Aflatoxin (AF) is the secondary metabolite of
Aflatoxin (AF) is the secondary metabolite of
The previous study showed that poultry aflatoxicosis causes lowered body weight, reduced feed intake and efficiency [
The most important is AF found as the residue in the chicken’s products that fed a diet contaminated with AF. As the residue, AF is resistant to food processing and possing risk for the human health [
The limitation of the previous study is not to evaluate the role of
All the experimental procedures are conducted in the Integrated Laboratory, Faculty of Health, the University of Muhammadiyah Sidoarjo from November 2017 until April 2018. The fresh leaf of
The SAP measured about 0.5 gram for each qualitative phytochemical test. The SAP is screened using the standard test for several components such as tannin, phenol, saponin, alkaloid, flavonoid, glycoside, and the carotenoid. Tannin is tested by the homogenising of the SAP with distilled water and filtered, then the filtrate drops with 1% ferric chloride. The phenol is tested by the similar method in the tannin; however the ferric chloride concentration was 5%. The saponin is by the demonstration of the frothing in the filtrate after boiling. The content of flavonoid is tested by the Shinoda’s, alkaloid by Mayer’s, and the glycoside by Borntrager’s method. However, the carotenoid content is tested by the mixing of the filtrate with chloroform and 85% sulphuric acid. The results of the qualitative phytochemical test are shown in the table (Table
Qualitative phytochemical analysis of SAP.
Variable | ||||||
---|---|---|---|---|---|---|
Tannin | Phenol | Saponin | Alkaloid | Flavonoid | Glycoside | Carotenoid |
+ | + | + | + | + | + | + |
+ = present; – = absent.
All the animal procedures in this study are approved by the ethical clearance committee from the Faculty of Veterinary Medicine, University of Gadjah Mada, Indonesia. A total of 108 one-day-old broiler chickens (DOC) strain Cobb were divided into 6 group. Chickens are treated in 24-hour light schedule (the light intensity reduced after 16 hours each day), 30°C temperature (and gradually decreased per week), and 65-70% humidity, with water and feed access ad libitum.
All chickens were fed with a broiler starter feed with 23% crude protein with 3200 kcal metabolizable energy. The feed composition was as described previously [
AF level in the chicken’s feed from days 0 to 21 (ppb) during the study.
Day | Group (AF level) | |||||
---|---|---|---|---|---|---|
I | II | III | IV | V | VI | |
0 | - | - | 25.21 | 20.00 | 51.02 | 110.07 |
3 | - | - | 27 | 29.08 | 63.77 | 129.82 |
6 | - | - | 23 | 17.85 | 70.00 | 107.73 |
9 | - | - | 27.12 | 23.19 | 74.30 | 126.25 |
12 | - | - | 26.84 | 29.41 | 78.26 | 136.15 |
15 | - | - | 33.01 | 35.24 | 78.26 | 144.52 |
18 | - | - | 40.15 | 39.44 | 81.10 | 138.66 |
21 | - | - | 42.87 | 44.98 | 84.16 | 141.00 |
Mean | - | - | 30.65 ± 7.30 | 29.89 ± 9.53 | 72.60 ± 10.84 | 129.27 ± 13.87 |
| ||||||
Feed treatments to reach the suitable AF level | ||||||
| ||||||
Temp. (°C) | 5 | 5 | 40 | 40 | 40 | 40 |
Humidity (%) | 50 | 50 | 90 | 90 | 90 | 90 |
Incubation period (day) | - | - | 7 | 7 | 14 | 21 |
| ||||||
SAP (%) | 0 | 5 | 0 | 5 | 5 | 5 |
– = AF not detectable/without incubation.
In this study, a total of 108 DOC were divided into 6 group and each contains 18 chickens. Each group is placed in the colony cage and consisting of three sampling periods of 6 chickens per period. Group I was a control group and fed with basal diet (AF not detectable). Group II fed with basal diet (AF not detectable) + 5% SAP; group III with AF (>1 ppb <50 ppb); group IV with AF (>1 ppb <50 ppb) + 5% SAP; group V with AF (>51 ppb <100 ppb) + 5% SAP; group VI with AF (>101 ppb <150 ppb) + 5% SAP. The treatment was conducted for 21 days. Six chickens from each group were euthanised on days 7, 14, and 21. This research design was conducted to determine the protection limit of SAP on the AF exposure in the low, middle, and high level of contamination.
