Comparative consequences of two sources of aflatoxins in ducklings experimental aflatoxicosis.

The purpose of this study was to investigate the effect of two aflatoxins (AFs) sources for experimental induction of aflatoxicosis in ducklings. Dietary supplementation of aflatoxin-contaminated grounded rice grains previously was cultured by Aspergillus parasiticus and dietary supplementation of aflatoxin methanolic extract of contaminated rice grains. A total number of 18 four-day-old ducklings were treated with varying sources of AFs. Treatments included: A: Control (basal diet without AFs), B: Contaminated feed with 0.20 mg kg-1 AFs (ground rice grains), C: Contaminated feed with 0.20 mg kg-1 AFs (methanol extract of contaminated rice grains). Results showed a significant decrease in serum levels of total protein, albumin, glucose, triglyceride, cholesterol, HDL, LDL and creatinine. The serum's hepatic enzymes levels were not affected in AFs-treated groups but lactate dehydrogenase (LDH) concentration increased by the two AFs sources. The relative weight of the liver and spleen was significantly increased in AFs-fed groups. Histopathological liver examination showed the vacuolar degeneration with small and large lipid droplets in hepatocytes in the AFs- contaminated groups. Dietary AFs resulted in a significant decrease of villus height, villus width and villus surface area of the small intestine compared to the control group. In conclusion, the results showed that the induction of experimental aflatoxicosis via two investigated AFs sources had slight differences concerning the studied parameters. It seems the group consumed ground rice grains indicated slightly fewer aflatoxicosis symptoms than the methanolic extract.

Introduction

Mycotoxins are secondary metabolites of fungal and aflatoxins are the most thoroughly studied mycotoxins, which were identified as a serious livestock problem in the 1950's. However, it was only in 1960 during the studies in the United Kingdom which was called turkey “x” disease causative agent. Aspergillus flavus and Aspergillus parasiticus were recognized as the organisms accountable for the expansion of the toxin in the feed.[1] The sensitivity to AFs varies according to species, dose, gender, age and nutritional status of the animal.[2] Oral administrations of AFs or aflatoxin B1 (AFB1) alone in the diet as a contaminated-rice powder,[3]-[5] dissolved in olive oil by stomach tube,[6],[7] or in drinking water by oral gavage[8] caused liver tumors (hepatocellular tumors) in all species tested. Many studies on AFs toxicity have been conducted; however, there are few researches on the efficiency of different sources and methods of application in vivo in animal models. Researchers investigated the production of AFs on the agricultural commodities including rice, wheat, corn, soybeans and sorghum, and found that rice grain was the best substrate.[9] Recently, extraction methods have been developed for the separation and purification of active compounds from a solid matrix and increases their access and influence compared to the raw substrate.[10] There are wide varieties of solvents used for the extraction of mycotoxins. The most common solvents used for the extraction of mycotoxins from the matrix such as herbal medicines are acetonitrile, methanol and chloroform.[11] Therefore, it was assumed in the current study that due to increased access to these active compounds, AFs extracted from a raw medium such as rice grain can effectively induce aflatoxicosis in a short time by describing the serum biochemical and intestinal morphological indices in an experimental animal model.

Materials and Methods

Experimental birds and diets. The animal use protocol was approved by the Institutional Animal Care of Tarbiat Modares University, Tehran, Iran (82/2067-March, 07, 2016). A total number of 18 four-day- old Pekin ducklings with an average initial body weight of 62.50 ± 5.00 g were obtained from a local hatchery (Moghadam Farm, Qom, Iran). The ducklings were then weighed, labeled, and randomly assigned to 18 battery cages (200 cm2 floor spaces per bird) equipped with nipple waterers in an environmentally controlled room. The two weeks experiment consisted of three treatments with six individual birds in a completely randomized experimental design. All diets were formulated to meet or exceed the National Research Council requirements for ducks from 4 to 18 days of age (Table 1).[12] Dietary treatments included: A: Control (basal diet without AFs), B: Contaminated feed with 0.20 mg kg-1 AFs (contaminated rice grains powder) C: Contaminated feed with 0.20 mg kg-1 AFs (methanol extract of contaminated rice grains). Feed and water were provided ad libitum during the period of study (4 to 18-day post-hatch). Ducklings were monitored daily for any health problems.

