Efficacy of Ginger and Nutritox® in counteracting aflatoxin effects on white Pekin ducklings

E-mail: walaaabuelela@hotmail.com Objective: To evaluate the anti-aflatoxin effects of Ginger and Nutritox® in white Pekin ducklings. Design: Randomized experimental study Animals: Nintyone-day-old white Pekin ducklings. Procedures: Ducklings were alienated randomly into six equal groups, each group in 3 replicates (each replicate, n=5). Control group G1) was fed on commercial broiler basal diet without any medications, while other experimental groups, namely 2, 3, 4, 5 and 6 were received diet containing: G2) basal diet (BD) + 300 ppb AF (aflatoxin); G3) BD + 300 ppb AF + 250 mg kg-1 Gr (Ginger); G4) BD +300 ppb AF +1gm/kg feed Nutritox®; G5) BD + 250 mg kg-1 mg/kg of B.W Gr; G6) BD + 1gm/kg feed Nutritox® during study (1-14) days respectively. Results: Results showed that, addition of Ginger and Nutritox® ameliorated in different degrees the adverse effects of aflatoxin and improved growth performance and some serum biochemical parameters [Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Alkaline phosphatase (ALP)]. They hasten the recovery of antioxidant enzymes (MDA and GSH) in tissue to their normal levels during certain periods of our experiments. Histopathological liver changes run in the same track with biochemical results. Results revealed that administration of Ginger or Nutritox® in diet prevents and/or reduce some adverse effects of aflatoxin in ducks. Conclusion and clinical relevance: Our study indicates that ginger, an alternative natural product can provide protection against aflatoxin as Nutritox®.


INTRODUCTION
Aflatoxins are hepatoxic metabolites of Aspergillus flavus and Aspergillus parasiticus as well as Penicillium puberulum [1] . In livestock and laboratory animal, the most biologically active form of AF is aflatoxin B1 (AFB1); it is responsible of increasing mortalities, decreasing performance, increasing liver lesions, and immunosuppression in poultry [2][3][4]. Although extensive data is obtainable on the effects of aflatoxin in livestock animals, yet there is less on ducklings; the most sensitive poultry species, who reveal 100% mortality at 1 ppm AF [5].
The adverse effects of mycotoxins in broiler chicks were ameliorated by many physical, chemical and biological methods [6]. Chemoprevention of toxins using antioxidant nutrients is being investigated because of their ability to reduce disease formation by induction or inhibition of key enzyme systems [7]. Numerous herbal products were believed to have therapeutic benefits with minimal side effects, easy viability and long lasting curative effects for hepatic fibrosis [8; 9]. The biological method, using microorganisms or their metabolites, was suggested as a favorable alternative to detoxify mycotoxins [10].
Ginger (Zingiber officinale), is herbaceous rhizomatous perennial consumed as a medicine, delicacy, or spice. Its family name is Zingiberaceae [11]. It used as a medicinal compound in various traditional systems of medicine to treat common illnesses such as gastrointestinal disturbances, rheumatic complaints, diarrhea, nausea and parasitic infestations [12]. Laboratory studies have shown that ginger possesses free radical-scavenging, antioxidative, antiinflammatory, antimicrobial, antiviral, antidiabetic, antihypertensive, anticancer, chemo preventive and immunomodulatory effects [12; 13]. Ginger works in many different ways: as a scavenger of free radicals, inhibits or breaks the chain of lipid peroxidation, possesses antiinflammatory effects, enhances antioxidant defense system and modulates detoxifying enzymes [14].
Probiotic is a live microbial feed supplement which increasingly used in poultry diets to improve feed utilization, enhance growth rate and control intestinal infections [15]. Many commercial products are used for detoxification containing mycotoxin-binders that holds promise for using in contaminated feeds [16]. Lactobacillus cultures [17], yeasts and lactic acid bacteria [18] act as biological adsorbents that Mans vet Med J 20:4 (2019) 21-28 prevent absorption of aflatoxin from intestine of humans and animals. Nutritox ® , one of a commercial mycotoxin binder products existing in the Egyptian market and used in broiler diet contaminated with aflatoxin [19; 20]. Therefore, the aim of the current investigations was to evaluate the ameliorative effect of herb (ginger) against aflatoxicosis in white Pekin duckling and to focus on the hepatoprotective of ginger (as a herbal compound) in prevention and treatment of hepatic damage induced by aflatoxin compared with Nutritox ® as a detoxifying commercial product mixture.

