Immune Parameters of Broilers Supplemented with Adsorbents and Quantification Challenged with Mycotoxins

Veterinary and Animal Sciences 2022; 10(4): 78-82 http://www.sciencepublishinggroup.com/j/avs doi: 10.11648/j.avs.20221004.11 ISSN: 2328-5842 (Print); ISSN: 2328-5850 (Online)

1. Introduction Globally, food and feed have been seriously contaminated with mycotoxins of which aflatoxin, zearalenone, fumonisin, deoxynivalenol and ochratoxin are the most common [23].

In farm animals consumption of mycotoxins, at levels that do not cause clinical mycotoxicosis, suppress immune functions and may decrease resistance to infectious diseases [1]. There are several relevant parameters for assessing the immune status of animals, including phagocytosis and interleukin assays. According to Sattler et al. [24], phagocytosis has become a central process of the first line of defense of the immune system against pathogens. Professional phagocytes take up microbes, kill and digest them, and activate the subsequent immune response. Essentially, cytokines represent an integrated network of cellular mediators capable of triggering diverse biological effects influenced by the prevailing state of the organism [2]. Avian cytokines, similar to mammalian ones, are influential in the host's immune response to pathogenic infection. Usually classified as pro-inflammatory or anti-inflammatory, cytokines are secreted by diverse cell populations after stimulation [21]. Notably, both pro-inflammatory and anti-inflammatory types elicit distinct responses to immunogens at different stages of an infection [3]. IL-6 is considered an early and sensitive indicator of inflammatory reactions. It is the basic stimulant of acute phase protein synthesis in the liver. In inflammatory conditions, the concentration of IL-6 in the serum of patients increases many times [5]. Interleukin IL10 is one of the main anti-inflammatory cytokines. Suppressing distinct functions of natural killer cells and mainly T lymphocytes, it prevents the production of IL-12 and other pro-inflammatory cytokines (such as TNFα, IL-6 and IL-1β) by APCs [20, 27]. IL-10 prevents the increased expression of several genes in phagocytic and dendritic cells that are normally induced by stimulation of TLRs (toll like receptor) [27]. The use of mycotoxin detoxifiers as feed additives aims to reduce the toxicity of mycotoxins in contaminated feed ingredients, allowing their use in animal feed formulation [16]. There is a wide range of mycotoxin detoxifiers, with an equivalent myriad of claims [12]. The aim of this study was to evaluate the effect of different mycotoxin adsorbents, with different compositions, on the immunological parameters of broiler chickens challenged with the mycotoxins aflatoxin, fumonisin and DON. With five treatments and six replicates, totaling 30 experimental units, each cage being an experimental unit composed of ten birds.

The experimental diets were formulated to meet the nutritional requirements, according to the NRC recommendations [22]. The diets were isocaloric, isoproteic and isovitaminic, according to the composition shown in table 1. The raw materials and experimental diets were analyzed for the presence of mycotoxins (aflatoxins, deoxynivalenol, diacetoxyscirpenol, umonisin, ochratoxin A, toxin T-2 and zearalenone), and no mycotoxins were detected in the raw materials used.   1 guarantee level per kg of product: Folic Acid: 140 mg/kg; Pantot Acid 1700 mg/kg; biotin: 15 mg/kg; Calcium: 30/130 g/kg; copper: 1410 mg/kg; choline: 40 g/kg DL-methionine: 260 g/kg; Enramycin: 1333 mg/kg; Iron: 8500 mg/kg Iodine: 150 mg/kg; Lysine: 50 g/kg; Manganese: 12 g/kg; Niacin: 5930 mg/kg; Selenium: 45 mg/kg; vit. A: 1800000 IU/kg, Vit. B1: 580mg/kg; vit. B12: 3000 mcg/kg; vit. B2: 960 mg/kg; vit. B6: 730 mg/kg; vit. D3: 300000 IU/kg; vit. E: 3750 IU/kg; vit. K3: 300 mg/kg, Zinc: 9170 mg/kg.

Table 1. Composition of diets.

