JWPR  
Poultry Research  
J. World Poult. Res. 10(2S): 263-277, June 14, 2020  
Journal of World’s  
Research Paper, PII: S2322455X2000032-10  
License: CC BY 4.0  
An Experimental Trial for Prevention of Necrotic Enteritis by  
Vaccination and Immune Enhancement of Broiler Chickens  
Sarah S. Helal1*, Hagar F.Gouda2, Noura M. khalaf3, Rehab I. Hamed4, Abd Elmoneim A. Ali5 and Mohammed A.Lebdah1  
1Avian and Rabbit Medicine Department, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig , Sharkia Governorate, Egypt.  
2Animal Wealth Development Department (Biostatistics), Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig , Sharkia Governorate, Egypt.  
3Veterinary Serum and Vaccine Research Institute, 11381, Abbassia, Cairo, Egypt  
4Reference Laboratory for Quality Control on Poultry Production (RLQP), Animal Health Research Institute (Sharkia Branch), Zagazig , Sharkia Governorate, Egypt.  
5Pathology Department, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig , Sharkia Governorate, Egypt  
*Corresponding author’s Email: dr.sarah.saied@gmail.com; ORCID: 0000-0003-3047-2239  
Received: 23 Feb. 2020  
Accepted: 30 Mar. 2020  
ABSTRACT  
Alternative strategies are applied for the prevention of Necrotic Enteritis (NE) particularly after the global  
perspective of the antibiotic ban. This study was a trial for NE control depending on vaccination by toxoid and/or  
immune enhancement by Nutri-lac IGA administration (a liquid mixture of fermentation by-product 80%, lactic acid  
10%, and formic acid 10%). A total of 120 one-day-old broiler chicks were randomly divided into four groups (30  
chicks/group). Group 1 (G1) was vaccinated with C. perfringens type A toxoid; Group 2 (G2) was toxoid-vaccinated  
and immune enhanced by Nutri-Lac IGA; Group 3 (G3) was immune enhanced by Nutri-Lac IGA and Group 4  
served as control. Each group was subdivided into two subgroups, one subgroup was challenged with C. perfringens  
and the other was kept unchallenged. No significant clinical signs were detected in birds and mortality was observed  
only among challenged controls. The thin and friable intestinal wall was observed in all challenged broilers which  
extended to ulceration only in the challenged control group. No prominent histopathological findings related to NE  
were detected except in challenged controls and the highest protection against the NE-histopathological changes  
vividly appeared in the challenged G2 group. Significant increase in body weight of G1 and G2 groups after  
challenge in comparison to before challenge. While body weight of chickens in both G3 and challenged control  
groups was lower after challenge than before challenge. Pre-challenge ELISA results indicated no significant  
difference in immunoglobulin (Ig) Y titer among all groups after the first dose of vaccination, while significant  
differences appeared after the booster dose. The highest IgY titer was recorded in the G2 group, followed by G1, and  
G3 group. Post-challenge ELISA results showed a highly significant difference among all challenged subgroups. The  
highest IgY titer was recorded in the G1, followed by G2, and G3 group. The serum neutralization test also  
demonstrated the highest mean antibody titer in G1 and G2 groups. In conclusion, this study confirmed that a toxoid-  
immunostimulant combination is effective in NE prevention only when it is accompanied by the absence of NE  
predisposing factors.  
Key words: Broiler chickens, Clostridium perfringens type A, Immunoglobulin Y, Lesion scoring, Necrotic enteritis,  
Toxoid.  
INTRODUCTION  
(NE) is considered one of the most threatening diseases in  
the poultry industry as it is associated with dramatic  
economic losses mainly due to high mortalities and  
reduced growth performance (Lovland and Kaldhusdal,  
2001; Skinner et al., 2010). NE is principally caused by  
Clostridium perfringens type A and rarely by type C  
The disease was previously controlled by the  
administration of antibiotic growth promoters (Lanckriet  
et al., 2010a), before the ban of antibiotics usage in poultry  
breeding by European Union (Casewell et al., 2003).  
