The effect of IMBO was investigated on humoral immune response to Newcastle disease vaccines in broiler chickens. Haemagglutination inhibition test and enzyme-linked immunosorbent assay were used to assess the immune response. Results showed that although IMBO significantly enhanced humoral immune response to live Newcastle disease vaccine, it did not decrease post virulent NDV challenge mortality

O. G. Mohammadamin

T. S. Qubih

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)     37 Effect of industrial product IMBO® on immunosuppressed broilers vaccinated with Newcastle disease vaccine  O. G. Mohammadamin* and T. S. Qubih  Department of Pathology and Poultry Diseases, College of Veterinary Medicine, University of Mosul, Mosul, Iraq  (Received May 13, 2009; Accepted October 5, 2009)   Abstract  The effect of IMBO was investigated on humoral immune response to Newcastle disease vaccines in broiler chickens. Haemagglutination inhibition test and enzyme-linked immunosorbent assay were used to assess the immune response. Results showed that although IMBO significantly enhanced humoral immune response to live Newcastle disease vaccine, it did not decrease post virulent NDV challenge mortality.  Keywords: Humoral immunity; Newcastle disease,Vaccine, IMBO. Available online at  http://www.vetmedmosul.org/ijvs   ￿￿￿￿￿ ￿￿  ￿￿￿￿￿￿￿ ￿￿￿￿ ￿￿￿￿￿ ® ￿￿￿   ￿￿￿￿￿ ￿￿￿￿￿￿￿ ￿￿￿￿￿￿ ￿￿￿￿￿￿ ￿￿￿￿￿ ￿￿￿￿  ￿￿￿￿￿￿￿     ￿￿￿￿ ￿￿￿ !￿"￿"  ￿￿￿ ￿  #￿$ ￿￿￿￿ %￿￿￿      ￿￿￿  ￿￿￿ ￿￿￿￿￿￿￿ ￿￿￿￿￿￿ ￿￿￿￿￿￿  ￿ ￿￿￿￿￿￿￿ ￿￿￿￿ ￿￿￿￿ ￿ ￿￿￿￿￿￿ ￿￿￿￿￿  ￿ ￿￿￿￿  ￿ ￿￿￿￿￿￿      &￿'(￿￿     ￿ ! "￿￿￿￿ ￿#￿￿￿￿￿  ￿￿ $%￿￿￿    ￿￿ &&￿￿￿￿ ￿￿'() *￿￿￿ ￿￿￿% +)   ￿￿￿￿￿   #,￿ -￿￿  "￿%￿.￿￿ ￿￿￿￿/￿￿￿￿￿￿*￿￿￿ ￿￿￿￿) #￿￿￿￿* ￿   0￿  1￿￿￿ ￿%￿￿￿ . ￿￿') ￿￿￿)/￿ ￿￿/)#￿ ￿   ￿ ￿&3)￿￿ ,￿  ￿ ￿&￿ ￿)#,￿ ￿￿￿.)￿  ￿￿￿￿*￿￿ ￿￿￿  . ￿#% ￿4 ￿￿￿￿￿ +)*￿ ￿￿ ￿#￿￿￿￿￿ "￿56￿ ￿￿￿￿)#,￿   ￿￿￿￿*￿￿￿ ￿/￿￿ ￿￿￿   -%￿￿ ￿#￿￿￿￿*￿￿ ￿￿￿ 7￿.￿￿    ￿￿ ￿￿￿ 6￿%￿￿ ￿￿8￿ ￿ .  ￿￿￿ "￿￿35￿￿ ￿￿  9￿￿￿￿:￿ ;￿%)￿￿ ;￿￿<￿￿ ￿#￿￿￿￿* .       Introduction  Severe  outbreaks  of  Newcastle  disease  often  occur  in areas  of°  intensive  poultry  production,  which is  reasoned mainly to break down in immunity. Although poor vaccine quality is one of several possible factors that could lead to vaccination  failures  (1).The  failure  of  protection  usually results  from:  (i)  mycotoxin  and/or  drug  induced immunomodulation (ii) cold or heat stress (iii) infectious agents (iv) malfunctioning of the host defense mechanism and (v) presence of high titers of maternal antibodies (1-7). Immunostimulation  of  a  bird  may  lead  to  increased antibody production, increased cellular immune responses,                                                  * Part of MSc thesis submitted by the first auther to the College of veterinary Medicine, Mosul University. and  increased  macrophage  phagocytic  ability  which positively  correlates  with  enhanced  resistance  to  various viral and bacterial infections (8,9). Probiotics are defined as direct feed microbials or microbial cell preparations with a beneficial effect on the health and well-being of the host (10). Probiotic represent one of the most recent examples of natural  substances  that  influence  adaptive  immune responses by activating the innate immune system (11), and enhancing the systemic antibody.response to some antigens in chickens (12). Recently, the beneficail effect of Biomin® C-X (Enterotococcus faecium + prebiotic+cell wall extract) on  humoral  immunity  to  Newcastle  disease  vaccine  of commercial broilers was studied (13). This experiment was conductd  to  investigate  the  effect  of  Biomin®IMBO (Biomin  G.T.I.  