Vaccine 30 (2012) 7193–7198 Contents lists available at SciVerse ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Safety and vaccine efficacy of a glycoprotein G deficient strain of infectious laryngotracheitis virus delivered in ovo Alistair R. Legione a,∗ , Mauricio J.C. Coppo a , Sang-Won Lee a , Amir H. Noormohammadi b , Carol A. Hartley a , Glenn F. Browning a , James R. Gilkerson a , Denise O’Rourke b , Joanne M. Devlin a a Asia-Pacific Centre for Animal Health, Faculty of Veterinary Science, The University of Melbourne, Victoria, 3010, Australia b Asia-Pacific Centre for Animal Health, Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria, 3030, Australia article info Article history: Received 5 June 2012 Received in revised form 21 August 2012 Accepted 5 October 2012 Available online 19 October 2012 Keywords: Herpesvirus ILTV Glycoprotein G In ovo Chicken abstract Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated vaccines. Glycoprotein G (gG) is a virulence factor in ILTV and a gG deficient strain of ILTV (gG-ILTV) has shown potential for use as a vaccine. In the poultry industry vaccination via drinking water is common, but technology is now available to allow quicker and more accurate in ovo vaccination of embryos at 18 days of incubation. In this study gG-ILTV was delivered to chicken embryos at three different doses (10 2 , 10 3 and 10 4 plaque forming units per egg) using manual in ovo vaccination. At 20 days after hatching, birds were challenged intra-tracheally with wild type ILTV and protection was measured. In ovo vaccination was shown to be safe, as there were no developmental differences between birds from hatching up to 20 days of age, as measured by weight gain. The highest dose of vaccine was the most efficacious, resulting in a weight gain not significantly different from unvaccinated/unchallenged birds seven days after challenge. In contrast, birds vaccinated with the lowest dose showed weight gains not significantly different from unvacci- nated/challenged birds. Gross pathology and histopathology of the trachea reflected these observations, with birds vaccinated with the highest dose having less severe lesions. However, qPCR results suggested the vaccine did not prevent the challenge virus replicating in the trachea. This study is the first to assess in ovo delivery of a live attenuated ILTV vaccine and shows that in ovo vaccination with gG-ILTV can be both safe and efficacious. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes a highly contagious respiratory disease of chickens and affects poultry worldwide [1]. Currently the spread of the disease is controlled, in part, by vaccination, using live attenuated strains of the virus [2] or recombinant vector vaccines [3]. It has been shown that live attenuated strains, obtained after multiple passages in embryonated eggs, are able to revert to virulence after bird to bird passages [4]. This has driven the development of genetically manip- ulated vaccine strains deficient in specific virulence factors as an alternative to conventionally attenuated strains [5–9]. Central to this concept has been the study of ILTV glycoproteins [6,7,10–12]. Abbreviations: ILTV, infectious laryngotracheitis virus; gG, glycoprotein G; gG-ILTV, gG deficient ILTV vaccine candidate; pfu, plaque forming units; qPCR, quantitative polymerase chain reaction; VTM, viral transport media; SPF, specific pathogen free; ELISA, enzyme linked immunosorbent assay; S.D., standard deviation. ∗ Corresponding author. Tel.: +61 3 8344 8095; fax: +61 3 8344 7374. E-mail address: legionea@unimelb.edu.au (A.R. Legione). Glycoprotein G (gG) is a viral chemokine binding protein in some alphaherpesviruses [13], and is a virulence factor in ILTV. Specifi- cally, gG inhibits leukocyte chemotaxis and encourages a humoral, rather than a cell mediated, immune response [14]. Previous stud- ies have shown that humoral immune responses are not protective against ILTV infection [15], whilst cell mediated immune responses are protective [16]. A strain of ILTV which was genetically modi- fied to remove the gG coding region (gG–ILTV) was shown to be an effective vaccine candidate [7]. The safety and efficacy of this vaccine have been tested after administration to chickens via dif- ferent methods of inoculation suitable for large-scale vaccination [17]. These methods have included drinking water, eye-drop and aerosol administration. Each of these methods are already used in industry for either ILTV vaccination [1] or vaccination against other pathogens. In recent years, machines have been developed to vaccinate birds via direct delivery into the egg (in ovo vaccination) [18,19]. This method can vaccinate up to 50,000 embryos an hour [18] and is currently used to vaccinate embryos at 18 days incubation against Marek’s disease [20]. Benefits of this route include earlier protec- tion compared to vaccinations delivered after hatching, uniform 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.10.023