Background

F1000Research

2046-1402

F1000 Research Limited

London, UK

10.12688/f1000research.158103.2

Research Article

Articles

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

[version 2; peer review: 4 approved with reservations, 1 not approved]

Safitri

Erma

Conceptualization Methodology Supervision Writing – Original Draft Preparation a 1 Purnobasuki

Hery

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 2 Lestari

Tita Damayanti

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 1 Utama

Suzanita

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 1 Erdhina

Merisa Wahyu

Data Curation Software Validation 3 Sadida

Maulida Ilma

Data Curation Software Validation 3 Hestianah

Eka Pramyrtha

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 4 Al Arif

Mohammad Anam

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 5 Nidom

Chairul Anwar

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 6 Mulyati

Sri

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 1 Rahmahani

Jola

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 7 Tacharina

Martia Rani

Data Curation Writing – Review & Editing 7 Hidanah

Sri

Data Curation Formal Analysis Investigation Resources Visualization Writing – Review & Editing 5 Rasad

Siti Darodjah

Conceptualization Methodology Supervision Writing – Original Draft Preparation 8 Jang

Goo

Conceptualization Methodology Supervision Writing – Original Draft Preparation 9 Takagi

Mitsuhiro

Conceptualization Methodology Supervision Writing – Original Draft Preparation 10 Purnama

Muhammad Thohawi Elziyad

Data Curation Writing – Review & Editing https://orcid.org/0000-0002-9496-0330 4 11 1Department of Veterinary Science, Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, 60115, Indonesia 2Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya, East Java, 60115, Indonesia 3Undergraduate Student, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, 60115, Indonesia 4Department of Veterinary Science, Division of Veterinary Anatomy, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, 60115, Indonesia 5Department of Veterinary Science, Division of Animal Husbandry, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, 60115, Indonesia 6Department of Veterinary Science, Division of Basic Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, 60115, Indonesia 7Department of Veterinary Science, Division of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, 60115, Indonesia 8Department of Animal Production, Faculty of Animal Science, Universitas Padjadjaran, Sumedang, West Java, 45363, Indonesia 9Department of Theriogenology, College of Veterinary Medicine, Seoul National University, Gwanak-gu, Seoul, 151-742, South Korea 10Laboratory of Theriogenology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Yamaguchi Prefecture, 753-8515, Japan 11Department of Biology, Graduate School of Natural and Applied Sciences, Eskisehir Osmangazi Universitesi, Eskişehir, Eskişehir Province, 26040, Turkey

a erma-s@fkh.unair.ac.id

No competing interests were disclosed.

27 1 2025

2024

1518

13 1 2025

2025

This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

In broiler farming, vaccination against Newcastle disease (ND) 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

A total of 20 broilers were randomly assigned into 4 groups with 5 replications i.e. (C-) broiler groups with basal feed, (C+) broiler groups with AFB1 and OTA feed contamination, (T1) and (T2) broiler groups with exposed AFB1, OTA, and toxin binders as feed additives with dose 1.1 g/kg and 1.6 g/kg feed, respectively. ND vaccination was carried out on day 7 and 21. Antibody titers were evaluated from serum samples of broiler on days 14, 28, and 35 for further hemagglutination inhibition (HI) test. Histopathology of the cecum and colon organs was evaluated using HE staining on day 36. HI test and histological scoring were analyzed using the One-Way ANOVA, followed by Duncan’s test with a p < 0.05 in SPSS v.26 software (IBM Corp., Armonk, NY).

Results

As a result, histopathological improvement of the cecum and colon was reported based on mucosal rupture, hemorrhage and necrosis on day 35. An increase in the mean antibody titer compared to days 14 and 28 was observed on day 35, 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).

Conclusions

This study revealed that a 1.6 g/kg toxin binder dose in feed can increase antibody titer and histopathology of cecum and colon in broiler chickens after ND vaccination fed with mycotoxin-contaminated feed.

aflatoxin ochratoxin antibody titer broiler toxin binders food production

This study was supported by funding from the Airlangga Research Fund 2023

No.254/UN3/2023datedFebruary23

2023

This study was supported by funding from the Airlangga Research Fund 2023 with the decree of the Rector of Universitas Airlangga No. 254/UN3/2023 dated February 23, 2023.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Revised Amendments from Version 1

For this version, we have considered and addressed all reviewer comments that can improve our manuscript. Amendments have been made by modifying the title according to reviewer recommendations; improving readability and understanding of the abstract; sorting keywords; rearranging the introduction to be more systematic and flowing; clarifying broiler chicken acclimatization methods, carcass handling, sample evaluation and statistics in the methods section; revising symbols and illustrations, p-value inclusion, data interpretation, and understanding in the results section; improving and adding references according to reviewer recommendations in the discussion section; slightly revising the conclusion; and arranging the order of references.

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 Ethics statement

This study was approved by the Ethical Committee of Animal Care and Use, Faculty of Veterinary Medicine, Universitas Airlangga (No. 1.KEH.033.02.2023) on February 10, 2023. This study was also reported according to the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines 2.0: author checklist. ⁴⁰ All efforts were made to ameliorate any suffering of animals.

Study period and location

All experiments in this study were conducted from February 27 to June 10, 2023 in an animal laboratory. Antibody titer evaluation was conducted in the microbiology laboratory, while histopathology evaluation was conducted in the pathology laboratory, Faculty of Veterinary Medicine, Universitas Airlangga.

Experimental animals

The five steps of preparation before acclimatization of day-old chicks (DOC) are as follows: 1. Clean and remove all physical materials from a chicken cage; 2. Wash and clean all equipment and the cage with soap and water, using scraping; 3. Spray liquid disinfectant on all equipment and the cage and close the cage for a week; 4. Spray solid disinfectant (lime) and mix it with water in the cage and close the cage for a week; 5. Fumigate with permanganate and formalin, close the cage, as this can irritate; and finally, wait a week before DOC arrives.

A total of 20 DOC broiler chickens Cobb strain were reared for 35 days as final stock following broiler rearing standards. Rearing standards for broiler experiments include: basic ventilation system (open house), rodent and flyproof, cleaning and disinfection protocol (five steps before DOC arrived), ambient temperature (18 to 24°C), relative humidity (50 to 70%), manual feeding system, watering system (manual, ad libitum, and groundwater), lighting program (light color at 20 lux intensity for the first 7 days and 17-20 hours duration), and lighting program. During rearing, the chickens were fed with a standard formulation for starter-stage broilers with the brand Hipprovite CP511 (Cat No. CP511-511, PT. Charoen Pokphand, Indonesia) and provided drinking water ad libitum. They were assigned into 4 treatment groups and 5 replications, i.e. (C-) was fed basal feed, (C+) was fed with AFB1 and OTA contamination, (T1) and (T2) were fed with AFB1 and OTA contamination and toxin binders (Mycofix ^®, Cat No. 20026289-FTS, PT. Biomin Indonesia) as feed additives with doses 1.1 g/kg and 1.6 g/kg feed, respectively.