Diet
Broiler diets (EL-Baraka Company) were analyzed in the Mycotoxins Central Lab and Food Safety of the National Research Center (NRC) to make sure that it was free from aflatoxins via high performance liquid chromatography (HPLC).

Artificially toxicated diet
Productions of aflatoxins were done by culturing of standard toxigenic strain of Aspergillus parasiticus NRRL2999, from fresh potato dextrose agar media (PDA) on rice according to the method of Shotwell et al. [21] in the laboratory of poultry diseases department, Faculty of Vet Med, Mansoura University. Moldy rice was autoclaved, dried, grounded to fine powder and analyzed for aflatoxins content by HPLC in mycotoxins central lab and food safety of the National Research Center (NRC). Ground rice was added to duckling basal diet to produce 300 ppb aflatoxin.

Body weight and feed intake
All experimental ducklings were daily observed for clinical signs, on day 7 and 14 all life birds were weighed individually and total feed intake were recorded for each pen. Feed conversion rate (FCR) was calculated according to the following formulae: FCR (g/ g) = FI (g)/ WG (g). Where FI is the Dry feed intake (g)

Serum samples
Blood samples were collected into a dry and clean centrifuge tubes without anticoagulant at day 7 and day 14. Clotted blood was centrifuged at 3000 rpm for 15 min and serum were separated and preserved at -20° C until submitted for biochemically analysis.

Liver samples
On 14 day 6 birds from each group were slaughtered, sacrificed and dead birds were subjected for postmortem examination. Liver was dissected out and divided into two parts; one part processed for histopathological examinations and the other used for liver homogenate.

Preparation of liver homogenate
Liver tissue was perfused with PBS (phosphate buffered saline) solution (pH 7.4) containing 0.16 mg / ml heparin to remove any red blood cells or clots. Tissue was homogenized in 10 ml cold buffer (i, e, 50 mM potassium phosphate, pH 7.5) per gram tissue, centrifuge (Hettich) at 4000 r-p-m for 15 minutes, then supernatant was removed and stored on ice for further analysis.

Histopathological examination
Liver specimens were placed in plastic cassette and immersed in 10% neutral buffered formalin. Five μm thick paraffin sections were prepared and stained with hematoxylin and eosin for histopathological examination [24].

Statistical analysis
All data were analyzed and expressed as means ± standard deviations of the means. Obtained data were analyzed by two ways analysis of variance (one ways ANOVA) using the SPSS software statistical package (SPSS version 16.0 for Window). Differences were considered statistically significant when P < 0.05.

Ducks performance
In the present study, the cumulative mortality was up to 13.3% and 20% at day 7 and 14, respectively ducklings fed Mans vet Med J 20:4 (2019) 21-28 on 300 ppb level of aflatoxin -contaminated diet (BAF). Only one bird died in ginger treated group in association with reduction of the severity of clinical signs, but no mortality was recorded in other groups.
As shown in tables 1 and 2, aflatoxin caused a sever reduction in body weight, weight gain, feed intake and growth performance. Weight gain of duckling fed Nutritox ® treated ration was significantly improved all over the experimental time points. Although the weight gain of duckling fed ginger (BAFGr) was significantly affected by aflatoxin in the 7 th day of life but not in the 14 th day. In table 2, FCR was significantly increased by aflatoxin (BAF) and significantly improved by ginger and Nutritox ® addition.

Biochemical parameters
As shown in table 4, aflatoxin spiked diet caused a significant increase in ALT, AST and ALP in the 7 th and 14 th day. Dietary supplementation of ginger to the aflatoxin contaminated diet (BAFGr) caused significant improvement in ALT levels in the 7 th and 14 th days. Serum activity of enzymes (ALT, AST and ALP) in ducks of (BAFNut) group revealed significant improvement in comparison to the normal picture at 7 th and 14 th days of experiment; except AST at 7 th day.
In table 5, feeding on aflatoxin spiked diet displayed significant increased and decrease MDA in and GSH level; respectively, when compared to control group. GSH level was improved (P < 0.05) and the elevated level of MDA was decreased significantly in BAFGr and BAFNut groups Coadministration of AF with ginger (BAFGr). No significant changed was found in (BNut) group fed only nutritox ® .