At the age of 3 days, all groups were vaccinated with Newcastle Disease (ND) using Medivac ND Hitchner B1 vaccine via ocular route according to the recommendation of the manufacturer.
The recording of chicken’s body weight and feed intake (FI) was conducted on days 7, 14, and 21. Comparison of feed efficiency was determined as feed conversion ratio (FCR) using the following formula:
Six chickens from each group were randomly selected, euthanised by cervical dislocation, and necropsied on days 7, 14, and 21. Only at day 21, the relative weight of the liver, kidney, and spleen, and BF was weighed and calculated using the following formula:
After the measurement, the liver, kidney, spleen, and BF were cut and separated into 2 part. The first part was stored in the 10% neutral buffered formalin (NBF) for histopathology and immunohistochemistry and the second one in the sterile plastic and kept in the refrigerator for AF residue test using ELISA.
Six blood samples were collected from each group on days 7, 14, and 21 before sacrifice. The blood was collected via the right jugular vein using 1 ml syringes (26G × 1/2” (0,45 × 13 mm)). The blood for the haematological profile examination was stored in the tubes with ethylenediaminetetraacetic acid (EDTA) and kept in the refrigerator at 4°C. The blood samples were also collected inside the tubes without EDTA and kept in the room temperature until clotted and then centrifuged at 3.000 rpm for 15 minutes. The serum was stored at -20°C until tested.
The blood samples were analysed for the haematological test such as total erythrocytes/red blood cells (RBC), total leucocytes/ white blood cells (WBC), haemoglobin (Hgb), packed cells volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and differential count of leucocytes. The blood smear examination was performed by Giemsa staining and observed under the light microscope with the 1000× magnification. All the haematological tests were examined using the standard methods.
The HI tests were performed on serum according to the OIE Manual of Standard Diagnostic Tests [
The ELISA was performed to count the total of AF level in the feed and organ of broiler chickens in this study. The samples weighed about 2 g, were extracted with methanol 70%, were homogenised in the room temperature, and were centrifuged. The supernatants were added in the well of the plates and diluted with phosphate buffer, then an aliquot (50
The organ (liver, kidney, spleen, and BF) from each group was fixed, dehydrated, and embedded in the paraffin. Thin sections (5
Two histopathologists analysed the histopathological slides under a blindfold condition to avoid bias. The assessment was performed using the semiquantitative scoring system from 0 to 4, as follows: (absence (0); minimal (1); mild (2); moderate (3); severe (4)). Each slide was analysed against several parameters (Table
Semiquantitative scoring systems for poultry aflatoxicosis.
Organ | Parameters |
---|---|
Liver | Necrosis, perilobular and inflammation, hydropic and/or fatty degeneration, bile-duct proliferation |
Kidney | Necrosis, inflammation, degeneration |
Spleen | Depletion of pulp, inflammation, congestion |
BF | Depletion of the lymphoid follicle, inflammation, congestion |
The immunohistochemical slides of spleen and BF were examined by ImageJ software against the percentages of the area that express the CD4+ and CD8+ lymphocytes. Then, the percentages area that immune-expression of CD4+ and CD8+ was measured as a ratio of CD4+/CD8+ lymphocytes.
The data were analysed by SPSS 16 and presented as the mean ± standard of deviation (SD). The body weight, haematological profile, haemagglutination inhibition (HI) titer, and AF residue were analysed with two-way ANOVA and post hoc test; the relative organ weight was analysed with one way ANOVA and post hoc test; on the other hands, the histopathological and immunohistochemical data were analysed with the Kruskal-Wallis test and Man-Whitney U test. A probability value (
The results showed that 5% SAP has a potential effect on the body weight of broiler chickens fed a diet with naturally contaminated with AF (
Effects of 5% SAP on the body weight, FI, and FCR of broiler chickens fed a diet naturally contaminated with AF at 7, 14, and 21 days of age.