ND vaccination was performed on seven-day-old broilers using La Sota inactivated ND vaccine (Medivac ^®, Cat No. 3526004001, PT. Medion Farma, Indonesia) via eye drops at a dose of 1 drop/head. Then 21-day-old broilers were booster vaccinated using the inactive ND Medivac ^® vaccine at a dose of 0.5 mL/head intramuscularly. The antibody titers were evaluated from serum samples on days 14, 28, and 35 for the hemagglutination inhibition (HI) test. The cecum and colon organs were collected on day 36 to evaluate histopathology using hematoxylin-eosin (HE) staining.

Hemagglutination (HA) and hemagglutination inhibition (HI) tests

Erythrocytes suspension was collected from ND virus antibody-negative donor chickens. Blood was drawn from the brachial vein and collected in tubes containing EDTA solution (BD Vacutainer ^® EDTA Tubes, Cat No. 366643, USA). The blood was washed thrice using PBS by centrifuging at 1.500 g for 10 minutes. The buffy coat and plasma fluid were discarded at each washing. The erythrocyte residue was then diluted with PBS to a concentration of 0.5%.

A total of 1 mL blood samples without anti-coagulant were collected through the brachial vein in 20 broiler chickens aged 14, 28, and 35 days. Blood was then allowed until collected serum and transferred into an Ependorf tube. The HA test was performed to determine the antigen titer that will be used for the HI test. This test begins with filling the microplate wells (A1–A12) with 25 μl of PBS. Furthermore, 25 μl of antigen was inserted into the A1 and A12 wells (antigen control). The antigen and PBS in the A1 well were then homogenized with the suction-blow technique using a 25 μl micropipette, then 25 μl of liquid was transferred from one well to the next well, and so on until it reached the A11 well. The remaining 25 μl in the micropipette was discarded. Furthermore, all A1–A12 wells were filled with 50 μl of 0.5% chicken erythrocyte suspension. The microplate was then shaken and incubated at room temperature for 30 minutes, and the titer was ready to evaluate.

The microplate was filled with 25 μl of PBS solution into number 1–5 wells in rows A and B (duplicate titration) using a micropipette. Then, the first well in rows A and B was filled with 25 μl of ND antigen. Antigen and PBS in 1 well were then homogenized by the suction-blowing technique using a 25 μl micropipette, then 25 μl of liquid was transferred from one well to the next well, and so on until reaching the 4 wells. Furthermore, all wells were filled with 50 μl of 0.5% chicken erythrocyte suspension. The microplate was then shaken and incubated at room temperature for 30 minutes, and the titer was ready to interpret.

The first until the twelfth microplate wells were filled with 25 μl of PBS using a micropipette. Then, the first and twelfth wells are filled with 25 μl of serum to be checked for titer. Serum and PBS were mixed in the first well by the suction-blowing technique using a micropipette, then 25 μl of liquid was transferred from the first well to the next well, and so on until the tenth well. Next, wells 1–10 were filled with 25 μl of ND antigen. The microplate was then shaken and incubated for 30 minutes at room temperature. Next, all wells were filled with 50 μl of 0.5% chicken erythrocyte suspension and incubated for 30 minutes for further evaluation.

Euthanasia in animals

On the 36th day, the cervical dislocation procedure was used to perform euthanasia. ¹⁸ All efforts were made to ameliorate any suffering of animals. In the event that the study’s chickens displayed signs of illness prior to the termination time, they received vitamin supplements to enhance their health and were treated in accordance with the disease’s symptoms. Nevertheless, the deceased chicken was taken out and insinerated if it died before the termination time.

The procedure of manual cervical dislocation (CD) involves twisting and stretching the neck with the hands. After being picked up, the chickens were fed to help them relax. As we gradually repositioned our hold, we waited for the chickens to calm down. The non-dominant hand was used to grasp the chicken legs. We slowly turned the chicken so that its feet were pointing up at our chest. To keep them motionless, the chicken feet were securely held close to the base of their rear end. Grasping the upper part of the chicken’s neck, where the soft, spongy vertebrae meet the brain stem, fingers were wrapped around them. Gently, the thumb and index finger were encircling this section of the chicken’s neck. After that, the neck of the fowl was angled 90 degrees downward. The chicken’s body was gradually moved to position its head away from the dominant side. Their feet were securely grasped while the head and neck of the chicken were arranged in an L configuration, the beak pointing downward. The twisting motion dislocates the vertebral column from the skull and ruptures or stretches the blood vessels, while the stretching action tears the neck muscles, ligaments, connective tissues, and blood vessels. The cessation of the pupillary light reflex and nictitating membrane reflex were death indicators in broiler chickens. ^{18,
19} The process of incineration involves burning carcasses or residues at temperatures as high as 850 °C to create an inorganic ash. All infectious agents are anticipated to be destroyed by the treatment. Ash usually makes about 1–5% of the initial volume of the carcass, though this will vary depending on the type of incinerator, the method, the fuel, and the species of animal. ²⁰

Histopathological evaluation

Sample collection was performed through abdominal dissection after euthanization. After the cecum and colon from 20 broilers were collected, the organs were fixed in pot containers using a 10% formalin solution to preserve histopathological preparations using hematoxylin-eosin (HE) staining. The histopathological evaluation was performed using a microscope (Nikon Eclipse E200, Japan) in five fields of view to investigate rupture, hemorrhage, and necrosis. The scoring method used the following criteria, (0) no change, (1) <25% pathological findings, (2) 26–50% pathological findings, (3) 51–75% pathological findings, and (4) >76% pathological findings. ²¹

Statistical analysis

All data were tabulated and then tested for normality using the Shapiro Wilk test. Normally distributed data were followed by One-Way analysis of variance (ANOVA) and Duncan’s multiple comparison post hoc test. All analyses were evaluated using SPSS v.26 for Windows (IBM Corp., Armonk, NY) with decision considerations using a 95% confidence level.

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

^a,bDifferent superscripts in the same column indicate significant differences (p < 0.05).

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

^a,b,cDifferent superscripts in the same column indicate significant differences (p < 0.05).

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

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

^a,b,cDifferent superscripts in the same column indicate significant differences (p < 0.05).