There is an emerging need for NE alternative control  
The poultry industry is one of the most vital sectors of the  
agriculture production system (Vaarst et al., 2015). Broiler  
chickens can be grown in an efficient and profitable  
breeding way by better management and adequate  
knowledge about infectious poultry diseases despite the  
continuous risk of developing diseases especially derived  
from normally inhabited microorganisms in the birds’ gut  
Enteric diseases can be caused by a wide range of  
etiological agents including Clostridium spp. (Cooper et  
al., 2013). Among clostridial diseases, Necrotic Enteritis  
To cite this paper: Helal SS, Gouda HF, khalaf NM, Hamed RI, Ali AEA and Lebdah MA (2020). An Experimental Trial for Prevention of Necrotic Enteritis by Vaccination and  
Immune Enhancement of Broiler Chickens. J. World Poult. Res., 10 (2S): 263-277. DOI: https://dx.doi.org/10.36380/jwpr.2020.32  
263  
Helal et al., 2020  
strategies particularly after problems related to the spread  
randomly divided into four groups (30 chicks/group):  
Group A was vaccinated; Group B was vaccinated and  
immune enhanced by Nutri-Lac IGA; Group C was  
immune enhanced by Nutri-Lac IGA and Group D was  
kept as control as shown in Table 1.  
of antibiotic-resistant microorganisms and antibiotic  
residues have appeared. The use of feed additives such as  
organic acids, essential oils, probiotics, prebiotics and  
symbiotics can partially decrease the NE occurrence in  
broilers without complete disease control (Timbermont et  
Recently, several studies have been focused on the  
development of vaccines against NE as active  
supernatants, formalin-inactivated toxoids and modified  
toxins either in I/M or S/C administration, in single or  
multiple dosage vaccination programs (Mot et al., 2013;  
This study was designed to prepare a formalized-  
killed vaccine (toxoid) from previously isolated toxigenic  
C. perfringens strain and to evaluate the efficacy of  
vaccine administration, Nutri-lac IGA treatment or the  
combination of both in NE controlling by using  
histopathology and measurement of humoral immune  
responses.  
Toxoid and immunostimulant administration  
regimen  
1st dose of vaccination: S/C injection of broiler  
chickens with 0.5 ml of prepared toxoid at 7 days of age.  
2nd dose of vaccination (booster dose): S/C injection of  
broiler chickens with 0.5 ml of prepared toxoid at 21 days  
of age. Nutri-Lac IGA liquid (Nutriad, Turnhout,  
Belgium), a liquid mixture of fermentation byproduct  
80%, lactic acid 10% and formic acid 10%, was given to  
broilers as immunostimulant at dose 3 ml/1 liter drinking  
water for five successive days at the 1st week of age and  
repeated at the 3rd week of age for 3-5 days according to  
the instruction of manufactured company.  
Experimental challenge  
MATERIALS & METHODS  
Before the experimental challenge, each group was  
subdivided into 2 subgroups (15 broilers/subgroup). One  
subgroup was challenged with C.perfringens whole culture  
and the other subgroup remained without challenge  
(Control) as shown in table 1.  
The challenge was carried out by oral administration  
of 1-2 ml of freshly prepared toxigenic NetB-negative  
C.perfringens whole culture (with bacterial concentration:  
109 CFU/ml and preformed toxin with Minimal Lethal  
Dose (MLD): 1/80) for 3 successive days (28th, 29th, and  
30th day of broilers` age). Mortalities and clinical signs  
were recorded during the experimental study.  
The mean body weight of all eight groups was  
measured twice before and after the experimental  
challenge comparing challenged birds and unchallenged  
controls. Blood samples were collected four times for  
measurement of anti-alpha toxin IgY: 1st time: before the  
1st dose of vaccination at 7th day of broilers` age; 2nd time:  
after the 1st dose of vaccination at 21st day of broilers` age;  
3rd time: after the booster dose of vaccination at 28th day of  
broilers` age and the last time: after the booster dose of  
vaccination and experimental challenge at 35th day of  
broilers` age. Humeral immune response was measured by  
ELISA according to the method described by Wood  
(1991) and Serum Neutralization Test (SNT) according to  
the method described by European pharmacopeia (1997).  
Lesion scoring was assessed according to the method  
described by Prescott et al. (1978). Lesions were scored  
from 0 to 4 (0: No apparent lesion; 1: thin friable small  
Ethical approval  
All animal care and experimental procedures were  
reviewed and approved by Zagazig University Institutional  
Animal Care and Use Committee (IACUC), and the  
IACUC gave this research the international criteria of the  
research  
ethics  
under  
the  
number  
(ZU-  
IACUC/2/F/102/2019).  
Toxoid preparation and evaluation  
The toxoid was prepared according to the method  
described by Gadalla et al. (1974) using C.perfringens  
type A highly toxigenic strain, fully identified by  
conventional and molecular methods by Helal et al.  
(2019). Thimerosal 10% (Sigma) was added as  
preservative and bactericidal agent. Aluminum hydroxide  
adsorbent gel 2% (Alliance Bio) was added at a  
concentration of 20% as an adjuvant.  
