2020, Scienceline Publication

JWPR

Poultry Research

J. World Poult. Res. 10(2S): 235-246, June 14, 2020

Journal of World’^s

Research Paper, PII: S2322455X2000029-10

License: CC BY 4.0

DOI: https://dx.doi.org/10.36380/jwpr.2020.29

Evaluation of the Effect of Mycotoxins in Naturally Contaminated

Feed on the Efficacy of Preventive Vaccine against Coccidiosis in

Broiler Chickens

Anwaar M. Elnabarawy^1*, Marwa M. Khalifa², Khaled S.Shaban¹and Walied S. Kotb³

¹Department of poultry diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

²Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

³Department of Pathology, Faculty of Veterinary Medicine, Kafr el-sheikh University, Egypt

*Corresponding author’s Email: anwaar.elnabarawy@gmail.com; ORCID: 0000-0003-1618-6842

Received: 18 Feb. 2020

Accepted: 29 Mar. 2020

ABSTRACT

This research was designed to evaluate the effect of naturally contaminated feed with mycotoxins on the efficacy of

vaccination against coccidiosis in broilers. Two hundred day-old Hubbard broiler chicks were divided into four

groups (50 chicks/group). Group 1 and 3 were kept on naturally contaminated diets containing 4 ppb aflatoxin, 3 ppb

ochratoxin, 1 ppm zearalenone and 2 ppb aflatoxin, 6 ppb ochratoxin and 1 ppm zearalenone in starter and grower

feed, respectively. Groups 2 and 4 were fed on diet without detectable levels of mycotoxins. Group 1 and 2 were

vaccinated with anticoccidial vaccine at 4 days of age. All groups were challenged with Eimeria tenella

(5×10⁴/chick) 14 days post-vaccination. Vaccinated mycotoxicated birds showed a significant reduction in body

weight, high mortality, significant oocysts shedding, severe hemorrhagic typhlitis, marked lymphoid depletion in

bursa of Fabricius and degenerative changes in liver and kidney. In addition, a remarkable decrease in length and

width of intestinal villi, mucosal length and crypt depth. Feed contamination with multi-mycotoxins in permissible

level caused vaccination failure and a remarkable decrease in intestinal morphometric histopathological parameters.

Key words: Coccidia Vaccine, Mycotoxins, Poultry Feed.

INTRODUCTION

immune responses including poor antibody titer and

lymphoid organ damage subsequently increased

susceptibility to different infections (Rosa and Santurio,

2005; EFSA, 2009; Resanovic et al., 2009 ).

Mycotoxicosis was firstly described by Forgacs and Carll

(1955) as toxicosis arising from fungus-infested feed.

They reported a hemorrhagic condition in poultry which

was associated with the ingestion of fungus and fungal

products in moldy feed. Later in the early 1960, an acute

hepatotoxic disease epidemic struck the turkey population

in England.

The presence of multiple mycotoxins simultaneously

in feed commonly occur as a result of the presence of

many fungal species in feed producing several different

mycotoxins simultaneously or due to formation of poultry

feed from different feed ingredient with different sources,

each of which is contaminated with a different mycotoxin

(Trenholm et al., 1989 ). The interactive effects of

mycotoxins, when occur in combinations, may be

synergistic, potentiated, or even antagonistic (Kubena et

al., 1988 ).

Immunosuppressive effects of mycotoxins are due to

effect on serum proteins, macrophages, complement and

interferon are because of inhibition of protein synthesis

and liver damages (Resanovic et al., 2009 ). Mycotoxins

also cause aplasia of bursa of Fabricius, thymus, and

spleen in chicken, which results in a marked decrease in

cellular and antibody responsiveness of immune system

(Karaman et al., 2005 ). Moreover, mycotoxins induced

marked morphological alteration in intestinal histology.