Figure 2. Histopathology of the cecum mucosal rupture ( <styled-content style="#00B050">➔</styled-content>) 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 ( <styled-content style="#00B0F0">➔</styled-content>) 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 ( <styled-content style="#00B050">➔</styled-content>) 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 ( <styled-content style="#00B0F0">➔</styled-content>) 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.

Data availability Underlying data

Figshare: Database of hemagglutination inhibition test data, histopathology scoring of cecum and colon, and original histopathology figures. https://doi.org/10.6084/m9.figshare.27248592.v2. ³⁹

The extended data set contains the following data: •

Antibody titer

•

Cecum histopathology scoring

•

Colon histopathology scoring

•

Histopathology figure of cecum

•

Histopathology figure of colon

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Reporting guidelines

Figshare: ARRIVE Guidelines 2.0 for Mycotoxin binders study. https://doi.org/10.6084/m9.figshare.27900534.v1. ⁴⁰

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Software availability

To analyse the quantitative data, SPSS version 26 (IBM Corp., Armonk, NY) was used. If readers are looking for an open-source substitute, JASP ( https://jasp-stats.org/) can be used to conduct comparable analyses.

Acknowledgments

The authors express their profound gratitude to Dr. Gadis Meinar Sari, dr., M. Kes, Chairman of the Institute for Research and Community Service, Universitas Airlangga, and Prof. Dr. Mirni Lamid, drh., M.P., the Dean of the Faculty of Veterinary Medicine, Universitas Airlangga Surabaya for providing the research facilities.

References 1

Samik

Safitri

: Mycotoxin binders potential on histological of ovary mice exposed by zearalenone. Vet. World. 2017;10(3):353–357. 28435200

10.14202/vetworld.2017.353-357

PMC5387665

Streit

Naehrer

Rodrigues

: Mycotoxin occurrence in feed and feed raw materials worldwide: long-term analysis with special focus on Europe and Asia. J. Sci. Food Agric. 2013;93(12):2892–2899. 23670211

10.1002/jsfa.6225

Hou

Gan

Zhou

: Immunotoxicity of ochratoxin A and aflatoxin B1 in combination is associated with the nuclear factor kappa B signaling pathway in 3D4/21 cells. Chemosphere. 2018;199:718–727. 29475160

10.1016/j.chemosphere.2018.02.009

Jalil

Samad

Islam

: Evaluation of maternally derived antibodies against Newcastle disease virus and its effect on vaccination in broiler chicks. Bangladesh J. Vet. Med. 2009;7:296–302. 10.3329/bjvm.v7i2.5995

Safitri

Purnobasuki

: Effectiveness of mesenchymal stem cells cultured under hypoxia to increase the fertility rate in rats ( Rattus norvegicus). Vet. World. 2021;14(11):3056–3064. 35017856

10.14202/vetworld.2021.3056-3064

PMC8743767

Murugesan

Ledoux

Naehrer

: Prevalence and effects of mycotoxins on poultry health and performance, and recent development in mycotoxin counteracting strategies. Poult. Sci. 2015;94(6):1298–1315. 25840963

10.3382/ps/pev075

PMC4988553

Campanella

Sorino

Bonfiglio

: Effects of weight change on all causes, digestive system and other causes mortality in southern Italy: A competing risk approach. Int. J. Obes. 2022;46(1):113–120. 34522001

10.1038/s41366-021-00954-8

Singh

Kim

: Effects of dietary fiber on nutrients utilization and gut health of poultry: a review of challenges and opportunities. Animals. 2021;11(1):181. 33466662

10.3390/ani11010181

PMC7828824

Mahmood

Guo

: Dietary fiber and chicken microbiome interaction: Where will it lead to? Anim. Nutr. 2020;6(1):1–8. 32211522

10.1016/j.aninu.2019.11.004

PMC7082689

Broom

: Mycotoxins and the intestine. Anim. Nutr. 2015;1(4):262–265. 29766989

10.1016/j.aninu.2015.11.001

PMC5940982

Wang

Chen

Zhai

: Ochratoxin A-induced apoptosis of IPEC-J2 cells through ROS-mediated mitochondrial permeability transition pore opening pathway. J. Agric. Food Chem. 2017;65(48):10630–10637. 29136370

10.1021/acs.jafc.7b04434

Liu

Wang

: Aflatoxin B1 impairs mitochondrial functions, activates ROS generation, induces apoptosis and involves Nrf2 signal pathway in primary broiler hepatocytes. Anim. Sci. J. 2016;87(12):1490–1500. 26997555

10.1111/asj.12550

McGavin

Zachary

: Pathologic basis of veterinary disease. Elsevier Health Sciences;2006;45.

Theumer

Cánepa

López

: Subchronic mycotoxicoses in Wistar rats: assessment of the in vivo and in vitro genotoxicity induced by fumonisins and aflatoxin B1, and oxidative stress biomarkers status. Toxicol. 2010;268(1–2):104–110. 20015463

10.1016/j.tox.2009.12.007

Devreese

De Backer

Croubels

: Different methods to counteract mycotoxin production and its impact on animal health. Vlaams Diergeneeskundig Tijdschrift. 2013;82(4). 10.21825/vdt.v82i4.16695

Alam

Nisa

Daud

: Mycotoxins in environment and its health implications. Hazardous Environmental Micro-pollutants, Health Impacts and Allied Treatment Technologies. Cham: Springer International Publishing;2022; pp.289–318. 10.1007/978-3-030-96523-5_12

Anilkumar

Sathyanarayana

Vijayasarathi

: Evaluation of ameliorating effect of vitamin E and selenium in experimental aflatoxicosis and ochratoxicosis in broiler chicken. Indian J. Anim. Sci. 2009;79(8).

Martin

McKeegan

Sparrey

: Comparison of novel mechanical cervical dislocation and a modified captive bolt for on-farm killing of poultry on behavioural reflex responses and anatomical pathology. Anim. Welf. 2016;25(2):227–241. 10.7120/09627286.25.2.227

Bandara

Torrey

Turner

: Efficacy of a novel mechanical cervical dislocation device in comparison to manual cervical dislocation in layer chickens. Anim. 2019;9(7):407. 10.3390/ani9070407

Gwyther

Williams

Golyshin

: The environmental and biosecurity characteristics of livestock carcass disposal methods: A review. Waste Manag. 2011;31(4):767–778. 21216585

10.1016/j.wasman.2010.12.005

Song

: Effect of microencapsulated sodium butyrate dietary supplementation on growth performance and intestinal barrier function of broiler chickens infected with necrotic enteritis. Anim. Feed Sci. Technol. 2017;232:6–15. 10.1016/j.anifeedsci.2017.07.009

