Mycotoxin binder for immune and intestinal histopathology ameliorations against Newcastle disease in vaccinated broilers

Introduction

The presence of immunosuppressant substances, such as mycotoxins including aflatoxins B1 (AFB1) and ochratoxins A (OTA),¹ and in tandem are the mycotoxin combination that has been shown to have high toxicity.² Which can decrease the body’s immune response to vaccination, may be the reason for antibody titer findings that indicate no protection against vaccination.³ Immunosuppressive disorders result from T cell proliferation failure carried on by immunotoxic processes, which prevents the body from responding to the incoming vaccination.^4,5 In the short term, OTA and AFB1 interacting to contaminate chicken feed will provide a synergistic negative effect.⁶ The digestive system has the highest risk of exposure since animal feed is the first point in the food chain where mix mycotoxin can enter directly through plant goods like grains.⁷ The process of digesting and absorbing dietary fibre in chickens is significantly aided by the large intestine.^8,9

Ochratoxin A profoundly alters the microbiota’s composition and metabolism in the large intestine, which has an impact on the process of fermentative digestion that occurs there.¹⁰ Aflatoxin B1 plays a significant role in the production of reactive oxygen species (ROS), which damage DNA and cause lesions in the mitochondria.^11,12 Damage to the membranes and nuclear structures of cells can result in necrosis or death of the cells.¹³ Mycotoxicosis is a poisoning that happens when an animal eats feed that is too toxic for their body to tolerate. When mix mycotoxin is exposed to the large intestine, it first disrupts the synthesis and breakdown of proteins, which alters the integrity of the intestine and damages the intestinal mucosa. Mix mycotoxin exposure will eventually cause oxidative damage. Large volumes of reactive oxygen species (ROS) are released that can injuring tissue and inhibit tissue repair, ultimately leading to cell necrosis.¹⁴ In the gastrointestinal system, mycotoxin binders generally act as adsorbents. One form of adsorbent that has been extensively researched in vivo and in vitro that is effective at removing mycotoxins from the body is bentonite (Dioctahedral montmorillonite). Because the fluid in the gastrointestinal system has a negative charge on its surface, bentonite will be distributed there and utilise cation exchange processes to absorb different biomolecules, including aflatoxin.¹⁵ Mycotoxin binder is a big molecular molecule that resembles a chemical sponge and is used to bind mixtures of mycotoxin in an animal’s digestive tract before it is expelled through its faeces. Mycotoxin binders have the ability to reduce the amount of mix mycotoxin in feed, which in turn lessens the amount of mix mycotoxin that penetrates the bloodstream and other bodily organs and causes mycotoxicosis.¹⁶ Mycotoxin binders in this research, also known as biotransforming agents, with different function in that they alter the chemical makeup of mycotoxins to be non-toxic molecules. This biotransforming agent was classified as yeast (Trichosporon mycotoxinivorans). As a biotransforming agent, the aforementioned yeast can hydrolise amide bonds in the chemical structure of OTA to break it down into new chemical and non-toxic molecules.¹⁷ Vaccination against Newcastle disease (ND) in broiler farming is essential. Nonetheless, during the post-vaccination phase, production may be negatively impacted by mycotoxin contamination in feed.

This study aimed to evaluate the effect of mycotoxin binders on immune and intestinal histopathology ameliorations against Newcastle disease in vaccinated broilers with aflatoxin B1 (AFB1) and ochratoxin A (OTA) toxication.

Methods

Results

Hemagglutination inhibition (HI) test

Results in this research have increased in the mean antibody titer observed on day 35 compared to days 14 and 28, with significant changes observed in serum samples based on the C+ group, which was significantly different from the C- and T2 groups (p < 0.05).

Based on hemagglutination inhibition (HI) test, as a result, erythrocyte sediment at the bottom of the microplate without a hemagglutination process results in a positive HI test; conversely, hemagglutination in the form of a diffused erythrocyte layer at the bottom of the microplate without an erythrocyte sediment results in a negative outcome. Based on Figure 1, presents the HI test, (A) depicts the absence of hemagglutination, which is defined as erythrocyte sedimentation when the microplate is tilted, and (B) illustrates the occurrence of hemagglutination, which is defined as erythrocyte absence at the microplate well’s bottom. The maximum serum dilution that could prevent hemagglutination against antigens was also used to calculate the HI titer.