Table 1

Ingredient composition and proximate analysis of diet.

Ingredients	Quantity (%)
Corn	56.29
Soybean meal	39.05
Soy oil	1.69
MCP	1.19
CaCO3	0.88
Common salt	0.34
DL-Methionine	0.06
Vitamin and mineral premix*	0.50
Total	100
Nutrients composition (%)
Metabolizable energy (kcal kg -1 )	2,900
Crude Protein	22.00
Calcium	0.65
Available phosphorus	0.40
Methionine	0.40
Methionine + cystine	0.76
Lysine	1.19
Threonine	0.83
Sodium	0.15

*Provided per kg of diet, Vitamin A: 8,000 IU, Vitamin D3: 1,200 IU, Vitamin E: 3.00 IU, Vitamin K3: 2.00 mg, Riboflavin: 8.00 mg, Nicotinic acid: 10.00 mg, Pantothenic acid: 150 mg, Copper: 2.00 mg, Iodine: 1.20 mg, Cobalt: 0.20 mg, Selenium: 0.10 mg.

Aflatoxins production. The AFs were produced via fermentation of rice by Aspergillus parasiticus PTCC-5286. The sterile substrate, placed in Erlenmeyer flasks, was inoculated with 2.00 mL of an aqueous suspension of the mold containing 107 spores mL-1. Cultures were allowed to grow for 7 days at 25.00 ˚C in darkness. On the seventh day, the Erlenmeyer flasks were autoclaved, and the culture material was dried for 48 hr at 40.00 ˚C in a forced-air oven and then ground to a fine powder.[13] The AFs levels in the ground culture material were measured by HPLC method.[14] The contaminated rice powder (raw substrate containing AFs) was incorporated into the basal diet to provide 0.20 mg AFs kg-1 of feed as the source 1, and the AFs extracted with methanol was sprayed on the basal diet to provide 0.20 mg AFs kg-1 of feed as source 2.

Sample collection. At the end of the experiment, ducklings were fasted for 12 hr. The body weight of each bird was recorded, and a 5.00-mL blood sample was collected from jugular vein in a test tube without anticoagulant. Blood samples were centrifuged at 2,500 g for 10 min, and the serum was separated and stored in 1.50 mL centrifuge tubes at −20.00 ˚C until analysis. After bleeding, birds were euthanized by decapitation for organ weight measurement, including heart, liver, spleen, kidney and bursa of Fabricius and their relative weights were calculated as (organ weight/live body weight) × 100.

Serum biochemistry and enzyme activity. Six replicate serum samples per treatment (18 samples in total) were analyzed for albumin, globulin, total protein, glucose, uric acid, creatinine, triglyceride, cholesterol, HDL, LDL, alanine aminotransferase (ALT), alkaline phos-phatase (ALP), aspartate aminotransferase (AST), lactate dehydrogenase (LDH). The analyses of the serum samples were performed by spectrophotometric methods using commercially available kits (Pars Azmun, Tehran, Iran).

Intestinal morphology. The digestive tract was carefully excised and sections were removed from the jejunum, the samples were flushed with physiological saline and plunged in 10.00% formalin, processed by the standard paraffin sectioning, stained by Hematoxylin-Eosin (H&E) and examined under a light microscope. Villus length was measured from the top of the villus to the upper part of the lamina propria, crypt depth was measured from the base upwards to the region of transition between the crypt and villus, villus width was measured at the widest area of each villus[15] and villus surface area was calculated using the formula:

Area (mm 2 ) 2π× (VW/2) ×VL

where, VW is the villus width (µm) and VL is the villus length (µm).[16] Reported values were means of 10 villi from each bird, six birds per treatment.

Liver histopathology. Parts of livers were fixed in 10.00% neutral buffered formalin and the fixed tissues were trimmed, embedded in paraffin, and stained with H&E for histopathological examination by light microscope.