Rozi

Rahmahani

Purnama

MTE

: Seroprevalensi Antibodi Newcastle Disease (ND) pada Itik di Desa Temuasri, Sempu, Banyuwangi. J. Med. Vet. 2020;3(1):108–113. 10.20473/jmv.vol3.iss1.2020.108-113

Lewis

: Cell cycle entry control in naïve and memory CD8+ T cells. Front. Cell Dev. Biol. 2021;9:727441. 34692683

10.3389/fcell.2021.727441

PMC8526999

Derksen

Tesselaar

Borghans

: Memories that last: Dynamics of memory T cells throughout the body. Immunol. Rev. 2023;316(1):38–51. 37114435

10.1111/imr.13211

Bugya

Prechl

Szénási

: Multiple levels of immunological memory and their association with vaccination. Vaccines. 2021;9(2):174. 33669597

10.3390/vaccines9020174

PMC7922266

Luo

Yin

: B cell response to vaccination. Immunol. Investig. 2021;50(7):780–801. 10.1080/08820139.2021.1903033

El-Sayed

Kamel

: Advances in nanomedical applications: diagnostic, therapeutic, immunization, and vaccine production. Environ. Sci. Pollut. Res. 2020;27(16):19200–19213. 31529348

10.1007/s11356-019-06459-2

Tesfamariam

De Boevre

Kolsteren

: Dietary mycotoxins exposure and child growth, immune system, morbidity, and mortality: A systematic literature review. Crit. Rev. Food Sci. Nutr. 2020;60(19):3321–3341. 31694387

10.1080/10408398.2019.1685455

Kraft

Buchenauer

Polte

: Mold, mycotoxins and a dysregulated immune system: a combination of concern? Int. J. Mol. Sci. 2021;22(22):12269. 34830149

10.3390/ijms222212269

PMC8619365

Wang

Hogan

: Performance effects of feed-borne Fusarium mycotoxins on broiler chickens: Influences of timing and duration of exposure. Anim. Nutr. 2019;5(1):32–40. 30899807

10.1016/j.aninu.2018.08.001

PMC6407085

Weaver

King

Verax

: Impact of chronic levels of naturally multi-contaminated feed with Fusarium mycotoxins on broiler chickens and evaluation of the mitigation properties of different titers of yeast cell wall extract. Toxins. 2020;12(10):636. 33019707

10.3390/toxins12100636

PMC7599674

Bhatti

Khan

Saleemi

: Impact of dietary Trichosporon mycotoxinivorans on ochratoxin A induced immunotoxicity; in vivo study. Food Chem. Toxicol. 2019;132:110696. 31344370

10.1016/j.fct.2019.110696

Zhang

Han

Zhao

: Removal of aflatoxin B1 and zearalenone by clay mineral materials: In the animal industry and environment. Appl. Clay Sci. 2022;228:106614. 10.1016/j.clay.2022.106614

Kihal

Rodríguez-Prado

Calsamiglia

: The efficacy of mycotoxin binders to control mycotoxins in feeds and the potential risk of interactions with nutrient: a review. J. Anim. Sci. 2022;100(11):skac328. 36208465

10.1093/jas/skac328

PMC9685567

Putra

Astuti

Respati

: Protective effects of feed additives on broiler chickens exposed to aflatoxins-contaminated feed: a systematic review and meta-analysis. Vet. Res. Commun. 2024;48(1):225–244. 37644237

10.1007/s11259-023-10199-7

Sadamatsu

Takahashi

Tashiro

: A rare case of Trichosporon mycotoxinivorans and Cryptococcus neoformans co-infection in lung. J. Infect. Chemother. 2020;26(8):838–842. 32249160

10.1016/j.jiac.2020.03.002

Jannah

Handayani

Lukiswanto

: Ivestigation of a multicomponent mycotoxin detoxifying agent for aflatoxin B1 and ochratoxin A induced blood profile in broiler chickens. Vet. World. 2024;17(5):1044–1051. 38911087

10.14202/vetworld.2024.1044-1051

PMC11188902

Wang

Hua

Shi

: Ochratoxin A: Occurrence and recent advances in detoxification. Toxicon. 2022;210:11–18. 35181402

10.1016/j.toxicon.2022.02.010

Purnama

MTE

Safitri

Purnobasuki

: Database of hemagglutination inhibition test data, histopathology scoring of cecum and colon, and original histopathology figures.Dataset. figshare. 2024. 10.6084/m9.figshare.27248592.v2

Safitri

Purnobasuki

Lestari

: ARRIVE Guidelines 2.0 for Mycotoxin binders study.Dataset. figshare. 2024. 10.6084/m9.figshare.27900534.v1

10.5256/f1000research.176969.r394912

Reviewer response for version 2

Sholikin

Mohammad Miftakhus

1 Referee https://orcid.org/0000-0001-8677-6982 1National Research and Innovation Agency (BRIN), Bogor, Indonesia

Competing interests: No competing interests were disclosed.

3 7 2025

2025

This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

recommendation

approve-with-reservations

The study presents an interesting topic; however, several aspects of the manuscript require revision. The following points highlight necessary improvements:

Despite these revisions, the research remains compelling. However, one crucial element appears absent from the article, and its inclusion is essential:

1. The specific type of mycotoxin binder remains unclear.

2. The contamination levels of AFB1 and OTA in the treated feed are not explicitly stated.

3. The source of the mycotoxins is also not explained.

The use of scientific phrases should also be reviewed, as the language currently resembles popular writing rather than scientific standards.

Suggested revisions:

1. Abstract: Consider revising the phrase:

"An increase in the mean antibody titer compared to days 14 and 28 was observed on day 35."

This sentence needs clarification for consistency and precision.

2. The abbreviation ROS appears to be defined more than once in the main text. Please verify and correct such redundancies.

3. Suggested rephrasing for scientific accuracy:

- Original: "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."

- Suggested: "Mycotoxin binders are typically composed of natural or synthetic adsorbent materials rather than single molecular structures. They function by capturing mycotoxins in the gastrointestinal tract and facilitating their excretion through feces."

4. The description of Mycofix in the methodology section lacks detail. Clarify its specifications, including key components and possibly the raw material origin.

5. The study used only 20 samples, with one broiler representing each replicate. Please justify the rationale behind this limited sample size, especially considering the collaboration with several institutions.

6. In Table 1, the replication statement should not appear in the table content. It is more appropriate as a table note. Moreover, ANOVA values are absent. If treatment effects are not significant, as seen on days 14 and 28 where all superscripts are "a," it is unnecessary to display them.

7. Tables 2 and 3 do not present ANOVA significance values or measures of variation such as SD, SE, or SEM. Please include these to strengthen data interpretation.

