Use of dust sampling to determine the success of live vaccination or for monitoring of pathogen levels in poultry flocks

Abstract

<p>The poultry industry is a rapidly growing sub-sector of agriculture all over the world. In response, the modern intensive industry houses very large numbers of chickens at high densities in individual houses or complexes with multiple houses. Effective control of infectious disease is critical to the success of these intensive systems and this is mostly based on the use of preventive measures such as biosecurity and vaccination, often using mass vaccination methods. When these methods break down outbreaks of infectious disease can occur with very significant consequences. Currently there is very little routine evaluation of pathogen status or vaccination success in the modern intensive chicken meat production system due to reliance on tests, often invasive, based on sampling individual chickens and the prohibitive costs associated with this. The development of effective economical populationlevel sampling and detection systems would allow routine monitoring of live vaccination success and/or flock pathogen status. Poultry house dust has been identified as a promising population level sample material for qPCR evaluation of certain pathogens or vaccine organisms of intensively reared broiler chickens. However considerable additional work is required to optimise the use of dust sampling, address questions about the nature of poultry dust and how this varies, and to expand the range of pathogens or live vaccine candidates for which population testing based on dust may be useful.</p> <p>In response to these needs this PhD project was developed with three broad objectives, namely 1) improving understanding of the composition of poultry dust, factors affecting this and whether qPCR detection of pathogens varies with different fractions of dust" 2) investigating the potential for qPCR based routine dust monitoring of ILTV to assess vaccination success and developing methods for preliminary investigations into detection of <i>Salmonella, Clostridium perfringens</i> and <i>Eimeria</i> spp. in dust samples" and 3) determining the extent of spatial and temporal variation in the levels of the above targets and the implications of this for dust sampling strategies. To achieve the first objective studies reported in Chapters 2 and 3 involved: (i) investigation into physical characteristics of poultry house dust using Scanning electron microscopy (SEM) and chemical characteristics using Inductively coupled plasmaoptical emission spectrometry (ICP-OES) and combustion analysis, accompanied by chemical data analysis using multivariate statistical techniques (chemometrics) (Chapter 2). (ii) Stereomicroscopy and image analysis to study the particle size of settled dust. Fractionation of settled dust based on particle size using a modified Andersen sampler and measurement of association of viral (fowl adenovirus, infectious laryngotracheitis virus, infectious bronchitis virus and Newcastle disease virus) genome copy (GC) load with different fractions by quantitative real-time PCR (qPCR) (Chapter 3). The second objective on dust monitoring of selected pathogens was addressed in studies reported in Chapters 4, 5 and 6. Assessment of ILTV vaccination outcomes involved comprehensive field investigation in commercial broiler chicken flocks following mass vaccination in water reported in Chapter 4. A preliminary investigation into detection pattern and shedding of: (i) <i>Salmonella</i> in dust under experimental conditions with different methods of extraction and enrichment is reported in Chapter 5 and detection of pathogenic <i>C. perfringens</i> and <i>Eimeria</i> spp. in dust under experimental and field conditions is reported in Chapter 6. The third objective involving investigation into spatiotemporal variation in GC load of the above targets in dust is reported in Chapters 4 and 6.</p> <p>The major findings from the PhD project are: 1) Settled poultry dust is comprised largely of very fine respirable particles with 54% of particles being of 5 µm or less and 18% being >5 to ≤10 µm. Excreta was found to be the main component of broiler chicken house dust, was associated with the finest particles and tended to increase as a proportion of dust with increasing broiler age. Age of birds and location of settle plate within the chicken house affected the amount of dust deposited in settle plate and particle size of settled dust but no significant variation in viral load was observed in different fractions of settled dust. 2) Assessment of ILTV mass vaccination outcome using qPCR using population-level monitoring of ILTV in dust post mass vaccination in water was successful. Results indicate that measurement of ILTV GC in single or pooled dust samples at 7–8 days post vaccination enables detection of poor vaccine take and provides a practical means of monitoring ILT vaccination outcomes. Longitudinal monitoring of <i>Salmonella, C. perfringens</i> and <i>Eimeria</i> spp. in experimentally inoculated or diseased flocks has revealed that dust has potential to be used as a populationlevel sampling tool for these targets. Improved methodology for detection of Salmonella was evaluated in dust and enrichment in buffered peptone water followed by extraction of DNA using the simple boiling method were found to be most effective. 3) Vaccine virus and/or pathogens load in dust changes over time and the optimum timing for monitoring will vary for each target. For ILTV vaccination monitoring an optimum timing of 7−8 days post vaccination was identified in this thesis, while for the other targets investigated, further work is required to define the optimum window for sampling. Location of dust collection within the poultry house had no significant effect on the detection rates and GC loads of any of the target organisms in dust samples indicating that a sample from any location in the poultry house is representative of the flock as any other location. This is consistent with the findings of several other contemporaneous published studies and indicates that while location of dust collection, primarily related to proximity to exhaust fans will affect the amount of dust collected and the particle size distribution of the dust, it is unlikely to affect the pathogen load detected in the dust.</p> <p>The outcomes of this thesis have improved our understanding of poultry dust and sampling methods for it, led to the development of a practical and economical method to assess ILT mass vaccination success in broiler flocks and identified potential for dust-based population-level monitoring to assist in the control of <i>Salmonella</i> spp., <i>C. perfringens</i> and <i>Eimeria</i> spp.</p>