Statistical analysis. All data were analyzed using the GLM procedure using SAS software (version 9.1; SAS Institute, Cary, USA) for completely randomized design. The means were compared by Duncan's multiple range tests (p ≤ 0.05).

Results

The results of HPLC analysis to assess the concentration of AFs in rice powder samples are presented in Table 2. The relative weights of heart, gizzard, bursa of Fabricius and proventriculus were not significantly affected by treatments (p > 0.05), (Table 3). The ducklings fed AFs contaminating diets by either source had higher relative weight of the spleen compared to control group (p ≤ 0.05), however, there was no significant difference between the AFs contaminated groups with various sources. Further-more, the AFs contaminate diets by the methanol extraction had higher relative weights of the liver compared to other experimental groups (p ≤ 0.05).

Table 2

HPLC analysis for rice powder samples

Aflatoxins	Concentration ( mg kg -1 )	Limit	LOQ
AFB 1	14.25	< 5.00	0.40
AFB 2	1.04	ND	0.08
AFG 1	ND	ND	0.40
AFG 2	ND	ND	0.08
Total	15.29	< 3000	-

LOQ: Limit of quantification, ND: Not detected.

Table 3

Effects of treatments on weight of viscera and jejunum morphology in ducklings (18-days old).

Parameter	Treatments			p -value
	A	B	C
	-	AFs (from raw rice grain powder)	AFs (from methanol extract)
Bursa of Fabricius (g per 100 g of BW)	0.13 ± 0.04	0.10 ± 0.04	0.11 ± 0.03	0.36
Liver (g per 100 g of BW)	5.29 ± 1.15b	5.62 ± 0.70ab	7.40 ± 1.40a	0.04
Spleen (g per 100 g of BW)	0.20 ± 0.06b	0.33 ± 0.02a	0.32 ± 0.07a	0.02
Heart (g per 100 g of BW)	0.93 ± 0.16	0.86 ± 0.14	0.88 ± 0.14	0.77
Gizzard (g per 100 g of BW)	6.65 ± 0.94	5.79 ± 1.46	6.56 ± 0.75	0.51
Villus height (µm)	767.80 ± 118.65a	663.30 ± 93.47b	548.30 ± 127.63 c	0.001
Villus width (µm)	108.50 ± 22.13	109.30 ± 30.11	101.60 ± 21.18	0.50
Crypt depth (µm)	5.30 ± 1.42b	6.90 ± 1.35a	5.50 ± 1.03b	0. 008
Goblet cell density (No. per 100 µm)	5.10 ± 1.46	4.40 ± 0.74	4.60 ± 0.92	0. 17
Villus surface area (mm 2 )	0.25 ± 0.07a	0.22 ± 0.06a	0.17 ± 0.05b	0.066

ab Means with the same superscript in the same row for each variable showing no significant difference (p ≤ 0.05).

Morphometric studies showed that feeding ducklings with AFs contaminated diets by both sources resulted in a significant decrease in jejunum villus height and villus surface area compared to the control group (p ≤ 0.05). Jejunum villus width and goblet cell counts were not significantly affected by treatments (p > 0.05). Feeding 0.20 mg kg-1 AFs in the form of contaminated rice powder and methanol extract resulted in the reduction of serum total protein, albumin, glucose, triglyceride, cholesterol, HDL, LDL, and creatinine (p ≤ 0.05). However, triglyceride and creatinine levels were not significantly different between the groups of methanol extract and control. There was no difference in ALT, AST, and ALP levels among the groups, however, LDH level was affected significantly by different dietary treatments (p ≤ 0.05), (Table 4). The group received contaminated rice powder showed a significant increase in comparison with the other dietary treatments (p ≤ 0.05). Histopathological examinations disclosed accumulation of large fat droplets replacing the nucleus and resulting in cell swelling (hydropic degeneration) in hepatocytes of duckling given dietary aflatoxin by the two AFs sources. In the control group, the hepatic tissue structures were normal and showed no sign of liver injury as judged by light microscopic observations (Fig. 1).