The most common pathogenic Eimeria species

affecting chickens are Eimeria necatrix, E tenella, E.

acervulina, E. maxima, and E. brunetti. Infection with

Eimeria spp. causes chicken coccidiosis that leads to

mortality, decreased weight gain and weights uniformity

of birds flock (McDougald and Fitz-Coy, 2013 ). This

protozoal disease causes enormous economic losses with a

global impact estimated to be over 3 billion USD per year

The most common clinical signs of mycotoxicosis in

broiler chickens are reduced feed intake, weight gain, poor

food conversion ratio, increased mortality and reduced

To cite this paper: Elnabarawy AM, Khalifa MM, Shaban KhS and Kotb WS (2020). Evaluation of the Effect of Mycotoxins in Naturally Contaminated Feed on the Efficacy of

Preventive Vaccine against Coccidiosis in Broiler Chickens. J. World Poult. Res., 10 (2S): 235-246. DOI: https://dx.doi.org/10.36380/jwpr.2020.29

235

Elnabarawy et al., 2020

in the poultry industry (Dalloul and Lillehoj, 2006 ).

Purification, identification and counting of oocysts

Purification of sporulated oocysts was done

Moreover, coccidiosis is considered an important factor

for the development of clostridial infection particularly

necrotic enteritis (Dahiya et al., 2006; Collier et al., 2008 ).

Poultry field protecting different poultry species and

performance from coccidiosis challenge by acquired

immunity (Shirley et al., 1995). So control of coccidiosis is

achieved by vaccination as an alternative to chemotherapy

as it is overcoming the problem of drug resistant resulting

from usage of chemotherapy. Anticoccidial vaccines are

composed of live oocysts of attenuated or non-attenuated

strains of Eimeria (Shirley et al., 2007). Therefore, this

study aimed to evaluate the effect of mycotoxins

determined in naturally contaminated broilers feed and

proven to be within the permissible levels, on the efficacy

of vaccines recommended against coccidiosis through

different parameters.

according to Khaier et al. (2015). The sporulated oocysts

were mainly identified as Eimeria tenella according to its

confined caecal part in naturally infected chickens and due

to its typical measurements of E. tenella according to

Khaier et al. (2015). Counting E. tenella oocysts was done

using McMaster Technique according to Soulsby (1982).

Infection and challenge

After count of sporulated oocysts (3 replicate), the

oocysts were allocated in separate doses each of 5x 10⁴.

Ten Birds from vaccinated and non-vaccinated groups were

inoculated intra-croup using suitable rubber syringe at the

recommended day of challenge (Velkers et al., 2010).

Oocysts output in feces of challenged birds were evaluated

weekly until the end of experiment, pooled samples from

dropping of the inoculated birds were collected (from each

group) as before.

MATERIAL AND METHODS

Sample collection

Fecal samples (droppings) were collected weekly

from all groups.

Ethical approval

This study was approved by Institutional Animal

Care and Use Committee (IACUC), Cairo University

(VetCU1010201903).

Histopathological samples

Tissue samples from intestine, bursa of Fabricius,

liver, and kidney were collected weekly from different

groups. These samples were fixed in 10% neutral buffered

formalin, sectioned at 5-6μm thicknesses and stained with

Hematoxylin and Eosin (H&E) stain (Bancroft et al., 1996 ).

Broilers feed

Commercial feed specified for broilers was analyzed

for detection and determination of contamination levels for

three important mycotoxins (aflatoxin, ochratoxin, and

zearalenone) in starter and grower feed types. Where feed

bags were thoroughly mixed to obtain representative feed

samples and fluorometer series 4 and protocol of manual

were used for quantitive determination of aflatoxin,

ochratoxin, and zearalenone according to AOAC (1995)

and FAO (2003).

Evaluation of parameters

Chicks were monitored daily for clinical symptoms,

mortality and post-mortem lesions. Counting of sheded

oocyst in both vaccinated, nonvaccinated groups. Body

weight, intestinal lesion scoring of morphometric

histopathological lesions with different treated groups were

recorded. Challenging parameters (mortality % and oocyst

shedding) were calculated.

Chicks

Two hundred day-old Hubbard broiler chicks were

employed in this experiment.

Experimental design and housing

Coccidial oocysts

Collection and sporulation of oocysts

The experiment was conducted in the poultry

experimental units of Poultry Diseases Department,

Faculty of Veterinary Medicine, Cairo University, after

cleaning and disinfection. Two hundred day-old Hubbard

broiler chicks and commercial diet specified for broilers

feeding free from anticoccidial and antimycotoxins were

employed in this study. The chicks were divided into four

groups (50 chicks/group). Group 1 and 3 (control positive

groups) were kept on naturally contaminated diet

Eimeria species oocysts used for challenge were

collected from ceci of dead naturally infected chickens. The

collected oocysts were cleaned and incubated for 48 hours

in 2.5% potassium dichromate (K₂Cr₂O₇) solution for

sporulation according to Khaier et al. (2015).