Parameter | Group | Day | ||
---|---|---|---|---|
7 | 14 | 21 | ||
Body weight (g) | I | 120.27 ± 2.70 | 338.67 ± 14.49 | 683.38 ± 13.32 |
II | 127.13 ± 3.03 | 353.40 ± 5.95 | 695.89 ± 12.45 | |
III | 108.95 ± 5.51 | 309.83 ± 7.26 | 641.19 ± 22.50 | |
IV | 125.44 ± 3.43 | 355.71 ± 4.79 | 695.80 ± 8.73 | |
V | 127.13 ± 2.23 | 353.22 ± 4.97 | 690.13 ± 16.55 | |
VI | 120.16 ± 2.44 | 337.22 ± 11.25 | 682.53 ± 14.11 | |
| ||||
FI | I | 171.00 ± 0.89 | 562.33 ± 2.33 | 1217.33 ± 11.69 |
II | 171.00 ± 0.89 | 563.00 ± 1.67 | 1213.00 ± 7.69 | |
III | 164.83 ± 4.07 | 539.83 ± 15.06 | 1155.16 ± 51.80 | |
IV | 170.83 ± 1.47 | 562.33 ± 1.86 | 1215.33 ± 14.50 | |
V | 169.33 ± 1.50 | 562.50 ± 1.51 | 1213.33 ± 4.30 | |
VI | 172.00 ± 2.00 | 564.17 ± 3.54 | 1226.00 ± 6.75 | |
| ||||
FCR | I | 1.42 ± 0.03 | 1.66 ± 0.07 | 1.78 ± 0.03 |
II | 1.34 ± 0.03 | 1.59 ± 0.02 | 1.74 ± 0.04 | |
III | 1.51 ± 0.09 | 1.74 ± 0.03 | 1.80 ± 0.06 | |
IV | 1.36 ± 0.03 | 1.58 ± 0.02 | 1.74 ± 0.02 | |
V | 1.33 ± 0.02 | 1.59 ± 0.02 | 1.75 ± 0.04 | |
VI | 1.43 ± 0.03 | 1.67 ± 0.05 | 1.79 ± 0.03 |
Macroscopically, there are no significant differences in the relative weight of liver in all groups (P > 0.05). Nevertheless, gross changing (swelling and pale) was found in the liver of group V (2/6 samples); yellowish colour in the liver of group VI (1/6 samples) and it was similar to group III (Figure
Effects of 5% SAP on the relative organ weight of broiler chickens fed a diet naturally contaminated with AF at 21 days of age.
Group | Relative organ weight (g/100 g body weight) | |||
---|---|---|---|---|
Liver | Kidney | Spleen | BF | |
I | 2.26 ± 0.06 | 0.64 ± 0.03 | 0.14 ± 0.03 | 0.30 ± 0.01 |
II | 2.29 ± 0.01 | 0.64 ± 0.02 | 0.14 ± 0.02 | 0.31 ± 0.02 |
III | 2.31 ± 0.02 | 0.71 ± 0.01 | 0.22 ± 0 | 0.18 ± 0.02 |
IV | 2.25 ± 0.04 | 0.65 ± 0.02 | 0.14 ± 0.02 | 0.30 ± 0.01 |
V | 2.26 ± 0.38 | 0.64 ± 0.03 | 0.15 ± 0.02 | 0.30 ± 0.05 |
VI | 2.29 ± 0.05 | 0.70 ± 0.01 | 0.20 ± 0.01 | 0.23 ± 0.05 |
Macroscopic examination of the chicken’s liver on day 21. Chicken’s liver of group II showed a normal appearance with brown colour and smooth surface (6/6 samples) (a); group III showed the enlargement, obtuse angle, pale, and hemorrhage on its surface (6/6 samples) (b); group V showed the enlargement, obtuse angle, and pale (2/6 samples) (c); group VI showed a yellowish colour, and it was suspected due to fatty degeneration (1/6 samples) (d).
This study showed that AF has potential effects on the haematological profile change in broiler chickens. Groups III and VI showed the significant differences compared with the other group regarding Hgb and MCH (
Effects of 5% SAP on the haematological profile (RBC, PCV, Hgb, MCV, MCH, and MCHC) of broiler chickens fed a diet naturally contaminated with AF at 7, 14, and 21 days of age.