GmbH.,  Ember  AG-Austria;  containing Enterotococcus faecium 5×1011 cfu /kg, prebiotic, cell wall Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)     38 and  algae  extracts)  as  a  potential  immunostimulator  to enhance  humoral  immune  response  to  live  and  killed Newcastle disease vaccines in broiler chickens.  Materials and methods  A total of 210 day-old Hubbard-Flex broiler chicks were procured from a local supplier. They were reared in cages in  a  separate  rooms  of  the  animal  house,  College  of Veterinary  Medicine,  University  of  Mosul  and  fed  ad libitum with a Hubbard-Flex recommended diet. Ambient temperature, lighting, ventilation and other environmental conditions fully met the requirements for management of Hubbard-Flex birds.  Biomin®IMBO  (Biomin G.T.I. GmbH., Ember AG-Austria, it contains Enterotococcus faecium 5×1011 cfu / kg, prebiotic, cell wall and algae extracts. IMBO was added to the feed free from antibiotics  and  administered  throughout  the  study  as recommended by the manufacturer 1.5g/kg.  Drugs  Cyclophosphamide  (CPA)  (Cycloxan®  manufaured  in India,  Biochem  Pharmaceutical  Industries  LTD)  was procured  from  a  local  pharmacy.  Day-old  chickens  of groups G2 and G5 were given 3 mg per chicken per day for 4 consecutive days intramuscularly into leg muscle (14).   Challenge virus One  day  before  challenge,  birds  in  G1  splited randomely to two halves; negative control group (G1); left wthout  challeneg  and  positive  control  group  (G8)  which submitted to challenge. At 39 days  of age chickens were intramuscularly inoculated with virulent field NDV strain (obtained  from  the  Microbiology  Department,  College  of Veterinary  Medicine,  Mosul  University).  The  virus  titer was determined to be 1×106.5 EID50 / 0.1ml.  Sampling  On  day  7(before  vaccination  0,  blood  samples  were taken  from  each  group  to  assess  the  maternal  immunity. Blood  samples  were  taken  at  weekly  intervals  after vaccination and challenge.  Serological Test  Antibodies  to  NDV  were  quantitified  by hemagglunation  inhibition  test  (HI)  using  the  diluted serum-constant  virus  procedure  (15)  and  by  indirect Enzyme-linked Immunosorbent Assay (ELISA).  Experimental design Chickens were randomly divided to 7 groups with 30 birds each. These groups consisted of: 1) Non IMBO + Non CPA + Non challenge referred to as negative control (G1), 2)  IMBO  +  CPA&L-K  (G2),  3)  IMBO  +  Non  CPA&L (G3), 4) IMBO + Non CPA&L-K (G4), 5) Non IMBO + CPA&L-K (G5), 6) Non IMBO + Non CPA&L (G6), 7) Non  IMBO  +  Non  CPA&L-K  (G7).  Birds  of  all  groups except negative and positive controls were vaccinated with live NDV (Cevac®Vitapest L; CEVA) at seven days old individually by oral route using 1 ml syringe. In addition, each  bird  in  groups  G2,G4,  G5,  and  G7  was intramuscularly injected with 0.1 ml of killed NDV vaccine (Cevac®Broiler NDK) at seven days of age. Revaccination with  live  ND  vaccine  LaSota  strain  (Cevac®  NEW L;CEVA) was done at 21 days of age by spraying.  Results  HI titer serum antibody response According  to  figure  (1),  chicks  contained  maternal antibody level before vaccination and gradually declined to low  levels  with  time.  At  28  days  of  age,  production  of antibody detected in all groups except G1 group. The G2 (vaccine + IMBO+CPA) and G5 (vaccine + CPA) groups produced significantly (P<0.05) lower levels of antibody in comparison  to  other  treatment  groups  indicating  that immunosuppression  occurred.  