8. Figure layout and labels overlap, reducing visual clarity. Adjust the formatting to improve readability.

9. In the Discussion section, avoid repeating statistical significance values such as "(p < 0.05)." The focus should be on interpreting the findings rather than restating results.

For example, in the sentence:

"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."

Instead of restating outcomes, explain the potential biological or experimental factors behind the observed patterns.

10. When mentioning the same species repeatedly, abbreviate it after the first mention. For instance, change Trichosporon mycotoxinivoran to T. mycotoxinivoran in subsequent mentions.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Animal Nutrition

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Purnama

Muhammad Thohawi Elziyad

Airlangga University, Indonesia

Competing interests: None

22 7 2025

Dear

Dr. Mohammad Miftakhus Sholikin, National Research and Innovation Agency (BRIN), Bogor, Indonesia

We would like to express our sincere gratitude to the reviewer for the valuable suggestions and constructive feedback provided on our manuscript. The comments have been instrumental in improving both the content and clarity of our work. We have revised the manuscript accordingly and hope that the changes meet the reviewer’s expectations and enhance the overall scientific contribution of this study.

---

The study presents an interesting topic; however, several aspects of the manuscript require revision. The following points highlight necessary improvements:

Despite these revisions, the research remains compelling. However, one crucial element appears absent from the article, and its inclusion is essential:

1. The specific type of mycotoxin binder remains unclear.

Author Response: The mycotoxin binder utilized in this study functions not only as an adsorbent but also as a biotransformation agent, converting toxic compounds into non-toxic metabolites. Specifically, the bentonite component effectively binds aflatoxin B1 (AFB1), which possesses a polar molecular structure, whereas ochratoxin A (OTA), being non-polar, requires enzymatic degradation into less toxic forms, a process facilitated by Trichoderma reesei. This statement has been reflected in the revised version of the manuscript.

2. The contamination levels of AFB1 and OTA in the treated feed are not explicitly stated.

Author Response: Revision has been implemented in the experimental animal and housing statement.

3. The source of the mycotoxins is also not explained.

Author Response: Revision has been implemented in the experimental animal and housing statement.

The use of scientific phrases should also be reviewed, as the language currently resembles popular writing rather than scientific standards.

Suggested revisions:

1. Abstract: Consider revising the phrase:

"An increase in the mean antibody titer compared to days 14 and 28 was observed on day 35."

This sentence needs clarification for consistency and precision.

Author Response: Revision has been implemented in abstract.

2. The abbreviation ROS appears to be defined more than once in the main text. Please verify and correct such redundancies.

Author Response: Revision has been implemented in the entire manuscript.

3. Suggested rephrasing for scientific accuracy:

Author Response: Revision has been implemented according to recommendation.

4. The description of Mycofix in the methodology section lacks detail. Clarify its specifications, including key components and possibly the raw material origin.

Author Response: Revision has been implemented in experimental animal and housing.

Author Response: The sample size was determined in accordance with ethical clearance guidelines, which mandate the minimal use of experimental animals. In addition to meeting ethical requirements, the sample size was calculated using the Federer formula, t(n − 1) ≥ 15, where t represents the number of treatment groups and n denotes the number of replicates per group. This statement has been reflected in the revised version of the manuscript.

Author Response: Revision has been implemented according to recommendation.

7. Tables 2 and 3 do not present ANOVA significance values or measures of variation such as SD, SE, or SEM. Please include these to strengthen data interpretation.

Author Response: Revision has been implemented according to recommendation.

8. Figure layout and labels overlap, reducing visual clarity. Adjust the formatting to improve readability.

Author Response: Improvisation of figures and layouts will be assisted by the publisher's technical team.

9. In the Discussion section, avoid repeating statistical significance values such as "(p < 0.05)." The focus should be on interpreting the findings rather than restating results.

For example, in the sentence:

Instead of restating outcomes, explain the potential biological or experimental factors behind the observed patterns.

Author Response: Revision has been implemented according to recommendation in discussion section.

10. When mentioning the same species repeatedly, abbreviate it after the first mention. For instance, change Trichosporon mycotoxinivoran to T. mycotoxinivoran in subsequent mentions.

Author Response: Revision has been implemented in the entire manuscript.

10.5256/f1000research.176969.r393150

Reviewer response for version 2

Al-Aalim

Ammar M.

1 Referee https://orcid.org/0000-0001-5734-8815 1University of Mosul, Mosul, Iraq

Competing interests: No competing interests were disclosed.

2 7 2025

2025

recommendation

approve-with-reservations

This manuscript deals with the mycotoxin binder effect on the vaccination

The materials and methods were not explained clearly, and so little information about raising chickens, with little information about how tests are conducted, and also why the author used inaccurate tests like HA and HI, while it could have used the ELISA test to detect antibody titer, which provides more sensitive and accurate results ???

The results, especially in Figure 1, need to be explained correctly, as well as all statistical analyses must be explained clearly.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Veterinary Immunology

Purnama

Muhammad Thohawi Elziyad

Airlangga University, Indonesia

Competing interests: None

22 7 2025

Dear

Dr. Ammar M. Al-Aalim, University of Mosul, Mosul, Iraq

---

This manuscript deals with the mycotoxin binder effect on the vaccination

Author Response: Revisions have been made to the sections on Methods, including Experimental Animals and Housing, Hemagglutination (HA) and Hemagglutination Inhibition (HI) Tests, and portions of the Histopathological Evaluation. The HA and HI tests were employed in this study due to their simplicity, cost-effectiveness, and reliability in measuring antibody titers, expressed in log₂ values.

The results, especially in Figure 1, need to be explained correctly, as well as all statistical analyses must be explained clearly.

Author Response: Additional caption has been provide in the Figure 1 legend. In addition, statistical analysis method statement has been improved properly.

Mahmmod

Ammar

Competing interests: No competing interests were found

8 8 2025

The author completed the revision note and the corrected manuscript was ok to published

Mahmmod

Ammar

Competing interests: No competing interests were disclosed.

9 8 2025

approved

10.5256/f1000research.176969.r372034

Reviewer response for version 2

Khan

Rifat Ullah

1 Referee 1Khyber Pakhtunkhwa Agricultural University, Khyber Pakhtunkhwa, Pakistan

Competing interests: No competing interests were disclosed.

23 4 2025

2025

recommendation

reject

This study evaluated the protective effects of a mycotoxin binder (1.6 g/kg feed) against aflatoxin B1 and ochratoxin A in broilers vaccinated against Newcastle disease. The binder significantly improved antibody titers and reduced histopathological damage (mucosal rupture, hemorrhage, necrosis) in the cecum and colon, highlighting its potential to mitigate immunosuppressive effects of mycotoxins and enhance poultry health.