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J. World Poult. Res.,10(2S): 235-246, 2020

containing 4 ppb, 3 ppb, 1ppm and 2 ppb, 6 ppb and 1

compared to mild hemorrhagic typhlitis in vaccinated

challenged control negative group (Figure 4). Vaccinated

challenged control negative group showed mild

ppm aflatoxin, ochratoxin and zearalenone in starter and

grower feed, respectively. GroupS 2 and 4 (control

negative groups) were fed on mycotoxins free diet. Groups

1 and 2 were vaccinated at age 4^thday with anticoccidial

vaccine via eye instillation. Group 3, 4 kept as control

positive and control negative non vaccinated groups.

Appropriate temperature, humidity, feeding, and lighting

program were followed according to standard

recommended by supplies. At 14^thday post-vaccination,

10 chicks from each group (1-4) were challenged with

5x10⁴live Eimeria tenella sporulated oocysts. All birds

were vaccinated at 7 days of age with Hitchner B via eye

instillation, at 10 days of age with inactivated avian

influenza and Newcastle disease virus (NDV) via

subcutaneous route at a dose of 0.5 ml per bird. At 13 days

of age, birds were vaccinated with live intermediate

Gumboro strain via eye instillation. Finally, all birds were

vaccinated by the NDV Lasota vaccine at 20 days of age

via eye instillation.

hemorrhagic

typhlitis.

Vaccinated

challenged

mycotoxicated group (control positive) showed severe

hemorrhagic typhlitis and challenged control negative

non-vaccinated group showed inspciated hemorrhagic

typhlitis (Figure 5). Moreover, pale yellow liver, marked

lobulation and paleness in kidney were constant

macroscopic lesions recorded in mycotoxicated groups

during the experimental period.

Statistical analysis

PASW Statistics, SPSS 18.0 software (SPSS Inc.,

Chicago, IL, USA) was used to analyze the data. Two-way

ANOVA was used to compare means between different

groups. Differences were considered statistically

significant at P-value < 0.05.

Figure 1. Reduction in body weight gain recorded in

broiler chicks fed on mycotoxin naturally contaminated

feed and vaccinated with anticoccidial vaccine (right)

compared to normal growth pattern in negative control

vaccinated group (left).

RESULTS

1. Clinical signs

Mycotoxicated groups (G1 and G3) showed un-

uniform growth pattern, whitish droppings, lameness and

inability to stand. Moreover, diarrhea tinged with blood

was recorded on 8-10 days post-vaccination in group 1

(control positive mycotoxicated vaccinated group). The

mortality rate was 40% in mycotoxicated vaccinated

group.

2. Postmortem lesions

Retardation in growth, severe hemorrhagic typhlitis

observed in control positive (mycotoxicated vaccinated

group) compared to few petechial hemorrhages on cecum

of control negative vaccinated group (Figures 1 and 2).

Petechial

hemorrhages

and

grayish-white

foci

Figure 3. Petechial hemorrhages and grayish-white foci

(schizogony) indicating Eimeria necatrix infection in

broiler chick fed on mycotoxin contaminated feed and

vaccinated with anticoccidial vaccine.

(schizogony) indicating Eimeria necatrix infection in

control positive (mycotoxicated vaccinated group 1)

(Figure 3). In addition to severe hemorrhagic typhlitis

observed in control positive vaccinated challenged group

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Elnabarawy et al., 2020

A

B

C

Figure 5. Post-mortem examination of broilers chickens

challenged with Eimera tenella oocysts (5×10⁴/chick) 14

days post-vaccination. (A) Mild hemorrhagic typhlitis

recorded in bird vaccinated with anticoccidial vaccine and

kept on mycotoxins free diet. (B) Severe hemorrhagic

typhlitis observed in bird vaccinated with anticoccidial

vaccine and fed with mycotoxins contaminated feed. (C)

Inspcisated hemorrhagic typhlitis observed in non-

vaccinated bird fed with mycotoxins free diet.

3. Body weight, Eimeria oocysts count

The mycotoxicated groups either vaccinated or non-

vaccinated (G 1 and G3) statistically recorded a significant

reduction in body weight compared to mycotoxins free

control negative groups (G 2 and G4) as showed in table 1.

