Enhancing The Immune Responses By Curcuma longa For ...kenanaonline.com/files/0085/85088/10119 - Hamada Elwan.pdf · contaminated diets improves the antioxidant, biotransformation,

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International Poultry Conference - Proceeding

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3 – 6 November 2014, Ain Sukhna, Red Sea – Egypt -(- 380 -)-

Enhancing The Immune Responses By Curcuma longa For Broiler chicks

Exposed to Aflatoxins Contaminated Diets

H. A. Elwan; A. H. EL- Bogdady; A. M. M. Hamdy; M. A. Toson; and S. A. Abdel-latif

Animal production Department, Faculty of Agriculture, Minia University, Egypt

Running Title: Alleviating the adverse effects of Aflatoxins by using herbal sources

CORRESPONDENCE TO: Dr. Hamada Elwan, Lecturer of Poultry Physiology

Work Address: Animal production Department, Faculty of Agriculture, Minia University,

Egypt

P.O. Box 61519

El-Minia, Egypt

Tel: 00201065656170

Fax: 0020862362182

E-mail: [email protected]

mailto:[email protected]

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Abstract:

Possible modulatory effect of Curcuma longa on immunological depression caused

by aflatoxins contamination had been examined. A total number of 160 unsexed, one day

old Ross broiler chicks were randomly distributed into four experimental groups for 40

birds each. The experimental basal diet contained 21.2%CP and 2950 kcal ME/Kg diet. The

rations of the four groups were; Birds in group 1 fed a basal diet and served as control;

those of group 2 fed a basal diet supplemented by 0.5% Curcuma longa; those of group 3

exposed to a basal diet contaminated with aflatoxins (AFs) 100 µg/ kg diet and birds in

group 4 received a contaminated basal diet supplemented with 0.5% Curcuma longa. The

experiment lasted for sex weeks of age.

Obtained observations reveal that Curcuma longa and AFs had significantly (P<

0.05) opposite directions on total and differential white blood cells count. A negative

precipitation test, total immunoglobulin and hemagglotination were decreased significantly

(P<0.05) due to AFs contamination. While by adding Curcuma longa they not only

recovered but recorded a significant (P< 0.05) improvement. Chemotactic movement of

white blood cells showed moderate (at 3 wks.) and weak at (6 wks.) for birds exposed to

AFs contaminated diet. However, white blood cells for birds received Curcuma longa

recorded fast (at 3 wks.) and very fast at (6wks.) chemotactic movement. In conclusion

both innate and humeral immune responses were enhanced (P<0.05) after three and six

weeks of age by the addition of Curcuma longa to AFs contaminated diet.

Key words: Aflatoxins, Curcuma longa, white blood cells, Chemotactic movement, Total

Immunoglobulin, Hemagglotination.

1. Introduction:

Birds’ commercial feed contains several cereals and oil seed, such as wheat, corn,

soybean, barley, oats, sorghum, and sunflower. These raw materials may be contaminated

with toxigenic fungi (genus Aspergillus, particularly A. flavusand A. parasiticus; Cotty

and Garcia, 2007) containing different mycotoxins; among them, aflatoxins (AFs) are the

prevalent toxic compounds of poultry feed (CAST, 2003). Twenty AFs have been

identified, the major ones being AFB1, AFB2, AFG1, and AFG2; which aflatoxin B1 (AFB1)

being the most common and toxic compound (Hussein and Brasel, 2001). The effects of

AFs are dose- and time-dependent, and 2 different forms of contamination with AFs

(aflatoxicosis) have been reported: acute and chronic (Osweiler, 1990). Liver is the main

target organ of AFs (Sawhney et al., 1973). Biochemical-hematological, immunological,

and pathological effects of AFs have also been well-described (Kiran et al., 1998; Qureshi

et al., 1998; Oğuz et al., 2000a). With increasing knowledge and awareness of AFs as a

potent source of health hazards to both man and farm animals, producers, researchers and

government organizations are making great effort to develop effective preventive

management and decontamination technologies to minimize the toxic effects of AFs

content in foods and feedstuffs. In order to reduce the toxic and economic impact of

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mycotoxins, established regulations and legislative limits have been set for AFs in poultry

feed. Many countries follow a maximum permissible level of 20 ppb for AFs in poultry

feed (CAST, 2003). Appropriate pre and post-harvest contamination can be reduced by

using appropriate agricultural practices. However, the contamination is often unavoidable

and still remains a serious problem associated with many important agricultural

commodities, which emphasizes the need for a suitable process to inactivate the toxin.

Besides the preventive management, several approaches have been employed including

physical (feed mill techniques, blending, extraction, irradiation, and heating), chemical

(acids, bases, alkali treatments and oxidizing agents) biological treatments (certain species

of fungi and bacteria) and solvent extraction to detoxify AFs in contaminated feeds and

feedstuffs (Piva et al., 1995; Parlat et al.,1999). Since the beginning of 1990s, the

adsorbent-based studies have also been reported to be effective in removing AFs from

contaminated feed and minimize the toxicity of AFs in poultry (Ibrahim et al., 2000).

Among several adsorbents commercially available in the market, Zeolites (Miazzo et al.,

2000), bentonites (Pasha et al, 2007) and clinoptilolite (CLI), (Oguz et al.,2000 b), were

preferred because of their high binding capacities for AFs and their reducing effect on AF-

absorption from the gastrointestinal tract. All these methods cannot be used in practical

feed manufacturing, because of the limitation of the nutrients decomposition, non-

availability of commercial methods and their residual effects. The increasing number of

reports on detoxification of AFs in poultry feed using different techniques has given rise to

a demand for practical and economical detoxification procedures. Some of the physical

treatments are reported to be relatively costly and may also remove or destroy essential

nutrients in feed. Natural compounds with antioxidant properties are potentially very

efficacious because of their ability to act as superoxide anion scavengers. Interesting results

have been obtained by food components contained in Curcuma longa, coffee, strawberries,

tea, pepper, grapes, Fava tonka, garlic, cabbage, and onions. Beneficial effects of

bioactive plant substances in animal nutrition may include the stimulation of appetite and

feed intake, the improvement of endogenous digestive enzyme secretion ,activation of

immune responses and antibacterial, antiviral and antioxidant action (Dorman and Deans,

2000; Brugalli, 2003; Hosseini_Vashan et al. 2011). Turmeric rhizome (Curcuma

longa) is an extensively used spice, food preservative and coloring material that has

biological actions and medicinal applications (Ishita et al., 2004, Akbarian et al.