Parameter | Group | Day | ||
---|---|---|---|---|
7 | 14 | 21 | ||
RBC | I | 2.17 ± 0.08 | 2.40 ± 0.03 | 2.74 ± 0.06 |
II | 2.25 ± 0.04 | 2.50 ± 0.06 | 2.85 ± 0.05 | |
III | 2.10 ± 0.17 | 2.09 ± 0.05 | 2.18 ± 0.09 | |
IV | 2.23 ± 0.03 | 2.39 ± 0.19 | 2.81 ± 0.07 | |
V | 2.13 ± 0.07 | 2.40 ± 0.08 | 2.64 ± 0.07 | |
VI | 2.24 ± 0.05 | 2.40 ± 0.08 | 2.63 ± 0.14 | |
| ||||
PCV (%) | I | 26.92 ± 0.48 | 27.41 ± 0.34 | 28.74 ± 0.41 |
II | 27.31 ± 0.40 | 28.23 ± 0.35 | 28.72 ± 0.32 | |
III | 27.27 ± 0.30 | 27.87 ± 0.19 | 27.25 ± 0.34 | |
IV | 27.26 ± 0.29 | 28.04 ± 0.45 | 28.46 ± 0.39 | |
V | 27.07 ± 0.28 | 28.21 ± 0.39 | 28.58 ± 0.30 | |
VI | 27.21 ± 0.33 | 28.08 ± 0.33 | 27.05 ± 0.84 | |
| ||||
Hgb (g/dl) | I | 10.36 ± 0.27 | 10.72 ± 0.18 | 11.22 ± 0.09 |
II | 10.52 ± 0.23 | 10.80 ± 0.08 | 11.33 ± 0.07 | |
III | 10.22 ± 0.13 | 10.25 ± 0.17 | 10.40 ± 0.30 | |
IV | 10.52 ± 0.24 | 10.77 ± 0.02 | 11.29 ± 0.15 | |
V | 10.52 ± 0.31 | 10.69 ± 0.31 | 11.16 ± 0.24 | |
VI | 10.36 ± 0.34 | 10.40 ± 0.26 | 10.90 ± 0.22 | |
| ||||
MCV (fl) | I | 124.16 ± 5.57 | 113.86 ± 1.79 | 104.96 ± 3.02 |
II | 121.44 ± 3.82 | 112.92 ± 3.79 | 100.84 ±2.75 | |
III | 129.67 ± 9.97 | 133.28 ± 4.30 | 125.09 ± 4.82 | |
IV | 122.20 ± 2.89 | 117.87 ± 11.57 | 101.18 ± 3.32 | |
V | 126.98 ± 5.84 | 117.64 ± 4.86 | 108.12 ± 3.10 | |
VI | 121.16 ± 2.59 | 117.16 ± 4.54 | 102.88 ± 5.16 | |
| ||||
MCH (Pg) | I | 47.76 ± 2.01 | 44.52 ± 0.69 | 40.97 ± 0.95 |
II | 46.78 ± 1.22 | 43.21 ± 1.35 | 39.76 ± 0.93 | |
III | 48.80 ± 4.04 | 49.00 ± 1.70 | 47.74 ± 2.31 | |
IV | 47.16 ± 1.31 | 45.25 ± 3.72 | 40.13 ± 0.96 | |
V | 49.35 ± 2.15 | 44.58 ± 1.40 | 42.24 ± 1.54 | |
VI | 46.15 ± 1.68 | 43.39 ± 1.86 | 41.47 ± 1.76 | |
| ||||
MCHC (%) | I | 38.47 ± 0.82 | 39.11 ± 0.50 | 39.04 ± 0.40 |
II | 38.54 ± 1.24 | 38.27 ± 0.47 | 39.44 ± 0.46 | |
III | 37.62 ± 0.46 | 36.76 ± 0.46 | 38.16 ± 1.02 | |
IV | 38.60 ± 0.85 | 38.44 ± 0.68 | 39.67 ± 1.00 | |
V | 38.88 ± 1.19 | 37.94 ± 1.57 | 39.06 ± 0.62 | |
VI | 38.09 ± 0.96 | 37.03 ± 0.56 | 40.34 ± 1.53 |
Photomicrographs of erythrocytes of the chickens fed a diet naturally contaminated with AF on day 21. The normal appearance of erythrocytes with a uniform size in group I (a); group II (b); pale colour and predominant small size of erythrocytes in group VI that indicates normocytic-hypochromic anaemia (c). Giemsa, 1000×.
The 5% SAP supplementation on the chicken’s feeds showed significant effects on the total WBC in groups II, IV, and V (
Effects of 5% SAP on the haematological profile (WBC and differential leucocytes count) of broiler chickens fed a diet naturally contaminated with AF at 7, 14, and 21 days of age.
