses were strengthened dose-dependent by the dietary AF contaminated levels. The cell and tissue were relevant to previous studies. Tuan et al (2002) found that liver exposed the 10 ppm AFB₁ or greater caused nuclei morphological changes with presenting of lipofushcin which imply to an unpleasant program cell death. The tilapia fish in this study exposed to high concentrations of AF demonstrated liver cell abnormalities. Usanno et al(2005) revealed that the necrotic hepatocytes of the fish with cell membrane damages, mitochondrial morphological changes and irregular arrangement of endoplasmic reticulum in the cytosol, and chromatin condensed in the nucleus could be stimulated by high oxidative stress or DNA impairment. Inclusion of TB as toxin adsorbent with different AF concentration in the feed displayed reduction of liver cell microscopic deformities, especially endoplasmic reticulum arrangement and mitochondrial cristae.

Besides improving tissue and cellular morphology of the liver as well as gill, when applied to investigate potential to prevent AFB₁₁ toxicities in vivo; 1%TB can enhance growth of tilapia during 8 weeks of toxin exposure, AST and ALT levels as indicators of liver cell function and immunity via level of antibody titers after bacterial vaccination. These data generally demonstrate a suitable manner to prevent toxic effects of AFB₁as in previous reported. Ellis et al (2000) concluded that using 2% bentonite in 20 ppb AF diet reduced aflatoxicosis effects in trout. Vekiru et al (2007) indicated that bentonite had potential to protect animas against toxicities of aflatoxin contaminated diet. Eya et al(2008) suggested that adding 5% bentonite and 2.5% modernite w/w enhanced growth rates of juvenile rainbow trout. Abdelaziz et al(2010) found that calcium bentonite possessed binding property to the mixed aflatoxin(less than 22 ppb) and ochratoxin(less than 15 ppb) in tilapia diet. Hassan et al (2010) revealed the efficiency of bentonite to increase total protein and albumin, to reduce structural chromosome aberrations and to prevent DNA damage by decrease AFB₁bioavailability. Furthermore, Zychowski et al (2013) demonstrated that aluminosilicate enhanced growth and feed efficiency as well as serum lyzozyme involving immune response, and reduced mortality rate and liver tissue lesions in red drum fish ( Sciaenopsocellatus) fed AF upto 5 ppm. Saei et al (2017) found that biotoxin, a toxin binder, increased survival rate, but not growth and blood parameters of the young rainbow trout fed diet contaminated with 1 ppm AFB₁

Conclusion

• The results indicated that 1% TB can reduce aflatoxicosis effects in terms of body weight, serum enzyme indicators, humoral immunity as well as morphology of the gill and liver of the Nile tilapia, and this approach can help lower the risk of human consumption of fish as good protein food source.

Acknowledgements

This research was supported by the Research Centre for Environmental and Hazardous Substance Management (EHSM), Khon Kaen University, and the Centre of Excellence on Hazardous Substance Management (HSM), Phatumwan, Bangkok, Thailand.

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Received 6 June 2018; Accepted 23 July 2018; Published 1 August 2018

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