2012). Curcumin is the main important bioactive ingredient responsible for biological

activity of Curcuma longa (Nouzarian et al. 2011). Curcumin has been shown to have

several biological effects, exhibiting antifammatory (Holt et al. 2005) antioxidant

(Hosseini_Vashan et al. 2012). It is used in gastrointestinal and respiratory disorders

(Anwarul et al. 2006). A number of studies have been conducted to evaluate its effect on

the performance of broiler chickens, laying hens and rabbits, (Suvanated et al. 2003;

Samarasinghe et al. 2003; Emadi and Kermanshahi, 2007; Zainali et al. 2009;

Nouzarian et al. 2011; Hosseini_Vashan et al. 2012). Additionally, some medicinal

herbs and plant extracts could potentially provide protection against AFB1. Plant

compounds like coumarins, flavonoids, and curcuminoids have inhibitory action on

biotransformation of AFs to their active epoxide derivatives. Turmeric (Curcuma longa), a

medicinal plant native to the Asian subcontinent, is known to possess antimicrobial and

antioxidant properties. The powder of dried roots and rhizomes of turmeric is used as one

of the spices in many countries. The curcuminoids, yellowish pigments present in turmeric

powder, have shown protective effects against AFB1 (Lee, et al., 2001). Supplementation

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of curcumin in the diet normalized the altered activities of LDH and ALT induced by AFs.

At molecular level, curcumin significantly reduced AFB (1)-N (7)-guanine adduct excretion

in the urine, DNA adduct in the liver and albumin adduct in the serum (Nayak and

Sashidhar, 2010). Supplementation of Curcuma powder in diets in chicks fed AFs

contaminated diets improves the antioxidant, biotransformation, and immune system genes

in livers of chicks fed AFB1(Surai, 2002). Therefore, the objective of the present study was

to evaluate the possible modulatory effect of Curcuma longa on immunological depression

caused by aflatoxins contamination.

2. Material and Methods

2.1. Preparation of Aflatoxins

Productions of AFs were carried out at laboratory of Animal Production, Faculty of

Agriculture, Minia University. Estimations of AFs were run at the laboratory of

Mycotoxins Central lab., & Food Safety National Research

Center, Cairo, Egypt. Production of AFs was prepared via fermentation of polluted ground

broiler diet by A. parasiticus and A. niger for 6 month. After that, fermented ground corn

dried and regrind to be fine powder. A sample of this fine powder was analyzed

spectrophotometerically for AFs content as reported by Nabney and Nesbitt, (1965) and

modified by Wisman et al., (1967).

To achieve the level of 100 µg of AF in the contaminated diets, the experimental

diets were formulated by replacing (4%) of the basal diet with contaminated ground broiler

diet, which contains 2500 µg AF / Kg diet. The fractions of AFs according to the chemical

analyses were (AFB1, AFG1, AFB2 and AFG2) 91.20, 4.48, 2.80 and 1.52 % respectively.

2.2. Experimental Birds

A total number of 160 unsexed, one day old Ross broiler chicks were randomly

distributed into four experimental groups of 40 birds, each. Each group contains four

replicates of 10 birds. The birds were housed in an open house in cleaned and fumigated

battery cages (1 x 0.4 x 0.6 meter as length, width and height). Feed and water were offered

to the bird’s ad-libitum during the experimental periods (0 – 6 weeks of age).

2.3. Experimental Diets:

Birds of all experimental groups were fed on commercial basal diet (21.2 %CP and

2950 kcal ME/Kg diet). The basal diet contained adequate levels of nutrients for growing

broiler chicks as recommended by the National Research Council, NRC, (2004). Birds of

all experimental groups were fed on a commercial basal diet supplemented with or without

100 µg AFs and/or 0.5 % Curcuma as follow:-

A- Basal diet (without AFs or Curcuma addition).

B- Basal diet + 0.5 % Curcuma.

C- Basal diet + 100 µg AFs /kg diet.

D- Basal diet + 100 µg AFs /kg diet +0.5 % Curcuma. .

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The chemical analysis of the experimental diets is shown in Table (1).

Table (1): The proximate nutritional analyses of the experimental diets.

Items

Diets

MO % DM % OM % CP % EE% CF % NFE% Ash%

A 6.85 93.15 87.40 21.20 2.78 2.67 67.60 5.75

B 6.83 93.17 87.35 21.15 2.77 2.66 67.58 5.82

C 6.84 93.16 87.51 21.29 2.80 2.67 67.60 5.65

D 6.84 93.16 87.59 21.30 2.82 2.67 67.62 5.57

Where: MO= Moisture, DM= Dry Matter, OM= Organic Matter, CP= Crude Protein, EE= Ether Extract, CF= Crude Fiber

and NFE= Nitrogen Free Extract.

2.4. Collection of blood and organs samples After 3 and 6 weeks of age, blood samples were collected from wing vein of

four chicks from each experimental group in two blood collected tubes. Blood sampling

time was fixed each time. It started at 7 am .

The first sample was collected in heparinized tube (2.25µ heparine / 5 ml blood)

for blood picture, tests of chemotaxis, chemokinesis and phagocytosis. The second

sample was collected in non–heparinized tube and centrifuged for 15 minutes at

3000 rpm to separate serum which stored at ( -20ºC ) for some humeral immune

responses tests (precipitation of antibody, hemagglutination test and total

immunoglobulin. After that, the birds were dissected, organs including spleen, thymus

and bursa of fibricius were removed, wiped with filter paper and fixed in 10% neutral

buffered formalin fixative for histopathological examination.

2.5. Total and differential leukocyte count

White blood cells were counted as described by (Campbell, 1995).