I have a few concerns and after specific revision, the paper is accepted for publication. Revise the introduction by dividing it into multiple paragraphs for better readability and incorporate the following citations for improved quality:

Ref: 1,2

Additionally, consider whether the sample size is sufficient, as it appears to be quite small. Please also include details on how the virulence of mycotoxins was confirmed. Clarify the conclusion

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Poultry science

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

References 1

: Fabrication of Polydopamine/hemin/TiO2 Composites with Enhanced Visible Light Absorption for Efficient Photocatalytic Degradation of Methylene Blue. Polymers (Basel) .2025;17(3) : 10.3390/polym17030311

39940513

10.3390/polym17030311

: Role of Quercetin in Diabetic Cardiomyopathy. Plants (Basel) .2024;14(1) : 10.3390/plants14010025

39795285

10.3390/plants14010025

Purnama

Muhammad Thohawi Elziyad

Airlangga University, Indonesia

Competing interests: No competing interests were disclosed.

22 7 2025

Dear

Dr. Rifat Ullah Khan, Khyber Pakhtunkhwa Agricultural University, Khyber Pakhtunkhwa, Pakistan

Author Response: Revision has been implemented in introduction by dividing it into multiple paragraphs.

Additionally, consider whether the sample size is sufficient, as it appears to be quite small.

Author Response: The determination of sample size was guided by ethical clearance requirements, which emphasize the reduction of experimental animal use to the minimum necessary. In addition to meeting ethical standards, the sample size was calculated using the Federer formula, t(n − 1) ≥ 15, where t represents the number of treatment groups and n the number of replicates per group. This statement has been reflected in the revised version of the manuscript.

Please also include details on how the virulence of mycotoxins was confirmed. Clarify the conclusion

Author Response: The virulence of the mycotoxins was confirmed by the presence of histopathological lesions—specifically mucosal rupture, hemorrhage, and necrosis—in the cecum and colon of the positive control group (C+). This statement has been reflected in the revised version of the manuscript.

References

1. Zhu Z, Zhou S, Tian D, Li GZ, et al.: Fabrication of Polydopamine/hemin/TiO2 Composites with Enhanced Visible Light Absorption for Efficient Photocatalytic Degradation of Methylene Blue. Polymers (Basel). 2025; 17 (3). PubMed Abstract | Publisher Full Text

2. Mustafa NH, Siti HN, Kamisah Y: Role of Quercetin in Diabetic Cardiomyopathy. Plants (Basel). 2024; 14 (1). PubMed Abstract | Publisher Full Text

Author Response: Both references have been added (Ref No. 41 and 42) along with the results of both studies.

Mahmmod

Ammar

Competing interests: no competing interests were found

9 8 2025

dear sir

The manuscript was approved for publication

10.5256/f1000research.173650.r349347

Reviewer response for version 1

Irawan

Agung

1 Referee https://orcid.org/0000-0003-1179-0469 1Universitas Sebelas Maret, Oregon State University,, Surakarta, Indonesia

Competing interests: No competing interests were disclosed.

6 1 2025

2025

recommendation

approve-with-reservations

Overall Assessment:

The authors evaluated the efficacy of mycotoxin binders on immunity and histopathology of broilers chickens. The topic of the manuscript is interesting and highly relevant to be disseminated. However, I found that in the current form, the manuscript requires substantial revision. Thorough the manuscript, it is poorly written, with significant issues in language, grammar, and coherence. Additionally, the scientific content is not presented in a manner consistent with standard academic practices, which undermines its clarity and impact.

Here are some major issues for authors’ attention:

The introduction lacks a logical structure for hypothesis formulation.

There is a lack of coherence between sentences.

The narrative is disjointed, making it difficult to follow the authors' rationale.

The results section contains many unnecessary descriptions that obscure the main findings.

A more concise and focused presentation of the data is required.

The discussion is poorly structured and hard to follow.

The findings are not effectively described, and there is a lack of chronological organization.

Critical interpretations are not adequately supported by references.

The authors mention a "hypothesis" in the conclusion but fail to clearly state it in the introduction.

The manuscript does not adequately discuss or revisit the hypothesis in the discussion section.

For more detailed review, please see below comments:

#Introduction

The introduction is not systematically written, there are too many issues with languages and grammar. Authors need to substantially revise the introduction both grammatically and scientific soundness. Between sentences’ connection is lacking in many parts of paragraphs, some unnecessary information is also found.

Some information requires more clarity, such as:

72% samples tested positive for…. -> please indicate what samples? It is not clear!

Are the mycotoxin combination that has been shown to have high toxicity -> please be more specific on the toxicity effects and indicate whether the study was in humans or animals (specify the animal)

There are some issues with the coherences of sentences in the first paragraph, making the reader a bit confused with the main idea of the paragraph. For instance, authors explain some molecular pathways associated with toxicity of AFB1 and OTA, and then jumped to failure of vaccination program etc. A large portion of the paragraph discuss ND vaccination issue while the importance of mycotoxin exposure issues is lost. Although they are relatable, but it is not written systematically. Please review and rewrite this part to be easier to understand.

At the end of 2 ^nd paragraph: plant goods -> what does that mean? I was unfortunately unable to understand. Can the authors please clarify?

The statement of the first sentence of 3 ^rd paragraph is not correct. Fiber digestion in poultry occurs primarily in the gizzard (mechanistic digestion) and only very little portion of the fiber is digested in the large intestine and this does not supply much energy for the bird. Please review this part and rewrite…

The statement of mycotoxicosis in person is not relevant in this part.

#Materials and Methods

Rpm should be converted into g to be more standardized.

In statistical analysis part, first sentence: followed -> analyzed. Authors mentioned Kruskal Wallis and Mann Whitney tests as well in this part, which one was actually used for the data analysis? Why Mann Whitney was used?

#Results

Please rewrite the results to be more concise. At the present form, there are many unnecessary descriptions. It would be better to consult with other experts or to copyedit the manuscript. Some examples are given below:

Please remove “as a result” in the first sentence of the section.

“Based on Figure 1, presents the HI test” -> Figure 1 (A) depicts

Paragraph 3: The investigation of 35-day-old broiler chickens’ antibody titers revealed statistically different results, with a p-value of 0.010 (p < 0.05). The fact that the value of p < 0.05 suggests that the administration of a mycotoxin binders agent has an impact on the antibody titer after ND vaccination -> Please change to “Antibody titers of broilers on day 35 differed (p = 0.010) between groups, suggesting an effect of mycotoxin binder on ND antibody titers establishment”.

#Discussion

The discussion is hard to follow. Some terms are not specified such as “organism enter the growth phase….” -> it is ambiguous what organism did the authors mean, whether bird or antigen?

