5th International Poultry Conference 10-13 March 2009.

Taba Egypt

D45

EFFECTS OF VITAMIN E, SELENIUM AND LEVAMISOLE ON

IMMUNE RESPONSE OF BROILER BIRDS ARTIFICIALLY
INFECTED WITH COCCIDIOSIS
T. Ahmad; M. M. Hussain; A. Yousaf; M. Z. Masood; and G. Muhammad
Fac. of Vet. Sci., Clinical Medicine & Surgery Dept., Uni. of Agri. Faisalabad, Pakistan
Email address of corresponding author: tvr_uaf@yahoo.com

Abstract
Coccidiosis is an economically important poultry disease causing heavy economic losses
and also causes immunosupression that predisposes the birds to other diseases. Keeping in view
the importance of this disease, the present study was planed to investigate the effect of vitamin E,
selenium and levamisole on immune response of broiler birds artificially infected with
coccidiosis. For this purpose, 135 day-old chicks were randomly divided into five equal groups
i.e. A, B, C, D and E having three replicates each. All groups received 60,000 sporulated oocysts
at the age of 15 days. The birds in group A served as infected and non-medicated control. Group
B was treated with vitamin E at dose rate of 300 mg/kg for a week before receiving the artificial
infection of sporulated oocysts. Group C received selenium at dose rate of 0.5 mg/kg of feed for a
week before receiving the artificial infection of sporulated oocysts. Group D received levamisole
at the dose rate of 10 mg/kg body weight orally and group Ereceived a combination of Vit. E,
selenium and levamisole for a week before receiving the artificial infection of sporulated oocysts
at the dose rate as mentioned above. The result showed that the weight average gain in group E
(2.05 Kg / bird) was highest and lowest in group A (1.45 Kg / bird). FCR was better in E group
(1.79) and worse in Group A (2.45) which was infected non medicated control. Least morbidity
(25%) and mortality (4%) was observed in Group E while highest morbidity (100%) and
mortality (41%) was observed in group A. Antibody titters were higher in the Group E as
compare to all other groups. Combination use of vitamin E, selenium and levamisole is
comparatively better to achieve strong immunity in broiler birds instead of there alone use.

Introduction
Poultry industry is one of the largest industries of Pakistan. It produces 463 million tons of meat
and 51037 million No. of eggs per year (Anonymous, 2006). It is still facing many problems. One
of the most important hurdles faced by this industry is prevalence of various deadly infectious
diseases like Newcastle disease (ND), Infectious bursal disease (IBD), Hydroparicardium
syndrome (HPS) and Coccidiosis (Qureshi, 1999).
Coccidiosis is one of the most important protozoan disease of poultry not only results in high
morbidity and mortality but also causes immunosupression thus paving way for the occurrence of
other diseases. The species of Eimeria genus are responsible for coccidiosis in chicken
(McDougald, 2003). All species except Eimeria tenella have been found associated with
intestinal coccidiosis while E. tenella causes caecal coccidiosis (Soulsby, 1982). Coccidiosis has
damaging effect on lymphoid tissue of thymus and bursa of fabricious resulting in reduced
immune response to routine vaccination (Wanis et al., 1991).
Vitamin E (- Tocopherol) is a fat soluble vitamin and well known antioxidant it protect the cell
membrane from free radicals attack. These free radicals generated during oxidative metabolism
and react with the fatty acid present in the phospholipids of the cell membrane and produce more
and more free radicals which can induce a chain reaction and at the end it causes the cell damage.
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5th International Poultry Conference 10-13 March 2009. Taba Egypt

