THE EFFECT OF HUMIC ACID AND ASCORBIC ACID ON IMMUNIZATION OF CHICKENS AGAINST INFECTIOUS BURSAL DISEASE

THE  EFFECT   OF  HUMIC  ACID  AND  ASCORBIC  ACID  ON  IMMUNIZATION    OF  CHICKENS   AGAINST   INFECTIOUS   BURSAL   DISEASE

                                                                                                                                                                    EL .Sayed.M.ELSayed.*Asawy.A.M.E **Shalaby .H .A.*** K.A.Deeb****

Animal health Institute                                                                                               

*Head  of  research(pharmacology) **Immunity    ***Biochemistry   ****Clinical  pathology   

Summary

                                                                                                                                                      This study was conducted on the impact  of  humic acid and  ascorbic acid supplementation on immunization against infectious bursal disease (IBD).Ninety  clinically healthy  avian  broiler chicks(one  day old )were  divided into three               equal    groups A,B and C.An attenuated live  IBDvirus(IBDV) vaccine was    administered at 14 day old in drinking water to all the experimental  chicks. Two        weeks post-vaccination, chicks of all groups were challenged with very virulent          IBDV   (field strain).Birds of group B were given humic acid (HA)and group C        ascorbic acid (AA)   from the   beginning till the end of the experiment as feed    supplement while group(A) kept  as control without any supplement .                                 

   Blood and serum   samples were collected during scarification on 39 and 49 day for hematological ,biochemical and immunological studies. Gross post-mortem finding; bursal indices ,average body weights and humeral ELISA antibody response to IBDVwere assessed

Groups (B&C)showed Leukocytosis,elevated antibodies ,lymphocytic transformation index and   phagocytosis  percentage    beside elevated transaminasis .The  uric acid and creatinine   were significantly  elevated .The bursa  of  fabricius was slightly congested and swollen .The liver was  slightly  enlarged  and dark .

                                       Introduction             

    Infectious bursal   disease is an acute ,highly contagious  disease of young  chicks caused by RNA virus belonging to family    Birnaviridae (Saif and Lukert ,2003).The virus targets  B-Lymphocytes  with  special  reference  to highly  proliferating  IgM  bearing  cells( Rodenberg et al.,1994 ).It remains the major contributor to economic losses in the poultry industry.The current favored strategy to  control IBDV is to vaccinate all chicks against IBDV with live vaccine during the first three weeks of  life (Winterfield et al.,1980 ). An ideal live vaccine should cause neither bursal lesions, nor immunosuppression, stimulate long lasting immunity and protect chicks against classical and variant strains of IBDV. Such vaccine is not available , thus other  ways are being investigated to increase protection afforded  by IBDV vaccine .The use of humic acid substances (HAS) in feed improve gut health for better nutrient utilization as well as improved the health status by working against pathogens by developing immunity ( Islam et al.,2005 ; Teravita ,2004;Kocabagli et al.,2002 and Faust ,1998 ) .HAS are able to  form a protective film on the mucous epithelia of the  gastrointestinal  tract against  infections and toxins (Kuhnert et al.,1991 ) also help to prevent excessive loss of water via the intestine as well as improving the immune  functions  ( Humin Tech.,2004) .Enhancing  IL-1 and  IL-3  production ( Haq et al.,2002 &Corder et al.,2003) .HAS have long been known to exhibit antiviral properties in particular against viruses and viral pathogens ( Thiel et al.,1977 ;Knoking .,1991 ;Laub .,2000 and Enviromate.,2002 ).

   Ascorbic acid (AA) has been investigated to improve disease resistance to viral disease by increasing IL-2 secration, increasing the number of T-cell (Wu et al., 2000) and enhancing interferon production ( Pardue et al .,1985).

     This work was planned to investigate and compare the effect of feed supplementation of (HAS) and ( AA ) on protection against Infectious bursal  disease evaluated by using immunological ,clinicopathological  and    biochemical means of investigation .

Material and Methods

Chicks:

   120 one day Avian broiler chicks were    obtained .The chicks possessed maternal IBDV antibodies ,housing and rearing procedure  and routine management were held under hygienic  measures .

    Immunostimulant            

   Humic acid were obtained commercially and thoroughly mixed with diet at a dose of 2.5kg /ton (Kocabagli et al.,2002) as sodium humate granules from (Humin tech.Heerdter .Germany).

    Ascorbic acid (vit.C.) was obtained commercially (COOPHAVET-France) and supplemented at 1000ppm   final dietary concentration (Amakye-Amin et al.,2000).

Vaccines :

     Commercial IBDV intermediate vaccine ,S706 (MERIAL- France L81192) was used. Each chick received 10 EID50  of the vaccine via drinking water at 14 day of age .

      Commercial Lasota (VG /GA strain MERIAL-France) was used for vaccination of chicks against Newcastle disease via drinking water.

