2020, Scienceline Publication

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

DOI: https://dx.doi.org/10.36380/jwpr.2020.32

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. Ali⁵and 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

(Keyburn et al., 2008; Van Immerseel et al., 2009;

Shojadoost et al., 2012).

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

(Timbermont et al., 2011 ).

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

al., 2010; Jerzsele et al., 2012).

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;

2014).

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

1^stdose of vaccination: S/C injection of broiler

chickens with 0.5 ml of prepared toxoid at 7 days of age.

2^nddose 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 1^stweek of age and

repeated at the 3^rdweek 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:

10⁹CFU/ml and preformed toxin with Minimal Lethal

Dose (MLD): 1/80) for 3 successive days (28^th, 29^th, and

30^thday 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: 1^sttime: before the

1^stdose of vaccination at 7^thday of broilers` age; 2^ndtime:

after the 1^stdose of vaccination at 21^stday of broilers` age;

3^rdtime: after the booster dose of vaccination at 28^thday of

broilers` age and the last time: after the booster dose of

vaccination and experimental challenge at 35^thday 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

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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

and normal hepatic cells were noticed in the liver (Figure

3 ). In positive control subgroup: Diffuse coagulative

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 7^thday and 21^stday 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 2^nddose of vaccination

at the 28^thday 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 2^nddose of vaccination on the

35^thday 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

35^thday of broilers age as shown in Figure 7 . The highest

IgY titer was reported at vaccinated subgroups either

challenged (3.64±0.06^a) or not (3.66±0.21^a). Also

vaccinated & Nutrilac IGA-treated challenged birds had

higher IgY titer (2.69±0.03^c) more than unvaccinated

challenged birds either Nutrilac IGA-treated (2.38±0.18 ^cd)

or positive control (2.10±0.11^d). Positive control had the

lowest antibody titer (2.10±0.11^d). It also had a lower

antibody titer than challenged Nutrilac IGA-treated group

(2.38±0.18^cd) but this difference wasn’t great to be

significant. Negative control nearly had the same antibody

titer (3.18±0.11^b) of the unchallenged Nutrilac IGA-treated

subgroup (3.17±0.25^b). 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 1^stdose 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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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 1^stdose of

vaccination at 21

days of age

After 2^nddose of

vaccination

at 28 days of age

After 2^nddose of

vaccination

at 35 days of age

Group

treatment

Subgroup

1

2

3

4

5

6

7

8

6

5

6

0

2

0

A

B

C

D

Vaccinated

0

4

3

0

5

6

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.

Figure 6 . Titers of anti-alpha toxin IgY detected by ELISA in different chicken groups before challenge with toxigenic

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 35^thdays 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

agreement with Du et al. (2016) and Li et al. (2018)

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

investigated by Broussard et al. (1986) and Kaldhusdal

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 10⁹CFU/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

agreement with Wilson et al. (2005); Olkowski et al.

(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

et al., 2007 ; Mikkelsen et al., 2009; Liu et al., 2010).

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 × 10⁸CFU/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 1^stand 2^ndBW 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

NE (Pedersen et al., 2008; Du et al., 2016; Fasina et al.,

2016; Fasina and Lillehoj, 2019). Although other

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 2^nddose 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 28^thday and 35^thday of age higher than the

mean IgY titer after 1^stdose of vaccination at 21^stday 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 2^nddose of

vaccination at 28^thday and 35^thday of broilers comparing

to the IgY titer after the 1^stdose of vaccination at 21^stday

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

et al., 2007; Cooper et al., 2009; Lanckriet et al., 2010b;

Saleh et al., 2011; Jang et al., 2012). SNT results also

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:

http://www.pelagiaresearchlibrary.com

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:

https://doi.org/10.1186/s40104-016-0079-7.

European pharmacopeia (1997). Veterinary vaccine, 3^rdedition.

Council of Europe, Strasburg.

Competing interests

The authors declare that they have no competing

interests.

European pharmacopeia (2001). Clostridium perfringens vaccine

for veterinary use, 3^rdedition. 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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