Ingredients	%
Corn	63,00
Soybean meal	29,80
Soy oil	3,00
lysine	0,10
Methionine	0,04
dicalcium phosphate	2,00
calcitic limestone	1,00
salt	0,46
premix1	0,60
Composition calculated
crude protein	20%
Metabolizable Energy	3050 Kcal/Kg
Met+ Cyst	0,95%
lysine	1,19%
Calcium	0,95%
phosphorus available	0,48%
Sodium	0,22%

2. Material and Methods 2.1. Animals This project was approved by the Commission for Ethics in the use of animals (CEUA) of the company SAMITEC – CEUA/SAMITEC.288.274. 300 male broiler chickens from Cobb's 500 line, with one day of age and an average weight of 41.88 grams, were used. The experimental test was carried out in an experimental room, 22 m2, with negative pressure, acclimatized. The animals were housed in experimental cages, each with a width of 0.5 m, a length of 0.5 m and a height of 0.33 m, arranged in four overlapping levels, each level with two cages. Each cage had a feeder-type drinker, nipple-type drinker with height adjustment. 2.2. Experimental Design and Diets Birds were distributed in a completely randomized design. Birds received feed and water ad libitum during the experimental period (1 – 21 days). Three mycotoxin adsorbents with different compositions were tested, which were added 2.5kg/kg, in diets contaminated with mycotoxins, using 1.0 PPM of aflatoxin + 50.0 ppm of fumonisin + 25.0 ppm of DON.

Aflatoxins (B1, B2, G1 and G2) were obtained from the cultivation of a toxin strain of Aspergillus parasiticus, and the concentrations used were B1: 93.8%, B2: 2.1%, G1: 3.4% and G2: 0.7%. Fumonisin (B1 and B2) was obtained from the cultivation of a toxin strain of Fusarium moniliforme, and the concentrations used were 95.8% of B1 and 4.2% of B2. And the mycotoxin deoxynivalenol (DON) was obtained from the cultivation of a toxin strain of Fusarium graminearum. We evaluated a control diet without contamination, mycotoxin-contaminated diet without absorbent, contaminated diet + SIM – FIX HP🇧🇷, contaminated diet + adsorbent A, contaminated diet + adsorbent B. The SIM adsorbent – FIX HP🇧🇷 presents itself in its composition: 1.3 and 1.6 beta-glucans, polycene bentonite, activated carbon, organic molecule, silymarin and organic selenium. And, as informed by the manufacturers, on the respective packaging, adsorbent A contains the following composition: dry brewer's yeast (glucomannans), Na and Ca silicate, and oyster flour. While adsorbent B is composed of: bentonite, diatomite, Eubacterium sp, seaweed flour, inactivated yeast, dried chicory pulp.

2.3. Interleukin IL 6 and IL 10 Gene Expression At the end of the experimental period, cecal tonsils were collected and the expression of pro-inflammatory (IL 6) and anti-inflammatory (IL 10) interleukins was determined by quantifying gene expression by RT-qPCR. Quantification of gene expression is performed by RT-qPCR, using specific primers (primers) for each target. In this type of analysis, each combination of target and sample generates a threshold value, Ct (threshold cycle), a measure of the concentration of target-specific messenger RNA (mRNA) in the sample. The Ct value needs to be normalized as a function of previously chosen reference genes, generating a deltaCt (dCt) value (Ct target-Ct reference gene). For RNA extraction, approximately 100mg of tissue was homogenized in TissueLyser (Qiagen), and the total RNA was purified by extraction with TRI® Reagent (Sigma) – chloroform. Extracts were treated with turboDNaseI (Ambion) and RNA was quantified with NanoDrop (Thermo Scientific). cDNAs were synthesized using the High Throughput cDNA Reverse Transcription kit (Applied Biosystems), using 1 ug of RNA per reaction. cDNAs were diluted 5x in sterile MilliQ water, and targets quantified using Bright-Green PCR Master Mix (Biotium) in a QuantStudio 3 thermocycler (Thermo). The cycling used was 95°C 10min, followed by 40 cycles of 95°C 15s and 60°C 1min. The Primer Express 3.0 program was used to design the oligonucleotide primers. The GAPDH and ACTB genes were used as internal controls, and the relative gene expression was determined using the 2-∆∆Ct method [17]. 2.4. Statistical analysis Data were subjected to analysis of variance and comparison of means by Tukey's test at 5%.   3. Results and discussion The results of the gene expression of the interleukins IL-6 and IL-10 are shown in Figure 1. A lower expression of IL-6 was observed in birds that received a diet contaminated with mycotoxin + adsorbent B, which serve to contain the production of pro molecules. -inflammatory to limit tissue damage and to maintain or restore tissue homeostasis.