The prepared toxoid was tested by sterility and safety  
tests which were carried out under the regulation of British  
Veterinary Pharmacopoeia (2007) and ensured that the  
prepared toxoid was free from any bacterial or fungal  
contamination and safe for animal use.  
Experimental design  
120 one-day-old broiler chicks were obtained from  
Al Dakahlia Company, Mitghamr County, Dakahlia  
Governorate, Egypt, floor- reared and fed commercial  
balanced ration without feed additives. The broilers were  
264  
J. World Poult. Res., 10(2S): 263-277, 2020  
intestine; 2: focal necrosis/ulceration or both; 3: patchy  
cell aggregation, congested blood vessels with apparently  
normal adjacent hepatic cells and hyperplastic Kupffer  
cells were common in liver (Figure 1). In vaccinated  
control: Intestinal mucosa and submucosa revealed  
lymphocytic cell aggregation with the fusion of some  
intestinal villi, In addition to the presence of mild villous  
enterocyte desquamation. Mild interstitial and portal  
lymphocytic aggregation within apparently normal hepatic  
parenchyma was seen in liver (Figure 1). In vaccinated &  
Nutrilac IGA-treated challenged subgroup: Partial  
desquamated superficial villous enterocytes with the  
fusion of some intestinal villi, intense inflammatory cells  
infiltration in mucosa and submucosa and hyperplastic  
necrosis and 4: severe extensive mucosal necrosis). Tissue  
samples were taken at 7 days post-challenge from all  
groups either challenged or unchallenged. Tissue sections  
were routinely stained with H&E and microscopically  
underwent for histopathological examination.  
Statistical analysis  
Data were statistically analyzed by SPSS version 24  
(IBM Corp, Armonk, NY). Results were expressed as  
mean ± SE. One-way ANOVA was used to test differences  
among body weights and differences in anti-alpha toxin  
titer. P < 0.05 was considered statistically significant.  
Duncan’s multiple range test and least significant  
difference tests were applied as post hock test after  
significant ANOVA results. Paired samples t-test was  
used to test differences between groups before and after  
administration of toxoid and/or Nutri-lac IGA.  
intestinal crypts were noticed.  
Intense heterophilic  
aggregation, congested blood vessels and proliferative bile  
ductules beside normal adjacent hepatic cells were evident  
in liver (Figure 2). In vaccinated & Nutrilac IGA-treated  
control subgroup: Intestinal mucosa showed normal villi  
lined with enterocytes with proliferative intestinal crypts  
and normal muscular coat. Mild dilated hepatic sinusoids,  
normal hepatic cells, hyperplastic Kupffer cells, and few  
portal lymphocytic infiltrations were common in liver  
(Figure 2). In Nutrilac IGA-treated challenged subgroup:  
Mild intestinal lesions represented by partial destruction  
and desquamation of villous epithelium which resulted in  
denuded villi. Other villi appeared broad with hyperplastic  
villous enterocytes from the proliferative intestinal crypts.  
Focal replacement of the hepatic parenchyma with  
leukocytic aggregation mainly heterophiles and  
lymphocytes. The adjacent hepatic parenchyma containing  
hyperplastic Kupffer cells were seen in liver (Figure 3).  
In the Nutrilac IGA-treated control subgroup: All the  
intestinal coats appeared normal with proliferative  
submucosal intestinal crypts. Mild portal lymphocytic and  
heterophilic infiltration with edematous portal vein wall  
necrosis containing bacterial colonies and inflammatory  
cells in the superficial mucosa sometimes with extension  
to the deeper mucosa. In addition to necrotic debris and  
inflammatory cells in the lumen were common. The  
adjacent intestinal crypts in the mucosa and submucosa  
were necrotic with edema and necrosis of the muscular  
coat in the intestine. Multiple necrotic areas were  
disseminated in the hepatic parenchyma with variable  
degenerative changes in the adjacent hepatic cells.  
Numerous heterophils were seen in the interstitial tissue  
and portal area (Figure 4). In the negative control  
subgroup, all the intestinal coats appeared within the  
RESULTS  
No marked clinical signs could be detected during this  
study in all subgroups either in challenged or non-  
challenged broilers. No mortalities could be recorded  
except in the positive control at which 3 out of 15 broiler  
chickens died at 4th and 5th day post-infection (20%).  