Histopathology
In the study, a significant increase in relative liver weight of sacrificed ducklings was noticed in BAF group, as shown in table 3. At day 14, the liver showed characteristic macrosteatosis, periportal intense inflammatory response, proliferation and fibrosis of the bile ductules, sometimes with apoptotic changes in their walls. Some of the hepatocytes and their nuclei were enlarged (megalocytosis). Marked peri-portal edema associated with compression atrophy of some bile ducts, icteric changes, multifocal periportal and midzonal coagulative necrosis together with adenomatous biliary proliferation were recorded (Fig.1).
Aflatoxin intoxicated ducklings treated with Nutritox ® showed Mild biliary proliferation, congestion of portal blood vessels , round cells infiltration (lymphoplasmacytic) and a small focal peri-portal areas of coagulative necrosis, infiltrated by lymphocytes and macrophages were seen, 14 days post-treatment (Fig.1).
Histological examination of liver sections from ducklings treated by Ginger (BGr) or Nutritox ® (BNut) for 7 or 14 days revealed, liver parenchyma covered by a thin connective tissue capsule. The hepatic parenchyma was arranged in hepatic lobules, composed of polygonal hepatocytes organized as irregular, radial interconnecting cords or groups of cells around a central vein and separated by blood sinusoids. In the boundary of each lobule a portal area was seen, consists of a branch of hepatic artery; one or more branches of hepatic vein and one to four branches of the bile ducts lined by cuboidal cells, such bile ducts, sometime contain a bile secretion. (Fig.1).