2.6. Chemokinesis assay

In an attempt to more precisely quantify the chemotactic response, a modified

Borden's chamber assay was used (Boyden, 1962). In a Borden’s chamber assay, cells

migrate through a micropore filter from a starting compartment into another containing

a chemoattractant. For the method employed here (Gearing and Rimmer, 1985), the

two compartments were formed by the wells of two microtitration plates on top of each

other, with cellulose acetate micropore filters (5 µm millipore 67/20 Molsheim, France)

partitioning the wells. The lower test wells were filled with chemoattractant (SRBCs)

or, as a control, saline (0.9% NaCl). Five-micrometer micropore filters were then placed

over all lower wells. The upper plate was inverted and placed over the lower plate, with

petroleum jelly around the perimeter joining the plates. Blood (0.3 ml) was introduced

into each chamber through a hole made in the base of each well. The whole plate was

then covered with aluminum foil to reduce evaporation and placed in humid incubator

at 37oC for 16-18 hours. Following incubation, the fluid was removed from the upper

wells with a syringe and needle and replaced with 0.3 ml of methanol to fix the cells.

After 20 minutes, the methanol was removed and the two plates were separated. The

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filter papers were then fixed in 10 % formalin for 20 minutes, stained in Delafield's

hematoxylin for 5 minutes, washed with distilled water and mounted in DPX mountant

on a microscopic glass slide with the bottom surface facing upward. Two counts were

made on each of 5 filters. The chemokinetic effect obtained was expressed using a

chemokinetic index calculated as: Mean number of cells per h.p. field with chemokinetic stimulus

Chemokinetic index = -------------------------------------------------------------------------------------

Mean number of cells per h.p. field with control stimulus

2.7. Agarose gel chemotaxis movement assay

A chemotaxis assay was used to study the chemotactic response of leukocytes in

response to SRBC's. A previously described method (Nelson et al., 1975, Comer et al.,

2005), in which cells migrate from a well cut into an agarose gel into a well containing

a chemotactic stimulus, was employed. The assay was performed in sterile 60 x 15 petri

dishes. Agarose gel was prepared in 100 ml volumes by dissolving 0.8 g of agarose

(BDH) in 38 ml of 0.9% saline solution in a boiling water bath for 20 minutes. One ml

of 1 % fetal calf serum (FCS; flow) and 1 ml of combined penicillin and streptomycin

(flow) were added to the gel as it cooled. The FCS was sterilized by filtering through a

0.22 mm disposable millex filter unit. The mixture was delivered to the petri dishes in

10 ml aliquots using a pre-warmed sterile pipette. The gel was then allowed to set on a

level table at room temperature and then stored at 4 oC until use. For each assay, two

groups of three 2.5 mm diameter wells separated by 2.5 mm were made in the gel.

Three petri dishes were used for each test, corresponding to six replicates for each. The

central wells received 10 µl of blood. The wells on one side were used as controls and

received 0.9 % saline solution. The plates were covered and incubated for 16-18 hours

at 38 –42 o C and 95 % humidity. The migrated cells were fixed in methanol for 30

minutes. The plates were coded and read independently by two separate observers. The

observed chemotactic effect was expressed using a chemotactic index calculated

similarly to the chemokinetic index.

2.8. Phagocytosis %: The phagocytic activity of macrophages as well as other phagocytic cells in

whole blood was examined using the binocular microscopy as follows-:

The whole blood was kept in cold saline solution (0.9 % Na Cl). One drop of

blood sample was mixed with a drop of paraffin oil and examined. Percentages of

Phagocytosis were calculated and any remarkable phagocytic state was photographed.

Abu El-Maged, (1991).

2.9. Turbidity test for estimation of total immunoglobulin level (Total Ig)

Two widely used procedures for measuring immunoglobulin levels

are single radial immunodiffusion (RID) (Meguire et al., 1976) and zinc sulfate

(ZnSO4) turbidity. The latter is a “salting out” procedure that depends on the

biochemical properties of immunoglobulin in relation to the characteristics of ZnSO4. In

this study, this technique was performed as previously described (Mcewan et al., 1970

and Pfeiffer et al., 1977).

2.10. Immunization and titration

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Detecting of agglutinating antibodies was assessed using passive hemagglutination

technique as described by (Hudson and Hay, 1980). Recognition of a positive response is

detected by the formation of a mat of agglutinated SRBCs on the bottom of the well. The

individual cells form a pellet on the base of the V – shaped wells, if no agglutination

occurs. The (-log 2) of the maximum dilution at which agglutination occurred was recorded

for each serum tested.

2.11. Precipitation test:

Precipitating antibodies were detected in control as well as infected birds' sera using

the immuno-double- diffusion technique. In this technique antigen and antibody were

allowed to migrate towards each other in a gel and a line of precipitation was formed where

the two reactants meet. As this precipitate is soluble in excess antigen, a sharp line is

produced at equivalence. This technique only identifies antigen and antibodies (Hudson

and Hay, 1980).

2.12. Statistical Analysis

Data are expressed as mean ± SE. Statistical analysis was performed using

one-way ANOVA. The general liner model (GLM) was applied to test the

differences among the eight experimental groups. P-values less than 0.05 were

considered to be statistically significant SAS Institute (2003).

The statistical analysis was calculated using the following equation:

Yij = μ+ Ti +Eij

Where:

Yij = Experiment observations.

μ = The overall mean.

Ti= The effect of dietary treatment.

i= T1, ----- T4.

Eij = The experimental error.

Duncan's test was used to examine the significance degrees among means (Duncan,

1955).

3. Results and Discussions: We observed that chicks treated with 100 µg Aflatoxins /kg diet were developed

symptoms of weakness, ruffled feathers, loss of appetite and stunted growth by 21 days of

age compared with other groups.

3.1. Curcuma longa supplementation enhances innate immunity of broiler chicks

during aflatoxicosis.

3.1.1. Absolute and relative weight of spleen, thymus and bursa of fibricius:

We monitored changes in immunological parameters related to innate immunity in

all groups throughout the experiment period. The absolute and relative weight of spleen,

thymus and bursa of fibricius are recorded in Table (2). Our results indicated that, adding

Curcuma to non-contaminated or contaminated diets decreased (P<0.05) absolute and

relative weights of spleen compared with other groups. Also, there were no significant

differences between T1 and T3 in absolute of spleen. We observe the same trend, in thymus

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weight. Likewise, bursa of fibricius becomes swollen by AFs contamination but the

addition of Curcuma curb AFs effect.