The effect of mycotoxins on the poultry immune system can be summarized as follows: depressed T- or B-lymphocyte activity (regressive bursa and thymus), suppressed immunoglobulin and antibody production, reduced complement or interferon activity, impaired macrophage-effector cell function , and reduction of antibody titers and serum concentration of antibiotics [11]. Basically, cytokines mediate the outcome of an effective immune response and serve as the interface between the two arms (ie, innate and adaptive elements) of an otherwise complex immune system [9]. IL-6 also has a pyrogenic effect. Together with IL-1, TNF and INF, this cytokine can significantly increase body temperature by stimulating the production of prostaglandins. The increase in IL-6 production and the maintenance of a high serum concentration of this cytokine promote the transition from an acute inflammatory reaction to the chronic phase [5]. The highest expression of IL-10, considering the scenario of contamination and addition of adsorbents, was observed in the challenged group and supplemented with the adsorbent YES – FIX HP. This fact is probably related to its composition and resulted from the synergy between its active ingredients. In addition to the raw materials responsible for the adsorption of the main mycotoxins found in the field, this adsorbent contains principles known to have immunomodulatory, antioxidant and anti-inflammatory action 1,3 and 1,6 beta-glucans from the yeast Sacharomyces cerevisiae, organic selenium and extract of milk thistle (silymarin) unlike the other adsorbents analyzed IL-10 is an immunoregulatory cytokine whose primary function is to limit inflammatory responses [6], with potent anti-inflammatory properties that play a central role in limiting the host's immune response to pathogens , thus preventing damage to the host and maintaining normal tissue homeostasis [13]. According to De Smedt et al. [7], IL-10 plays an essential role in controlling the immune response, balancing the response between Th1 and Th2 by regulating the synthesis of cytokines by cells that present antigens and reducing them, differing from other treatments. Which, when unbalanced, could indicate immunosuppression of the birds' defense system. The other treatments did not differ statistically from each other.

The immune response to pathogens involves rapid activation of pro-inflammatory cytokines that serve to initiate host defense against microbial invasion. However, excess inflammation can give rise to systemic metabolic and hemodynamic disturbances that are harmful to the host. As a result, the immune system has evolved in parallel with anti-inflammatory tissue damage mechanisms. The function of the beta-glucan present in Yes-Fix HP is mainly the adsorption of mycotoxins, especially zearalenone [30]. Furthermore, β-glucans are known as modulators of the immune system, acting mainly on macrophages, exerting a beneficial effect against a variety of bacteria, viruses, fungi and parasites [19], which can reduce the release of pro-inflammatory cytokines [ 28].

As b-glucans are not present in animal cells, they are seen as “foreign” by the immune system and act as a microbe-associated molecular pattern (MAMP), which primarily activates members of innate immunity [26]. However, it is notable that the immunomodulatory attributes of these molecules, which induce regulatory responses of greater or lesser intensity, are probably related to their degree of purification and the biotechnologies involved in their production process.

According to Zabriskie et al. [31], the immunoregulation mechanism mediated by beta-glucan depends on its interaction with immune cells located in the intestine, which recognize these oligosaccharides. An excessive inflammatory response is associated with oxidative stress [15]; selenium regulates the activation of NF-κB, a transcription factor, which plays a key role in the regulation of inflammatory pathways [14]. Selenium can inhibit NF-κB from turning on inflammation-related genes that eventually reduce the expression of pro-inflammatory cytokines [8]. The anti-inflammatory function of Se may be due to the presence of specific selenoproteins, such as glutathione peroxidase (GPx), which reduces the inflammatory changes induced by oxidation in the liver [18].