- This paper may be useful as a reference: Putra RP et al. (2024 [Ref-1]) https://link.springer.com/article/10.1007/s11259-023-10199-7

#Conclusion: Authors mentioned the “hypothesis” but never stated their hypothesis in the introduction and never discussed it.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Nutritional biochemistry of poultry and ruminants related to feed, feed technology, and feeding.

References 1

: Protective effects of feed additives on broiler chickens exposed to aflatoxins-contaminated feed: a systematic review and meta-analysis. Vet Res Commun .2024;48(1) : 10.1007/s11259-023-10199-7 225-244

37644237

10.1007/s11259-023-10199-7

Purnama

Muhammad Thohawi Elziyad

Airlangga University, Indonesia

Competing interests: No competing interests were disclosed.

12 1 2025

Dear reviewers,

We appreciate your advice on our manuscript. Your suggestions have been addressed and the manuscript has been updated. In our opinion, these changes result in a much better manuscript, and we hope that it is now suitable for publication.

Overall Assessment:

Here are some major issues for authors’ attention:

The introduction lacks a logical structure for hypothesis formulation

Author Response: Revision has been implemented, We changed the hypothesis formulation with aims the research

There is a lack of coherence between sentences

Author Response: Revision has been implemented

The narrative is disjointed, making it difficult to follow the authors' rationale.

Author Response: Revision has been implemented

The results section contains many unnecessary descriptions that obscure the main findings.

Author Response: Revision has been implemented

A more concise and focused presentation of the data is required.

Author Response: Revision has been implemented

The discussion is poorly structured and hard to follow.

Author Response: Revision has been implemented

The findings are not effectively described, and there is a lack of chronological organization. Author Response: Revision has been implemented

Critical interpretations are not adequately supported by references.

Author Response: Revision has been implemented

The authors mention a "hypothesis" in the conclusion but fail to clearly state it in the introduction.

Author Response: We have revised the word “hypothesis” in conclusion

The manuscript does not adequately discuss or revisit the hypothesis in the discussion section.

Author Response: Revision has been implemented

For more detailed review, please see below comments:

#Introduction

Author Response: Revision has been implemented

Some information requires more clarity, such as:

72% samples tested positive for…. -> please indicate what samples? It is not clear!

Author Response: Revision has been implemented

Are the mycotoxin combination that has been shown to have high toxicity -> please be more specific on the toxicity effects and indicate whether the study was in humans or animals (specify the animal)

Author Response: We have rewrited

Author Response: We have revised in paragraph 1 & 2 introduction

At the end of 2 ^nd paragraph: plant goods -> what does that mean? I was unfortunately unable to understand. Can the authors please clarify?

Author Response: We have changed the statement with this : The digestive system has the highest risk of exposure since animal feed is the first point in the food chain where mixed mycotoxins can enter directly through grains crop

Author Response: The process of digesting and absorbing dietary fibre in chickens is significantly aided by the gizzard (mechanistic digestion) and ends in the large intestine. (This sentence is at the end of the first paragraph)

The statement of mycotoxicosis in person is not relevant in this part.

Author Response: We have changed the statement with this : Mycotoxicosis is poisoning that occurs when animals eat feed containing mycotoxins, where the toxins from these mycotoxins cannot be tolerated by the body

In the last paragraph, I do not see how the hypothesis was built, there is missing connection between the scientific background, gap of knowledge, and the objective being pursued in this study. Also, Trichosporon mycotoxinivorans is mentioned with no reason! Author Response: We have changed the hypothesis with aim statement. Also about Trichosporon mycotoxinivorans, we have rewrited

#Materials and Methods

Rpm should be converted into g to be more standardized.

Author Response: Revision has been implemented

#Results

Please remove “as a result” in the first sentence of the section.

Author Response: Revision has been implemented

“Based on Figure 1, presents the HI test” -> Figure 1 (A) depicts

Author Response: We have revised

Author Response: I've changed it according to reviewer's suggestion

#Discussion

The discussion is hard to follow. Some terms are not specified such as “organism enter the growth phase….” -> it is ambiguous what organism did the authors mean, whether bird or antigen?

Author Response: We have rewrited

- This paper may be useful as a reference: Putra RP et al. (2024 [Ref-1]) https://link.springer.com/article/10.1007/s11259-023-10199-7

Putra RP, Astuti D, Respati AN, Ningsih N, et al.: Protective effects of feed additives on broiler chickens exposed to aflatoxins-contaminated feed: a systematic review and meta-analysis. Vet Res Commun. 2024; 48 (1): 225-244

Author Response: We have added in Reference number 35

#Conclusion: Authors mentioned the “hypothesis” but never stated their hypothesis in the introduction and never discussed it.

Author Response: We have deleted the word “hypothesis” in conclusion

10.5256/f1000research.173650.r349350

Reviewer response for version 1

Soliman

Essam S

1 2 Referee 1Food Control and Hygiene, King Salman International University, Ras Sedr, South Sinia, Egypt 2Suez Canal University, Ismailia, Egypt

Competing interests: No competing interests were disclosed.

26 12 2024

2024

recommendation

approve-with-reservations

Manuscript F1KR00CDE F1R-VER173650-R entitled “ Implementation of mycotoxin binders to enhance immune response and large intestine histopathology against Newcastle disease in vaccinated broiler chickens fed naturally contaminated diet with mixed mycotoxins”. Please notice the following:

General view: The manuscript highlighted that a toxin binder dose of 1.6 g/kg can increase antibody titer and histopathology of cecum and colon in broiler chickens after ND vaccination fed with mycotoxin-contaminated feed. The manuscript was expressed using moderate English and grammar. A certain degree of copyediting & proofreading, proper display of the introduction, and declaring some explanations of the materials and methods have to be made to achieve better readability & understanding, and publication value.

Title: Very long. Preferred to be modified for clarity and simplification into “ Mycotoxin Immune and Intestinal Histopathology Ameliorations Against Newcastle Disease in Vaccinated Broilers”.

Abstract: Please notice the following:

1. Clear to a great extent.

2. Some modifications must be made to enhance the readability and understanding of the text.

Keywords: Re-arrange in alphabetical order as follows: “Aflatoxin, Antibody titer, Broilers, Food production, Ochratoxin, Toxin binders”.

Introduction: Please notice the following:

Improperly arranged into four paragraphs.

The introduction has to be re-arranged into three paragraphs only i.e. 1. Introduction 2. Significance of the study, and 3. Aim of the study.

Some modifications must be made to enhance the readability and understanding of the text.

The aim: Clear to a certain extent. Some modifications must be made to enhance the readability and understanding of the text.