Vitamin E reacts with these free radicals and form more stable vitamin E derivative. (Azzi et. al,
2002)
The immunomodulatory effect of vitamin E is due to its influence on cell function, homeostasis
and prostaglandin synthesis through a number of inter-related processes mediating the immune
function (Moriguchi and Muraga, 2000). It also improves the bird ability to cope with various
diseases by enhancing local mucosal production of Ig A (Muir et al., 2000).
Selenium is a trace element which is required in the diet of many species, including humans.
Selenium can combine with proteins e.g. Selenomethionine and selenocystien. It has structural
and enzymatic roles and is involved in important regulatory and protective functions such as
immunomodulation and detoxification of heavy metals and carcinogenic organic compounds
(Arthur, 2000). This element is needed for the proper functioning of neutrophils, macrophages,
NK cells, T-lymphocytes and some other immune mechanisms (McKenzie et al., 1998). It has
ability to elevate antibody titre against ND vaccine when used alone or in combination with
vitamin E (Swain et al., 2000).
Levamisole is widely use as anthelmintics; it is a synthetic chemical which has thymomimetic
activity compound. Stimulates the precursor of T-lymphocyte and phagocytes (Symoens et.
al,1992). It has effect on humoral immunity as suggested by the finding that levamisole increases
the number of immunoglobulin M antibody-forming cells (direct plaque forming cells) after
intravenous immunization with sheep erythrocytes ( Renoux et al 1978). It enhanced the
vaccination response in chicken by enhancing activity of peritoneal macrophages and serum
antibody titers (Onaga et al., 1984).
Keeping in view the immunosuppressive effects of coccidiosis and possibility of their reversal by
using commonly available immunomodulators, this study has been designed to;
Evaluate immune response to vaccination against ND, IBD and HPS in birds suffering from
coccidiosis treated with immunomodulators.
Investigate the comparative effect of vitamin E, selenium and levamisole on the immune response
to vaccination against ND, IBD and HPS in the birds concurrently suffering from Coccidiosis

Materials and Methods

Collection of Samples
A survey of different poultry forms of Faisalabad was conducted. Intestine of the broiler chicks
infected with coccidiosis were obtained from different poultry farms of Faisalabad .the caeca and
intestines were opened and contents were examined (Soulsby, 1982). The contents of positive
guts were collected and stored in 2.5% potassium dichromate solution. Oocysts from positive
contents were separated, concentrated by flotation technique (Hayat and Akhtar, 1999) and stored
in 2.5% potassium dichromate solution for further use.
Sporulation of coccidial oocysts
Sporulation of coccidial oocysts was performed as describe by Davies et al 1963).
Experimental Design
A total of 135 day-old broiler chicks were purchased from a commercial hatchery. The birds
were divided randomly into 5 equal groups viz. A, B, C, D, and E, and each groups were
subdivided into 3 subgroups having 9 birds each.

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5th International Poultry Conference 10-13 March 2009. Taba Egypt

Group A served as infected control .Group B treated with Vitamin E at the dose rate of 300 mg/kg
of feed for a week before receiving the artificial infection of sporulated oocysts. Group C
received selenium at the dose rate of 0.5 mg /kg of feed for a week before receiving the artificial
infection of sporulated oocysts. The birds in group E received levamisole at the dose rate of 10
mg/kg body weight orally and group D received a combination of vitamin E, selenium and
levamisole in combination for a week before receiving the artificial infection of sporulated
oocysts at the dose rate as mentioned above. Coccidiosis infection was induced in the all groups
by giving 60,000 sporulated oocysts orally by crop tube method at the age of 15 days (Murad,
1999)
Coccidial oocyst challenge
The birds in all groups were induced with coccidial infection by giving 1ml of suspension
containing 60,000 sporulated oocysts orally (Murad, 1999) by crop tube method at day 15.
Vaccination
All the birds in each group were vaccinated as per schedule given in table: 3.1
Sampling

Blood

About 5ml of blood was collected after slaughtering of the birds at the end of 2nd, 3rd and same
volume of blood was drawn from wing vein of the broiler birds at the end of 4th and 5th weeks.
Evaluation Parameters
Evaluation criteria included feed conversion ratio, weight gain, morbidity, mortality and
antibody titers for Newcastle disease, infectious bursal disease and hydroparicardium syndrome.
Weight gain
Weight gain was calculated by weighing the birds at the end of every week.
Feed conversion ratio
Feed conversion ratio was calculated by using the following formula
Total feed consumption
FCR =

-----------------------------

100

Total weight gain

Morbidity
Morbidity was calculated by using the following formula
No. of morbid birds
Morbidity =

-------------------------

Total No. of Birds

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5th International Poultry Conference 10-13 March 2009. Taba Egypt

Mortality
Mortality was calculated by using the following formula

Mortality =

No. of dead

100

Total No. of Birds

Determination of Antibody titers Serum collection
Antibody titres against NDV were measured in experimental birds through
haemagglutination inhibition (HI) test) and antibody titres against IBD and HPS vaccines were
measured in experimental birds through indirect haemagglutination (IHA) test described by
Hussain et al. (2003).
Statistical analysis
HI antibody titres against NDV and IHA antibody titres against IBD vaccine were
statistically converted into Geometric Mean Titres (GMT) for each group as described by Burgh,
(1976).

