V. Perez et al. Merial Avian Bulletin 3 (2008) Page 3 to 7 Laboratory Clinical Study Anatomopathological analysis of lymphoid organs and serological analysis of chickens vaccinated with a turkey Herpesvirus vectored vaccine containing the VP2 gene of the IBD virus (VAXXITEK 3 HVT+IBD) Victor Perez P. 1, Alejandro Rojas O. 1, Usiles Revelo S. 1, Jorge Medina P. 1, Ramon Ochoa M. 1, Victor Petrone G. 2 1 MERIAL DE MEXICO S.A., avenida de las Fuentes 66, Querétaro CP 76246 México 2 UNAM Facultad de Estudios Superiores, Departamento de Ciencias Pecuarias San Sebastián Xhala, Cuautitlán Izcalli CP 54714 México Summary The purpose of this trial was to evaluate macroscopically and microscopically the bursa, and antibodies against IBD in commercial broilers, when vaccinated at 18 days of embryonation or hatched, with vectored commercial vaccine. All groups at 18 days were challenged with IBD intermediate strain. Mortality, gross and microscopic lesion, bursal index, clinical signs and immune response (ELISA) were evaluated. Some morphological characteristics and benefits were founded inherent to vaccination. VAXXITEK is a registered trademark of Merial in the United States of America and elsewhere. Avian Bulletin: Volume 1, Number 3, Page 7 of 94
V. Perez et al. Merial Avian Bulletin 3 (2008) Page 4 to 7 Introduction The IBD virus infects the lymphocytes of the cloacal bursa producing immunosuppression. The live vaccines against the IBD replicate in the cloacal bursa producing macroscopic and histological lesions, as well as serological response. The HVT virus permanently infects the bursal lymphocytes. Therefore, the HVT virus, containing the VP2 gene, presents the antigen of IBD, without causing bursal lesions. The objective of this study is to evaluate the serological response and the serological response and the macroscopic and histological integrity of the bursa in birds vaccinated with a HVT vectored IBD vaccine, administered both in ovo and subcutaneously, and challenged with an IBD virus of intermediate virulence at 18 days of age. Methods and material 150 embryonated chicken eggs and 150 one day-old broiler chickens were divided into three groups respectively. The birds were vaccineted with a vectored vaccine or bivalent vaccines in ovo at 18 days of embryonation or at one day of age by subcutaneous route. 1). Table1. Treatments by age, type of vaccine and vaccination route. Turkey herpesvirus vectored vaccine contains the VP2 gene of IBD virus (VAXXITEK 4 ) (Merial Limited). Bivalent Vaccine is composed of HVT and IBD (Lukert strain) (Merial Limited). BURSA-VAC (Schering-Plough Animal Health, Millsboro, Delaware, USA); an IBD vaccine strain of VAXXITEK HVT+IBD is a registered trademark of Merial in the United States of America and elsewhere. similar intermediate virulence to the field strains in Mexico, with a titre EID 50% = 10 4.5 was used as the challenge virus. Serums for analysis were taken for ELISA. The morphometric index (MI) was determined by dividing the weight of the cloacal bursa by the weight of the bird (Table 2). The histological study was done and the diameter of the lymphoid follicles of cloacal bursa was measured. Results The ELISA titers (Figure 1) at 23 and 31 days of age, the morphometric index (Figure 2) and the diameter of the lymphoid bursa follicles (Figure 3) at 31 and 43 days of age were higher in the birds vaccinated with the vectored vaccine (P<0.05). At 43 days of age, the birds vaccinated with bivalent vaccine in ovo had higher titers (P<0.05) (Figure 1). Conclusions Maternal antibodies did not influence the serological response produced by the vectored vaccine or the bivalent vaccine. In the face of the challenge, the vectored vaccine had a better serological response than the bivalent vaccine. Also, the vectored vaccine resulted in better bursal integrity both macroscopically and histologically. References Snyder D.B., 1990, Changes in the field status of infectious bursal disease virus, Vet. Avian Path., 19, 419-423. Mazariegos L.A., Lukert P.D. & Brown J., 1990, Pathogenicity and immunosuppressive properties of infectious Avian Bulletin: Volume 1, Number 3, Page 8 of 94
V. Perez et al. Merial Avian Bulletin 3 (2008) Page 5 to 7 bursal disease Intermediate strains, Avian Dis., 34 203-208. Saif Y.M., 1991, Immunosuppression induced by infection bursal disease virus, Vet. Immunol. Immunopathol., 30, 45-50. Banda A., Villegas P., El-Attrache J. & Estévez C., 2001, Molecular characterization of seven field isolates of infectious bursal disease virus obtained from commercial broiler chickens, Avian Dis., 45, 620-630. Banda A. & Villegas P., 2004, Genetic characterization of very virulent Infectious Bursal Disease virus from Latin America, Avian Dis., 48, 540-549. Hamoud M.M. & Villegas P., 2006, Identification of Infectious Bursal Disease viruses from RNA Extracted from paraffin-embedded tissue, Avian Dis., 50, 476-482. Rautenschlein S., Kraemer C., Vanmarcke J. & Montiel E., 2005, Protective efficacy of intermediate and intermediate plus Infectious Bursal Disease virus (IBDV) vaccines against very virulent IBDV in commercial broilers, Avian Dis., 49, 231-237. Morales O. & Boclair W., 1993, Morphometric relations Bursa/Spleen in Infectious Bursal Disease, 42 nd WPDC, Sacramento, CA, United States. Tsukamoto K., Saito S., Saeki S., Sato T., Tanimura N., Isobe T., Mase M., Imada T., Yuasa N. & Yamagushi S., 2002, Complete, long-lasting protection against lethal Infectious Bursal Disease virus challenge by a single vaccination with an avian Herpesvirus vector expressing VP2 antigens, J. Virol., 76, 5637-5645. Table 1. Treatments by age, type of vaccine and vaccination route. Group Date of vaccination Vaccine Dose Bird / age A (bivalent) B (vectored vaccine) C (Control) Marek vaccine diluent D (bivalent) E (vectored vaccine) F (Control) Marek vaccine diluent Avian Bulletin: Volume 1, Number 3, Page 9 of 94
V. Perez et al. Merial Avian Bulletin 3 (2008) Page 6 to 7 Table 2; Analysis of histopathology and serology of vaccinated broiler chickens by in ovo and sub-cutaneous routes. Evaluation Samples Quantity Age (days) Serology Serum 10 3, 12, 23, 31 and 43 Morphometric index Histopathological examination Cloacal bursa, thymus, and spleen Cloacal bursa, thymus and spleen 5 5 12, 24, 32 and 43 3, 12, 23, 31 and 43 Figure 1. Titres of ELISA of immunized birds in ovo and sub-cutaneous routes with vectored and bivalent vaccines, challenged at 18 days with virus of IBD. Figure 2. Morphometric index of immunized birds in ovo and sub-cutaneous routes with vectored and bivalent vaccines, challenged at 18 days with virus of IBD. Avian Bulletin: Volume 1, Number 3, Page 10 of 94
V. Perez et al. Merial Avian Bulletin 3 (2008) Page 7 to 7 Figure 3: Diameter of the lymphoid bursa follicles of immunized birds in ovo and sub-cutaneous routes with vectored and bivalent vaccines, challenged at 18 days with virus of IBD. Avian Bulletin: Volume 1, Number 3, Page 11 of 94