Lesion scoring  
No apparent lesion could be detected in all  
unchallenged subgroups. Lesion score was 0 in vaccinated,  
vaccinated and Nutrilac IGA-treated, Nutrilac IGA-  
treated, and negative controls. Slight congestion in the  
liver could be seen in all controls. Thinning and friability  
of intestinal wall could be appeared in challenged  
vaccinated, vaccinated & Nutrilac IGA-treated and  
Nutrilac IGA-treated subgroups (lesion score =1). Besides,  
ballooning of intestine, congested mucosa at some  
intestinal parts and congested liver could be also detected  
in all challenged subgroups. Positive control showed gross  
lesions varying from thin and friable intestinal wall (lesion  
score =1) to necrosis and/or ulceration in the intestinal  
mucosa (lesion score =2). Severely congested liver with or  
without necrosis and congested intestinal wall with  
ballooning could be also detected.  
Histopathological examination  
In vaccinated challenged subgroup: Focal necrotic  
intestinal mucosa with few inflammatory cells and the  
surrounding intestinal crypts were hyperplastic to replace  
and regenerate the destructed mucosa. Portal mononuclear  
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Helal et al., 2020  
normal morphological picture while the hepatic  
parenchyma appeared normal (Figure 4).  
at the 7th day and 21st day of broilers` age (P>0.05) as  
shown in Figure 6. One-Way ANOVA results showed that  
there was a highly significant difference among the four  
groups in ELISA readings after the 2nd dose of vaccination  
at the 28th day of broilers` age. The highest antibody titer  
was recorded in vaccinated and Nutrilac IGA-treated  
group (2.95±0.21) followed by the vaccinated group  
(2.55±0.21). Nutrilac IGA-treated group had antibody titer  
lower (2.32±0.20) than vaccinated & Nutrilac IGA-treated  
group and the small decrease than the vaccinated group  
was not great to be significant. The negative control had  
the lowest antibody titer (1.64±0.11) as shown in Figure 6.  
One-Way ANOVA results showed that there was a non-  
significant difference among the unchallenged subgroups  
in ELISA readings after the 2nd dose of vaccination on the  
35th day of broilers age (P>0.05) as shown in Figure 6.  
Average body weight  
The difference in body weight (BW) among different  
subgroups was statistically insignificant (P>0.05) before  
the experimental challenge but there was a highly  
statistical difference in BW of all birds after experimental  
challenge as shown in Figure 5. Paired samples t-test  
results indicated that the highest increase in BW was  
detected in the vaccinated challenged subgroup as the  
average BW of examined broilers was significantly higher  
after challenge (1994.00 ± 66.69) than before (1460.00 ±  
50.99) P<0.01. Also in the vaccinated & Nutrilac IGA-  
treated subgroup, the significant difference before and  
after the challenge was recorded as average BW was  
higher after challenge (1566.00 ± 84.79) than before  
(1290.00 ± 30.98) P< 0.05.  
Humeral immune response after challenge  
One-Way ANOVA results showed that there was a  
highly significant difference among all subgroups in  
ELISA readings after the experimental challenge on the  
35th day of broilers age as shown in Figure 7. The highest  
IgY titer was reported at vaccinated subgroups either  
challenged (3.64±0.06a) or not (3.66±0.21a). Also  
vaccinated & Nutrilac IGA-treated challenged birds had  
higher IgY titer (2.69±0.03c) more than unvaccinated  
challenged birds either Nutrilac IGA-treated (2.38±0.18 cd)  
or positive control (2.10±0.11d). Positive control had the  
lowest antibody titer (2.10±0.11d). It also had a lower  
antibody titer than challenged Nutrilac IGA-treated group  
(2.38±0.18cd) but this difference wasn’t great to be  
significant. Negative control nearly had the same antibody  
titer (3.18±0.11b) of the unchallenged Nutrilac IGA-treated  
subgroup (3.17±0.25b). SNT results showed variation in  
the mean antibody titer among different groups as shown  
in table 2.  
There was a decrease in BW of broiler chickens in  
the Nutrilac IGA-treated subgroup before and after  
challenge (1430.00 ± 66.33 and 1334.00 ± 100.72),  
respectively. The same result was detected in the positive  
control (1432.00±39.67 and 1423.00±62.86) even if this  
decrease was statistically insignificant (P>0.05) Figure 5.  
Significant increase in BW among all unchallenged birds  
(vaccinated & Nutrilac IGA-treated (P<0.01), Nutrilac  
IGA-treated and negative control (P< 0.05) and vaccinated  
broilers (P>0.05) even if this increase was statistically  
neglected in the last subgroup) (Figure 5).  
Humeral immune response before challenge  
One-Way ANOVA results showed that there was a non-  
significant difference among the four groups in ELISA  
readings after the administration of 1st dose of vaccination  
Table 1. Experimental design  
Group  
Subgroup  
Vaccination  
Immunostimulant  
Experimental challenge  
1
+
+
+
+
-
-
-
+
-
Group A  
Group B  
Group C  
2
3
+
+
+
+
-
+
-
4
5
6
+
-
-
7 (positive control)  
8 (negative control)  
-
+
-
Group D  
-
-
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J. World Poult. Res., 10(2S): 263-277, 2020  
Table 2. Mean anti-alpha toxin titer (IU/ml) in all experimental chicken groups using serum neutralization test.  