Results
Clinical sings
On 3rd day post infection of coccidiosis approximately all birds off feed. Birds in group A having
severe bloody diarrhoea. Birds in group B, C, D and F having mild bloody diarrhoea. On 5 th day
post challenge 5 birds in group A were observed died and post-mortem was conducted. There
were bloody lesions in the intestine and in caeca specifically.
Weight Gain
Weight gain was measure every week there was no significant difference in body weight of the
birds in first week. After 2nd week there was a slight difference in the weight gain in the groups.
After 3rd and 4th week there was a significant difference in the weight gain among all groups but
group E was having maximum weight gain. Table 4.1
Feed conversion Ratio
The FCR of birds at the end of every week was maximum in group E and is given in table: 2
Morbidity All birds in group A were morbid while least number of birds were morbid in Group E
(Table 3).
Mortality
Highest mortality rate (41%) was observed which was infected with coccidiosis and lowest in the
group E (Table4)
Antibody titers
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5th International Poultry Conference 10-13 March 2009. Taba Egypt

Highest antibody titers against ND, IBD and HPS were in group E and lowest antibody titers were
observed in group A (Table5;6 and 7) .

Discussion
The immune system is a highly sophisticated and flexible defense system, designed by nature to
protect the body against pathogenic organism and any other cell, tissue organ, or particle which is
characterized as nonself. Immune status of birds in the context of its significance has always
been a subject of prime interest. Immunological titres can be achieved immediately and most
successfully
by
the
application
of
medicines
enhancing
immunity which are commercially available. The interaction bet-ween immunomodu-lators and
immune system has been of great interest to immunologists and nutritionists. Selenium (Biomin,
Austria), theoretically, is more effective because it is claimed to increase FCR, increase immune
response. The present study was, therefore, conducted to know the effect of Selenium
supplementation on humoral immune response against infectious bursal disease, Newcastle
disease vaccines, its effect on feed conversion ratio (FCR) and on mortality rate in broilers.
The results of the present study were in agreement with the findings of Romach et al.
(1993) who reported that vitamins A, E, and D have regulatory role in cells of the immune
system. The results of the present study were in agreement with the findings of Klasing, (1997)
who studied that the some nutrients (e.g., fatty acids and vitamins A, D, and E) have direct
regulatory actions on leukocytes by binding to intracellular receptors or by modifying the release
of second messengers. The results of present study are in line with the findings of Friedman et al.
(1991) who reported that High levels of vitamin E (greater than 10 times the required level) have
been found to be immunostimulatory.
The results of the present study were in line with the findings of Franchini et al. (1995) who
reported that when vitamin E added to inactivated and emulsified vaccines e. g. Newcastle
disease and infectious bursal disease vaccines it enhanced the immune response to viral antigens
in chicken. The results of the present study coincide with the findings of Dalloul et al. (2003) they
reported that Humoral and local cellular mediated immunity enhanced with vitamin first time.
The results of the present study were in line with the findings of the G o r e a n d Q u r e s h i ,
( 1 9 9 7 ) t h e y r e p o r t e d t h a t different levels of vitamin E (VE) enhanced antibody and
macrophage response. The results of the present study were in agreement with the findings of
Muir et al. (2002) they reported that dietary vitamin E supplementation could act as an
immunomodulator of total and antigen-specific IgA antibody.
References
A r t h u r, J.R. 2000, The glutathione peroxidases. CellMol. Life Sci., 57, 1825-1835
Azzi. A., r. Ricciarelli and J. M . Ziingg, 2002. Non-antioxidant
molecular function of alpha -tocoferol(Vitamin E) J. Sci. Direct 519: 1 -3
Burgh, M.
A., 1998. Simple method for
serological data. Avian Dis., 2: 362 -365.

recording and analyzing

Dalloul, R. A., H. S. Lillehoj, T. A. Shellem, and J. A. Docrr.2003. Enhancedmucosal immunity

against Eimeria acervulina in broilers fed a Lactobacillus-based probiotic. Poult Sci 82:62-6
Davies , S.F. M., L.P. Joyner and S.B. Kendal,1963, cocidiosis. Oliver and boyd ltd. London. Pp
223-225
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5th International Poultry Conference 10-13 March 2009. Taba Egypt