Thymus and bursa of fabricius have responsibility in both cellular and humeral

immunity in broilers and these organs must be functional throughout their life (Qureshi et

al., 1998). Regression of thymus and bursal developments would appear in the poor

immune response in chicks and may cause failure in vaccination. In our investigation,

histopathologic changes (Fig. 1to 24) such as atrophy and lymphoid cell depletion were

observed in thymuses of broiler chicks fed a AFs diet alone in most cases. Chicks fed AFs

alone showed slight cell depletion in the spleen. These spleen lesions also support the

immunotoxic and haematotoxic effect of AFs (Azzam and Gabal 1998, Ibrahim et al.,

2000). They concluded that, the AFs exposure could affect the cellular and humeral

immunity of chicks and AFs would impair immunological performance and increase

susceptibility of animals to environmental and infectious agents. The conspicuous findings

obtained by the histopatological examinations of the bursa of fabricius and thymus (nearly

disappeared cortex-medulla zone in the thymus, lymphocytosis in the bursa) showed that

this compound AFs had an immunosuppressive effect (Ogguz et al., 2000a). Hassan et al.,

(2006) found that the relative weights of spleen increased, while those of bursa of fabricus

and thymus glands decreased by aflatoxicosis. However, the bird fed on aflatoxic diet

supplemented with Curcuma alleviated the adverse effects of AFs. Contrary, AL-Sultan,

(2003) found that, the higher bursa and thymus weight indices were detected in birds

received diet containing 0.5% Curcuma, while the higher spleen weight index was observed

in birds received feed contained 1.0% Curcuma.

3.1.2. Total and differential count of white blood cell:

From Table (3) it is clear that, the addition of Curcuma to non-contaminated or

contaminated diets enhance (P<0.05) WBC's of broiler chicks where, it increase the total

number of WBC's at 3 and 6 weeks of age compared with T1 and T3. Also, we monitored

severe reduction in WBC's count by feeding AFs diet at 6 week (1.91x 103) as a result of

cumulative effect of AFs. There was intense decreased on heterophil % at 3 and 6 week of

age while addition of Curcuma clearly promote it nearly to control group. Conversely,

lymphocyte % recorded highest % by AFs contamination and the addition of Curcuma

become as control group. H/L ratio decreased (P<0.05) at 3 week of age by feeding broiler

chicks on contaminated diets which supplemented or non-supplemented with Curcuma

compared with other groups. At 3 weeks of age addition of Curcuma to non-contaminated

diet increased monocyte % compared with other groups. Contrary, adding Curcuma to

contaminated diet failed (P<0.05) in enhancing monocyte % which decreased by feeding

AFs contaminated diet. However, at 6 weeks of age the increasing of monocyte % did not

continued by the addition of Curcuma to non-contaminated diet. Furthermore, the decreases

on monocyte were continued for AFs groups. Eosinophil and basophil % was increased as a

result of feeding broiler chicks on AFs contaminated diet and control diet whether at 3 or 6

weeks of age. There was no significant difference between Curcuma supplemented groups

which non-contaminated or contaminated groups.

Heterophillia that appeared may be due to increase granulopiosis as result of

Curcuma supplementation to the diet., Further H/L constitute the first line of defense with

efficient Chemotactic response, it suggested that birds of treated group were better

equipped for the non –specific cellular response when invaded by foreign agents viable or

innate. On the other hand, significantly (P˂0.05) higher level of globulin (data not showed),

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suggested that birds of treated group had potential for better humeral immune status, as that

in control group these results agree with (Raghdad, 2012).

The lower percentage of heterophils % and monocytes % in the AFs groups may

suggest an effect of AFs on the phagocytic cells (Table 4). These results were in agreement

with those obtained by Del Bianchi et al., (2005) who's observed that the highest

percentage of heterophils in birds fed the control diet was 46.25%, and the lowest (P <

0.05) percentage was found for treatments (50 µg) of AFB1/kg diet + 10 mg of FB1/kg diet

(25.25%), (2,450 µg) of AFB1/kg (24.75%) and (2,450 µg) of AFB1/kg + (10 mg) of

FB1/kg (27.00%).

3.1.3. Chemokinesis indexes, Chemotaxsis and phagocytic activity:

There were significantly (P<0.05) decrease (P<0.05) at 3 weeks of age, and there

after it was increased (P<0.05) in chemokinesis indexes Table (4). Adding Curcuma longa

enhance (P<0.05) chemokinesis indexes after 3 and 6 week of age. Birds fed on T1 and T2

diets had the highest chemokinesis indexes compared with other contaminated groups.

Furthermore, addition of Curcuma significantly (P<0.05) improve chemotaxsis compared

with other groups. Phagocytic activity was improved (P<0.05) by using curcuma with non-

contaminated or with contaminated diets compared with other groups.

It is well known that chemotaxis and subsequent phagocytosis are two integral

components of the inflammatory response and innate immunity. Chemotactic migration of

leucocytes to the site of inflammation is an active and directional event. The leucocyte is

moving from the blood to the inflamed tissue along chemical gradients originating at the

site of inflammation. The factors stimulating this migration may be microbial or host origin.

When leucocytes migration is directional along an increasing concentration gradient of an

attracting substance, it is termed chemotaxis. While, if the response to the substance has

increased speed or frequency of migration it is termed chemokinesis. Chemotactic

migration resulting in accumulation of leucocytes at sites of tissue injury is one of a number

of recognized components of the inflammatory responses.

Phagocytosis is the first step of the macrophage response to invading

microorganism while curcumin elevated the innate immune response in the body by

increasing the ability of heterophils and mature monocytes to engulfing antigens

(phagocytosis). It is known that macrophages produce some Reactive Oxygen Species

(ROS) during phagocytic process. Accordingly, oxygen radical production indicates the

actual killing capacity of the macrophages. It is suggested that hydrogen peroxide (H202)

and Nitric Oxide (NO) by macrophages may contribute to inflammation and tissue injury

(Loskin and Laskin, 1996). Several studies have shown that curcumin has a strong

capability for scavenging superoxide radicals, hydrogen peroxide and nitric oxide from

activated macrophages, reducing iron complex and inhibiting lipid peroxidation (Ali et al.,

2006; Nisarani et al., 2009; Sankar et al., 2012 and Cai et al., 2012).