Table 2 presents that mean coccidial oocysts count

shed from mycotoxicated vaccinated group significantly

higher than those shed from control negative vaccinated

group. In control negative and control positive non-

vaccinated groups (G3 and G4), none of oocysts were

detected along the experimental time. A two-way analysis

of variance yielded a main effect for the groups, F (1, 16)

= 39.963, p<0.0001, the oocyst shedding was significantly

higher for group 1 (M = 2.8×10⁶, SE = 4.9×10⁵) than for

group 2 (M = 1.5×10⁶, SE = 3.0×10⁵). The main effect of

weeks was significant, F (3, 16) = 45.027, p<0.0001.

Moreover, the interaction effect was significant, F (3, 16)

= 3.395, p=0.044, There was a statistically significant

interaction between the effects of groups and weeks on the

shedding of coccidial oocyst.

Figure 2. Severe hemorrhagic typhlitis observed in 14-day

old broiler chicks fed on mycotoxin-contaminated feed

and vaccinated with anticoccidial vaccine (up) compared

to few petechial hemorrhages on cecum of negative

control vaccinated birds (down).

Table 3 showing oocyst coccidial count shed from

vaccinated challenged and non-vaccinated challenged

groups significantly higher number in group 1, 3 than

group 2, 4. A two-way analysis of variance yielded a main

effect for the groups, F (3, 16) = 12.228, p<0.0001, the

oocyst shedding was significantly lower for group 2

(M=1.0×10⁶, SE=3.9×10⁵) than for groups 1, 3 and 4. The

main effect of weeks was significant, F (1, 16) = 280.688,

p<0.0001. Moreover, the interaction effect was significant,

Figure 4. Severe hemorrhagic typhlitis was observed in

birds fed on mycotoxin contaminated feed and vaccinated

with anticoccidial vaccine and challenged with Eimera

tenella oocysts (5×10⁴/chick) 14 days post vaccination

(left). Mild hemorrhagic typhlitis was observed in birds

fed on mycotoxin free diet, vaccinated with anticoccidial

vaccine and challenged with Eimera tenella oocysts

(5×10⁴/chick) 14 days post vaccination (right).

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F (3, 16)=4.184, p=0.023, There was a statistically

significant interaction between the effects of groups and

weeks on the shedding of coccidial oocysts.

lining and necrotic core. There was also a remarkable

decrease in intestinal parameters.

Vaccination of the challenged birds showed

improvement in intestinal parameters. While mycotoxin

supplementation in diet of diseased birds with previous

vaccination demonstrated marked retardation of jejunal

morphometric parameters.

The cecum showed more prominent lesions than

other intestinal sections (Figure 7) including the jejunum.

The bird vaccinated with live coccidial vaccine (G2)

showed a mild degree of necrotic typhlitis associated with

the presence of different coccidial stages within the

mucosal cell lining. The chicken kept on mycotoxins -

contaminated ratio and vaccinated with the same vaccine

(G1) revealed a marked degree of necrotic enteritis,

typhlitis accompanied by interstitial hemorrhage with a

high number of different coccidial stages.

4-Mortality pattern

Mortality rate recorded in different groups was 40%

in control positive vaccinated group, 0% in control

negative vaccinated group and control negative vaccinated

challenged, 60% in vaccinated challenged control positive

group. Eighty percent in control positive non vaccinated

challenged group and 20% in control negative non

vaccinated challenged group.

5-Result of histopathological examination

5.1. Histopathological scoring of the intestinal

parameters and pathological alteration lesions of

jejunum and cecum within the different treated groups.

The histopathological score as illustrated in the table

4. Pathological alteration lesions (Figure 6) were recorded

in groups G2, G1 (control negative and control positive

vaccinated groups). The jejunum of chicken supplemented

with basal diet and vaccinated with the live attenuated

coccidial vaccine (G2) showed the feature of catarrhal

enteritis associated with mucosal lining degeneration,

goblet cell proliferation and marked lymphocytic cells

infiltration. The jejunum of chicken supplemented with

mycotoxin-contaminated ration and vaccinated with the

same vaccine (G1) revealed marked aggravation the

inflammatory grade to reach to some cases to necrotic

enteritis accompanied with focal ulceration of the lining

mucosa. Also, a decrease in the intestinal morphometric

parameters in comparison with previous group. Most of

the jejunal villi showed marked blunting associated with

decrease their length and decrease the crypt depth.

