INFLUENCE OF MYCOTOXINS ON IMMUNE RESPONSES AGAINST SALMONELLA TYPHIMURIUM INFECTION OF BROILER CHICKENS

CFU/ml ), I/V, and 4 th group was inoculated I/V,1ml sterile normal saline and served as control negative group , all chicks were sacrificed at 3 weeks post infection, it was recorded that mycotoxin suppress the cellular and humoral immune responses , phagocytic activity ,in addition to high mortality rate were found in chicks fed contaminated diet with and without immunization.


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
. The immune response against Salmonella infection is very complicated and required interaction of innate and acquired immune responses (1), which are not completely understood and they required further researches, Poultry are commonly exposed to chronic form of mycotoxins due to intake of low concentration of these toxin for long period and they express chronic symptoms of mycotoxins such as poor growth, poor feed efficiency, decline in their production but acute mycotoxicosis may occur in poultry post-ingestion high concentration of the toxin that lead to acute clinical symptoms due to toxic effects on their vital organs ,immune response and other their physiological features in addition to high mortality (9). However, impair immune responses were recorded in human and animals consumption food contaminated with mycotoxins resulting in increase susceptibility of the host to infectious pathogens such as Salmonella spp. in addition the toxins themselves can change the virulence factors of the pathogens that associated with increasing microbial toxicity and its invasiveness ability to varies host tissue include immune cells (4). The mycotoxin can cause immunotoxic in the birds associated with suppresses cell mediated immunity CMI, humoral immunity and inflammatory response (5), there is few researches about the immunotoxic effects of mycotoxins in broiler therefore the aim of the present study was to determine the influence of mycotoxin on immune response against infection by S.typhimurium in broiler chickens.

MATERIALS AND METHODS
A total of forty broiler chicks both (male and female), 1 day of age. were obtained from a local commercial hatchery and then wingbanded and raised in a floor pens with clean wood shaving in an environmentally controlled electricallyheated room (the room temperature was maintained at 32 c ͦ from d 1 to 7 (with 35-45% relative humidity) and then gradually reduced 2 c ͦ per week. All chicks were vaccinated with Newcastle disease (ND) via drinking water at day 7 followed by booster dose of ND virus vaccine at day 17 and with Gomboro vaccine at day 14. Experimental period extended to 8 weeks. Mycotoxin analysis (AFB1, OTA, T-2). All samples were homogenized to obtained 10 commercially poultry diet and a total 10 representive samples (1 kg per sample) were analysis for mycotoxin by ELISA assay kits in veterinary laboratories of general authority of veterinary medicine in Al-Nahta region. The detection limit for these mycotoxins was (10 ppb of AFB1), (1.4 ppb of Ochratoxin A), and (129.9 ppb of T-2 toxin).

Bacterial strain
The strain of S.typhimurium was isolated from naturally infected chickens diagnosed by routine bacterial methods according to (22) and activated according to (7). Whole sonicated S.typhimurium antigens prepared according to (26). Activity of phagocytic cells of immunized groups and control group has been done according to the method of (15). Experimental Design: Forty broilers (Rose), one day old, both sex, were randomly divided into four equal groups and treated as the following -First group was fed diet contaminate with mycotoxin at age 7 days for 7 weeks and at 2 weeks age, it was immunized S/C with 0.5 ml of whole sonicated S.typhimurium antigens (WSS Ags) (protein conc. 1.89 mg/ml), two does, two weeks intervals.
-Second group was immunized with (WSSAgs) only treated as in 1 st group with normal diet. -Third group was fed contaminated food with mycotoxins as 1 st group -Fourth group was served as control negative group. At 4 weeks post immunization, skin test was done in the immunized groups in all groups and blood samples were collected in nonheparinized tubes by brachial vein puncture to determine the level of IgG in serum, as well as phagocytic activity, then 1 st , 2 nd , and 3 rd groups were inoculated with high dose of bacterial suspension of Salmonella typhimurium containing (1 × 10 12 ) CFU/ml (1 ml/ I.V) in wing vein Clinical symptoms and mortality rates were determine during the experiment course, and then all animals were sacrificed at 3 weeks post infection. Gross examination of internal organs was done, and small pieces of heart, spleen, liver, kidney, lung , bone , bursa and thigh muscle were fixed in 10% neutral buffer formalin for histopathological examination RESULTS AND DISCUSSION Delayed type hypersensitivity (DTH): At 24hr post examination, the results of skin test showed that the mean skin thickness in the 1 st and 2 nd were 2.32 and 3.06±0.05, respectively and these values were decrease at 48hr post examination as following: 1.58±0.11 and 1.85±0.25 in the 1 st and 2 nd groups respectively (Table: 1) There were significant differences (P<0.05) between the mean values of two groups after 24 hr post-examination, in which G2 (3.06±0.05) showed the highest mean value