Furthermore, no  significant differences  were  found  when  the  two  groups  were compared at different time points post booster vaccination. The data also revealed that GMT of G3 ( live vaccine + IMBO)  group  was  significantly  (P<0.05) higher  than  G6 (live vaccine alone) group while GMT of G4 (live& killed vaccine  +  IMBO)  group  was  statistically  (P>0.05)  not different  compared  with  G7  (live&  killed  vaccines). Furthermore,  when  GMTs  of  G3  and  G4  groups  were compared no significant (P>0.05) difference were found.   ELISA titer immune response The results of ELISA test are presented in figure (2). On day  28,  only  birds  in  G3  and  G7  groups  showed seroconversions,  however  their  mean  titers  were  not significantly (P>0.05) different. The data also demonstrated that  the  antibody  titer  of  G3  was  significantly  higher (P<0.05) when compared with titer of G6, meanwhile the titer of G4  was not significantly (P>0.05) different when compared  with  G7.  The  titers  of  G2  and  G5  groups remained low and did not differ signficantly.  Mortality The post-challenge test results are shown in Table (1). The table shows that the protection rate in G2, G3,G4, G5, G6, G7 and G8 were 28.6, 85.71%, 96.29%, 31.8%, 83.87, 96.66%, and 0% respectively. No significant differences in mortality  were  found  between  probiotic  fed  and  their corresponding control groups.  Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)     39      Fig. 1: Geometric mean HI antibody titer (log2) in chickens with or without IMBO supplementation.    Fig.  2:  Mean  ELISA  antibody  titer  in  chickens  with  or without IMBO supplementation.  Table  1:  Response  to  challenge  with  virulent  Newcastle disease virus in chickens  Groups No dead/No challenged No survived/No challenged Negative control group  (G1)  0/14  14/14 a (100)B IMBO,CPA &L-K vaccine (G2)  20/28  8/28 c (28.57) IMBO&live vaccine (G3)  4/28  24/28 a (85.71) IMBO&L-K vaccine (G4)  1/27  26/27 a (96.29) CPA &L-K vaccine (G5)  15/22  7/22 c (31.81) Live vaccine  (G6)  5/31  26/31 a (83.87) L-K vaccine  (G7)  1/30  29/30 a (96.66) Positive control group G8)  13/13  0b Discussion  In this experiment, significantly higher HI and ELISA titers were seen in birds received live NDV +IMBO(G3). This is in agreement with finding of (13), however, IMBO had  no  effect  in  birds  immunized  with  live  and  killed vaccine  G4.  The  data  on  the  effect  of  probiotics  on immunity are extremely controversial due to the variety of variables  reported  (16).  More  over(17)  reported  treating with  just  one  bacterial type  may  not  be  as  effective  and increasing the types of bacteria in the mix could enhance the  efficacy  of  probiotic  functions.  The  post-challenge mortality rates observed in immunosuppressed (G2 and G5 groups)  were  higher  compared  with  immune-competent birds (G3, G4, G6 and G7 groups). The protection rate did not  differ  significantly  in  G3  compared  with  G6  despite enhancement  of  humoral  immune  response  which contradicts  preivious  report  (18).  The  different  results might be due to twofold increase in titer observed in latter study  and  in  addition,  they  challenged  birds  orally  with virulent  NDV  compared  with  IM  challenge  used  in  our study,  in  addition  Leghorn  male  chickens  were  used compared to broilers in the present experiment. 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Effect of industrial product IMBO® on immunosuppressed broilers vaccinated with Newcastle disease vaccine

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