(p<0.05). The jejunum of chicken supplemented with basal

diet then challenged Showed mild degree of necrotic

enteritis. While challenged birds subjected to

mycotoxicated-diet revealed a marked degree of necrotic

enteritis associated with necrosis, sloughing of mucosal

5.2. Histopathological findings of bursa of

Fabricius and kidney in control negative and

mycotoxicated control positive groups.

In Figure 8, the bursa of Fabricius in control

negative bird showed normal bursal compartments with an

increase of lymphoid elements (A) while the bursa of

control positive birds showed separated follicles,

edematous background and marked germinal centers

necrosis associated with endodermal hyperplasia (B). The

kidney of control negative bird revealed mild renal tubular

degeneration mostly of granular eosinophilic cell swelling

of the renal tubular epithelium (C) compared to kidney of

control positive birds showed marked tubular degeneration

accompanied with marked vacuolation of the renal tubules

and interstitial inflammatory reaction mostly mononuclear

cells (D). Later on, liver of control negative bird showed

normal hepatic tissues (E) compared to liver of control

positive birds showed hepatic degenerative changes

represented by marked hydropic degeneration to

multifocal hepatic necrosis associated with marked

lymphocytic cells infiltration (F).

Table 1. The effect mycotoxin-contaminated feed on body weight of broiler chickens in vaccinated and non-vaccinated groups

against coccidiosis.

Overall

Mean±SE

389.38±35.35^b

464.88±47.24^a

377.00±33.09^b

436.38±40.58^a

Groups

Week 1

128.00±5.28 266.00±10.92 466.50±19.90

133.50±4.15 275.50±5.60 567.50±13.48

Week 2

Week 3

Week 4

G1 (mycotoxicated, vaccinated group)

697.00±25.64

883.00±40.76

628.50±23.71

748.50±25.85

G2 (Non-mycotoxicated, vaccinated group)

G3 (mycotoxicated, non-vaccinated group)

G4 (Non-mycotoxicated, non-vaccinated group)

110.00±6.32 282.50±20.87 487.00±25.65

116.00±7.59 303.00±14.32 578.00±34.27

^a,bDifferent superscripts indicate significant difference at p<0.05; SE: Standard error

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Elnabarawy et al., 2020

Table 2. The effect mycotoxin-contaminated feed on Eimeria oocysts count of broiler chickens in vaccinated and non-vaccinated

groups against coccidiosis

Overall Mean±SE of

Groups

Time 1

Time 2

Time 3

Time 4

times/group

G1 (mycotoxicated &vaccinated group )

G2 (Non-mycotoxicated, vaccinated group)

G3 (mycotoxicated, non-vaccinated group)

4.6×10⁶±6.7×10⁵3.5×10⁶±1.7×10⁵2.8×10⁶±5.9×10⁴

2.8×10⁶±4.1×10⁵1.6×10⁶±2.3×10⁵1.5×10⁶±1.2×10⁵

3.9×10⁵±5.8×10⁴

1.4×10⁵±1.2×10⁴

0.0±0.0

2.8×10⁶±4.9×10^5a

1.5×10⁶±3.0×10^5b

0.0±0.0

G4 (Non-mycotoxicated, non-vaccinated group)

p-value

^a,bDifferent superscripts indicate significant difference at p<0.05

0.0±0.0

< 0.0001

Table 3. The effect mycotoxin-contaminated feed on Eimeria oocysts count of broiler chickens in vaccinated with anticoccidial

vaccine and non-vaccinated groups post-challenge with Eimera tenella oocysts (5×10⁴/chick) 14 days post vaccination.