Before vaccine  
administration  
at 7 days of age  
After 1st dose of  
vaccination at 21  
days of age  
After 2nddose of  
vaccination  
at 28 days of age  
After 2nd dose of  
vaccination  
at 35 days of age  
Group  
treatment  
Subgroup  
1
2
3
4
5
6
7
8
6
5
6
6
0
2
0
0
A
B
C
D
Vaccinated  
0
0
0
0
4
3
0
0
5
6
0
0
Vaccinated & Nutrilac  
IGA-treated  
Nutrilac IGA-treated  
Control  
Subgroups 1,3,5 and 7: challenged Subgroups 2,4,6 and 8: unchallenged  
Figure 1. Histopathological examination of liver and intestine of chickens vaccinated with Clostridium perfringens type A toxoid in  
challenged or unchallenged groups with toxigenic C. perfringens culture. (1a) the intestine of vaccinated and challenged chicken  
showing focal necrosis, few inflammatory cells and hyperplastic intestinal crypts, (1b) liver of vaccinated and challenged chicken  
showing portal mononuclear cell aggregation, congested blood vessels, apparently normal adjacent hepatic cells and hyperplastic  
Kuepfer cells, (1c) Intestine of vaccinated and unchallenged chicken showing intense mucosal and submucosal lymphocytic cell  
aggregation and mild villous enterocyte desquamation, (1d) liver of vaccinated and unchallenged chicken showing mild portal and  
interstitial lymphocytic aggregation within apparently normal adjacent hepatic parenchyma. (H&E ×400)  
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Helal et al., 2020  
Figure 2. Histopathological examination of intestine and liver of Clostridium perfringens type A toxoid-vaccinated and  
Nutrilac IGA-treated chickens in challenged or unchallenged groups with toxigenic C. perfringens culture. (2a) intestine of  
vaccinated, Nutrilac IGA-treated (challenged) chicken showing partial desquamated enterocytes, intense infiltrated  
inflammatory cells and hyperplastic intestinal crypts, (2b) liver of vaccinated, Nutrilac IGA-treated (challenged) chicken  
showing intense heterophilic aggregation, congested blood vessels and proliferative bile ductules in portal area, (2c) Intestine  
of vaccinated and Nutrilac IGA-treated (unchallenged) chicken showing normal villi lined with enterocytes, proliferative  
intestinal crypts, and normal muscular coat, (2d) liver of vaccinated and Nutrilac IGA-treated (unchallenged) chicken showing  
mild dilated hepatic sinusoids, normal hepatic cells and hyperplastic Kuepfer cells. (H&E ×400)  
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Figure 3. Histopathological examination of intestine and liver of Nutrilac IGA-treated chickens in challenged or unchallenged  
groups with toxigenic Clostridium perfringens culture. (3a) intestine of Nutrilac IGA-treated chicken (challenged) showing  
partial destruction and desquamation of villous epithelium which resulted in denuded villi, (3b) liver of Nutrilac IGA-treated  
chicken (challenged) showing focal replacement of the hepatic parenchyma with leukocyte aggregation mainly heterophiles  
and lymphocytes, (3c) Intestine of Nutrilac IGA-treated chicken (unchallenged) showing normal intestinal coats with  
proliferative submucosal intestinal crypts, (3d) liver of Nutrilac IGA-treated chicken (unchallenged) showing mild portal  
lymphocytes and heterophiles infiltration with edema in the portal vein and normal hepatic cells. (H&E ×400)  
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Helal et al., 2020  
Figure 4. Histopathological examination of liver and intestine of unvaccinated and untreated chickens in challenged or  
unchallenged groups with toxigenic Clostridium perfringens culture. (4a) intestine of positive control (challenged) chicken  
showing diffuse coagulative necrosis containing bacterial colonies, inflammatory cells in the superficial mucosa and necrotic  
intestinal crypts, (4b) liver of positive control (challenged) chicken showing multiple necrotic areas disseminated in the  
hepatic parenchyma with variable degenerative changes in the adjacent hepatic cells, (4c) Intestine of negative control  
(unchallenged) chicken showing all intestinal coats within the normal morphological picture, (4d) liver of negative control  
(unchallenged) chicken showing normal hepatic parenchyma. (H&E ×400)  
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Figure 5. Changes in the average body weight (g) among different chicken groups (before and after challenge with toxigenic  
Clostridium perfringens culture) during the experiment.  