Friedman, A., 1991. Induction of immune response to protein antigens by subcutaneous coinjection of water-miscible vitamin A derivatives. Vaccine 9:122128.
Gore, A. B., and M. A. Qureshi. 1997. Enhancement of humoral and cellular immunity by
vitamin E after embryonic exposure. Poult. Sci. 76:984991.
Hassan., N. Barhoum, 1991. Effect of infection with Eimeria necatrix on the response of chickens
to ND vaccine using Mesogenic Komarov K strain of NDV. Assiut Vet. Med. J. 26: 124-137.
Hussain, I., M. A. Zahoor, M. H. Rasoole, M. S. Mehmood, M. K. Mansoor and M. N. Riaz,
2003. Detection of serum antibody levels against
infectious bursal disease using indirect
haemagglutination test. Int. J. Poult. Sci., 2: 442-445.
Hayat ,C.S. and M.A.
Islamabad,Pakistan.,Pp16-21

Akhtar,19999.parasitic

diagnosis.1st

ED.uni.grant

comm..,

Klasing, K. C. 1997. Interaction between nutrition and infectious disease. Pages 7380 in:
Diseases of Poultry, B. W. Calnek, ed. Iowa State University Press, Ames, IA.
McDougald L.R., 2003. Dise ases of Poultry. 11th Ed., Iowa State Press.
Pp: 974-990

McKenzie, R.C., T.S. Rafferty, G.J. Beckett1998, Selenium: an essential element for immune
functions.Immunology Today, 19. 342-345.
Moriguchi, S. and M. Muraga, 2000. Vitamin E and immunity, Vitamins and Hormones. 59: 305336
Muir, W. I., W. L Bryden, and C. V. Husband, 2000. Immunity, vaccination and the avian
intestinal tract. Develop. Comp. Immunol., 24: 325-332.
Murad,A.K.,19999 CELL mediated immune response of chicken against sonicated coccidial
oocytes ,M.sc. thesis, Deptt. Ot parasito., univ.of agri. Faisalabad.
Onaga, H., M.Tajima and T. Isshii, 1984. Effect of levamisole on immune response of infected
with Eimeria tenella. Zentrailbl Bakteriol Mikrobio. Hyg., 3: 323-327
Swain, B. K., T. S. Johri., and S. Majumdar, 2000. Effect of supplementation of Vitamin E,
Selenium and their different combination on the performance and immune response of broilers.
Brit. Poult. Sci. 41: 278-92
Symoens, J., M. Rosenthal, M. D. Brabander, 1979. Immuoregulation with levamisole. Springer
Semin Immunopathol. 2:49-68.
Soulsby, E. J. L., 1982. Helminths, Arthopods and Protozoa of Domesticated Animals, 7 th Ed.
Bailliere Tindall London. Pp: 630-633.
Qureshi M. A., 1999. Gumboro disease in Pakistan. Poult. Int. 38: 42-43.
Renoux, G, 1978. Modulation of immunity by levamisole. responses in
angus and brahman X angus feeder calves. Jour Anim Sci. 59:576 -583.

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5th International Poultry Conference 10-13 March 2009. Taba Egypt

Romach, E., S. Kidao, B. Sanders, and K. Kline, 1993. Effects of RRR--tocopheryl succinate on
IL-1 and PGE2 production by macrophages. Nutr. Cancer 20:205214
Wanis E. I., E. L. Dehali, A. M. Nassar, E. L Ebiary, E. Asely, N.
Table 1:

Vaccination schedule

Diseases

Primary (day)

Booste
r (day)

Newcastle
disease

21

Infectious
bursal disease

22

Hydroparicar
dium syndrome

17

---

Table 2

Weight gain (grams) per bird

Groups
weeks

1st

203

224

213

215

220

2nd

481

488

425

456

497

3rd

850

885

901

805

920

4th

1237

1223

1300

1350

1500

Table: 3 Feed Conversion Ratio

Groups
weeks

1st

2.38

2.30

2.46

2.52

2.48

2nd

2.35

2.15

2.00

2.36

2.10

3rd

2.50

2.12

2.16

2.24

2.11

4th

2.30

2.22

2.22

2.18

2.17

5th

2.20

2.22

2.06

2.18

2.18

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Table: 4 % Morbidity and mortality