Parameter | Group | Day | ||
---|---|---|---|---|
7 | 14 | 21 | ||
WBC (× 103/ | I | 20.96 ± 6.24 | 22.70 ± 1.74 | 24.11 ± 3.92 |
II | 22.47 ± 3.13 | 24.00 ± 2.96 | 25.35 ± 3.72 | |
III | 19.39 ± 48.40 | 20.70 ± 42.89 | 21.36 ± 43.66 | |
IV | 22.37 ± 3.43 | 24.03 ± 3.55 | 25.40 ± 5.25 | |
V | 22.05 ± 7.06 | 24.05 ± 2.42 | 24.98 ± 4.70 | |
VI | 21.20 ± 4.97 | 22.45 ± 1.33 | 24.11 ± 5.84 | |
| ||||
Heterophils (× 103/ | I | 4.37 ± 2.55 | 5.07 ± 2.96 | 5.58 ± 4.91 |
II | 4.98 ± 3.06 | 5.59 ± 3.32 | 5.99 ± 2.15 | |
III | 4.10 ± 4.54 | 4.51 ± 2.85 | 4.94 ± 3.09 | |
IV | 4.73 ± 2.57 | 5.48 ± 2.99 | 6.09 ± 1.40 | |
V | 4.81 ± 2.08 | 5.73 ± 1.18 | 5.58 ± 4.11 | |
VI | 4.55 ± 3.36 | 5.65 ± 1.57 | 6.62 ± 9.17 | |
| ||||
Lymphocytes (× 103/ | I | 12.47 ± 3.22 | 13.73 ± 4.02 | 14.23 ± 4.57 |
II | 14.15 ± 3.50 | 14.96 ± 4.09 | 15.79 ± 3.31 | |
III | 11.25 ± 3.96 | 12.34 ± 2.38 | 12.78 ± 4.23 | |
IV | 13.98 ± 6.24 | 14.90 ± 3.07 | 15.87 ± 5.01 | |
V | 12.61 ± 7.71 | 14.54 ± 1.34 | 15.24 ± 5.19 | |
VI | 12.37 ± 5.54 | 13.66 ± 2.95 | 14.90 ± 2.98 | |
| ||||
Ratio H/L | I | 0.35 ± 0.01 | 0.36 ± 0.02 | 0.39 ± 0.03 |
II | 0.35 ± 0.02 | 0.37 ± 0.01 | 0.37 ± 0.01 | |
III | 0.36 ± 0.03 | 0.36 ± 0.02 | 0.38 ± 0.03 | |
IV | 0.33 ± 0.02 | 0.36 ± 0.01 | 0.38 ± 0.01 | |
V | 0.38 ± 0.02 | 0.39 ± 0.00 | 0.36 ± 0.03 | |
VI | 0.36 ± 0.03 | 0.41 ± 0.01 | 0.44 ± 0.02 | |
| ||||
Monocytes (× 103/ | I | 2.09 ± 1.77 | 2.11 ± 1.15 | 2.41 ± 2.22 |
II | 1.94 ± 1.07 | 1.91 ± 4.80 | 1.56 ± 0.96 | |
III | 1.97 ± 1.36 | 2.37 ± 2.30 | 2.17 ± 0.94 | |
IV | 1.56 ± 3.24 | 1.76 ± 1.25 | 1.60 ± 1.35 | |
V | 2.16 ± 2.23 | 2.04 ± 1.46 | 2.16 ± 3.98 | |
VI | 2.08 ± 1.93 | 2.28 ± 2.24 | 2.09 ± 4.06 | |
| ||||
Eosinophils (× 103/ | I | 1.77 ± 1.52 | 1.55 ± 2.25 | 1.76 ± 4.92 |
II | 1.23 ± 3.92 | 1.43 ± 2.11 | 1.73 ± 2.01 | |
III | 1.70 ± 4.40 | 1.03 ± 2.41 | 0.99 ± 3.98 | |
IV | 1.71 ± 2.61 | 1.80 ± 2.78 | 1.61 ± 3.96 | |
V | 2.05 ± 1.51 | 1.60 ± 3.01 | 1.74 ± 2.74 | |
VI | 1.90 ± 3.09 | 0.86 ± 1.67 | 0.48 ± 3.47 | |
| ||||
Basophils (× 103/ | I | 0.24 ± 2.09 | 0.22 ± 2.04 | 1.12 ± 2.03 |
II | 0.14 ± 1.15 | 0.08 ± 1.25 | 0.25 ± 2.30 | |
III | 3.56 ± 2.83 | 4.17 ± 2.65 | 4.63 ± 3.66 | |
IV | 0.36 ± 5.83 | 0.08 ± 1.25 | 0.21 ± 3.02 | |
V | 0.40 ± 2.43 | 0.11 ± 1.31 | 0.24 ± 2.21 | |
VI | 0.28 ± 2.88 | 0 ± 0 | 0 ± 0 |
On day 7, the chickens in group II showed maximally increasing of the GMT against ND vaccine compared with the other groups. It is followed by groups IV and V, despite the increasing of GMT slower than group II. Surprisingly, GMT against ND vaccine in group I and group VI does not show any differences. This result proved that 5% SAP has potential effects to maintain the stability regarding titer ND production postvaccination. On the other hands, group III shows the lowest response postvaccination regarding the GMT. It is suspected caused by the impacts of AF on the broiler’s lymphoid organ that impair the antibody production in group III (Figure