During toxification, free radical damage may be produced, not only by a direct

production of oxygen radicals by phagocytes, but also by a Tumor Necrosis Factor (TNF)-

mediated generation in target cells. Antioxidants have demonstrated protective capacity for

TNF cytotoxicity (Ferlat and Favier, 1993). A redox imbalance caused by an over

production of pro-oxidants or a decrease in antioxidants seems to play an important role in

the normal physiological function. Following activation, lymphocytes production increased

levels of OS. Lymphocytes from such individuals were more prone to undergo apoptosis or

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cell death in vitro (Buttke and Sandstorm, 1995). Antioxidant supplementation

significantly improves some measures of oxidative defence (Batterham et at., 2001).

Curcumin enhances the immune response of broiler (Emadi and Kermanshahi, 2007).

Curcumin with its potential properties is a favorite additive. Using curcumin with wide

ranges of positive activities might be a suitable additive in poultry that may alternate means

of oxidative stresses and improves immunity responses in broiler chickens.

Chicken macrophages are susceptible to both environmental and toxicological

insult. For example, macrophage viability and functions are adversely affected by

mycotoxins such as aflatoxins (Neldon-Ortiz and Qureshi, 1991), Fumonisin-B1

(Qureshi and Hagler, 1992), T-2 toxin (Kidd et al., 1997).

3.2. Humeral immune responses

It were significantly (P<0.05) decreased firstly at 3 weeks of age, and then followed

with increased (P<0.05) in total immunoglobulin Table (5). Adding Curcuma longa

enhance (P<0.05) total immunoglobulin after 3 and 6 week of age. Birds fed on T1 and T2

diets had the highest total immunoglobulin and positive precipitation tack place (Figs. 1, 2,

4, 5, 6 and 8) compared with other contaminated groups. Furthermore, addition of

Curcuma improve (P<0.05) hemagglotination compared with other groups. Where as, there

was no precipitation tack place for birds fed aflatoxicated diets compared with other groups

(Figs. 3 to 7). Generally, the immunotoxic dose of AFs is considered as less than the

dose required to eliciting the reduction in birds performance. Though several

contradictory reports are available, the threshold dose of AFs may be generalized to

be 0.4 and 1 mg/kg for the negative effects on cell mediated and humoral

immunity, respectively. However, the question regarding susceptibility of modern

broiler immunotoxicity remains yet to be answered. Furthermore, there is evidence

regarding biphasic nature of the effects of AFs on humoral immunity. In this regard

our recent data indicate that humoral immune response from broilers could increase

and decrease depending upon the level and length of exposure to the toxin. The underlying

mechanisms for this temporary increase in humoral immune response are not known

yet. As a matter of fact, the exact mechanisms of even immunosuppression during

aflatoxicosis are not clearly understood in spite of 50 years of research on the mycotoxin.

In this regard, Surai and Dvorska, (2005) have reviewed some aspects of AFB1-induced

immunotoxicity. A brief but comprehensive discussion on the subject can also be

found in an article by (Celik et al. 2000). Curcuma longa had humoral

immunostimulating potential. This finding is in accordance with the earlier work of

Kurkure et al., (2000) who have reported that Curcuma has restored the reduced humoral

response of AFs induced immunosuppression, thus carry the humoral immunostimulatory

potential.

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3.4. Histopathological changes of some lymphatic organs:

3.4.1. Spleen:

A- Non-contaminated groups:

Section of Non-contaminated groups stained with (H&E) showed that the splenic

parenchyma was composed of the white and red pulp. It is surrounded by a capsule of dense

connective tissue which sent out trabecular dividing the splenic parenchyma into

incomplete compartments. The connective tissue of the capsule and trabeculae contained

some smooth muscles cells. The white pulp consisted of lymphatic tissue arranged in

sheaths around central arteries as well as lymphoid nodules appended to the sheaths (the

per-arterial lymphatic sheaths). These sheaths were formed mainly of dense accumulation

of small lymphocytes with deeply stained nuclei. The lymphatic follicles were spherical or

ovoid structures. They presented as either primary follicles which were formed mainly of

densely packed small lymphocytes, or secondary follicles which could be differentiated into

an outer dark zone formed of small lymphocytes and inner light one called germinal center

containing large and medium sized lymphocytes, dendritic cells and few macrophages. The

germinal zone between the white pulp and red pulp, which consisted of loose lymphoid

tissues, many active phagocytes and large number of red blood cells. The red pulp is a

reticular tissue which differentiated into splenic cords and blood sinusoids. The cords

consisted of macrophages, monocytes, lymphocytes, plasma cells, and many blood

elements (erythrocytes, platelets and granulocytes). The sinusoids have a dilated large,

irregular lumen lined with flat endothelial cells (Fig. 9, 10, 13 and 14).

B- Contaminated groups:

Spleen sections of birds fed diet with (100 µg AFs / kg diet) at 3 and 6 weeks of age

showed that the white pulps were increased in size with low density of the lymphocytic

population. The wall of central arterioles becomes ruptured, their lumen become more

narrowing. The red pulp was congested with red blood cells. The number of bundles like

trabeculae was increased. Some lymphocytes showed pyknotic pictures (Figs. 11 and 15).

Spleen sections of birds treated with curcuma and AFs together showed thin bundle of

fibers like trabeculae, pyknotic nuclei, macrophages, and plasma cells (Fig. 12 and 16).

3.4.2. Thymus:


The thymus of birds is fowl strings along each side of the neck are close to the

jugular vein and their number is different in birds from one to another. Histological

structure of the thymus of the fowl resembles that of the mammal. Each lobe consists of

lobules, which are partly separated by connective tissue. The lobule consists of an outer

dark cortex and inner pale medulla. Islands of reticular cells occur in the medulla; the cells

are usually vacuolated rather than laminated unlike those of mammalian corpuscles.

