There is a need for an effective, efficient way to monitor the disease status of production facilities while minimizing the stress caused to an animal by blood sampling and other invasive diagnostic techniques. The purpose of this work is to develop an air sampler and collection media that will allow the detection of PRRSV non-invasively. OBJECTIVES: The objective of this Phase I work is to determine the feasibility of developing a low cost air sampler and attendant collection medium to efficiently collect and preserve viable viral pathogens present in livestock facilities. Feasibility will be demonstrated by determining the collection efficiency of the sampler and medium for collecting a viable virus. The efficiency of the collector will determine the minimum amount of pathogen required in the sampled air that will result in detectable sample. It is important that a detectable amount of pathogen can be collected quickly when small amounts are present in the environment. This is critical to minimize false negatives in the sample collected. To demonstrate the feasibility of collecting viral pathogens present in livestock facilities, the following four Technical Objectives will be pursued in Phase I: 1) Modify an existing air sampler prototype to include volumetric flow control. This will allow the flow rates to be varied to determine the optimum flow rate for PRRSV collection. 2) Construct a delivery system capable of providing a known quantity of live virus in aerosolized form. It will be important to know how much PRRSV is delivered to the sampler in order to determine efficiency. 3) Identify an efficient collection media / flow rate combination for viable PRRSV collection. The media must be capable of removing a large percentage of PRRSV from sampled air and preserving it for later measurement. 4) Determine the minimum concentration of viable PRRS virus in air that can be collected in a reasonable amount of time and detected by VI and/or RT-PCR. The modifications to the air sampler in Technical Objective 1 will become the basis for the prototype handheld collector design to be developed in the Phase II work. Attention will be given to making the collector low cost, which is necessary for successful commercialization of a system for early PRRSV detection. The buffer media that performs the best in Technical Objective 3 will be marketed with the collector in the commercialization phase, while additional buffer media for other pathogens will also be developed in the Phase II work. APPROACH: A PC controlled air sampler will be constructed that is based on a previously developed proof of concept prototype. It consists of a proprietary collection module, and a PC based control system to control the volumetric flow rate of air through the collection module. Attention will be given to optimizing the design of the sampler for manufacturability and cost effectiveness. The collection module will be designed using commercial-off-the-shelf (COTS) components where possible, and any custom components will be designed to minimize the cost of manufacture. To test the sampler, an apparatus to deliver a known quantity of PRRS virus to the sampler in the form of an aerosol will be developed. It consists of a filtered containment chamber to filter environmental contaminants and to prevent releasing the PRRS material into the environment, and a six-jet Collison nebulizer, which will be placed along with the air sampler inside the containment chamber. The nebulizer will be used to aerosolize the PRRS material and its exhaust will be directed into the inlet of the sampler so that all generated aerosols are introduced to the sampler. The containment chamber will be placed inside a temperature and humidity chamber for all experiments. This will allow ambient conditions inside the containment chamber to be controlled at 68 degrees fahrenheit and 60 percent relative humidity. Evaluating seven potential media mixtures will identify a collection media, which efficiently collects PRRSV and preserves it for testing. These mixtures are based on the work of others as reported in the literature. Known amounts of PRRSV will be introduced to the air sampler for each media mixture and VI and RT-PCR testing will be used to determine the amount of viral material collected. The media with the best performance will be tested to determine the minimum detectable concentration of PRRS virus the sampler can collect in 5 minutes. From a starting concentration of 104 TCID50/ml the concentration to be aerosolized will be decreased by 10 fold until virus cannot be detected from the sampler collection buffer. The 95% detectable limit and 95% confidence interval on that limit will be determined by a dose response curve. The shape of the dose response curve will be chosen from the standard probability distributions (such as probit, logit, weibull, etc.) based on likelihood considerations and goodness of fit criteria