Clostridium perfringens culture.  
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Helal et al., 2020  
Figure 7. Comparison of anti-alpha toxin IgY detected by ELISA among different challenged (with toxigenic Clostridium  
perfringens culture) and non-challenged chicken groups at 35th days of age.  
experimental challenge without combination of any  
nutritional factors, Eimeria co-infection or IBD vaccines,  
resulted in the development of a subclinical form of the  
disease with no clinical signs. The reason for exclusion of  
any predisposing factors from this study was interpreted  
by Shojadoost et al. (2012) who stated that NE  
reproduction without the aid of any predisposing factors is  
an important element in case of testing a vaccine or a  
specific drug against NE to avoid the induction of  
extremely severe disease. No apparently macroscopic  
lesions in all unchallenged broilers (lesion score =0) in  
indicating no exogenous infection affecting the  
experiment`s results. Thin and friable intestinal wall in all  
challenged subgroups either vaccinated, Nutri Lac-IGA or  
both of them (lesion score =1) and lesions in positive  
control varying from thin and friable intestine (lesion  
score =1) to ulceration (lesion score =2) with severely  
enlarged and congested liver with or without necrosis.  
Thinning and friability of intestinal wall with gas  
ballooning at some intestinal parts were previously  
and Hofshagen (1992) who declared that typical  
subclinical form of NE contained ulceration with  
discolored material adhering to the mucosa and Løvland  
and Kaldhusdal (1999) who detected severely enlarged  
DISCUSSION  
In this study, birds were experimentally challenged by oral  
administration of freshly prepared whole C.perfringens  
culture with a bacterial concentration of 109 CFU/ml and  
preformed toxin with MLD 1/80. In agreement with  
Shojadoost et al. (2012) who declared that it is preferable  
to experimentally induce NE using whole C. perfringens  
culture containing preformed toxin which initiates  
intestinal damage rather than waiting for the toxin to be  
produced by pathogenic C. perfringens strain in the  
intestine. Besides, the strain used for NE challenge was  
NetB-negative C.perfringens isolate, thus, this study  
suggested that the NetB is not sufficient alone for disease  
development and its critical role in NE occurrence  
depends on further factors. This study is consistent with  
Martin and Smyth (2009) who declared that NetB is not an  
essential factor for NE induction in all cases, but  
inconsistent with Rood et al. (2016) who declared that  
NetB toxin is an important virulence factor in NE  
occurrence.  
There were no marked clinical signs detected in the  
experimental study even in the positive control. The  
challenged birds showed the subclinical form of NE in  
(2008) and Pedersen et al. (2008) who ensured that NE  
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J. World Poult. Res., 10(2S): 263-277, 2020  
livers sometimes with pale necrotic foci in subclinical NE  
cases.  
The moderate total mortality (20%) among positive  
infected birds compared with uninfected birds (Chalmers  
The results also showed a marked elevation in BW  
gain after challenge in vaccinated (P<0.01) and vaccinated  
& Nutrilac IGA-treated subgroups (P<0.05) respectively.  
The effect of vaccine administration on BW gain was  
previously investigated by Jang et al. (2012) who  
suggested that vaccination with C.perfringens recombinant  
proteins, particularly NetB toxin or perfringolysin PFO  
improved BW gain and protective immunity against  
experimental NE induction in broilers. Dietary  
supplementation with Nutrilac IGA (immunostimulant  
contains formic acid 10% and lactic acid 10 %) had an  
overt impact on growth performance only among  
unchallenged birds in agreement with Rosen (2007);  
Abdel-Fattah et al. (2008); Dizaji et al. (2012) and  
Hedayati et al. (2013) who administrated several organic  
control subgroups, along with the low lesion scores  
observed in the challenged birds was indicative of the  
established subclinical NE in chickens during this study.  
Similar results were obtained by Li et al. (2018) who  
orally challenged broiler chickens with C. Perfringens  
culture (2 × 108 CFU/ml, 1.0 ml/bird) and reported that  
challenged birds had 20% mortality, while no mortalities  
were recorded in the control group.  
There was variation in protection due to vaccination  
and/or Nutri-Lac treatment against NE histopathological  
detrimental changes with the highest degree of protection  
in vaccinated & Nutrilac IGA-treated subgroup and the  
lowest one in Nutrilac IGA-treated subgroup. No  
significant histopathological findings could be detected in  
euthanized birds except in positive control subgroup which  
showed revealed diffuse coagulative necrosis containing  
bacterial colonies and inflammatory cells in the superficial  
mucosa with extension to the deeper mucosa. Besides, the  
presence of necrotic debris and inflammatory cells in the  
lumen was common. In agreement with Olkowski et al.  