Overall Mean±SE of

Groups

Time 1

Time 2

times/group

G1 (mycotoxicated &vaccinated group )

G2 (Non-mycotoxicated, vaccinated group)

G3 (mycotoxicated, non-vaccinated group

3.7×10⁶±2.4×10⁵

1.9×10⁶±1.2×10⁵

3.5×10⁶±2.3×10⁴

4.9×10⁵±5.8×10⁴

1.6×10⁵±2.0×10⁴

8.5×10⁵±1.8×10⁵

2.1×10⁶±7.2×10^5a

1.0×10⁶±3.9×10^5b

2.2×10⁶±5.9×10^5a

G4 (Non-mycotoxicated, non-vaccinated group)

2.9×10⁶±4.8×10⁵

7.1×10⁵±5.8×10⁴

1.8×10⁶±5.5×10^5a

^a,bDifferent superscripts indicate significant difference at p < 0.05

Table 4. Histopathological scoring of the intestinal parameters (jejunum and cecum) within the different groups of broiler chickens

Treatment

Jejunum

Cecum

Vaccination

against

coccidiosis

Mucosal

length

(µm)

Groups

Villi length

(µm)

Villi width

(µm)

Crypt depth

(µm)

No. of

Diet

Mycotoxicated

Basal

Challenge *

oocytes/mm²

Unchallenged

Challenged

583.04±60.93

486.06±58.38

728.34±86.96

662.90±65.96

79.44±17.73

68.40±23.74

37.35±5.41

51.26±7.77

74.45±11.12

59.86±10.21

136.82±27.72

96.56±8.13

506.62±35.05

427.40±32.00

615.05±69.45

552.02±61.35

39.75±1.71

60.50±4.43

17.50±1.91

30.75±3.30

G1

G2

Vaccinated

Unchallenged

Challenged

G3

G4

Mycotoxicated

Basal

Nonvaccinated

Challenged

467.49±49.94

69.47±21.58

62.51±13.90

94.74±11.69

363.79±70.75

501.49±19.66

73.50±5.80

43.75±3.77

Challenged

588.53±61.07

53.12±17.90

*Broiler chicks were challenged with Eimeria tenella oocyst (5*10⁴/chick) 14 days post-vaccination.

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Figure 6. Intestine (jejunum section) of different chicken groups (2^ndweek); A) chicken supplemented with basal diet and

vaccinated with live anti-coccidial vaccine showing normal intestinal villi (arrow indicates normal mucosal lining); B) chicken

supplemented with mycotoxin-contaminated ration and vaccinated with the same vaccine showing blunting of the intestinal villi and

decrease of their length; C) chicken supplemented with basal diet and then challenged with E. tenella oocysts revealing marked

degenerative changes within the covering mucosa (arrow); D) chicken supplemented with mycotoxin-contaminated ration and challenged

showing severe catarrhal enteritis (arrow indicates marked inflammatory cells infiltration mostly mononuclear cells); E) chicken

supplemented with normal ration, vaccinated and challenged showing decrease the degenerative and desquamative changes and with

improvement of villi length; F) chicken supplemented with mycotoxin-contaminated ration, vaccinated and challenged showing necrosis and

sloughing of the mucosal lining (arrow). H&E, X200.

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Figure 7. Cecal section of different groups (2^ndweek); A) chicken supplemented with basal diet and vaccinated with live anti-

coccidial vaccine showing presence of few numbers of coccidial oocysts (arrow) with slight intestinal crypt degeneration; B) chicken

supplemented with mycotoxins-contaminated ration and vaccinated with the same vaccine showing presence of remarkable number of

parasitic oocysts within the interstitial tissue and glandular epithelium mucosa (arrows); C) chicken supplemented with basal diet and then

challenged revealing marked degenerative and hyperplastic changes within the crypt epithelium associated with presence of the different

coccidial stages within the epithelium (arrows); D) chicken supplemented with mycotoxins-contaminated ration and challenged showing

severee necrotic typhilitis (arrowheads indicates necrosis of the intestinal crypts) and marked interstitial hemorrhage (curved-arrow)

associated with coccidial stages (arrows); E) chicken supplemented with normal ration, vaccinated and challenged showing a marked

decrease of coccidial stages with the intestinal mucosa (arrows) and with subsequent decrease intestinal degeneration and necrosis; F)

chicken supplemented with mycotoxin-contaminated ration, vaccinated and challenged showing superficial sloughing of the mucosal lining

(arrowheads) and crypt necrosis accompanied with coccidial parasites (arrows). H&E, X200.

