PREPARATION OF CELL-ASSOCIATED VACCINE FROM ATTENUATED INFECTIOUS LARYNGEOTRACHEITIS ( ILT ) VIRUS

The present work was carried out to develop cell-associated (CA) vaccine prepared from chicken embryo fibroblast (CEF) cells infected with the tissue-culturemodified strain of infectious laryngotracheitis (ILT). The titer of prepared vaccine was 6 log10 TCID50 before and 5.7 following preservation. The vaccine was free from bacteria and other detectable viral agents by all the usual safety tests. Immunization was successful by using the prepared ILT vaccine subcutaneously in one-day-old specified pathogen-free (SPF) chicks. No clinical signs, gross and histopathological lesions related to ILT were observed in vaccinated group, as well as contact and control groups, in addition, the CA vaccine was safe when chickens were inoculated with 10 doses. The use of serum neutralization test and ELISA indicated that chicks inoculated with prepared ILT vaccine acquired high virus-neutralizing antibody titers at 14, and 21 days postvaccination and these titers persisted in protective levels up to ten weeks post vaccination. On challenge, 93.3% protection was achieved in vaccinated groups. On the contrary all the birds in control group and chicks contact-exposed to vaccinated groups showed sever clinical signs and microscopic tracheal lesions with high mortality rates post-challenge. It is concluded that this experimental vaccine was effective for chickens, eliciting protection effect against challenge with a virulent strain of ILT virus without any clinical signs. INTRODUCTION Infectious laryngotracheitis (ILT) is an acute viral respiratory disease, primarily of chickens. Economic losses attributable to ILT affect many poultry-producing areas throughout the world. Despite efforts to control the disease by vaccination, prolonged epidemics of ILT remain a threat to the poultry industry (Ivomar and Maricarmen 2007). Infectious laryngotracheitis virus (ILTV) is classified as a member of the family Herpesviridae in the subfamily Alphaherpesvirinae and continues to cause sporadic cases of respiratory disease in chickens world-wide. The virus is taxonomically identified as Gallid herpesvirus 1 (GaHV-1), genus Iltovirus, which genome is a linear double stranded DNA with 155 kb. Their exist strains of different virule246 Amany Saleh. nce and pathogenicity, but all isolates belong to one serotype (Guy and Bagust 2003). Despite improved biosecurity and vaccination programs in recent years, ILT continues to emerge in the field on a regular basis in poultry producing areas. Evidence is mounting that most field outbreaks are caused by viruses indistinguishable from chicken-embryoorigin vaccine strains and, for that reason, broiler outbreaks in the field are often referred to be as "vaccinal laryngotracheitis" (VLT) (Dufour, 2008). Guy and Bagust (1991) and Kotiw et al., (1995) found that the modified-live (ML) ILTV viruses, both of tissue-culture-origin (TCO) and chicken-em-bryo-origin (CEO), when passaged 20 times in specificpathogen-free chickens (serial bird-to-bird passage), increased vir-ulence was observed for CEO virus but not TCO virus. Increased mortality and increa-sed severity and duration of respiratory disease were observed in chickens inocu-lated with chicken-passaged CEO viruses; only mild respiratory disease (no morta-lity) occurred in chickens inoculated with chickenpassaged TCO viruses. Chicken embryo fibroblast (CEF) cells has shown high levels of cytopathic effect (CPE) following ILTV infection, although the CPEs were different from those of either LMH (a chicken liver tumor line, which is used as the standard plaque assay for ILTV infection) or primary CEK cells that produce multinucleated syncytia (Byung and Jeong 2008). Sakai, et al (1991) studied the safety and efficacy of the cell-associated (CA) vaccine prepared by chicken embryo fibroblast (CEF) cells infected with the modified-tissue-culture strain of ILTV in chickens. Over seventy percent of chickens inocu-lated with the C-A vaccine by the subcuta-neous (S.C.) or intramuscular (I.M.) route at 1 day of age were protected against chall-enge with a virulent strain of ILT virus without any clinical signs. Chickens vacci-nated with the C-A vaccine at 1 day of age acquired immunity within 6 days after va-ccination, and the protection rate mainta-ined more than 60% until 10 weeks post-vaccination. In addition, the CA vaccine was safe when chickens were inoculated with 10 doses. On the same manner Honda, et al (1994a) observed that Chickens inoculated with the cell-associated (CA) vaccine acquired higher protective immunity to ILT. As well as, in chickens vaccinated with CA or cell-free (CF) vaccine, respectively, virus-neutralizing and IgGand IgM-ELISA antibodies were detected in the serum, but no antibody was detected in the tracheal washes of the vaccinated chickens. More apparent antibody response was seen in chickens vaccinated with the CA vaccine than with the CF vaccine. However the cell-mediated immune responses of the chickens inoculated with the cell-associated (CA) ILT vaccine were studied by Honda, et al (1994b). Lymphocyte blastogenic response was tested by MTT assay with spleen, thymus, bursa of Fabricius and peripheral blood lymphocytes of the SCVMJ, IVX (1) 2009 247 vaccinated chickens. The CA vaccine induced an immunity satisfactorily in bursectomized chickens indicated that the immune mechanism with CA vaccine against ILT involves mainly cell-mediated immunity. The purpose of the present study is a trial to prepare a live ILTV vaccine comprising as the effec-tive component culture cell infected with the virus obtained by cultivation of the attenuated virus using chicken embryo fib-roblast culture cells.