(2006) who discussed the histopathological changes  
accompanied by NE has including hyperemic mucosa  
which was infiltrated with numerous inflammatory cells  
mainly heterophiles and Van Hoek (2013) who found  
large and gram-positive rod-shaped bacilli attached to  
submucosa after the sloughing of necrotic mucosa.  
The statistical results revealed that the BW of  
broilers in all subgroups did not statistically differ  
(P>0.05) before the experimental challenge. As all broiler  
chickens were reared under the same managemental  
conditions. Also, there was an obvious increase in weight  
gain among all unchallenged birds during the period  
between 1st and 2nd BW measurement (vaccinated &  
Nutrilac IGA-treated P<0.01, Nutrilac IGA-treated and  
negative controls P<0.05), even if it was neglected in  
vaccinated control sub-group (P>0.05) during the  
experiment, which was highly revealing of the absence of  
any exogenous infection which could badly affect the  
growth performance of tested broilers during the  
experiment. There was an insignificant decrease in BW  
after challenge in the positive control subgroup (P>0.05),  
in agreement with other investigators who recorded the  
neglected decrease in BW after the subclinical infection of  
investigators declared a significant decrease in BW in  
acids  
in broilers  
as  
growth promoter  
and  
immunomodulator. Besides, early access to Nutrilac IGA  
showed a significant elevation in BW in vaccinated &  
Nutrilac IGA-treated challenged sub-group (P<0.05).  
Although Nutrilac IGA administration significantly  
elevated BW in unchallenged birds either when it  
administrated alone (P<0.05) or concurrently with vaccine  
(P<0.01), it failed to improve weight gain in challenged  
birds when it administrated alone. Closely-related results  
obtained by Ao et al. (2012) who indicated that early  
dietary supplementation of MOS and/or organic acids  
improved the intestinal absorption, increased the villus  
height/crypt depth ratio and enhanced the chicken growth  
performance before C. perfringens challenge. However,  
neither of these feed additives gave the broilers the same  
degree of protection against C. perfringens challenge as  
any antibiotic did and failed to prevent the NE  
consequences as high lesion scores and low weight gain.  
Vaccination can provide a valuable tool for the prevention  
of NE under field conditions (Keyburn et al., 2013). One-  
Way ANOVA results showed that after challenge, the  
vaccinated challenged birds either with or without  
Nutrilac IGA treatment, had higher antibody titer more  
than unvaccinated challenged birds either Nutrilac IGA-  
treated or positive control. This result came in contact with  
Cooper et al. (2009) who declared that anti-alpha toxin  
IgY titer in vaccinated chickens was 5-fold greater than  
that in non-vaccinated chickens. Moreover, anti-alpha  
toxin IgY titer elevated after challenge in vaccinated birds  
and was 15-fold higher than that in non-vaccinated birds.  
These results suggested that alpha toxin can produce an  
effective immune response in addition to its role in  
pathogenesis. SNT results revealed that the prepared  
273  
Helal et al., 2020  
toxoid gave protective mean IgY titer after 2nd dose of  
positive control but this difference was too little to be  
neglected (P>0.05). The same indication can be concluded  
from SNT results as the mean anti-alpha titer in challenged  
Nutrilac IGA-treated subgroup was (0 IU/ml) compared  
with unchallenged (2 IU/ml). This study closely related to  
a study done by Ao et al. (2012) who found stronger  
immune response in birds fed on organic acid-  
supplemented diet but none of the supplemented feed  
additives could achieve full protection against NE  
challenge. Higher immune response could also be detected  
in Nutrilac IGA-treated birds in this study but under  
C.perfringens challenge, it failed to fully protect  
challenged birds alone. On the other hand, Combination of  
vaccine and Nutrilac IGA (as immunostimulant) in this  
study gave promising results in NE control in agreement  
with Lohakare et al. (2005) who achieved better results in  
post-vaccine immune response when organic acids were  
supplemented in poultry diets  
vaccination at 28th day and 35th day of age higher than the  
mean IgY titer after 1st dose of vaccination at 21st day of  
age in both vaccinated and vaccinated &Nutrilac IGA-  
treated groups. ELISA readings revealed the same result as  
a significant elevation in the IgY titer after the 2nd dose of  
vaccination at 28th day and 35th day of broilers comparing  
to the IgY titer after the 1st dose of vaccination at 21st day  
of broiler age also in both vaccinated and vaccinated  
Nutrilac IGA-treated group respectively. These results  
ensured the significance of booster dose of vaccination in  
immune enhancement for a longer period and multiple  
vaccination regimens can markedly reduce NE lesions in  
challenged birds in agreement with other reports (Kulkarni  
showed the measured mean anti alpha toxin in all  
vaccinated groups exceeded the international standard (0.5  
IU/ml) which was determined by European pharmacopeia,  
(2001) while low (2 IU/ml) or no (0 IU/ml) mean anti-  
alpha titer could be detected in both Nutrilac IGA-treated  
control and challenged subgroups respectively. Generally,  
vaccinated birds were more resistant to experimental  
challenge and the NE induction compared with the  
unvaccinated tested broilers. All vaccinated chickens  
could produce anti-alpha toxin antibodies in serum (IgY)  
and intestine (IgY and IgA), regardless of their degree of  
immune protection according to Lee et al. (2011).  
