Elsevier

Vaccine

Volume 36, Issue 18, 25 April 2018, Pages 2487-2493
Vaccine

Evaluation of Mycoplasma gallisepticum (MG) ts-304 vaccine as a live attenuated vaccine in turkeys

https://doi.org/10.1016/j.vaccine.2018.02.117Get rights and content

Highlights

  • A GapA positive clone designated MG ts-304 can colonise turkeys.

  • MG ts-304 is a safe, efficacious vaccine that can protect turkeys against challenge with wild-type strain Ap3AS.

  • MG ts-304 strain has the potential to enable turkeys to be protected against MG infection for the first time.

Abstract

Mycoplasma gallisepticum (MG) is an important pathogen of poultry worldwide that causes chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys. Vaxsafe MG (strain ts-11) is a live attenuated temperature sensitive vaccine that has been proven to be effective in controlling CRD in chickens, but it is not efficacious in turkeys. The gapA gene, which encodes a mature cytadhesin protein with a molecular weight of approximately 105 kDa, is not expressed in strain ts-11 because a 20 base pair reiterated sequence introduces a frame shift and causes premature truncation of the translated peptide. A GapA positive clone, MG ts-304, isolated from strain ts-11 has been shown to have enhanced efficacy in chickens. Here we describe studies we conducted to assess the safety and efficacy of the MG ts-304 vaccine candidate in turkeys. We found that MG ts-304 was able to colonise the trachea of 3-week-old turkeys and was safe, even at a tenfold overdose, inducing no adverse clinical signs of respiratory disease or significant gross lesions in the respiratory tract (air sacs or trachea), and was poorly transmissible to in-contact birds. We also showed that it was efficacious when administered to 3-week-old turkeys, inducing protective immunity against challenge with the M. gallisepticum wild-type strain Ap3AS. MG ts-304 is therefore a promising live attenuated vaccine candidate for use in turkeys.

Introduction

Mycoplasma gallisepticum (MG) is an important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys [16], [31]. The clinical signs of MG infection in turkeys include laboured breathing, nasal and ocular discharge, swollen sinuses and depression, as well as tracheitis and airsacculitis [18]. M. gallisepticum can be transmitted by inhalation of contaminated aerosol droplets or via the egg [25], resulting in rapid disease transmission throughout a flock. Three live attenuated M. gallisepticum vaccines, ts-11, F-strain and 6/85, are commercially available and have been shown to have a high degree of efficacy in controlling the dissemination of this pathogen in chickens [3], [5], [33], [34], but they have not been found to be suitable for use in turkeys. The live attenuated temperature sensitive vaccine strain ts-11 (Vaxsafe MG, Bioproperties Australia Pty. Ltd., Glenorie, NSW, Australia) has been used to control and even eradicate M. gallisepticum infection in commercial chicken flocks [29]. It is administered by eye drop, persists in the chicken for long periods (approximately 3 months post vaccination) and stimulates a detectable, although variable, systemic antibody response [23]. However, in turkeys the vaccine colonises the respiratory tract poorly. Vaccination of turkeys with Vaxsafe MG (strain ts-11) has also failed to induce protection from field strains of M. gallisepticum [1], [17]. The F strain vaccine has been shown to be highly pathogenic in turkeys following experimental infection [21], and it has been associated with outbreaks in breeder and meat turkeys under field conditions [19]. Studies on the strain 6/85 vaccine found it to be safe in turkeys but protection against challenge with virulent M. gallisepticum was not assessed [5]. A preliminary study demonstrated that MG strain K5054, which was originally isolated from turkeys exhibiting mild signs of mycoplasmosis, was safe when administered to turkeys and induced protection against tracheal lesions and partial protection against air sac lesions following challenge with virulent R strain [6], but there have been no further studies reported on this strain.