Groups

% Morbidity

% Mortality

100

41

25.02

12

25.02

12

29.19

21.85

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5th International Poultry Conference 10-13 March 2009. Taba Egypt

Table 6 TEMPORAL DISTRIBUTIO N OF ANTIBODY TITRES

AGAINST NEWCASTLE DISEASE VACCINE (NDV) IN
AFTER EVERY WEEK
Gro u p s

H I A a n ti bo dy titre s
16
2

32
1

64

B
C

1
1

1
2

At d 1 4
A

BROILERS

GM T
128

256

512

1024
2 0 .1 5
3 1 .9 8
5 0 .7 7
2 5 .3 9

4 0 .3 0

At d 2 1
A

2
1

2 5 .3 9
1

6 3 .6 3

8 3 .5 6

1 0 0 .4

1 2 7 .2

At d 2 8
A

1
2

5 0 .7 7
1

1 3 1 .9 8

1 5 8 .4

1 0 0 .4

1 5 8 .4

At d 3 5
A

5 0 .7 7

3 1 6 .2

2 5 1 .1

3 1 6 .2

E
Gro up
Gro up
Gro up
Gro up
Gro up

A=.i n f ec ted c o n tr o l
B=V i t . E 3 0 0 m g / k g o f f e e d
C= Se .0.5mg/kg of feed
D = Le v a mi so le1 0 mg / k g o f b o d y we i g ht
E = co mb i nat io n o f Vi t.E ,s e a nd le va mi s o le

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3 9 8 .1

5th International Poultry Conference 10-13 March 2009. Taba Egypt

Table 7 TEMPORAL DISTRIBUTIO N OF ANTIBODY TITRES

AGAINST INFECTIOUS BURSAL
BROILERS AFTER EVERY WEEK
Gro u p s

DISEASE

VACCINE

(IBD)

H I A a n ti bo dy titre s
8
2

16
1

B
C

1
2

2
1

1 2 .6 6
1 0 .0 3

1 0 .0 3

At d 1 4
A

32

64

GM T
128

256

512

1024
1 0 .0 3

2 0 .4 6

At d 2 1
A

1
1

1 0 .0 3
1

3 1 .9 8

5 0 .7 7

5 0 .7 7

4 3 .1 8

At d 2 8
A
B

1
1

1 0 .0 3
1

6 3 .6 3

8 0 .1 6

6 3 .6 3

8 0 .1 6

3 1 .9 8

8 0 .1 6

6 3 .6 3

6 3 .6 3

C
D

E
At d 3 5
A

E
Gro up
Gro up
Gro up
Gro up
Gro up

A=.i n f ec ted co n tr o l
B=V i t . E 3 0 0 m g / k g o f f e e d
C= Se .0.5mg/kg of feed
D = Le v a mi so le1 0 mg / k g o f b o d y we i g ht
E = co mb i nat io n o f Vi t.E ,S e a nd le va mi s o le

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1 2 6 .4

IN

5th International Poultry Conference 10-13 March 2009. Taba Egypt

Table 8 TEMPORAL DISTRIBUTION OF ANTIBODY TITRES AGAINST

HYDROPARICARDIUM SYNDROM VACCINE (HPS) IN BROILERS
EVRRY WEEK .
Gro u p s

H I A a n ti bo dy titre s
8
1

16
1

32
1

B
C

2
1

1
1

At d 1 4
A

64

GM T
128

256

512

1024
1 5 .9 9
1 0 .0 3
1 5 .9
1 0 .0 3

2 0 .1 5

At d 2 1
A

1 5 .9 9

1 0 .0 3

2 5 .3 9

2 0 .1 5

5 0 .7 7

At d 2 8
A
B

1
1

1 0 .0 3
1

3 1 .9 8

4 3 .1 8

3 1 .9 8

5 0 .7 7

1 5 .9 9

5 0 .7 7

3 1 .9 8

6 5 .5 6

C
D

E
At d 3 5
A

E
Gro up
Gro up
Gro up
Gro up
Gro up

A=.i n f ec ted co n tr o l
B=V i t . E 3 0 0 m g / k g o f f e e d
C= Se .0.5mg/kg of feed
D = Le v a mi so le1 0 mg / k g o f b o d y we i g ht
E = co mb i nat io n o f Vi t.E ,S e a nd le va mi s o le

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6 5 .5 6