Figure 1. Hemagglutination inhibition test on microplate, with (A) positive HI test, and (B) negative HI test.

There were no discernible changes between groups C-, C+, T1, and T2 when the antibody titer of broiler chickens was examined at 14 days of age, or 7 days after the initial ND vaccination. Analysis by average revealed that all treatment groups’ antibody titers seemed to be moderate and did not yet exceed ND-protective titers (≥7 HI log 2).

On day 35, broiler antibody titers varied (p = 0.010) between groups, indicating that mycotoxin binder may have an impact on the development of ND antibody titers. According to the results of the Duncan test, there were no significant differences between the treatment groups C- with T1, C- with T2, C+ with T1, and T1 with T2, but the C+ with C- and C+ with T2 groups were significantly different, as shown by different letter superscripts in the same column ( Table 1). On the other hand, compared to the average antibody titer of chickens at 14 and 28 days of age, the antibody titer of broilers at 35 days of age increased significantly. The immunological response to the vaccine has only recently started to emerge; the immune system is still in the stage of recognizing the first entered antigen, which accounts for the low mean of antibody titer in 14-day-old broilers (7 days after the initial ND vaccination). The main immune response is the body’s immunological reaction to antigens that initially enter the body.²²

Table 1. Antibody titers in 14, 28, and 35-day-old broilers.

Treatment group	Replication	Antibody Titers (log 2) (Mean ± SD)
		14th Day	28th Day	35th Day
C-	5	3.2a ± 0.83	4.2a ± 0.44	7.4b ± 0.89
C+	5	2.8a ± 1.09	3.6a ± 2.07	5.8a ± 0.83
T1	5	2.8a ± 0.44	4.4a ± 1.14	7.2ab ± 1.48
T2	5	3.2a ± 1.30	4.0a ± 0.70	8.6b ± 1.14

Histopathological evaluation

Histopathological improvement of the cecum and colon was reported based on reduced mucosal rupture, hemorrhage and necrosis on day 35.

The findings of the scoring analysis of the broiler chickens’ mucosal rupture, necrosis, and hemorrhage in the cecum ( Table 2) and colon ( Table 3). The control group (C-), had the lowest mean rank. The C+ treatment group, had the highest mean rank. The T1 treatment group and Treatment group T2 as indicated in Table 2. Furthermore, the T2 group demonstrated a progressive improvement in the histological differences related to mucosal rupture, hemorrhage, and necrosis in the cecum organ ( Figures 2–4).

Table 2. Histopathological evaluation of the cecum at the end of the study.

Treatment group	Parameters (Mean rank)
	Mucosal rupture	Hemorrhage	Necrosis
C-	3.8c	4.7c	5.0c
C+	17.9a	17.6a	17.5a
T1	13.1b	13.1b	12.9b
T2	7.2c	6.6c	6.6c

Table 3. Histopathological evaluation of the colon at the end of the study.

Treatment group	Parameters (Mean rank)
	Mucosal rupture	Hemorrhage	Necrosis
C-	4.5c	5.0c	4.2c
C+	17.9a	18.0a	17.3a
T1	12.8b	12.8b	13.2b
T2	6.8c	6.2c	7.3c

Figure 2. Histopathology of the cecum mucosal rupture (➔) using 100× and 400× magnification with HE staining.

Figure 3. Histopathology of the cecum hemorrhage (yellow area) using 100× and 400× magnification with HE staining.

Figure 4. Histopathology of the cecum necrosis (➔) using 100× and 400× magnification with HE staining.

The control group, treatment group C-, had the lowest mean rank. Treatment group C+, had the highest mean rank. The T1 treatment group. Table 3 shows that the T2 treatment group demonstrated a progressive improvement in the histological differences of mucosal rupture, haemorrhage, and necrosis in the colon organ ( Figures 5–7).

Figure 5. Histopathology of the colon mucosal rupture (➔) using 100× and 400× magnification with HE staining.

Figure 6. Histopathology of the colon hemorrhage (yellow area) using 100× and 400× magnification with HE staining.

Figure 7. Histopathology of the colon necrosis (➔) using 100× and 400× magnification with HE staining.