Clinical symptoms and mortality
The main clinical symptoms expressed by life chickens, post infection with high dose in 1 st , 2 nd and 3 rd group , were white diarrhea, decrease feed intake, increase water intake and lowered grwth rate. In addition to depression, dull feathers (loss of shining, paralysis of the legs (Fig:1) associated with inability to eat and drink as well as exited yellow liquids from mouth of the some infected birds, nervous cramps, whistling, and high mortality rates in broiler fed contaminated diets with and without immunization, these symptoms were moderated in the life immunized animals with moderate mortality (Table :4   Histopathological results of infected groups G1 (Immunized with Mycotoxin).: The spleen showed depletion of white pulp with hemorrhage and congested red pulp (Fig.:2) the liver revealed granulomatous lesion in their parenchyma, with dilated sinusoids (Fig.:3) and fibrin networks deposition, Also there was edema between cardiac muscle fiber, while section of the lung were showed fibrin deposition in the interstitial tissue with congested blood vessels, increase thickness of the epithelial layer of parabronchi , heterophils infiltration, and edema in the wall of the bronchi. The lesion in the kidney characterized by presence of mononuclear cells aggregation in the interstitial tissue and severe congestion of blood vessels (Fig.:4).with no clear lesion in bone.

G2 (Immunized only without Mycotoxin group)
Histopathological changes were observed in liver characterized by presence of moderate to marked mononuclear cells aggregation around congested blood vessels with fibrin deposition in parenchyma of liver and dilated sinusoids (Fig.:5), Also inflammatory cells particularly heterophils infiltration between fragment fibers of skeletal muscle (Fig.:6), but there was no clear lesion in heart and in bones. In addition to above changes, sections from kidneys also were showed hemorrhage in the interstitial tissue, mononuclear cells infiltration between glomeruli, mononuclear cells aggregation in the wall of congested blood vessels with acute cellular degeneration of tubular epithelial lining (Fig.:7). Also infiltrated of a few of MNCs in the wall of the bronchiole of the birds lungs. The spleen showed amyloid like substance deposition around white pulp. While the bursa of Fabricius showed focal necrosis of lymphoid follicle (Fig.: 8). Animals fed contaminated diet and infection with S.typhimurium. The heart shows fibrin deposition, edema and inflammatory cells infiltration in the pericardium in addition to inflammatory cells infiltration between cardiac muscle fiber (Fig.: 9), while in kidney shows severe hemorrhage in the interstitial tissue with acute cellular degeneration (Fig.:10), also severe hemorrhage in the capsular region with severe vacuolar degeneration of epithelial cells may noticed in other animals , While the lesion of bursa revealed severe depletion of lymphoid follicles with cystic formation (Fig.:11), The spleen shows severe depletion of white pulp with bacterial colonies in spleen ( Fig.:12), the main lesion of the liver in bird of this group consisting of severe destruction of hepatocytes with large necrotic area accompanied with granulomatous lesion ,in another chicken severe portal fibrosis of liver parenchyma (Fig.:13), and skeletal muscle showed inflammatory cells infiltration between fragment muscle fiber, In the lung section revealed inflammatory cells infiltration in the interstitial tissue with calcium particles deposition in the atrial tissue in addition to hemorrhage in the parabronchial tree in another animal.  control -(without treatment) Histopathological examination revealed that the spleen expressed normal white pulp with mononuclear cells lining sinus of red pulp, and the bursa showed folded mucosa ,epithelial layer , cortex and medulla of lymphoid follicles, also it was recorded that the lung normal with parabronchial tree lining by single epithelial cells and atria around alveolar duct (Fig.:14), mononuclear cells aggregated in portal area of the liver (Fig.:15), few mononuclear cells were seen between mucosal glands of intestine, in addition to normal skeletal muscular fiber and normal structure of the kidney and heart.

