Shedding, Kinetics, Molecular Epidemiology and Improved Surveillance of Inclusion Body Hepatitis (IBH) Caused by Fowl Adenovirus Type-8b in Broiler Chicken

Abstract

<p>Whilst working as the livestock manager of an integrated poultry company in Fiji the author was faced with the difficult task of managing the loss of production returns due to Inclusion body hepatitis (IBH) caused by Fowl Adenovirus of serotype 8b (FAdV-8b) from the Aviadenoviridae family of the Adenoviridae genus. At the same time, a Poultry CRC project looking at methods to quantify and inactivate viruses in poultry litter to allow safe re-use of spent litter was being formulated in Australia. As a result, this doctoral project aimed to investigate the viral kinetics in the host, the effect of the virus on the immune-related organs, the shedding patterns of FAdV-8b from the host and contamination of the litter associated with such shedding and, the time-temperature associations required for the inactivation of this virus in the litter. Another objective of this thesis was to determine whether FAdV-8b viral genome could be readily detected from poultry house dust samples and whether this information would have some diagnostic and surveillance value, allowing for non-invasive sampling methods that do not require a cold chain logistics for shipment to laboratories for developing countries. The final aim was to determine the main risk factors for IBH during an outbreak in Fiji.</p> <p>To address these aims, six different experiments were designed, implemented and the results analysed and evaluated. These included development and optimisation of methods of sample collection and extraction of nucleic acids and optimisation of a quantitative PCR that enabled detection and measure absolute quantification of FAdV-8b genome copies (GC) in tissues, faeces, dust and litter. This was preceded by a thorough search in the literature for potential gaps in the knowledge relating to the objectives being addressed. Analysis of a significant production dataset comprising 1623 broiler flocks between April 2008 and July 2014, kindly made available by the integrated poultry company from Fiji revealed the negative effects IBH disease had on production and identified as major risk factors multi-age production systems and inadequate shed rest times. Two animal challenge experiments, involving FAdV-8b maternal antibody (MAb) negative and MAb+ chickens infected soon after hatch (0 or 3 d.o.a) and 14 days later, were carried out revealing clear shedding profiles of FAdV-8b in faeces for the first time. In MAb- chicken, shedding of FAdV-8b was detected from 3 dpi and for both challenge ages the peak was at 5 days post inoculation. However, the difference was that the shedding in the group challenged at 3 days of age continued post 28 dpi whereas in those inoculated 14 days later, the viral GC was undetectable by 21 dpi. For MAb+ chicken, shedding was detected at 7 dpi and both inoculated groups displayed a similar pattern of shedding peaking between 14 and 19 dpi and continuing post 28 dpi. The level of shedding was significantly higher in 16 d.o. inoculated group. </p> <p>This is the first study to report detection and quantification of FAdV-8b GC in poultry house dust. The high FAdV-8b GC observed in dust samples over time is suggestive of accumulation and persistence of FAdV-8b in the environment. In this sense it may not be reflective of the actual current shedding pattern in the birds. Strong positive associations showing increases in FAdV-8b GC in liver resulting in corresponding increases in FAdV-8b GC in faeces and dust was encouraging as it suggested the potential of using dust samples as a monitoring tool and using this information in developing a sound biosecurity program for the farm to prevent future outbreaks. The detection of typical IBH lesions by histopathology, by an independent pathology laboratory, together with the significant FAdV-8b GC load in liver samples demonstrated that FAdV-8b is transmissible via contaminated air.</p> <p>In MAb- chickens, the total GC numbers per organ also demonstrated that younger birds were capable of producing large amounts of virus in their organs when compared to older birds. This however was different for MAb+ chickens where birds infected at 16 d.o had significantly higher levels of viral GC in liver than those infected at 0 d.o.</p> <p>In MAb- chickens, age resistance was observed particularly in terms of resistance to bursa and thymus atrophy and bodyweight. Chickens that were exposed to FAdV-8b infection earlier in life exhibited typical clinical signs to IBH and tended to have more severe effects on the immune organs and had more severely impaired performance parameters. In MAb+ chickens, the role of maternal antibodies in damping early clinical disease development prior to agerelated resistance is clearly seen. Chickens exposed to FAdV-8b infection at both ages were able to tolerate infection without showing any clinical signs. The absence of clinical syndromes is a marked difference to the results from experiment involving MAb- chickens where classical IBH signs were noticed in chickens infected with the same inoculant and at the same dosage. This is more evidence to support age-related resistance to IBH infection, as the birds are able to tolerate infection without showing any clinical signs.</p> <p>Two final experiments investigated time-temperature effects on inactivation of FAdV-8b in broiler litter using a chick bioassay of infectivity and qPCR detection of GC in litter as endpoints. Clear effects were demonstrated that FAdV-8b can be inactivated to safe levels when litter is exposed to temperatures of 45˚C or higher for at least 5 days. There was a clear overall correlation of detection of viral genome by qPCR with loss of viral infectivity for FAdV-8b. Enumeration of GC was also affected by temperature and time with marked reductions at higher temperatures except when litter was dry and had been stored frozen for a long period (11 months). Loss of detection of viral nucleic acids lagged behind loss of infectivity so was not an accurate direct measure of it, particularly in dry litter, but showed some promise as a marker of infective virus content in fresher litter. These findings enable more accurate prediction of viral inactivation rate in litter pasteurised by heaping, for which spatial and temporal variation in temperature has been well modelled. Effective pasteurisation of used litter making it safe for multi-batch re-use will reduce costs of production and the carbon footprint of poultry production.</p> <p>Additional findings included demonstration of a clear temporary immunosuppressive effect of FAdV-8b in SPF chickens, manifest as reduced relative bursal weight. The detection of high levels of FAdV-8b in darkling beetle adults and larvae from infected farms in Fiji flag the potential of darkling beetles as a vector for FAdV-8b. These findings improve our understanding of IBH infection in chickens, factors affecting this, provide some alternative sampling methods for monitoring the disease, and provide new information on the persistence of FAdV-8b in litter under different temperature conditions that can assist in better decision making for prevention and management of this disease.</p>