Discussion

The primary immune responses that initiated by cell-mediated immunity (CMI) often follow a more gradual course than secondary immunological responses. T cells that serve the role of CMI will develop into effector T cells, or T helper (CD4+) and T cytotoxic (CD8+). Both CD8+ and CD4+ go through three phases in their response to antigens: the expansion phase, the death phase, and the memory phase.²³ Seven days after the antigen (organism) enters the body, the organism enters the growth phase, which is followed by the death phase, in which homeostatic processes cause the death of over 90% of active T cells.²⁴

The investigation of antibody titer evaluation in this research performed seven days after booster vaccination in 28-day-old broilers following ND vaccination revealed no significant difference between the C-, C+, T1, and T2 groups. In contrast to the antibody titer at 14 days of age, the average antibody titer in the C-, C+, T1, and T2 groups increased. Immunoglobulin or antibody levels tend to be low seven days after the first vaccination when the HI test is conducted because of this mechanism, which prevents IL-2 produced by CD4+ and CD8+ from signaling B cells to perform maximum cell differentiation. In order to remember and identify antigens to contribute to the secondary immune response during booster immunization, the remaining active T cells will go into the memory phase.²⁵

The development of a secondary immune response could be the cause of the increase in antibody titer at 28 days of age. Booster vaccinations containing the same antigen can accelerate the proliferation and differentiation of B cells by activating memory cells produced from the main immune response.²⁶

In all treatment groups, the average antibody titer appears to have increased, but it is still below protective levels (≥ 7 HI log 2). Because the body’s production of antibodies against the inactive vaccine has not yet reached its maximal titer, this is conceivable. According to a prior study, because inactive vaccines contain an oil adjuvant, vaccination with them will result in the highest antibody titer occurring 14–21 days after the vaccination.²³ In order to improve the immune response, adjuvants are substances that are added to vaccines. By slowing down the body’s process of destroying antigens, adjuvants prolong the time that the vaccination comes into touch with macrophages and lymphocytes, which delays the vaccine’s ability to induce the development of antibodies.²⁷

At 35 days of age, the analysis of antibody titers revealed significant differences (p < 0.05) between the C+ group with C- and C+ with T2. In contrast, there was no significant difference between the C- and T1 groups, C- and T2 groups, C+ and T1 groups, and T1 and T2 groups. The broiler group with the lowest average antibody titer among the other treatment groups is C+, which was exposed to AFB1 and OTA without the use of any additional mycotoxin binder agents. According to a prior study, immunotoxic diseases might arise in the body as a result of mycotoxin exposure.²⁸ The primary immune cell apoptotic pathways, autophagy, and oxidative stress will all play a role in the immunotoxic mechanism. These cells include B lymphocytes, dendritic cells, macrophages, neutrophils, and T cells. Moreover, the body experiences immunosuppression, autoimmunity, immunological dysfunction, and hypersensitivity reactions as a result of mycotoxin exposure.²⁹

The impact of chronic mycotoxicosis conditions linked to weight loss, cancer, immunosuppression, and pathological conditions with other slow onset times is known as immunosuppression. In this study, the effect of immunosuppression resulting from mycotoxin exposure on post-vaccination antibody titers has not been observed significantly at 14 and 28 days of age examination. The type, duration, dose, age, and species of the mycotoxin all affect the intensity and symptoms of exposure. If mycotoxins are given to chickens for longer than three to four weeks, the result is chronic mycotoxicosis.^30,31

The analysis of antibody titers at 35 days of age revealed significantly different findings in group C+ with T2 (p < 0.05) in addition to group C+ with C-. T2 is a group of broilers given commercial feed that has been exposed to AFB1 and OTA as well as a mycotoxin binder agent at a dose of 1.6 g/kg feed. In order to prevent the toxicity of mycotoxins from spreading throughout the body, mycotoxin binder agents as feed additives typically function by lowering the bioavailability of mycotoxins while they are still in the gastrointestinal tract. Mycotoxin binders, such as bentonite clay, and mycotoxin modifiers, such as Trichosporon mycotoxinivorans yeast, are two key components found in mycotoxin binder agent products. When it comes to removing mycotoxin exposure from the body, the two of them collaborate well.³²