Materials and methods: Please notice the following:

1. MUST provide details on the microclimatic conditions used for rearing the experimental broilers such as ambient temperature, relative humidity, feeding system (manual/automated), watering system (mechanism and source & type of water), lighting program (Color, duration, and intensity), ventilation system, rodentproof, flyproof, cleaning procedures (Provide the active principle of the detergent used), disinfection protocol (Provide the active principle of the disinfectant used), and treatment applied if any.

2. Provide the total number of blood samples collected by the end of the study and specify their types (With or without anticoagulant).

3. Provide a reference for the Euthanasia technique used in broilers.

4. Explain how the carcasses were disposed of hygienically after sampling.

5. Specify the number of the histopathological samples collected.

6. Provide the statistical model exhausted in the statistical analysis.

7. Some modifications must be made to enhance the readability and understanding of the text.

Results: Please notice the following:

1. MUST not list the P-value for the non-significant results.

2. Do not list the details of the treatment groups all over and use the symbols instead.

3. Reduce the illustration of numbers as much as possible as they are listed in tables and express them in your own words.

4. Some modifications must be made to enhance the readability and understanding of the text.

Discussion: Please notice the following:

1. Clear to a certain extent.

2. A moderate degree of speculation and comparison was used.

3. Some modifications must be made to enhance the readability and understanding of the text.

Conclusion: Clear and concise.

Authors’ contributions: Clear.

Acknowledgment: Clear.

References: Sufficient as 62.1% (23 out of 37) were published in the last five years.

Tables: Properly designed and well organized.

Figures: Properly designed and well organized.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Reviewer Expertise:

1. Preventive medicine and prophylactics of animals (Large and small), poultry, and fish. 2. Biosafety and biosecurity measures, plans, and strategies in animal and poultry farms. 3. Green environment and waste management and reuse. 4. Combating means of infectious and contagious diseases. 5. Air and water quality, and environmental sanitation. 6. Epidemiology of infectious and contagious disease and measurement of disease occurrence.

Purnama

Muhammad Thohawi Elziyad

Airlangga University, Indonesia

Competing interests: No competing interests were disclosed.

12 1 2025

Dear reviewers,

Title: Very long. Preferred to be modified for clarity and simplification into “Mycotoxin Immune and Intestinal Histopathology Ameliorations Against Newcastle Disease in Vaccinated Broilers”.

Author Response: Yes, We agree to changed the title modification according to the reviewer's suggestion

Abstract: Please notice the following:

1. Clear to a great extent. Author Response: Revision has been implemented.

2. Some modifications must be made to enhance the readability and understanding of the text.

Author Response: We have revised in text

Keywords: Re-arrange in alphabetical order as follows: “Aflatoxin, Antibody titer, Broilers, Food production, Ochratoxin, Toxin binders”.

Author Response: Yes, We agree to re-arranged in alphabetical, according to the reviewer's suggestion

Introduction: Please notice the following:

Improperly arranged into four paragraphs.

The introduction has to be re-arranged into three paragraphs only i.e. 1. Introduction 2. Significance of the study, and 3. Aim of the study.

Some modifications must be made to enhance the readability and understanding of the text. The aim: Clear to a certain extent. Some modifications must be made to enhance the readability and understanding of the text.

Author Response: We have revised in text

Materials and methods: Please notice the following:

--> The five steps of preparation before acclimatization of day-old chicks (DOC) are as follows: 1. Clean and remove all physical materials from a chicken cage; 2. Wash and clean all equipment and the cage with soap and water, using scraping; 3. Spray liquid disinfectant on all equipment and the cage and close the cage for a week; 4. Spray solid disinfectant (lime) and mix it with water in the cage and close the cage for a week; 5. Fumigate with permanganate and formalin, close the cage, as this can irritate; and finally, wait a week before DOC arrives.

--> A total of 20 DOC broiler chickens Cobb strain were reared for 35 days as final stock following broiler rearing standards. Rearing standards for broiler experiments include: basic ventilation system (open house), rodent and flyproof, cleaning and disinfection protocol (five steps before DOC arrived), ambient temperature (18 to 24°C), relative humidity (50 to 70%), manual feeding system, watering system (manual, ad libitum, and groundwater), lighting program (light color at 20 lux intensity for the first 7 days and 17-20 hours duration), and lighting program.

2. Provide the total number of blood samples collected by the end of the study and specify their types (With or without anticoagulant).

Author Response: We have revised in text

--> A total of 1 mL blood samples without anti-coagulant were collected through the brachial vein in 20 broiler chickens aged 14, 28, and 35 days. Blood was then allowed until collected serum and transferred into an Ependorf tube.

3. Provide a reference for the Euthanasia technique used in broilers.

Author Response: Reference number 18

4. Explain how the carcasses were disposed of hygienically after sampling.

Author Response: The process of incineration involves burning carcasses or residues at temperatures as high as 850 °C to create an inorganic ash. All infectious agents are anticipated to be destroyed by the treatment. Ash usually makes about 1–5% of the initial volume of the carcass, though this will vary depending on the type of incinerator, the method, the fuel, and the species of animal.

5. Specify the number of the histopathological samples collected.

Author Response: After the cecum and colon from 20 broilers were collected

6. Provide the statistical model exhausted in the statistical analysis.

Author Response: Revision has been implemented

7. Some modifications must be made to enhance the readability and understanding of the text.

Author Response: We have revised in text

Results: Please notice the following:

1. MUST not list the P-value for the non-significant results.

Author Response: We have deleted in text

2. Do not list the details of the treatment groups all over and use the symbols instead.

Author Response: We have revised

3. Reduce the illustration of numbers as much as possible as they are listed in tables and express them in your own words.

Author Response: Revision has been implemented.

4. Some modifications must be made to enhance the readability and understanding of the text.

Author Response: Revision has been implemented.

Discussion: Please notice the following:

1. Clear to a certain extent.

Author Response: Revision has been implemented.

2. A moderate degree of speculation and comparison was used.

Author Response: Revision has been implemented.

3. Some modifications must be made to enhance the readability and understanding of the text.

Author Response: Revision has been implemented.

Conclusion: Clear and concise.

Author Response: Thank you for your recommendation.

Authors’ contributions: Clear.

Author Response: Thank you for your recommendation.

Acknowledgment: Clear.

Author Response: Thank you for your recommendation.

References: Sufficient as 62.1% (23 out of 37) were published in the last five years.

Author Response: Now 65,7 % (25 out of 38)

Tables: Properly designed and well organized.

Author Response: Thank you for your recommendation.

Figures: Properly designed and well organized.

Author Response: Thank you for your recommendation.