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

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.⁴⁴ All efforts were made to ameliorate any suffering of animals. Animal handling was carried out in accordance with institutional guidelines and the American Veterinary Medical Association standards, 2020 (AVMA, 2020).

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 design

This study employed a true experimental design to assess the effects of a dietary mycotoxin binder (Mycofix^®, Cat No. 20026289-FTS, PT. Biomin Indonesia) on antibody titers and intestinal histopathology (cecum and colon) in broiler chickens exposed to AFB1 and OTA following ND vaccination. A completely randomized design (CRD) was utilized. Sample size determination was based on the Federer formula: t(n − 1) ≥ 15, where t represents the number of treatment groups and n the number of replicates per treatment. Accordingly, twenty Cobb-strain broiler chickens were randomly assigned to four treatment groups, with five birds per group (n = 5).

Experimental animals and housing

Day-old male Cobb broiler chicks (sourced from PT. Charoen Pokphand Indonesia, Jakarta, Indonesia) were sexed based on feather morphology. The chicks were housed in sanitized battery cages (30.5 × 35 cm) under controlled temperature and photoperiod conditions, with unrestricted access to feed and water. A 7-day acclimatization period was provided prior to the initiation of experimental treatments. All birds received vaccinations against ND and avian influenza (AI) on days 7 and 21.

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. From day 8 to day 35, the chicks were subjected to one of four dietary treatment regimens. They were randomly allocated into four treatment groups, each comprising five replicates. The negative control group (C−) received only the commercial basal diet (CP511 starter and CP512 finisher; PT. Charoen Pokphand Indonesia). The positive control group (C+) was fed the same basal diet contaminated with AFB1 at 0.16 mg/kg (Fermentek Ltd., Jerusalem, Israel) and OTA at 0.16 mg/kg (GLP BIO, Shanghai, China). Treatment group 1 (T1) was provided the contaminated basal diet supplemented with 1.1 g/kg of a commercial mycotoxin binder comprising bentonite, Bacillus subtilis, T. mycotoxinivorans, and phytogenic compounds (Mycofix^®, Cat No. 20026289-FTS, PT. Biomin Indonesia). Treatment group 2 (T2) received the same contaminated diet supplemented with 1.6 g/kg of the mycotoxin binder. All feed formulations and mixing procedures were conducted at the Feed Laboratory, Faculty of Veterinary Medicine, Universitas Airlangga. Feed was administered twice daily, while water was provided ad libitum throughout the experimental period.

The mycotoxin binder used in this study not only adsorbs mycotoxins but also biotransforms them into non-toxic metabolites. Bentonite effectively binds the polar AFB1, whereas OTA, which is non-polar, requires biotransformation into less toxic forms, a process facilitated by Trichoderma reesei.

Newcastle Disease (ND) vaccination was administered to seven-day-old broiler chickens using the La Sota inactivated ND vaccine (Medivac^®, Cat No. 3526004001, PT. Medion Farma, Indonesia) via ocular instillation at a dose of one drop per bird. A booster vaccination was given on day 21 using the same inactivated vaccine at a dose of 0.5 mL per bird via intramuscular injection. Antibody titers were assessed through the hemagglutination inhibition (HI) test using serum samples collected on days 14, 28, and 35. Additionally, cecum and colon tissues were harvested on day 36 for histopathological examination 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

Tissue sampling was conducted via abdominal dissection following euthanasia. The cecum and colon from a total of 20 broiler chickens were excised and immediately fixed in 10% formalin solution within labeled containers to preserve the tissues for histopathological analysis using hematoxylin-eosin (HE) staining. The fixed tissues were embedded in paraffin, sectioned at a thickness of 5 μm, and subsequently stained with HE. Histopathological evaluation was performed using a light microscope (Nikon Eclipse E200, Japan), with inflammatory cell infiltration assessed at 100× magnification and morphological alterations in the cecum and colon examined at 400× magnification. For each sample, five randomly selected fields were analyzed and scored by blinded observers to minimize bias. The extent of pathological alterations was quantified using a standardized scoring system based on the proportion of affected tissue area: score 0 indicated no observable changes, score 1 corresponded to pathological findings in less than 25% of the field, score 2 represented involvement of 26–50%, score 3 indicated 51–75% affected, and score 4 denoted more than 76% pathological changes within the examined tissue. This method allowed for a systematic and semi-quantitative assessment of histopathological severity.²¹

Statistical analysis

The hemagglutination inhibition (HI) test data were organized and assessed for normality using the Shapiro-Wilk test. Data conforming to a normal distribution were subsequently analyzed via one-way analysis of variance (ANOVA), followed by Duncan’s multiple range post hoc test for pairwise comparisons. Histopathological scores, which were non-parametric, were analyzed using the Kruskal–Wallis test, with pairwise comparisons conducted using the Mann–Whitney U test. All statistical analyses were performed using SPSS version 26.0 for Windows (IBM Corp., Armonk, NY, USA), with significance determined at a 95% confidence interval.

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.

Panel (A) demonstrates the absence of hemagglutination, characterized by the sedimentation of erythrocytes upon tilting the microplate. In contrast, panel (B) illustrates the presence of hemagglutination, indicated by the lack of erythrocyte sedimentation at the bottom of the microplate well.

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). Conversely, a significant increase in antibody titer was observed in broilers at 35 days of age compared to the mean titers recorded at 14 and 28 days. The relatively low antibody titer at 14 days of age—seven days following the initial ND vaccination—can be attributed to the early phase of the immune response, during which the immune system is still in the process of recognizing and responding to the initial antigen exposure. This stage, known as the primary immune response, represents the body’s initial immunological reaction to a novel antigen.^22,23

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).

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.