Colonisation of the respiratory epithelium by M. gallisepticum has been shown to be mediated, in part, by GapA, a protein of approximately 105 kDa that is thought to be its primary cytadhesin [11], [14]. Previous work in our laboratory identified two ts-11 variants within the commercial vaccine Vaxsafe MG ts-11, one with an intact and fully functional gapA gene and a second with a 20 bp sequence duplication within the gapA gene that induced a frame shift and resulted in premature termination of translation [13]. A GapA + clone, MG ts-304, was derived from ts-11 and shown to have potential as a vaccine for the control of MG in chickens [26]. The ts-304 strain was administered to 4-week-old specific pathogen-free (SPF) chickens at a dose of 105.5 colour changing units (CCU) and shown to be safe, with no clinical signs or respiratory tract lesions seen in the vaccinated birds [26]. The ts-304 vaccine was shown to protect chickens at a 100-fold lower dose than ts-11, and antibody against M. gallisepticum was more readily detectable in the serum of birds vaccinated with ts-304. These studies showed that the attenuation of ts-11 was not dependent on the duplication in the gapA gene, but that expression of GapA was important for infectivity and continued colonisation. Preliminary studies with MG ts-304 have indicated its potential as a safe and efficacious live vaccine candidate for use in 3-week-old turkeys [36]. The aims of the studies reported here were to further evaluate the safety of the MG ts-304 vaccine and to determine the minimum dose required to protect turkeys from disease induced by challenge with virulent M. gallisepticum strain Ap3AS [27], [32]. Efficacy was determined using the infection model described previously [35], which simulates the natural route of infection seen in the field and thus the pathogenesis of naturally occurring disease [32]. Dissemination of the MG ts-304 vaccine within the host and the propensity of the vaccine candidate to revert to virulence were also investigated.

Section snippets

Mycoplasma gallisepticum strains

The GapA + Mycoplasma gallisepticum strain MG ts-304 was obtained from the Vaxsafe ts-11 MG vaccine as previously described [13], [26]. For vaccination, the MG ts-304 vaccine was administered by eye-drop using a calibrated pipette. For challenge, an ampoule of the virulent M. gallisepticum Ap3AS culture, the characteristics of which have been previously described [27], was thawed at 37 °C, diluted 1:10 in mycoplasma broth (MB) and incubated for 24 h at 37 °C. Prior to challenge, the actively

Experiment 1 - Safety and efficacy of different doses of ts-304

The average concentration of the MG ts-304 culture used for vaccination was 1.15 × 109 CCU/ml. The concentration of the Ap3AS culture used for challenge was 3.44 × 107 CCU/ml.

All the turkeys in the unvaccinated, unchallenged control groups remained seronegative to MG, MS and MM throughout the experiment. Two birds from Groups 3 and 6 (20%), 4 birds from Group 4 (40%) and 5 birds from Group 5 (50%) were positive (RSA score ≥ 0.5) at 14 days post-inoculation. Birds in groups 2, 3, 4 and 5 were

Discussion

Live attenuated vaccines for mycoplasma diseases should confer a strong level of immunity, be highly efficacious, induce a long duration of immunity, and be stable and affordable. In this study, the safety and efficacy of MG ts-304 as a live vaccine for use in turkeys was evaluated. Vaccination of turkeys with MG ts-304 did not result in clinical signs or significant lesions indicative of infection with virulent MG. All the doses of the MG ts-304 vaccine (106.7, 107.2 and 107.7 CCU/0.03 ml)

Conflict of interest

Gregory J. Underwood is an employee of Bioproperties Pty. Ltd. The other authors have no potential conflicts of interest (financial, professional or personal) related to the research reported here.

Ethics

The animal experiment was conducted with the approval of The University of Melbourne Animal Ethics Committee, number 1423167.

Acknowledgements

The authors gratefully acknowledge the assistance of Cheryl Colson, Pollob Shil, June Daly and Jenece Wheeler for care of the animals, and Faye Docherty and Paul John Benham for assistance with histopathology. The investigators would like to acknowledge the assistance of the staff of Bioproperties Pty. Ltd., including Fabian Carter, Sarika Kuchipudi, Gamini Jayawardane, Matthew Heslop, Petrina Young, Ross Henderson, Hilda Joubert and Phil Wordsworth.

This work was partially supported by the

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