In addition to β-glucan and organic selenium, another particularity of the composition of Yes – Fix HP is the presence of silymarin. Silymarin is a natural product, extracted from the seeds and fruits of the plant Silybum marianum (milk thistle), and its effectiveness has been attributed to antioxidant, anti-inflammatory and immunomodulatory mechanisms that act on various cell signaling pathways [25, 4] . Wang et al. [29] observed a reduction in the expression of pro-inflammatory interleukins with the administration of silymarin before the challenge with acute hepatotoxicity induced by triploids. According to Esmaeila et al. [10], silymarin inhibits factor-kappaB activation by suppressing inhibitory kappa B (IκB) degradation and suppresses the inflammatory response, oxidative stress. Furthermore, silymarin, by suppressing STAT3 and ERK1/2 signaling pathways, inhibits oncogenesis, cell proliferation, cell migration and iNOS gene expression.

[ IL-6 relative gene expression THE NEW. P<0.0001 1.5 | 1.0 | 0.5 | 0.0 Adsorbent Fix HP Control Control with mycotoxin Adsorbent A Adsorbent B ]

[ IL-10 relative gene expression ANOVA, P<0.0001 1.5 | 1.0 |0.5 | 0.0 Control Control with mycotoxin Adsorbent Fix HP tAbsorbent A Absorbent B ]

Figure 1. Gene expression of IL-6 and IL-10 interleukins in cecal tonsils of broiler chickens challenged with mycotoxins and different adsorbents.

4. Conclusion Supplementation with SIM adsorbent – FIX HP🇧🇷 allowed a greater expression of IL-10, which is possibly related to its composition and resulted from the synergy between its active principles. In addition to the raw materials responsible for the adsorption of the main mycotoxins found in the field, this adsorbent contains known principles of immunomodulatory, antioxidant and anti-inflammatory action, which can contribute to strengthening the health of animals. References 1. Aly, E., Madbouly, Y. (2016). The Effect of Mycotoxins on Immune Response of Broilers to the Live ND Vaccines Applied by Different Routes. Middle East Journal of Applied Sciences, 06 (01), 51-58. 2. Arseniy EY, Anton GK (2014). interleukins in Cancer Biology: Their heterogeneous scroll🇧🇷 Elsevier; London, UK: Academic Press; Cambridge, MA, USA. 3. Bello RO, Chin VK, Abd Rachman Isnadi MF et al. (2018). The Role, Involvement and Function(s) of Interleukin-35 and Interleukin-37 in Disease Pathogenesis. international Journal of Molecular Sciences. 19 (4). 4. Bijak, M. (2017). Silybin, a Major Bioactive Component of Milk Thistle (Silybum marianum L. Gaernt.)—Chemistry, Bioavailability, and Metabolism. molecules, 22, 1-11. Borzecki, P.

5. Borzecka A., Chylinska-Wrzos P., Lis-Sochocka M., Wawryk-Gawda E., Jodlowska-Jedrych. (2019). Evaluation of interleukin-6 concentration in the liver of Albino Swiss mice after intoxication with various doses of patulin. Curr. Issues Pharm. Med. Sci., 32 (1), 34-39. 6. Cho, MJ, Ellebrecht, CT, Payne, AS (2015). The dual nature of interleukin-10 in pemphigus vulgaris. Cytokine, 73 (2), 335-341. 7. De Smedt, T., van Mechelen, M., De Becker, G., Urbain, J., Leo, O. and Moser, M. (1997). Effect of interleukin-10 on dendritic cell maturation and function. European Journal of Immunology. 27, 1229-1235. 8. Duntas, LH (2012). The evolving role of selenium in the treatment of Graves' disease and ophthalmopathy. J thyroid res, 736161. 9. Duke AG, & Descoteaux A. (2014). Macrophage Cytokines: Involvement in Immunity and Infectious Diseases. Front. immunol, 5, 491. 10. Esmaeila N., Anarakib BS, Gharagozlooc M., Moayedia B. (2017). Silymarin impacts on immune system as an immunomodulator: One key for many locks. international Immunopharmacology, 50, 194-201.