Effect of 5% SAP on the geometric mean titer (GMT) value against ND vaccine in the broiler chicken fed a diet with naturally contaminated with AF.
The AF residues in the organ (liver, kidney, spleen, and BF) in groups I, II, IV are not detected on days 7, 14, and 21. The similar results of AF residue in the organ of groups III, V, and VI were not detected at days 7, 14, and 21, except the liver at day 21. There was a detectable residue of AF in the livers of groups III, V, and VI on day 21. It may because of the accumulation effects of AF in these group. However, the detected AF level in the liver of group V was 0.10 ppb ± 0.09 (4/6 samples), and in group VI was 0.24 ppb ± 0.09 (6/6 samples). On the other hands, group III shows the highest AF residue in 0.28 ppb ± 0.58 (6/6 samples). The supplementation of 5% SAP on the chicken’s feed may have resulted in the reduction of AF residue in the liver and the other organs.
In this study, group III with AF (>1 ppb < 50 ppb) shows severe liver necrosis and degeneration compared with the others (
Effects of 5% SAP on the liver histopathology of broiler chickens fed a diet naturally contaminated with AF at 7, 14, and 21 days of age.
Parameter | Group | Day | ||
---|---|---|---|---|
7 | 14 | 21 | ||
Necrosis | I | 0 ± 0 | 1.00 ± 1.09 | 0.50 ± 0.54 |
II | 0 ± 0 | 0.66 ± 0.51 | 0.50 ± 0.54 | |
III | 0.33 ± 0.51 | 0.83 ± 0.98 | 2.16 ± 1.83 | |
IV | 0 ± 0 | 1.00 ± 0.89 | 0.50 ± 0.54 | |
V | 0 ± 0 | 0.50 ± 0.54 | 0.50 ± 0.54 | |
VI | 0 ± 0 | 0.50 ± 0.54 | 1.33 ± 1.62 | |
| ||||
Perilobular inflammation | I | 0 ± 0 | 0.33 ± 0.51 | 0.16 ± 0.40 |
II | 0 ± 0 | 0.16 ± 0.40 | 0.16 ± 0.40 | |
III | 0 ± 0 | 0.83 ± 0.75 | 2.33 ± 1.86 | |
IV | 0 ± 0 | 0.33 ± 0.51 | 0.16 ± 0.40 | |
V | 0 ± 0 | 0.33 ± 0.51 | 1.16 ± 1.47 | |
VI | 0 ± 0 | 0.33 ± 0.51 | 1.50 ± 1.37 | |
| ||||
Interlobular inflammation | I | 0 ± 0 | 0 ± 0 | 0 ± 0 |
II | 0 ± 0 | 0 ± 0 | 0 ± 0 | |
III | 0 ± 0 | 0.50 ± 0.54 | 1.66 ± 1.96 | |
IV | 0 ± 0 | 0 ± 0 | 0 ± 0 | |
V | 0 ± 0 | 0 ± 0 | 0.83 ± 1.60 | |
VI | 0 ± 0 | 0 ± 0 | 1.16 ± 1.47 | |
| ||||
Hydrophic and/ or fatty degeneration | I | 0.50 ± 1.22 | 0.66 ± 1.63 | 0.33 ± 0.51 |
II | 0 ± 0 | 0.16 ± 0.40 | 0.33 ± 0.51 | |
III | 0.50 ± 0.54 | 1.00 ± 0.89 | 2.00 ± 1.09 | |
IV | 0.16 ± 0.40 | 0.16 ± 0.40 | 0.66 ± 0.81 | |
V | 0.16 ± 0.40 | 0.16 ± 0.40 | 1.33 ± 1.50 | |
VI | 0.16 ± 0.40 | 0.16 ± 0.40 | 1.33 ± 1.50 | |
| ||||
Bile-duct proliferation | I | 0 ± 0 | 0 ± 0 | 0.33 ± 0.81 |
II | 0 ± 0 | 0 ± 0 | 0.16 ± 0.40 | |
III | 0 ± 0 | 0.66 ± 1.03 | 3.50 ± 0.83 | |
IV | 0 ± 0 | 0 ± 0 | 0.16 ± 0.40 | |
V | 0 ± 0 | 0 ± 0 | 0.83 ± 0.98 | |
VI | 0 ± 0 | 0 ± 0 | 2.00 ± 1.67 |