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Each thymic lobule consisted of a dark staining peripheral zone (the cortex) and

light staining central zone (the medulla). Both the cortical and medullary zones have the

same cellular types (Fig. 17, 18, 21 and 22).The cortex consisted mainly of small and

packed lymphocytes arranged in a continuous layer which passed from one lobule to the

other. The medulla consisted of large number of reticuloepithelial cells, thus it stained

lightly. Fewer numbers of thymic lymphocytes were found in the medulla, but these cells

are fully mature and smaller than those found in the cortex.


Thymus sections of birds fed diet with (100 µg AFs / kg diet) at 3 weeks showed

lymphocytic depletion, invasion of lymphocytes, fibroblasts, and occasional eosinophilic

cells as well as pyknotic nuclei and focal areas of macrophage activity (Figs. 19 and 23).

Thymus of birds treated with curcuma and AFs showed improvement in

lymphocytic population although haemolized area was recorded (Fig. 20 and 24).

3.4.3. Bursa of Fabricius:


The bursa of Fabricius (BF) is an immunological organ that plays a primordial role

in the poultry immunity. The different aggressions of the environment such as stress, bad

hygiene, vaccination and pathologies influence the anatomical and physiological

development of BF.

Bursa of Fabricius sections showed that the wall of the organ consist of tunica

mucosa, tunica muscularis and tunica serosa. It was determined that tunica mucosa forms

the plicae at different length and thickness which prolongate towards lumen. The surface of

plicae was surrounded by pseudo stratified epithelium. Epithelium cells had usually an

ovoid nucleus located below the midline of the cell and more than one nucleolus. Among

these cells, more granulated P.A.S. positive cells and goblet cells were observed. The

epithelium cells were surrounded by a P.A.S. positive basement membrane (Fig. 25, 26, 29

and 30). Each plica was completely filled with follicles separated by connective tissue. The

forms and sizes of follicles were different from one to another. The follicles had an inner

pale medulla and outer dark cortex.


Bursa of Fabricius sections of birds fed diet with (100 µg AFs / kg diet) showed,

lymphocytic depletion, invasion of lymphocytes, fibroblasts, and pyknotic nuclei (Figs. 27

and 31).

Bursa of Fabricius of birds treated with curcuma and AFs showed improvement in

lymphocytic population although haemolized area was recorded (Fig. 28 and 32).

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Results concerning immunological parameters were confirmed and supported by

organs histological findings.

In the present study, liver from chicks treated with mold-contaminated feed showed

liver lesion including severe vacuolar degeneration of hepatocytes, which is consistent with

(Banlunara et al., 2005; Dvorska et al., 2007) studies.

Mycotoxin inducing hepatotoxicity may be due to many complex reasons. One of

the important reasons may be that mycotoxins cause oxidative stress in liver, the formation

of potent cytotoxic oxidant peroxy nitrite is increased, and these events may culminate in

lipid peroxidation, cell death and hepatic injury (Gowda et al., 2008).

Denli et al., (2009) showed that histological analysis indicate significant damage in

the liver tissues of broilers receiving AFB1 alone. Liver tissue from this treatment had

vacuolar degeneration of hepatocytes, per-lobular inflammation (mainly mononuclear

cells), bile duct hyperplasia, and hypertrophy compared with the tissue of birds fed on the

uncontaminated diet. Zhengquan, et al., (2011) found that, liver from chicks treated with

mold-contaminated feed showed liver lesion including severe vacuolar degeneration of

hepatocytes.

They concluded that concerning serum biological and hematological parameters were

confirmed and supported by liver histological findings.

Conclusion:

Application of 0.5 % Curcuma in the management of poultry health, promising to

yield better immune response, so better disease resistance and health would be possible.

Under the conditions of this study, it was concluded that turmeric rhizome powder

(Curcuma) might have some positive effects on immunity response and may be used

directly in the diet as immune system fortification in broiler chickens.

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تحسين اإلسجابات المناعية لبدارى اللحم المغذاه على عالئق ملوثة باألفالتوكسينات باستخدام الكركم

محمود , أكرم زين العابدين محمود حمدى, عطيات حسين البغدادى, حمادة عبدالحميد مهنى علوان عباس طوسون و شاكر عبدالتواب عبداللطيف

جامعة المنيا– كلية الزراعة –قسم اإلنتاج الحيوانى

حيث .بمزرعة اإلنتاج الحيواني والدواجن بكلية الزراعة جامعة المنياى هذا البحث أجر

من مسحوق الكركم الى العالئك الملوثة أو الغير ( %0.5)صممت التجربة لدراسة تأثير إضافة

لدراسة تأثير إضافة الكركم الى . (كجم عليمة/ ميكرو جرام افالتوكسين 100)ملوثة باألفالتوكسينات

. على واألستجابه المناعيه و الهستولوجية لبدارى التسمينعالئك بدارى اللحم الملوثة باألفالتوكسينات وأجريت التحاليل الكيميائيه بمعامل لسم اإلنتاج الحيوانى بكليه الزراعه جامعه المنيا و المركز المومى

عمر (سرو) كتكوت تسمين 160 تم إستخدام اهفيو .للبحوث لسم السموم الفطرية و سالمة الغذاء

؛ ( طائر لكل مكررة10 طائر لكل معامله في أربع مكررات 40) معامالت 4يوم؛ تم تمسيمهم إلى

: وتم تمسيم المعامالت كاألتى

بدون ) ى إضافات أ تم تغذيتها على عليمة تجارية بدون:(كمجموعة مقارنة)المعامله األولى .1

. (كركم و بدون افالتوكسين

كركم طوال مدة % 0.5 تم تغذيتها على عليمة تجارية مضاف اليها :المعامله الثانيه .2

.(6 – 0 )التجربة 100 تم تغذيتها على عليمة تجارية ملوثة بـاألفالتوكسينات بمعدل :الثالثةالمعامله .3

.(6 – 0 )كيلوا جرام عليمة طوال مدة التجربة/ ميكروجرام ميكرو 100كركم مع % 0.5 تم تغذيتها على عليمة تجارية مضاف اليها :الرابعةالمعامله .4

.(6 – 0 )جرام من األفالتوكسينات طوال مدة التجربة: كركم الى العالئق الملوثة او الغير ملوثة باألفالتوكسينات مايلى % 0.5أوضحت نتائج إضافه