                                                                                               Challenge virus:

      Field very virulent IBDV (vvIBDV) was obtained from veterinary vaccine &sera Institute, Abassia, Egypt .Chicks were challenged with 10  EID50 /0.1ml of the vvIBDV via eye drop instillation (Giambrone   and Closser .,1990)         Determination of antibody titere to IBDv :

       Antibody  titers  to  IBDV  was  assessed  using  commercially  available  ELISA   kits ( IDEXX ,Weestbrook ,Marine 0492 USA ) .

                                                                                                                                                                                                                                                                                                                                                                                             Bursal  index :                                                                                                      On 39 &49 days of age, the chicks were weighted, bursae were removed and weighted. The bursal indices were calculated according to (Sharma et al .,1989).

  Statistical analysis:

         The obtained data were statistically analyzed according to    (Snedecor  &Cochran .,1982 ) .

 Experimental design :

        120 one day old Avian broiler chicks (Cairo comp. for poultry) were divided into 3 equal groups (A,B &C ) .The chicks were kept under strict hygienic conditions .The drinking water and ration were supplied ad libitum .Group (A) was kept as control without feed additive ,group (B)feed a ration containing(HA) and group (C )feed with ration containing (AA ) .

       All groups were   vaccinated against Newcastle  disease by (VG /GA strains ) on days 7 ,21 and 31 in drinking water .furthermore ,the chicks were vaccinated at the age of 14day against IBD with 10 EID50 /0.1ml of IBDV intermediate vaccine (S706 ) via drinking water .Two weeks post-vaccination ,all chicks were challenged with vvIBDV containing 10  EID50 /0.1 ml via  eye drop instillation .

        After challenge chicks were observed daily for morbidity and mortality .Half of birds from each group were weighted and scarified on the day 39 and the other half on the day 49 of age for post-mortem examination .Bursas were removed  and weighted for calculation of bursal indices .

 Sampling :

    On the days of  39 & 49 of  age samples of whole blood on heparin by cardiac puncture and serum from coagulated blood in plain tube by centrifugation were collected .

  A-Immunological studies :Hepranized blood samples were used for lymphocytic transformation according to(Raiel-Balhaa et al.,1985) and phagocytosis assay according  to (Woldehiwat andRowan.,1990) .The serum antibody titers were determined   using ELISA kits according to (Hoshi et al.,1995 ).

   B-Hematological studies: Hepranized whole blood was used for total          leukocytic count according to (Coles ,1986) and blood smears stained with Wright  stain for differential leukocytic count according to (Schalm et al.,1975 ).

 C-Biochemical studies : The harvested serum was used for measuring the total protein (Peters.,1968 ) albumin & globulins (Drupt.,1972) ,the activities of alanine aminotransferase (AST) and aspartate aminotransferase (AST) (Reitman andFrankel .,1957) ,gamma glutamyl transferase (Bernard.,1991) ,creatinine(Seeling and Wust.,1969) and uric acid (Fossat et al .,1980) .

Results and  Discussion

 On day39 the leukogram revealed significant lekocytosis due to significant lymphocytosis and non significant heterophilia and monocytosis  in group (B) , while in  group (C)there was   significant leukocytosis due to significant lymphocytosis ,heterophilia and monocytosis .On day 49 leukocytosis was due to significant lymphocytosis ,heterophilia and monocytosis in group (B) ,while in   group (C) significant   leukocytosis was due to significant  lymphocytosis and non significant heterophilia and monocytosis (table 1&2).

  Serological response  to IBDV vaccine ,there was elevation in antibody titers in both group(B&C) but no significant differences were observed  between (HA &AA) treated groups (table 3&4).

  At the end of the experiment ,non  significant  difference in  the average body weight was detected between  group (B&C) but  both  groups showed high significant mean  body weights in  comparison to  control group (table 5).

  Bursal index: The control group (A)showed the lowest bursa index ,but no  significant differences   were found between treated groups (B & C) .

  In the present study two natural immunstimulant ,(HA &AA)were used . Vaccination was  done at  14  days of age after waning of maternal immunity to IBDV , to overcome its adverse effect on vaccination (Van den berg .,2000) .Both (HA & AA) resulted in  increased post-vaccination immunity to IBDV . this was indicated by significant increase of anti-IBDV antibody in both treated groups( B & C) post-vaccination ,in comparison to the control untreated one  .Such results confirms the findings that ascorbic acid possess potent immunostimulant activity (McCorkle et al.,1980 ;Pardue et al.,1985 and Haq .,2002 ) .

  The significant increase in total leukocytic count ,heterophils ,lymphocytes and monocytes in group (B &C) coincides with the  findings obtained by (Perdigon et al .,1998 ) who suggested that lactic acid producing bacteria could ineract with M-cells which activates payer s patchs lymphocytes that  might be liberated from the intestine and reach   systemic circulation .