Effect of 5% SAP on the liver histopathology in the broiler chicken fed a diet with naturally contaminated with AF on day 21. The normal appearance of liver hepatocytes in group II (a); severe lipid degeneration with mononuclear cells infiltration in group III (6/6 samples) (b); lipid droplet (arrow) inside the cytoplasm of hepatocytes due to lipid degeneration in group IV (1/6 samples) (c); lipid degeneration (arrow) with sinusoid congestion (c) in group V (2/6 samples) (d); lipid degeneration (arrow) and hydrophobic degeneration (arrowhead) with mononuclear cells infiltration (m) in group V (2/6 samples) (e); bile-duct proliferation (p) and mononuclear cells infiltration (m) on the portal triad area in group VI (3/6 samples) (f); local mononuclear cells infiltration (m) in group VI (3/6 samples) (g). H&E, 10× (b, f); 40× (e); 100× (c, g); 1000× (a, d).
Group III shows the most severe kidney histopathological change (necrosis, degeneration, and inflammation) compared with the others (
Effects of 5% SAP on the kidney histopathology of broiler chickens fed a diet naturally contaminated with AF at 7, 14, and 21 days of age.
Parameter | Group | Day | ||
---|---|---|---|---|
7 | 14 | 21 | ||
Necrosis | I | 0 ± 0 | 0.16 ± 0.40 | 0.50 ± 0.54 |
II | 0 ± 0 | 0.16 ± 0.40 | 0.33 ± 0.51 | |
III | 0 ± 0 | 1.00 ± 0.89 | 2.16 ± 0.75 | |
IV | 0 ± 0 | 0.16 ± 0.40 | 0.50 ± 0.54 | |
IV | 0 ± 0 | 0.16 ± 0.40 | 0.66 ± 0.81 | |
VI | 0 ± 0 | 0.16 ± 0.40 | 0.66 ± 0.81 | |
| ||||
Degeneration | I | 0 ± 0 | 0.33 ± 0.51 | 0.50 ± 0.54 |
II | 0 ± 0 | 0.16 ± 0.40 | 0.33 ± 0.51 | |
III | 0 ± 0 | 0.83 ± 0.75 | 1.50 ± 1.04 | |
IV | 0 ± 0 | 0.50 ± 0.83 | 0.66 ± 1.21 | |
V | 0 ± 0 | 0.33 ± 0.51 | 0.50 ± 0.83 | |
VI | 0 ± 0 | 0.33 ± 0.51 | 0.50 ± 0.83 | |
| ||||
Inflammation | I | 0 ± 0 | 0 ± 0 | 0 ± 0 |
II | 0 ± 0 | 0 ± 0 | 0 ± 0 | |
III | 0.16 ± 0.40 | 1.00 ± 0.89 | 1.33 ± 1.21 | |
IV | 0 ± 0 | 0 ± 0 | 0.16 ± 0.40 | |
V | 0 ± 0 | 0.50 ± 0.83 | 0.50 ± 0.83 | |
VI | 0 ± 0 | 0.50 ± 0.54 | 0.50 ± 0.54 |
Effect of 5% SAP on the kidney, spleen, and BF histopathology in the broiler chicken fed a diet with naturally contaminated with AF on day 21. The normal appearance of kidney in group II (a); mononuclear cells infiltration (m) in the interstitial with necrosis (arrow) of the tubules epithelial of kidney in group III (b); and group V (c); necrosis (arrow) and degeneration (arrowhead) of the tubules epithelial of kidney in group VI (d); severe depletion (arrow) in the white pulp of the spleen in group III (e); massive congestion (c), depletion of lymphocytes (arrow), with polymorphonuclear cells infiltration (p) in the white pulp of the spleen in group VI (f); depletion of lymphoid follicle (arrow), extensive mononuclear cells infiltration (m), and congestion (c) of the BF in group III (g); irregular size of lymphoid follicles due to depletion of lymphocytes (arrow) with polymorphonuclear cells infiltration (p) in the BF in group VI (h). H&E, 40× (b); 100× (d, e, g); 1000× (a, c).
