Sam Abraham

Environmental decontamination is a key management strategy to maintaining good respiratory herd health in piggeries. Actinobacillus pleuropneumoniae (A. pp) is one of the most important endemic respiratory pathogens of Australian pigs currently controlled by in-feed antibiotic medication and other bacterial species associated with porcine respiratory disease complex (PRDC) are important secondary pathogens. Livestock-associated MRSA has been identified as a major public health issue for intensive animal production, particularly for in-contact workers and their families. Disinfection of the farm environment to eliminate these pathogenic organisms remains a key component of disease control and production efficiency. Of the many disinfectants used in farm decontamination, quaternary ammonium compounds (QAC) have been shown to prevent aerosol transmission of A. pp in a previous CRC project. However, QAC and many other currently used disinfectants have significant drawbacks such as being hazardous to the environment and potentially toxic to animals and piggery workers, whilst also being responsible for co-selection of resistance to antibiotics (class 1 integrons, which are major drivers of the evolution of multidrug-resistance, always contain a QAC resistance gene in their structure).

Therefore, in this project, an electrochemically activated solution (ECAS), an anolyte generated by electrolysing a dilute sodium chloride solution in a four-chamber electrolytic cell (Ecas4 Australia), was tested as an aerosol sanitiser. The Ecas4 anolyte has a neutral pH and is 100% safe; it is currently used for sanitisation of Legionella contamination in hospital water supplies, disinfection of dairy manufacturing equipment, and in seafood and fresh food produce as both a wash and dry fog. In vitro antimicrobial susceptibility testing of ECAS against field strains of A. pp representing the main serotypes and MRSA isolated from Australian pigs, confirmed that at very low concentrations of the anolyte in water (0.19-3.13% v/v or 0.48-7.8 ppm of active chlorine) ECAS was equally effective at killing both A. pp and MRSA within 30 s of exposure.

SYBR green-dye-based real-time quantitative PCR was optimised to detect and quantify low levels of total bacteria and A. pp targeting the 16S rRNA and apxIVA genes, respectively, using previously published primer sets. Using the Coriolis air sampler device, the sample collection protocol to capture A. pp. from the farm environment was optimised and very low levels of A. pp (5.1×105 genomic units) were detected in both weaner rooms and grower/finisher sheds at a continuous flow farm with endemic pleuropneumonia. Furthermore, a novel step was introduced into the qPCR by treating samples with 50 µm propidium monoazide to differentially quantify live and dead bacterial cells in the sample, an ideal rapid quantitative assay for determining the effectiveness of aerosol disinfection methods.

To examine the effectiveness of Ecas4 dry fogging in eliminating A. pp and other aerosolised bacteria, a proof-of-concept trial was conducted in a recently vacated weaner room at the same continuous flow piggery using a protocol adapted from Dr Peter McKenzie’s Pork CRC project 2C-117. A 1-log10 reduction in total bacterial count was observed after the first hour of fogging, a 2-log10 reduction was observed after fogging for two and three hours, while 99.9% (3.7-log10) of total bacteria were effectively inactivated by Ecas4 dry fogging after five hours of discontinuous treatment. However, since we were not able to conduct fogging trials with pigs in the weaner room, due to the potential for creating a large number of immunologically naïve animals entering the high-risk environment of the grower shed, we could not capture A. pp. from the weaner room environmental air samples. Hence, disinfection efficacy on farm against A. pp could not be determined. Nevertheless, the pilot results with total bacteria are encouraging enough to upscale to a much larger trial in the grower shed in a future project.

The outcomes of this innovative project will support application of Ecas4 technology specifically in disinfection of respiratory pathogens in the farm environment and fulfil the aims of Sub-Program 2C: Replacement of Antibiotics with Effective Integrated Health Strategies. An added bonus of this project is that we have optimised a diagnostic assay to detect low levels of A. pp (as little as 0.1 pg of DNA) from the air of the farm environment and a novel qPCR method to discern live bacteria from dead bacteria, addressing another Pork CRC Sub Program 2A: Novel Disease Diagnostics.