DISCUSSION
Although extensive information is available on the effects of aflatoxins in other livestock animals, yet there is less information regarding ducklings; the most sensitive poultry species [1; 5; 25]. One-day-old white Pekin ducklings were used in this experiment to evaluate the protective efficacy of ginger and Nutritox ® against aflatoxicosis.
In the present research work, the cumulative mortality was 13.3% and 20% at 7 th and 14 th day, respectively, in ducklings fed on 300 ppb level of aflatoxin -contaminated diet (BAF). LD 50 were assumed to be 1 ppm in white Pekin duckling [1]. Similar finding has been recorded in a previous report of Abbasi et al. [26] who recorded that mortality was significantly increased at level of 200 μg/kg AFB1 in diets from day 1 to 14. On contrary Chen et al. [27] reported that dietary AFB1 (0.21 mg/kg) did not affect duck mortality which averaged 3.5% across all treatments at 14 d of age. Interestingly, ginger administration in (BAFGr) reduced mortality rate. This result is in accordance with that of Doaa and Ghada. [28]. Ginger reduced the severity of clinical signs through improving protein digesting enzymes and decreasing pathogenic bacteria [29]. AFB1 in diet caused significant reduction in the growth of 3 weeks old mule duckling [30] and 14 day Pekin duckling [27; 31] with no beneficial effects in average daily gain. Adverse effects of aflatoxin were species and dosedependent, and might be due to the deleterious effects of aflatoxin B1 on protein, carbohydrate, lipid metabolism and pancreatic enzymes activity [10]. In accordance with that of previous reports [28; 32], weight gain of duckling fed Nutritox ® supplemented diet was significantly improved all over the experimental time points. Ginger increased the body weight and this may be due to its content of volatile oils as gingrol, zingibaine and zingiberol which accelerate digestion, improve protein digesting enzymes, stimulates lactic acid bacteria and decreases pathogenic bacteria [29; 33]. Our findings revealed that dietary supplementation of ginger significantly increased the feed intake. This improvement indicated that pungent taste and strong aromatic odor of ginger did not affect the feed intake [29; 34].
Interestingly, FCR was significantly increased by aflatoxin (BAF) [31] and significantly improved by ginger and Nutritox ® addition. Dietary supplementations of ginger increased fiber content in diet leading to decrease the feed energy concentration with better growth performance in chickens [35].
Liver function tests as evaluated by alanine aminotransferases (ALT), serum aspartate aminotransferases (AST) and alkaline phosphatase (ALP), indicated a liver damage as described by Kubena et al. and Ortatali et al. [36; 37]. Aflatoxin in diet caused a significant increase in ALT, AST and ALP. Changing in those enzymes during aflatoxicosis has been recorded previously by Mahmoud and Khorshom; Walaa et al. and Ostrowski-Meissner [1; 31]. Aflatoxin was reported to elevate serum levels of AST, ALT and ALP due to muscular trauma, liver damage [30; 36], hepatocellular damage in ducks, biliary cholestasis and hyperplasia of bile duct [27; 38]. Significant improvement in ALT levels was recorded in (BAFGr) group. These results were in harmony with Doaa and Ghada. [28]; where broiler chicks fed on ginger revealed positive effects on liver enzymes through a reduction in AST, ALT and ALP levels. Insignificant improvement in AST levels occurs in the 14 th day of aflatoxin administration. ALT is a specific cytoplasmic liver enzyme increased in hepatic diseases and fatty changes, while increase serum levels of AST alone is not indicative to specific organs [39]. Vipin et al. [40] found that ALP was not affected by ginger addition. Serum biochemical enzymes (ALT, AST and ALP) of ducks fed on aflatoxin plus Nutritox ® (BAFNut) revealed significant improvement when compared with aflatoxicated ducks (BAF). These results agree with those of Abdalla et al. and Walaa et al. [19;31]. This may be attributed to the beneficial effect of L. form of bacteria that reduce aflatoxin and improve health status [41].
GSH and MDA levels were analyzed to determine antioxidant status and the oxidative damage. MDA is a reactive aldehyde that appears during the peroxidation and degradation of polyunsaturated fatty acid in cell membrane, so it considered as an intermediate product of lipid peroxidation [42]. Aflatoxin spiked diet increased MDA level significantly and decreased GSH level significantly. Our results are consistent with Ayoub et al., 2010; Yang et al. and Vipin et al. [40;43;44]. Increase hepatic level of MDA could be attributed to AFB1 metabolites by cytochrome P450 enzyme to form AFB1-8,9-epxide, which in turn reacts with macromolecules such as DNA and lipid, which leads to lipid peroxidation and cellular injury. Depletion of liver GSH may be related to its conjugation with the electrophilic metabolites [40; 45].
Ginger is a good scavenger of peroxide radicals, and contains polyphenols that increase the antioxidant enzyme gene expression and derive the intracellular response against oxidative stress [46] that leads to increase GSH level, enhanced the detoxification by neutralization of reactive oxygen sources (ROS) and it protect the liver from injury [40].
Supplementation of nutritox ® with AF (BAFNut) significantly improved the decreased level of GSH and elevated level of MDA. These results are in accordance with those of [10; 47]. Serum and hepatic MDA level was relatively decreased, and the activity of GSH-Px increased markedly in liver when ducks fed on aflatoxin-contaminated diets containing Bacillus subtilis (B. subtilis) [47]. Additionally, dietary supplementation of yeast probiotic in broiler chicken enhance serum antioxidant enzyme activities [10].
The liver is the major target organ for aflatoxicosis [4]. Ahmed [21][22][23][24][25][26][27][28] to severe fatty changes, loss of architecture, mild to severe aggregation of lymphoid cell and heterophils in periportal and parenchymatous areas in addition to multifocal necrotic foci in liver of broiler chicken. The observed dystrophic and necrotic changes in liver, in our results, can be attributed to the altered primary macromolecules (lipids, proteins and DNA) provoked by the oxidative stress-induced DNA damage and lipid peroxidation. Aflatoxins undergo bioactivation to reactive 8,9-epoxide, which then binds to DNA and proteins [44; 50; 51]. Moreover, Aflatoxin В1 is cytotoxic for hepatocytes and inhibits their proliferation. The hyperplasia of bile duct epithelium occurs consequently to the direct toxic effect of AFB1 on bile duct epithelium or excessive production of prostaglandins due to AFB1-induced lipid peroxidation [52]. Marked micro-hepatosteatosis, moderate portal biliary proliferation, portal and interstitial lymphoplasmacytic aggregations together with peri-portal coagulative necrosis in BAFGr group. Our results are parallel with Doaa and Ghada. [28], who stated that coadminstration of ginger with aflatoxin, liver at 21 day showed foci of lymphohistiocytic exudate replaced hepatic exudates in broilers and also in rats [40]. Ginger improved the histoarchitecture of liver with well-formed polygonal hepatocyte arrangement and nearly normal sinusoids [53]. Ginger extract could be able to enhance hepatocytes regeneration, protect against membrane fragility, and decrease leakage of the enzymes into circulation [54].
In this study, Nutritox ® treated aflatoxicated ducklings (BAFNut) showed mild biliary proliferation, congestion of portal blood vessels, perei-portal edema, acute cholangitis and vacuolation of moderate number of hepatocytes. Nutritox ® ameliorates the adverse effect of AF and this attributed to its excellent content of essential micronutrients, organic acids and their salts and Lactobacillus acidophilus extract [19], which have adsorbing capacity and play an important role in preventing mycotoxins absorption through the gastrointestinal tract, so help in getting rid of these toxins outside the bird body [20].

Conclusion
Administrations of Ginger and Nutritox ® in diet could ameliorate the adverse effects of aflatoxin in ducks diet. Although we can consider ginger as a natural alternative anti-aflaoxin, yet Nutritox ® is still giving much higher protection against aflatoxicosis than ginger.