Spleen and BF was the prominent immunological organ. Inside the spleen and BF parenchyma the T-cells subsets can be found, such as CD4+ and CD8+ lymphocytes. The minimal doses of AF (>1 ppb <50 ppb) exposure affect the immune-expression of both CD4+ and CD8+ lymphocytes. It is proved by the results of this study that show the decrease in CD4+ and CD8+ lymphocytes in the spleen and bursa of Fabricius (
Effects of 5% SAP on the immune-expression of CD4+ (a) and CD8+ (b) lymphocytes in the spleen of broiler chickens fed a diet naturally contaminated with AF at 7, 14, and 21 days of age.
Effects of 5% SAP on the immune-expression of CD4+ (a) and CD8+ (b) lymphocytes in the BF of broiler chickens fed a diet naturally contaminated with AF at 7, 14, and 21 days of age.
Effects of 5% SAP on the ratio of CD4+/CD8+ lymphocytes in the spleen (a) and BF (b) of broiler chickens fed a diet naturally contaminated with AF at 7, 14, and 21 days of age.
Effect of 5% SAP on the immune-expression of CD4+ and CD8+ lymphocytes in the spleen and BF of the broiler chicken fed a diet with naturally contaminated with AF on day 21. High immune-expression of CD4+ lymphocytes (arrow) (a); low immune-expression of CD8+ lymphocytes (b) in the white pulp of the chicken’s spleen in group V; high immune-expression of CD4+ lymphocytes (arrow) (a); low immune-expression of CD8+ lymphocytes (b) in the lymphoid follicle of the chicken’s BF in group V. IHC antibody anti-CD4+, DAB, 100× (a), 1000× (b); IHC antibody anti-CD8+, DAB, 100× (c), 1000× (d).
AF is a mycotoxin produced by
One of the AF potential effects is mutagenic, teratogenic, and hepatocarcinogenic [
Every nutrient produced through the metabolism process is circulated by the RBC. In the poultry aflatoxicosis, the AF depressing the haemopoietic tissues might result in decreasing of RBC production [
In contrast to erythrocytes, leucocytes do not exhibit lifespan in the circulatory system but rather leave the circulatory system and infiltrate to the tissues at random times in response to chemoattractant stimuli [
The liver is the primary organ in detoxification during aflatoxicosis. In the liver, the toxin especially AF is absorbed and transformed into its metabolites form. The metabolites form of AF binds the nucleic acids and protein of the hepatocytes, causing the liver residue [
The low levels of AF contamination in the feed (50 and 100 ppb) with chronic exposure were reported as a source of liver histopathological change [
Both CD4+ and CD8+ lymphocytes have a prominent role in the activation of the immunological complex via major histocompatibility complex (MHC) class II and class I. In aflatoxicosis, there is a changing of T-cells subpopulation in the primary lymphoid tissues (thymus and BF) [
This study clearly demonstrated that 5%
The data used to support the findings of this study are available from the corresponding author upon request.
This research has no funded by the third party and this work is a part of the author’s academic project.
The author declared that there are no conflicts of interest.
Mrs. Marina Dwi Nurhayati (Department of Clinical Pathology), Dr. Dewi Pratamasari, and Mr. Soetopo (Department of Pathology) from Disease Investigation Center (DIC), Yogyakarta, Indonesia, were acknowledged for their assistant during this study.