نمصا (الثالةالمجموعة )ظهرت الكتاكيت المغذاه على العليمة الملوثة أسابيع أ 6 و 3عند عمر .1

يات الدم البيضاء المتعادلة ممارنة ببالى رمعنويا فى العدد الكلى لكريات الدم البيضاء و ن 3عند عمر (الثانية و الرابعة)ختالفات معنوية بين المجموعات إلم تكن هنان , بينما. المجموعات

تجاها إخذت الكريات اللمفاوية و األحادية و الحامضية و الماعدية أ, لى ذالنإضافة إ. اسابيع . معاكسا لذالن

لل ليم أ( الثالثةالمجموعة )ظهرت الكتاكيت المغذاه على العالئك الملوثة أ اسابيع 6 و 3عند عمر .2

هجرة كريات الدم البيضاء عبر الغشية شبة المنفذة و كذا عبر األجاروز و )فى المناعة الخلوية كركم مع % 0.5 ةكما كان هنان تحسنا جزئيا باضاف. ( لكريات الدم البيضاءالنشاط البلعمى

األجسام المضادة الكلية و الدفع )تجاة كان للمناعة الخليطة إلنفس ا. التلوث باألفالتوكسينات . ( و لدرة األجسام المضادة على ترسيب األجسام الغريبة ضد كريات الدم الحمراء للغنمالمناعى

التغيرات النسيجية المرضية لكل من الطحال و الغدة الثيموثية و غدة فابريشيس لكل المامعالت تم .3

حيث كانت التغيرات النسيجية الطحال و الغدة الثيموثية و غدة . اسابيع6 و 3اجرائها عند عمر

لريبة من التركيب (األولى و الثانية)فابريشيس للمجموعات التى غذيت على العالئك الغير ملوثة

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كانت التغيرات النسيجية (الثالثة)المجموعات التى غذيت على العالئك الملوثة , بينما. الطبيعى لهاحتمان شديد فى األوعية الدموية و تليف و فراغات سيتوبالزمية و فمد فى الكريات إلها ممثلة فى

كركم % 0.5المعاملة ب , نأفى حين . نزفة دمويةأاللمفاوية و تكاثف لكروماتين األنوية و

.خففت من حدة هذة التغيرات (الرابعةالمجموعة ) باألفالتوكسينات هلوثمالو: الخالصــــــــة .4

يستنتج , تمدم ومن خالل البيانات والنتائج التي تم الحصول عليها من هذه الدراسة بناء على ما للطيور المغذاه على دى الى تحسنا معنويا فى معظم المياسات المناعيهأكركم % 0.5استخدام ;أن

كركم كمضاد % 0.5لذا يمكن استخدام . كجم عليمة/ ميكروجرام األفالتوكسينات100

األضافة ياتعمك من حيث مسوأكما تحتاج لدراسة . فالتوكسينات ذات التركيزات المنخفضةألل. التلوثياتومستو

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Fig. 1 to 4: Photomicrographs of precipitation test at 3 weeks of age:

Fig. 5 to 8: Photomicrographs of precipitation test at 6 weeks of age:

Fig. (3): Precipitation of Antibodies of birds in T3. Note. The

absent of sharp precipitating line (UV 360nm wave length).


formation of sharp precipitating line (Arrow) (UV 360 nm

wave length).


formation of sharp precipitating line (UV 360nm wave

length).


formation of sharp precipitating line (UV 360 nm wave

length).

Fig. (7): Precipitation of Antibodies of birds in T3.

Note. The absent of sharp precipitating line (UV 360

nm wave length).


Note. The formation of sharp precipitating line (UV

360 nm wave length).







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Fig. 9 to 12: Histopathological changes of spleen sections as affected by dietary treatments at 3 weeks of

age.

Fig. (11): A photomicrograph in the spleen section for T3,

showing red pulp and white pulp which have few lymphocytes and congested sinusoids and blood vessels with blood (wavy,

curve and head arrows) (H&E. x100).

Fig. (9): A photomicrograph in the spleen section for

T1, showing red pulp (head arrow) white pulp (thick

arrow) and trabeculae (wavy arrow) (H&E. x100).

Fig. (12): A photomicrograph in the spleen section for T4,

showing red pulp (head arrow), white pulp (thick arrow) and

blood vessels (curve arrow). (H&E. x100).

Fig. (10): A photomicrograph in the spleen section

for T2, showing red pulp (head arrow) white pulp

(thick arrow) and blood vessels (curve arrow). (H&E. x100).

Fig. 13 to 16: Histopathological changes of spleen sections as affected by dietary treatments at 6 weeks

of age.

Fig. (15): A photomicrograph in the spleen section for T3, showing red pulp and white pulp which have few

lymphocytes and congested sinusoids and blood vessels

with blood (thick, curve and head arrows) (H&E.

x100).



arrow). (H&E. x100).

Fig. (16): A photomicrograph in the spleen section for T4, showing red pulp (head arrow) white pulp (thick



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arrow) and few numbers of lymphocytes (H&E. x100). arrow) and blood vessel (curve arrow). (H&E. x100).

Fig. 17 to 20: Histopathological changes of thymus sections as affected by dietary treatments at 3 weeks

of age.

Fig. (19): A photomicrograph in the thymus section for T3, showing the cortex (thick arrow) and medulla

(head arrow). Note, Hassle's corpuscle (zigzag

arrow), hemorrhages (curve arrow) and invasion of fibroblasts pink to rose area. (H&E. x500).

Fig. (17): A photomicrograph in the thymus section for T1,

showing the cortex (thick arrow) and medulla (curve arrow).

Note, Hassle's corpuscle (pink to red area). (H&E. x500).

Fig. (20): A photomicrograph in the thymus section

for T4, showing the cortex (thick arrow), medulla

(head arrow), invasion of fibroblasts (curve area), Note, Hassle's corpuscle the highly curve arrow and

connective tissues (wavy arrow). (H&E. x500).


showing the cortex (thick arrow) and medulla (head arrow).

Note, Hassle's corpuscle (pink to red area). (H&E. x500).