This study did not apply an experimental model  
depending on birds' exposure to predisposing factors  
which considered as an aid for NE induction. As a result  
of this, the vaccination regimen was able to successfully  
protect the tested chickens, to some extent, from the  
disease development. In agreement with Zahoor et al.  
(2018) who concluded that the incidence of NE can be  
minimized up to some extent by minimal exposure of  
chickens to predisposing factors as well as vaccinating the  
birds with C. perfringens and/or its toxoids.  
This study used vaccine preparation depending on  
alpha toxin for immunization against induced NE (alpha  
toxin which converted to toxoid), and that contradicted  
several investigators who declared that there were antigens  
other than alpha toxin play a critical role in protection  
against NE including NetB toxin (Jang et al., 2012; Lee et  
al., 2012 and Keyburn et al., 2013). In a study made by  
Kulkarni et al. (2007) comparing the degree of  
immunization among different immunogenic proteins  
secreted by virulent C.perfringens including alpha protein,  
hypothetical protein, pyruvate ferredoxin oxidoreductase,  
glyceraldehyde-3-phosphate dehydrogenase, and fructose  
1,6-biphosphate aldolase. The study found that all proteins  
give protection against challenge. ELISA results indicated  
a high titer of antibody could be detected in unchallenged  
Nutrilac IGA-treated birds either when it administrated  
alone or with the toxoid comparing with challenged  
Nutrilac IGA-treated birds either when it administrated  
alone or with the toxoid. Moreover, challenged Nutrilac  
IGA-treated subgroup had anti-alpha titer higher than the  
CONCLUSION  
The present study demonstrated that Nutrilac IGA  
treatment alone could not control necrotic enteritis.  
Multiple vaccination regimen provides higher protection  
level against necrotic enteritis than single vaccination  
regimen. Vaccine accompanied by Nutrilac IGA was  
effective in the prevention of necrotic enteritis only when  
it was accompanied by the absence of predisposing factors  
of necrotic enteritis.  
DECLARATIONS  
Acknowledgment  
The authors would like to acknowledge the Department of  
Anaerobe at Veterinary Serum and Vaccine Research  
Institute, Abbassia, Cairo, Egypt for logistic supports.  
274  
J. World Poult. Res., 10(2S): 263-277, 2020  
Dizaji BR, Hejazi S and Zakeri A (2012). Effects of dietary  
Authors' contributions  
supplementations of prebiotics, probiotics, synbiotics and  
acidifiers on growth performance and organs weights of  
broiler chicken. European Journal of Experimental Biology,  
S.S. Helal applied the practical part of experimental  
work and wrote the manuscript. H.F.Gouda made the  
statistical analysis for all results of the experiment and  
helped in the practical work. N.M. Khalaf prepared and  
evaluated toxoid, prepared the toxigenic culture and  
helped in experimental tests. R.I. Hamed helped in  
manuscript writing. A.A. Ali made the histopathological  
work. M.A. Lebdah designed the experiment and  
supervised the work.  
2:  
2125-2129.  
Available  
at:  
Du E, Wang W, Gan L, Li Z, Guo S and Guo Y (2016).  
Effects of thymol and carvacrol supplementation on  
intestinal integrity and immune responses of broiler  
chickens challenged with Clostridium perfringens. Journal  
of Animal Science and Biotechnology, 7: 19. DOI:  
European pharmacopeia (1997). Veterinary vaccine, 3rd edition.  
Council of Europe, Strasburg.  
Competing interests  
The authors declare that they have no competing  
interests.  
European pharmacopeia (2001). Clostridium perfringens vaccine  
for veterinary use, 3rd edition. Council of Europe, Strasburg,  
pp. 649-651.  
Fasina YO and Lillehoj HS (2019). Characterization of  
intestinal immune response to Clostridium perfringens  
infection in broiler chickens. Poultry Science, 98: 188-198.  
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