Kinetics and Transmission of Virulent and Vaccine Strains of Infectious Laryngotracheitis Virus in Meat Chickens

Abstract

<p>Infectious laryngotracheitis virus (ILTV) causes a highly contagious upper respiratory and conjunctival disease of poultry. ILTV strains are antigenically similar when serotyped but there is substantial variation in pathogenicity. Disease outbreaks are controlled by application of live attenuated vaccines in Australia and this has resulted in the ongoing emergence of new virulent strains due to natural recombination. The recombinant ILTV strains have dominated field outbreaks and this is thought to have occurred due to improved fitness based on replication kinetics and enhanced transmissibility associated with their high virulence. ILTV is thought to exit the host in respiratory aerosols and enter by inhalation of these. Moderate to high levels of ILTV DNA have been detected in excreta, dust, blood or plasma and in various organs outside the respiratory tract; raising the possibility of alternative routes of shedding from the host. Wind borne transmission and dust are also implicated in the epidemiology of ILT. However, despite the widespread acceptance of airborne transmission of ILTV there appear to have been no controlled experiments to investigate the efficiency of airborne transmission or the infectivity of ILTV detected in dust, excreta and blood or plasma of infected chickens by qPCR.</p> <p>In response to these gaps in understanding this Ph.D. project was developed with two broad objectives, namely 1) To compare three vaccine and two recent Australian virulent recombinant ILTV isolates with regard to pathogenicity and viral kinetics in the upper respiratory tract, blood and excreta determined by qPCR and 2) To improve our understanding of the means by which virulent and vaccine strains of ILTV transmit between chickens by determining the role of airborne transmission, direct contact between chickens, and exposure to excreta, dust, blood or plasma from infected chickens. To achieve the first objective, the study reported in Chapter 3 compared the pathogenicity of field and vaccine strains of ILTV in commercial meat chickens and assessed detection and kinetics of infection from conventional and novel sources including blood components and excreta using quantitative real-time PCR (qPCR). The second objective on the means by which ILTV transmitted between chickens and their roles was addressed in studies reported in Chapters 4 and 5. Investigation of airborne transmission of field and vaccine ILTV strains between eye drop-infected birds in isolators and those receiving air ducted from the infected birds through a 2 m air duct is reported in Chapter 4 together with investigations into the in vivo infectivity of extracts of excreta from infected chickens. The comparison of transmission of ILTV between birds in direct contact with each other or sharing a common airspace without contact is reported in Chapter 5. Also reported in this chapter are the results of testing for in vivo infectivity of fresh dust, extracts of dust, blood and plasma, together with results of a preliminary investigation into vaccine take by coarse spray vaccination.</p> <p>With regard to objective 1 Class 9 and Class 10 field recombinant isolates exhibited severe pathogenicity in meat chickens relative to the vaccine strains and excreta proved to be unsuitable as a non-invasive sample type to monitor ILTV status in the flock due to poor sensitivity of detection. Vaccine strains caused no (A20) or mild (SA2 and Serva) clinical signs while infection with Class 9 and Class 10 resulted in severe clinical signs. The dominant clinical sign observed was conjunctivitis. Higher overall viral load was observed in choanal cleft swabs of SA2 (5.71±0.28 log10 genome copies [GC]/reaction) infected birds compared to Class 9 (3.97±0.29) and Class 10 (4.60±0.31) indicating lack of strong association between pathogenicity and viral load in choanal cleft swabs. In excreta, ILTV GC was higher for the virulent field strains groups compared to the vaccine strains but levels were 2-4 log lower than detected in choanal cleft swabs. This resulted in reduced detection of positive samples, this being 28% for excreta samples and 87% for choanal cleft samples. Viral DNA was detected in whole blood and blood fractions collected from all groups of chickens infected with field and vaccine ILTV strains, with a trend towards a higher ILTV GC in plasma and leucocyte fractions rather than the erythrocyte fraction. Again sensitivity of detection (56%) was lower than observed for choanal cleft swabs.</p> <p>With regard to objective 2 all strains of ILTV (Class 9, Class 10, SA2, Serva and A20) transmitted by the airborne route, whether through a 2 m air hose between isolators, or by sharing a common airspace without physical contact. Wild type virus transmitted very effectively by this route, but transmission of the vaccine viruses was significantly less efficient, particularly when via the 2 m air hose. The field viruses induced clinical signs, pathology, and greatly elevated ILTV genome copies in airborne exposed birds. However, clinical signs were less severe and delayed compared to birds infected by eye drop, or in direct contact with infected birds. This was also reflected in reduced and delayed ILTV GC in choanal cleft swabs. When birds shared a common airspace, transmission rate was higher for the Class 9 virus (100% by 14 dpe) and Serva (100% by 17 dpe) but inefficient for A20 with only 27% of birds infected by 21 dpe. Birds incontact with eye drop inoculated birds had 100% transmission by 6 dpe and significantly higher mean of ILTV GC (4.78 ± 0.16) than birds sharing a common airspace (2.89 ± 0.12), indicating the importance of degree of contact between chickens on the transmission of ILTV. There was no transmission of ILTV to susceptible chickens using extracts of excreta, dust or its extracts and blood or plasma from ILTV infected birds at different stages of infection. None of the birds inoculated with these materials showed clinical signs and choanal cleft swabs were negative for ILTV by qPCR. While it is possible that the methods used failed to detect low levels of infective virus in these materials it is clear the majority, if not all of the ILTV GC detected in them reflects the presence of inactivated virus or fragments. ILTV GC in excreta most likely represents non-infective virus inactivated by passage through the gut with no role in ILTV transmission. As excreta is the main component in poultry dust, this inactivated ILTV may explain the failure to transmit ILTV in dust or extracts of dust from infected birds. Similarly, the detection of ILTV GC in blood seems to be due to circulation of a non-infective form of ILTV and/or presence of very low levels of circulating infective virus. Preliminary investigation into coarse spray vaccination with the A20 and Serva vaccines did not induce an undesirable vaccine reaction but resulted in an inadequate infection rate with the methodology used. Significant additional work would be required to optimize this approach. </p> <p>The outcomes of this thesis improve our understanding of the mechanisms of transmission of ILTV and the differences between virulent and attenuated ILTV strains. They have potential to improve monitoring and control measures for this complex and difficult to control disease. </p>