Fig. 21 to 24: Histopathological changes of thymus sections as affected by dietary treatments at 6

weeks of age.


showing the cortex (thick arrow) and medulla (head arrow), focal areas of macrophage activity (thin arrow) and

hemorrhage (curve arrow). (H&E. x500).


showing the cortex (thick arrow) and medulla (curve

arrow). (H&E. x500).

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showing the cortex (thick arrow) and medulla (head arrow)

and focal areas of macrophage activity (wavy arrow). (H&E. x500).

Fig. (22): A photomicrograph in the thymus section for T2, showing the cortex (thick arrow) and medulla (head

arrow), hemorrhage (wavy arrow) and connective tissues

(zigzag arrow). (H&E.x500). Fig. 25 to 28: Histopathological changes of bursa of fabricius sections as affected by dietary

treatments at 3 weeks of age.

Fig. (27): A photomicrograph in the Bursa of Fabricius

section for T3, showing degeneration within the lobules,

epithelial cells and losing lymphocytes. (H&E. x500).

Fig. (25): A photomicrograph in the Bursa of

Fabricius section for T1, showing the normal structure

of epithelial cells and lymphocytes. (H&E. x500).

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Fig. 29 to 32: Histopathological changes of bursa of fabricius sections as affected by dietary treatments

at 6 weeks of age.


section for T4, showing degeneration within the lobules,

epithelial cells and little losing lymphocytes. (H&E. x500).

Fig. (26): A photomicrograph in the Bursa of

Fabricius section for T2, showing the normal structure

of epithelial cells, lymphocytes and normal bursal

lobules. (H&E. x500).


section for T3, showing severs degeneration within the

lobules, epithelial cells and losing lymphocytes. (H&E.

x500).


section for T1, showing the normal structure of epithelial

cells and lymphocytes in bursal lobules. (H&E. x500).


section for T4, showing protection from the degeneration

within bursal lobules, epithelial cells, normal lymphocytes

with some hemorrhage and little losing of lymphocytes.

(H&E. x500).


section for T2, showing normal structure of epithelial cells,

lymphocytes in bursal lobules and normal connective

tissues. (H&E. x500).

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Table (2): Effect of Curcuma longa and /or Aflatoxins on Absolut and relative weight of some lymphoid organs.

a-d Values under the same column have different superscripts are significantly different (p<0.05). **= (P<0.01)

Absolut and relative weight of

bursa of fabricius at 6 week of age


thymus at 6 week of age


spleen at 6 week of age

Traits

Treatments

Relative Absolut Relative Absolut relative Absolut

0.10d

1.98d

0.19b

3.55b

0.16b

2.97a

T1

0.12c

2.32c

0.14d

2.50d

0.13c

2.33c

T2

0.44a

7.00a

0.23a

3.66a

0.19a

3.02a

T3

0.27b

5.68b

0.15c

3.17c

0.13c

2.62b

T4

0.01 0.21 0.01 0.12 0.01 0.07 ± SE

** ** ** ** ** ** Significance

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Table (3): Effect of Curcuma longa and /or Aflatoxins on total and differential quaint of white blood cells.

Traits

Treatments

WBC's

n×103 / micro liter

Heterophils % Lymphocyte

%

H/L Ratio

3 Wk 6 Wk 3 Wk 6 Wk 3 Wk 6 Wk 3 Wk 6 Wk

T1 2.10b 3.19

c 35.96

a 33.07

b 51.05

c 52.52

d 0.70

a 0.62

a

T2 3.70a 4.56

a 34.26

b 32.45

c 52.00

c 54.08

c 0.66

a 0.60

c

T3 2.16b 1.91

d 25.06

d 20.40

d 62.13

a 68.13

a 0.40

b 0.30

d

T4 3.69a 3.97

b 33.83

c 34.58

a 56.91

b 56.41

b 0.59

b 0.61

b

± SE 0.096 0.053 0.422 0.262 0.354 0.182 0.09 0.06

Significance ** ** ** ** ** ** ** **

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a-d Values under the same column have different superscripts are significantly different (p<0.05). **= (P<0.01)

Traits

Treatments

Monocytes % Esinophils % Basophils %

3 Wk 6 Wk 3 Wk 6 Wk 3 Wk 6 Wk

T1 11.38b 12.90

a 1.010

b 0.982

b 0.600

b 0.528

c

T2 12.50a 12.17

b 0.875

c 0.805

c 0.365

c 0.495

c

T3 10.01c 8.66

c 1.74

a 1.755

a 1.060

a 1.055

a

T4 7.70d 7.45

d 0.870

c 0.860

bc 0.690

b 0.700

b

± SE 0.447 0.195 0.057 0.049 0.050 0.053

Significance ** ** ** ** ** **

Traits

Treatments

Chemokinesis index Chemotaxis Phagocytic activity %

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Table (4): Effect of Curcuma longa and /or Aflatoxins on cell medeatimmune responses.

a-d Values under the same column have different superscripts are significantly different (P<0.05).

**= (P<0.01), * = (P< 0.05)

± = No chemotactic movement. + =Weak

chemotactic movement. ++= Moderate chemotactic movement . +++ = Strong chemotactic movement.

Wk3 Wk6 Wk3 Wk6 Wk3 Wk6

T1 4.08a 4.10

a ± ± 36.12

c 36.00

c

T2 3.77b 3.83

b +++ +++ 45.00

a 49.00

a

T3 2.04d 2.20

d + + 27.50

d 30.00

d

T4 2.96c 3.35

c ++ +++ 40.50

b 42.00

b

± SE 0.051 0.073 0.699 0.565

Significance ** ** * **

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Table (5): Effect of Curcuma longa and /or Aflatoxins on humoral immunoresponses.

a-d Values under the same column have different superscripts are significantly different (P<0.05).

**= (P<0.01), + = Precipitation take place. - = No Precipitation take

place.

Traits

Treatments

Total immunoglobulin (mg/dl) Hemagglutintion Ab.

(log2)

Precipitation test

Wk3 Wk6 Wk6 Wk3 Wk6

T1 115.56b 179.50

c 3.55

b + +

T2 122.71a 193.53

a 4.06

a + +

T3 91.59d 149.92

d 2.54

d - -

T4 108.23c 189.13

b 3.47

d + +

± SE 0.216 0.194 0.022

Significance ** ** **

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