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Figure 8. The bursal, renal and hepatic lesions in control negative and positive group according to mycotoxins

supplementation. A represents the bursa of Fabricius in control negative bird showing mild reactive lymphoid hyperplasia (arrow); B) the

bursa of control positive birds that showing marked lymphoid depletion of the germinal centers (arrow); C) kidney of control negative bird

that showing mild renal tubular degeneration (arrow indicates granular eosinophilic cell swelling); D) the kidney of control positive birds

that showing marked tubular degeneration (arrow); E) liver of control negative bird that showing mild hepatic vacuolation (arrow); F) the

liver of control positive birds that showing focal hepatic necrosis associated with marked lymphocytic cells infiltration (arrow). H&E,

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Elnabarawy et al., 2020

multi contamination with mycotoxins altered immune-

DISCUSSION

mediated components. In addition, immunosuppression

induced by mycotoxins causing a decrease in host

resistance and consequently increase susceptibility to

infectious diseases and reduce vaccine efficacy.

While Pier (1992) confirmed that the vaccinal

immunity in properly vaccinated flocks is broken down

due to the contamination of mycotoxins in feed.

Avian mycotoxicosis is a great constraint in the poultry

industry due to the development of immunosuppression,

hepatotoxicity, and nephrotoxicity. Mycotoxins can

transfer through chicken meat and egg to human,

therefore, avian mycotoxicosis also is considered a public

health issue (Adeniran et al., 2013).

The obtained result revealed that a significant

reduction in body weight of groups 1 and 3 in comparison

to the other groups. In the same respect, Aravind et al.

(2003) stated that naturally mycotoxin contaminated feed

at starter and growing period can affect broiler growth

performance. Moreover, Girish and Smith (2008) and

Yang et al. (2012) recorded a reduction in feed

consumption and nutrient digestibility. They referred to

these effects due to alterations caused by mycotoxins on

intestinal morphology. Rosa and Santurio (2005); EFSA

(2009) and Resanovic et al. (2009) reported that the most

common clinical signs of mycotoxins in broiler chickens

are reduced feed intake and weight gain, poor food

conversation ratio, increase mortality, reduced immune

response, organ damage, meat discoloration, and skeletal

abnormalities as tibial dyschondroplasia, articular gout. In

the same respect, Andretta et al. (2011) stated that the

mycotoxins presence in diets reduces weight gain by 14%

when compared with the control groups.

The effect of different mycotoxins in gut health was

reported by Liew and Redzwan (2018) as they described

the different actions of aflatoxin, ochratoxin, and

zearalenone and they include growth retardation,

immunosuppression, and genotoxicity. They also revealed

gut changes due to previously mentioned mycotoxins and

those are alterations in nutrient absorption, inhibition of

cell growth, increase lactate dehydrogenase activity and

caused genetic damages that mean disruption of intestinal

barrier, cell proliferation as the development of

subepithelial space and villi degeneration, cell apoptosis

a1nd immune system.

In the same respect, Desjardins (2006) found that

Fusarium mycotoxins are affecting different cellular and

molecular levels those resulting in adverse effect on

proliferation and differentiation of immune system cells.

The explanation of that mycotoxins are immune

suppressive resulting in inhibition of protein synthesis or

impairment of the activity or secretory functions of

immune system cells as well as synthesis of cytokines that

regulate the communication network of the immune

system (Swamy et al., 2004; Oswald et al., 2006 ).

Moreover, Oswald et al. (2006) reported that in vitro

phagocytosis, intracellular killers were inhibited by

aflatoxin B1. Gastrointestinal tract (GIT) function is feed

ingestion, digestion, energy, and nutrients absorption, as

well as elimination of waste products (Celi et al., 2017 ).