Table 4

Effects of treatments on serum biochemical parameters and hepatic enzymes activity (18 days old)

Parameter	Treatments			p -value
	A	B	C
	-	AFs (from raw rice grain powder)	AFs (from methanol extract)
Protein (g dL -1 ) Total	4.80 ± 0.11a	4.49 ± 0.26ab	4.42 ± 0.34 b	0.05
Albumin (g dL -1 )	1.86 ± 0.05a	1.55 ± 0.07b	1.61 ± 0.06b	0.001
Globulin (g dL -1 )	2.94 ± 0.09	2.87 ± 0.28	2.88 ± 0.20	0. 81
Triglyceride (mg dL -1 )	155.16 ± 8.67a	132.59 ± 12.71b	146.55 ± 14.25ab	0.01
Cholesterol (mg dL -1 )	181.47 ± 14.76a	135.45 ± 10.91b	125.70 ± 8.24b	0.04
HDL (mg dL -1 )	47.39 ± 2.92a	40.72 ± 4.33b	41.56 ± 4.87b	0.03
LDL (mg dL -1 )	134.08 ± 12.81a	95.22 ± 7.22b	84.14 ± 4.35c	0.001
Glucose (mg dL -1 )	139.88 ± 7.98a	132.44 ± 11.75ab	121.85 ± 13.63b	0.04
Uric acid (mg dL -1 )	5.66 ± 0.36b	6.38 ± 0.53a	6.13 ± 0.35ab	0.03
Alanine aminotransferase (IU L -1 )	46.64 ± 18.26	53.60 ± 19.50	49.18 ± 26.66	0.94
Aspartate aminotransferase (IU L -1 )	48.74 ± 13.89	59.66 ± 26.80	70.14 ± 5.55	0.15
Lactate dehydrogenase (IU L -1 )	780.40 ± 212.04b	1375.60 ± 391.04a	1012.90 ± 244.82b	0.01
Alkaline phosphatase (IU L -1 )	167.41 ± 39.78	191.92 ± 58.46	153.93 ± 14.58	0.30

ab Means with the same superscript in the same row for each variable showing no significant difference (p ≤ 0.05).

Fig. 1

Photomicrographs (optical microscopy) of Hematoxylin and Eosin-stained ducklings liver sections from different treatments A) Normal histological appearance of the liver in the control group, B) Vacuolar degeneration with small and large fat droplets (black arrows) in ducklings given dietary AFs at concentration of 0.20 ppm via methanol extract and , via rice powder (C)

Discussion

Aflatoxins have been reported to affect the various body organs and increase the size of liver, kidney, gizzard and spleen and decrease the weight of bursa of Fabricius and thymus.[17] The addition of AFs in the diet of broilers showed a significant increase in relative weights of liver, kidney, gizzard and spleen.[18],[19] Regardless of the atrophy of the bursa of Fabricius and thymus glands, the apparent alteration of splenic function is also of diagnostic significance and implies the alteration in the birds with aflatoxicosis.[17] The liver is intended the target organ for AFs because it is the organ where most AFs are converted to the reactive 8,9-epoxide form, which is found to bind DNA and proteins, damaging the liver cell and increasing liver weight.[20],[21] Our results were in agreement with previous reports that AFB1 reported to cause pallor discoloration and enlargement of the liver in broiler and ducklings.[22],[23]

The gastrointestinal tract is the principal route of entry of mycotoxin- contaminated diet also the main path of excretion aflatoxin metabolites from the bile.[24] A larger small intestine volume or surface area (the surface area is further increased by villi and microvilli) presumably allows more area over which these nutrients can be digested and absorbed and also the villus surface area is an important factor for gastrointestinal absorption.[25] According to results of the present study absorptive surface jejunum was declined by consumption of AFs sources.