As an enterosorbent, bentonite can bind polar mycotoxins like AFB1 in a certain way. It disperses into the gastrointestinal tract’s fluid, becoming negatively charged. This negative charge can then absorb aflatoxins through cation exchange reactions or positive charges from AFB1 dicarbonyl bonds.³³ The pH, binder content, mycotoxin kind and dosage, and incubation duration are just a few of the numerous variables that impact an adsorbent’s capacity to interact with mycotoxins.^34,35 Unlike bentonite, the yeast Trichosporon mycotoxinivorans has the capacity to decrease the bioavailability of ochratoxin A (OTA) by the hydrolysis of the amide bond structure that links the hydro-isocoumarin ring and the phenylalanine group. Two less hazardous chemicals, L-β-phenylalanine and α-ochratoxin (OTα), will be produced if the amide link is effectively hydrolysed.^36,37

Using mycotoxin binder chemicals as feed additives to bind mix mycotoxin can help prevent uncontrollably contaminated mycotoxin mixes. Using mycotoxin binders, the mix of mycotoxin is detoxified according to its specific features. In this work, the polar toxin known as aflatoxin B1 is detoxified using bentonite, an adsorbent that will boost enzyme activity and prevent exposure to the toxin in the digestive system. Mycotoxin binders function by biotransformation, which is the process of converting mixed mycotoxin into non-toxic metabolites, as opposed to ochratoxin A, which is non-polar.^37,38

Mycotoxin binder is an option that can be utilised as a feed additive in the large intestine, with an ideal dosage of 1.6 g/kg feed. The integrity of the intestinal mucosa will be impacted if the toxicity of mix mycotoxin declines. It will result in fewer goblet cells in the large intestine, which will make the intestinal mucosa less susceptible to injury and mucosal rupture.¹⁰

In an attempt to detoxify the toxins created by mixed mycotoxin and lessen their toxicity, a mycotoxin binder is utilized, with an ideal dosage of 1.6 g/kg feed. Aflatoxin B1 will be adsorbed by the mycotoxin binder, but ochratoxin A will be biotransformed. By detoxifying the poison created by mixing mycotoxin, a mycotoxin binder will lower the amount of toxin generated. Lower toxin levels will undoubtedly have an impact on endothelial damage brought on by exposure to mixed mycotoxin; as endothelial damage declines, the endothelium’s role as a mediator of fluid or plasma release that aids in hemostasis will rise.^10,12

As a mycotoxin deactivator for Aflatoxin B1 and Ochratoxin A, mycotoxin binder, with an ideal dosage of 1.6 g/kg feed, is one of the things that can be done. The varied target mycotoxin will determine how the mycotoxin binder functions. Because it has a polar chemical structure and a strong, stable specific bond that will deactivate it, aflatoxin B1 functions as a mycotoxin binder by adsorption. When employing common adsorbents like aluminum silicate, which break down and expand when combined with water, this cannot occur. At pH 3, zeolite will also no longer have its adsorption qualities. The mycotoxin binder in Ochratoxin A, on the other hand, will work as an enzymatic breakdown with biotransformation to break down the mycotoxin into non-toxic metabolites. The combination mycotoxin won’t have a harmful impact, which causes oxidative stress in the form of necrosis or cell death if the toxin exposure has been eliminated.^12,21

Conclusions

The study’s findings revealed that providing a mycotoxin binder agent to broilers exposed to AFB1 and OTA at a dose of 1.6 g/kg influences the development of antibody titers following ND vaccination and also guards against rupture, hemorrhage, and necrosis in cecum and colon.

Author contributions

Erma Safitri, Siti Darodjah Rasad, Goo Jang, and Mitsuhiro Takagi: Supervision, Research Conceptualization, Methodology and Research Observation, Writing—Original Draft Preparation; Hery Purnobasuki, Tita Damayanti Lestari, Suzanita Utama, Eka Pramyrtha Hestianah, Mohammad Anam Al Arif, Chairul Anwar Nidom, Sri Mulyati, Sri Hidanah, and Jola Rahmahani: Curation for Data, Analysis and Formal Investigation, Resources of References, Visualization, Writing—Review and Editing; Merisa Wahyu Erdhina and Maulida Ilma Sadida: Curation for Data, Software and Validation; Martia Rani Tacharina and Muhammad Thohawi Elziyad Purnama: Curation for Data, Proof Read, Review and Editing.