11. Hofstetter, U. (2007). The negative effects of various mycotoxins on the immune system of poultry. BIOMIN Newsletter, 5-46. 12. Holland, DM, Kim, SW (2020). Efficacy of Mycotoxin Detoxifiers on Health and Growth of Newly-Weaned Pigs under Chronic Dietary Challenge of Deoxynivalenol. toxins, 12, 311. 13. Iyer, SS, Cheng, G. (2012). Role of Interleukin 10 Transcriptional Regulation in Inflammation and Autoimmune Disease. Crit Rev Immunol. 32(1), 23-63.Khan, AZ 14. Khan, IU, Khan, S. …Ran L. (2019). Selenium-enriched probiotics improve hepatic protection by regulating pro-inflammatory cytokines and antioxidant capacity in broilers under heat stress conditions Journal of Advanced Veterinary and Animal Research. 6 (3), 355-361. 15. Kim, SH, Oh, DS, Oh, J.Y, Son, TG, Yuk, D.Y, Jung, S. (2016). Silymarin prevents restraint stress-induced acute liver injury by ameliorating oxidative stress and reducing inflammatory response. molecules 21,443. 16. Lauwers M, Croubels S, Letor B, Gougoulias C, Devreese M (2019). Biomarkers for exposure as a tool for efficacy testing of a mycotoxin detoxifier in broiler chickens and pigs. toxins, 11, 187. 17. Livak, KJ, Schmittgen, TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta C(T)) Method. Methods, 25 (4), 402-8. 18. Liu Y, Liu Q, Ye G, Khan A, Liu J, Gan F, et al. (2015). Protective effects of Selenium-enriched probiotics on carbon tetrachloride-induced liver fibrosis in rats. J Agricultural Food Chem; 63, 242. 19. Mantovani MS, Bellini MF, Angeli JP, Oliveira RJ, Silva AF, Ribeiro LR (2008). Beta-Glucans in promoting health: prevention against mutation and cancer. MutatRes., 658 (3), 154-61. 20. Moore, KW, Waal Malefyt, R.de, Coffman, RL, & O'Garra, A. (2001). Interleukin-10 and the interleukin-10 receptor. Annual review of immunology, 19, 683-765.

21. Ning X., Jian Z., Wang W. (2015). Low Serum Levels of Interleukin 35 in Patients with Rheumatoid Arthritis. Tohoku J. Exp. Med, 237, 77-82. 22. National Research Council L. Nutrient requirements of poultry. 9 th ed. Washington, DC: National Academy of Science, 1994. 154 p. 23. Qu D., Huang X., Han J., Man N. (2017). Efficacy of mixed adsorbent in ameliorating ochratoxicosis in broilers fed ochratoxin A contaminated diets. Italian J Anim Sci. 16, 573-579. 24. Sattler N., Monroy R., Soldati T. (2013) Quantitative Analysis of Phagocytosis and Phagosome Maturation. In: Eichinger L., Rivero F. (eds) Dictyostelium discoideum Protocols. Methods in Molecular Biology (Methods and Protocols), vol 983. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-302-2_21 25. Shahbazi, F., Dashti-Khavidaki, S., Khalili, H., Lessan-Pezeshki, M., (2012). Potential renoprotective effects of silymarin against nephrotoxic drugs: A review of literature. Journal of Pharmacy and Pharmaceutical Sciences, 15, 112-123. 26. Soltanian, S., Stuyven, E., Cox, E., Sorgeloos, P. and Bossier, P. (2009) Beta-glucans as immunostimulant in vertebrates and invertebrates. Crit. Rev. Microbiol., 35, 109-138. 27. Trinchieri, G. (2007). Interleukin-10 production by effector T cells: Th1 cells show self-control. The Journal of experimental medicine, 204 (2), 239-43. 28. Tzianabos, AO (2000). Polysaccharide immunomodulators as therapeutic agents: structural aspects and biologic function. Clinic. Microbiol., 13, 523-533. 29. Wang L, Huang QH, Li YX, et al. Protective effects of silymarin on triptolide-induced acute hepatotoxicity in rats. Mol Med Rep. 2018; 17(1): 789-800. doi: 10.3892/mmr.2017.7958. 30. Yiannikouris A., Francois J., Poughon L., Dussap CG, Bertin G., Jeminet G., Jouany JP (2004). Adsorption of zearalenone by beta-D-glucans in the Saccharomyces cerevisiae cell wall. J. FoodProt. 67, 1195-1200. 31. Zabriskie, Hannah A., Julia C. Blumkaitis, Jessica M. Moon, Brad S. Currier, Riley Stefan, Kayla Ratliff, and Chad M. Kerksick (2020). Yeast Beta-Glucan Supplementation Downregulates Markers of Systemic Inflammation after Heated Treadmill Exercise. Nutrients, 12 (4), 1144.