Intestinal epithelial cells can be exposed to high concentrations of toxins by ingestion of aflatoxins-contaminated feed and direct intestinal injury can be applied by the biological action of mycotoxins.[26] Epithelium cells lining the small intestine have a high turnover, as it is necessary to hold the natural balance. Investigation showed that intestinal morphology (intestinal crypt depth) and the specific activity of intestinal disaccharidase and maltase were also altered by AFB1 ingestion.[27] Broilers fed diets contaminated with 0.50 mg deoxynivalenol kg-1 had shorter and thinner villi which resulted in lighter small intestines compared to birds fed control diets.[28]

According to the results obtained in the current study ducklings exposed to two various sources of AFs showed a decline in the jejunum villus height and subsequently a decrease in the villus surface area. The small intestine provides the main way for excretion of xenobiotics.[29] Enzymatic biotransformation in intestinal mucosal cells has the potential to remove these generally hydrophobic xenobiotics by directly making easy their repulsion to the intestinal lumen, or by conjugation with subsequent excretion.[28] However, the biotransformation could also potentially activate some xenobiotics such as AFs with toxic effects on the organism. One of the important structures on the apical surface of small intestines epithelial cells is microvilli.[29] Investigators reported that intestine function and growth performance of animals are associated with the morphology of the villi and crypts.[30] The involvement of microvilli has been established in various functions such as secretion, mechanotransduction, absorption, and cellular adhesion. On the other hand, epithelial cell mitochondrion plays important roles not only in producing ATP but in controlling apoptosis and contributing to the calcium homeostasis process of cells.[31] Damage to the mitochondria and microvilli by aflatoxins can cause dysfunction of these structures resulting in functional disorders of absorptive cells in the small intestine because it can induce reactive oxygen species to cause oxidative stress by damaging cells and DNA and induce genetic alterations leading to DNA damage and mitochondrial permeability alterations.[32]

Aflatoxicosis is related to serum biochemical changes involving decreased total protein, albumin, cholesterol and triglyceride values[33] which are the results of impaired carbohydrate and lipid metabolism.[34]

The liver is the primary site for protein synthesis, enzymatic metabolism, detoxification protective functions, AFs activation and their toxicity.[35] The AFB1 adducts with biomolecules cause damage to liver cells that have a negative effect on metabolic functions of the liver during AFB1 exposure. This is represented by AFB1 that reduced total serum protein levels, as the liver is responsible for the production of most proteins and aflatoxicosis negatively affects albumin, globulin, cholesterol, and triglyceride levels in serum.[33],[36] Protein content may be decreased because AFB1-DNA adducts prevent transcription or translation and AFB1-lysine adducts result in protein degradation or excretion. AFB1-exposed chickens exhibited a reduction in hepatic fatty acid synthesis which could be accountable for the lower production of serum cholesterol and triglycerides.[1] The results of this study in regards to reduction in serum total protein, albumin, cholesterol, and triglyceride were in agreement with previous results on broilers[34] and ducks.[37]

Aflatoxin has a marked effect on hepatic enzyme activities that reflect liver damage and leakage of enzymes in the bloodstream.[36] Previous researchers showed that consuming aflatoxin increased the concentration of liver enzymes especially AST, ALT and LDH in broilers[34],[38] and ducks.[36] In the present study, the increase in LDH activity due to aflatoxins consumption by two sources was found to be significant. It has been reported that LDH level may be elevated due to cell necrosis in many diseases.[39]

Liver is the major target organ in terms of the pathological effects of the mycotoxin.[15] AFs can induce reactive oxygen species generation leading to oxidative stress and liver injury.[40] Hepatocytes control hepatic biochemical and metabolic functions in the liver, including triglyceride metabolism.[41] Disorder of the release in hepatic triglyceride to the plasma due to aflatoxicosis resulted in the accumulation of large lipid droplets in liver tissue and changes in plasma lipid profile.[42] In the present study, significant changes were observed in histopathology in birds fed AFs contaminated diets by two sources, although no changes were observed in control group that were in agreement with previous investigations.[43]-[45]

In conclusion, the current study demonstrated that feeding ducklings with AFs-contaminated diets by the two applied sources from 4 to 18 day of age-induced deleterious effects on organ development, intestinal morphology and biochemical parameters. According to the results obtained from the analysis of target parameters it seemed that diets contaminated with two sources exerted harmful effects, although in the methanol extract consumer group was slightly higher. A possible hypothesis for these observations was that the methanolic extract could provide quick access to effective compounds such as AFs compared to raw rice grain powder.