weeks, shows mononuclear cells aggregation in portal area (arrow) (H &E stain 100X)
The present study showed increased thickness of the skin in chicks immunized with WSSAgs ,this result may indicated this type of antigen can stimulated cell mediated immune response, since DTH reaction was considered one sign of cell mediated immune response that stimulated by Th1 cytokines such as IFN-y, this idea was agreement with (20) who demonstrated that WSSAgs stimulated both humoral and cell mediated immune response (24). The current result revealed that immunized chicks with WSSAgs and fed diet contaminated with mycotoxins expressed low skin thickness and serum antibody titers as compared with those value in the immunized chicks with WSSAgs only, these result may indicated that the mycotoxin can cause suppression of humoral and cell mediated immune response which is dependent on activation of CD4 T cells by proper presenting antigen cells APCs, these idea was agreement with (19) who demonstrated that aflatoxin B1 (AFB1) can induce dysregulation of the antigen presenting capacity of dendritic cells associated with impair cell mediated immunity. The mean value of phagocytic index in G2 was higher than the other group this result may indicate (WSSAgs) stimulate immune response through production interferon gamma that stimulate activity of macrophage this idea agreement with (12) who was reported both of INF-γ and TNF-α can stimulate the bactericidal activity of macrophages. Also the phagocytic activity in G1 were less than G2 this result indicate that mycotoxin can suppress activity of phagocytic cell. This idea agreement with (5) who demonstrate the mycotoxin can decrease innate and acquired immune response. The high mortality rate in G3 post infection with S.typhimurium may indicate that the isolated strain highly virulent and avoid the immune system and disseminated to all the organs causes septicemia and dead of animal this idea agreement with (11) who demonstrated infection with salmonella particularly S.Gallinarum, progresses anaemia and septicaemia occur with salmonella shed back result in death of the animal. Also the highly mortality 80% of G1 and G3 may indicate that the mycotoxin increase susceptibility to infection to S. Typhimurium as result to depression of immune response and produce oxidative stress this idea in coincidence with result of immune response, results this idea was agreement with (30) who recorded that mycotoxins can suppress immune responses and result in increased susceptibility to infection, also high mortality rates in broiler chickens in group four as compared with immunized animals fed normal diet may indicated that the mycotoxin can inhibit immune response elicited by WSSAgs, these idea was agreement with (14) and (14) who demonstrated that T-2 can inhibit protein and DNA synthesis and immunosuppression associated with impair antibodies production and inhibit DCs maturation and development else, it was recorded that T 2 can decrease function of the innate immune response (13).
The severe pathological lesions in the examined organs of chicks fed diet contaminated with mycotoxin may due to cytotoxic effect of the mycotoxins ,that cause liver and kidney damage (23) that associated with increase susceptibility of bird to infection (10) as well as cellular apoptosis (2), also these mycotoxin can cause oxidative damage of the cells consist such as lipid, proteins and nucleic acids (18), in addition to it was recorded that T-2 can reduce Tlymphocytes proliferation and antibody production (21). The severe lesions in the lymphatic tissue associated with dead most of broiler fed diet contaminated with mycotoxin after infection with S.typhimurium may indicated that this toxin act as predisposal factor for Salmonella infection , these idea was agreement with (17) who recorded that the poultry fed diet contaminated with T 2 expressed severe economic losses as a result infection by different disease outbreaks, It was recorded that the spleen was more sensitive to AFB1 as a result that this toxin cause severe damage to lymphoid tissue (6). The depletion of bursa of fabricia in the broiler infected with S.typhimurium in the present study may indicated that the toxin of this pathogen cause damage of these organs ,this idea was agreement with (25) who found that various infectious disease such as E.coli and Salmonellosis can induced acute atrophy these organs.