  The increased  immune response was represented by a significant increase in ELISA titer , lymphocytic transformation index and phagocytosis percentage .Such findings are inagreement  with (Nemcova et al.,1999 ;Perdigon et al 1998 and Simone et al. ,1989 ) who confirmed  the immunostimulating effect of probiotics on cell mediated immunity , initiated the induction of lymphokinse and immunoglobulinse ,stimulation of non specific host

defense mechanism , as well as immunological means of prevention of gastrointestinal  infection and increasing the antibacterial activity of lymphocytes in the payer s patches of lymphatic system .

  The significant increase in the enzymatic activities of transaminases and  -glutamyltranspeptidase on both groups (B & C) may be due to the effect of the IBDV challenge on liver resulting in alteration of membrane permeability or damage of the hepatic cells leading to escape of these enzymes to the   plasma (Coles .,1986 and Peters .,1967 ) whose stated  that the elevated   enzymatic activities and changes in the liver  and kidneys to IBDV infection are  non specific table (  7 &  8 ) .

  The challenge of chicks with  IBDV resulting in an increased level of creatinine and  uric acid .The elevation of uric acid and creatinine are expected in  birds with impaired renal function (Halliwal.,1981 and Kaneko.,1980) .Such explanation gets along with our finding because the renal congestion interferes with the excretion of uric acid creatinine with consequent increase of their levels in the serum .

  It could be concluded that the addition of immunostimulant to the broiler chicks , alleviated the IBDV effect by ameliorating the host defense against infection .

   Table (1 )Leukogram in chicks  scarified on the 39^th day   :

         * significant  ( p<0.05 )                                                                                                                     TLC        : total leukocyte                       H           : heterophile           E  :eosinophile     

               M            :monocyte                               L            :lymphocyte

      Table (2 ):Leukogram in chicks scarified on the 49^th day :   

* significant  (   p<0.05 )                                                                                       

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Table (3) :ELISA  antibody  titer ,lymphocytic transformation index and phagocytosis percentage in chicken sacrified on the 39^th day .                                             

  * significant ( P  <0.05 )                                                                                               

                                                                                                                                  Table (4 ) :ELISA antibody titer ,lymphocytic transformation index and                

 Phagocytosis percentage in chicken sacrified on the 49^th day .               

  * significant ( P  <0.05 )                                                                                                

Table (5) :The mean bursal indices ( BI )in IBDV vaccinated groups post-challenge .

  * significant ( P  <0.05 )                                                                                               

          Table ( 6 ):some biochemical parameters in chicken sacrified on the 39^th day .

Significant ( P<0.05 )*

ALT =Alanine  aminotransaminase.              AST =Aspartate aminotranspeptidase.

                                    .             T.P. =Total  protein. GGT =gamma glut amyl transferase

                                       ALB =Albumin.                                             GLO=Globulin.    

           Table ( 7 ):some biochemical parameters in chicken sacrified on the 49^th day .

                                                                                                                                            Significant ( P<0.05 ):                          *

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الملخص العربى

       تأثيراضافة حمض الهيومک وحمض الأسکوربيک للعلف على الأستجابة المناعية عند تحصين الدواجن ضد مرض الجامبورو.

ا0د/السيد مصطفى السيد ؛   أبوالخيرمحمدابراهيم عيسوى  ؛ حا مد عبد المجيدالأمام شلبى؛ خالدعبدالرحمن الغريب د يب .

معهد بحوث صحة الحيوان –المنصورة.

    أجريت هذه الدراسة على عدد 120(مائة وعشرون) کتکوت  عمر يوم  من کتاکيت التسمين قسمت إلى ثلاثة مجاميع  متساوية  ( أ، ب ، ج ) تم تحصينها  ضد مرض الجامبوروا  عند عمر14 يوم  فى ماء الشرب . تم إضا فة حمض الهيو ميک  لکتاکيت المجموعة (ب) و حمض الأسکوربيک لکتاکيت المجموعة (ج)   فى العليقة منذ  بد اية التجربة بينما إستخدمت کتاکيت المجموعة ( أ ) کضا بط بدون أى إضافات .

   تم إجراء اختبا ر التحدى بعد  اسبوعين من التحصين . أخذ ت عينا ت دم و مصل عند عمر 39 ،49 يوم  من جميع   طيور التجربة وذلک لإجراء الفحوص الدموية و البيوکيميا ئية و المناعية  بجا نب الفحص الداخلى .

   بعد تحليل النتا ئج إحصا ئيا والفحص وجد أن إستخدام حمض الهيوميک والأسکوربيک له تأ ثير منشط للمنا عة مما أدىإلى تخطى اختبا رالتحدى بدون حدوث  تغيرات مرضية  فى الاختبارات سا لفة الذ کر مع تغيرات طفيفة فى غدة فابريشى والکبد .