Epithelial layer is the inner most of intestinal mucosa of

vital importance. As they contains enteroendocrines,

enterocytes and gablet cells at villi whereas the paneth

cells, located under the crypts (Fink and Koa, 2016 ). This

epithelium layer working as normal barrier to prevent the

entry of pathogens and toxins moreover, it is the site for

nutrient absorption including electrocytes (Constantinescu

and chon, 201 6). Desmosomes tight junctions and adherent

junctions are connecting intestinal epithelial cells. These

junctions controlling the intercellular space and regulate

selective paracellular ionic solute transport (Capaldo et al.,

2014 ). Zearalenone well recognized to be implicated in

reproductive disorders. Zhou et al. (2017) indicated that

zearalenone has hepatotoxic, hematotoxic, immunotoxic

and genotoxic effect. The effect of zearalenone on GIT is

that, inducing cell death without affecting cell integrity

(Marin et al., 2015). Ochratoxin the immunsuppresive,

teratogenic and nephrotoxic substance reflected faster and

more harmful parasite infection induced by E. acervulina

and E. adenoides in OTA treated chicks and turkeys

(Laderia et al., 2017 ). As Manafi et al. (2011) indicated

that high lesion and oocyst indices in the intestine due to

Eimeria infection caused more damage for mucosa and

this is attributed to increasing intestinal permeability

(McLaughlin et al., 2004 ). Solcan et al. (2015) reported

that OTA fed broilers caused a decrease in villi height and

Mycotoxins are potent immune suppressive factors

and produced a negative effect on both humoral and cell-

mediated immune response to live New Castle disease

viral vaccine resulting in pronounced lowering in

protection rate against infection with VVND

(viscerotropic velogenic New Castle disease virus). These

aforementioned results were obtained during experimental

work carried out by Anwaar et al. (2016).

Oswald et al. (2006) stated that mycotoxins ingestion

impaired the acquired immunity through vaccination and

244

J. World Poult. Res.,10(2S): 235-246, 2020

mycotoxicosis in naturally contaminated feed on performance and

serum biochemical and hematological parameters in broilers.

Poultry Science, 82:571-576. DOI:

https://dx.doi.org /10.1093/ps/82.4.571.

increase apoptosis of intestinal epithelial cells. Numerous

studies on broilers fed with aflatoxin B contaminated diet

showed that reduction in the density (weight/ length) of

intestine (Hossein and Gurbuz, 2015). Moreover, the

increased apoptosis was corresponded to lower jejuneal

villi height (Peng et al., 2014 ).

Bancroft JD, Stevens A and Turner DR (1996). Theory and practice of

Histological Technique. Fourth Ed., Churchill Livingstone, New

York, London, San Francisco, Tokyo. Available at:

https://trove.nla.gov.au/work/10963990

Capaldo CT, Farkas AE and Nusrat A (2014). Epithelial adhesive

junctions. F1000Prime Report, 6:13. DOI: https://doi: 10.12703/

P6-1.

DECLARATION

Celi P, Cowieson A, Fru-Nji F, Steinert R, Kluenter AM and Verlhac V

(2017). Gastrointestinal functionality in animal nutrition and health:

new opportunities for sustainable animal production. Animal Feed

Science and Technology, 234: 88-100. DOI: https:// 10.1016/

j.anifeedsci. 2017. 09.012.

Acknowledgment

This study was financially supported by scientific

research sector of Cairo University through the project

Collier CT, Hofacre CL, Payne AM, Anderson DB, Kaiser P, Mackie RI,

and Gaskins HR (2008). Coccidia-induced mucogenesis promotes

the onset of necrotic enteritis by supporting Clostridium perfringens

growth. Veterinary Immunology and Immunopathology, 122:104-

115. DOI: https://doi.org/10.1016/j.vetimm.2007.10.014.

titled

“Mycotoxicosis,

the

natural

potent

immunosuppressive carcinogen of veterinary and public

health concern".

Constantinescu CS and Chou IJ (2016). Intestinal bacterial antigens,

toxin induced pathogenesis and immune cross-reactivity in

neuromyelitis optica and multiple sclerosis, in Neuro-Immuno-

Gastroenterology, eds C. S. Constantinescu R, Arsenescu and V.

Arsenescu (Cham: Springer), 227-236. DOI: https:// 10.1007/978-3-

319-28609-9-13.

Authors’ contributions

Anwaar M. Elnabarawy designed the experiment,

provided facilities and material needed, performed

mycotoxin detection and determination, collected results,

and wrote and revised the manuscript. Marwa M. Khalifa

prepared the challenging doses of Eimeria oocysts,

collected dropping samples and counted numbers of

shaded oocysts in at least 40 samples, contributed to

manuscript writing. Khaled S. Shaban recorded the body

weight, daily observation for clinical symptoms, mortality

and contributed to detection and determination of

mycotoxins levels in feed. Walied S. Kotb prepared and

examined histopathological sections.

Dahiya JP, Wilkie DC, Van Kessel AG and Drew MD (2006). Potential

strategies for controlling necrotic enteritis in broiler chickens in

post-antibiotic era. Animal Feed Science and Technology, 129:60-

88 DOI: https://doi.org/10.1016/j.anifeedsci.2005.12.003.

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