SHIC-funded APP Study Defines Outbreak Dynamics and Environmental Stability of Bacteria

In response to a severe 2021 outbreak of Actinobacillus pleuropneumoniae (APP) serotype 15 in Iowa finishers, SHIC funded research to define the risk and mitigation of this emerging swine disease strain. Drs. Marcelo Almeida and Alyona Michael, Iowa State University, recently completed three objectives as part of the one-year research project, including 1) understanding the APP serological status of sows that supplied the finishers involved in the outbreak, 2) characterizing APP persistence in finishers who had recovered from the disease, and 3) comparing the environmental stability of two APP15 strains with an APP1 strain under laboratory conditions. This study provides information regarding the role of sows in epidemiology of APP, the recommended sample types for accurate diagnosis, and the bacterial stability across different temperatures and surfaces. Through responding to the APP15 strain emergence, this SHIC-funded study adds key information for improved APP surveillance and implementation of biosecurity practices on the farm.

In late November 2021, several Iowa finisher sites across multiple unrelated systems exhibited high mortality, reaching up to 51% in a matter of days due to an outbreak of virulent APP. Clinical signs associated with the outbreak included rapid onset of high fever, coughing and respiratory distress with mortalities exhibiting frothy, red discharge from both the nose and mouth. This outbreak challenged several assumptions about APP dynamics in the US, such as the prevalence of virulent strains, the risk factors for APP lateral transmission, and environmental persistence. Preliminary sequencing efforts of APP isolates did not identify a clear source herd and further investigation into APP15 was warranted.

Objective 1 of the investigation included cross-sectional sampling to determine the serologic status of sow farms supplying pigs to the APP15 affected finisher sites. Serum samples, nasal swabs, and tonsil scrapings were collected from 30 sows (parity 0 and 1) in each sow herd. Serum samples were tested for APP15 antibodies at the ISU Veterinary Diagnostic Lab, using routine procedures. For the sow farms that submitted samples, 15 of 19 farms were serologically negative for APP serogroup 3-6-8-15 suggesting the majority of sow farms providing pigs for sites experiencing outbreaks were free of APP15. The sites receiving pigs from those sources may have been laterally infected with APP15 during the post-weaning period. Unfortunately, incomplete serologic screening of farms and lack of culture and sequencing follow-up made it impossible to confirm whether the isolate originated from an endemically infected sow farm, as opposed to originating from an alternative source of lateral transmission.

For Objective 2, a prospective longitudinal study of individually identified finishers was conducted at one Iowa site following a confirmed recent APP15 outbreak. Individually identified pigs were repeatedly sampled weekly to evaluate APP15 persistence in the nasal cavity and tonsils and monitor the development of humoral immunity. These results were compared to oral fluids and environmental swab samples to understand population shedding dynamics and environmental persistence. Environmental swab samples included internal locations including feeders, water nipples, and floors and external locations including rendering area and office door handles.

During the finisher investigations, tonsil scrapings had a higher detection rate than nasal swabs or oral fluid, with positive detection in 95% of sampled pigs across six weekly sampling events ending at eight weeks post-outbreak. PCR of tonsil scrapings were overwhelmingly the most sensitive means of screening for individual carriers long-term. APP detection rate in oral fluids was over 10% by PCR up to eight weeks after the reported outbreak. These results are significant in that they suggest that oral fluids have a more temporally robust utility for post-outbreak population surveillance than previously reported.

Environmental sampling for APP15 genetic material was primarily detected in avenues of human traffic (door handles, barn entry floor) and deadstock collection sites (rendering pile).  Except for week eight, APP was not detected by PCR in any feeders or waterers. None of the PCR-positive environmental samples yielded isolates on culture. The environmental viability of APP could not be adequately determined due to the high degree of environmental bacterial contamination and further laboratory investigation of APP viability was performed.

Objective 3 compared the longitudinal viability of APP15, for outbreak and historical strains, to APP1 at different temperatures, surfaces, and organic matrices. Survival of each strain on different surfaces (concrete, stainless steel, and rubber) and in different substrates (water, fecal slurry, and horse serum-NAD) was evaluated at six time points post-inoculation (four and eight hours, one, two, three, and seven days) at four temperature set points (-20°C, 4°C, 25°C, 37°C).

For the laboratory comparison of APP serotypes, this study is the first to observe differences in stability across serotypes and the impact of solid substrates on stability when exposed to cooler or freezing conditions. Concrete surfaces exhibited the longest stability for APP15 and APP1 with all strains surviving up to seven days post inoculation at -20ºC, and up to 48 hours at 4ºC with APP1 and APP15h surviving up to 72 hours. Rubber and stainless-steel surfaces exhibited viability at 4ºC and 25°C, but not at 37°C. The results of this benchtop study support previous APP persistence research showing decreased stability in warmer conditions, with relatively prolonged persistence at 4°C and -20°C.

While the outbreak strain of APP15 did not exhibit enhanced persistence compared to other serotypes, contributing factors to the spread and geographic persistence of this bacterium could include PCR detection at rendering piles, increased survival on concrete and rubber surfaces under laboratory conditions, and cold ambient temperatures during the winter 2021 outbreak. Together, the studies reported here generate new important knowledge related to APP ecology and epidemiology that can have important implications for disease diagnosis, monitoring and surveillance, and biosecurity practices.

The Swine Health Information Center, launched in 2015 with Pork Checkoff funding, protects and enhances the health of the US swine herd by minimizing the impact of emerging disease threats through preparedness, coordinated communications, global disease monitoring, analysis of swine health data, and targeted research investments. As a conduit of information and research, SHIC encourages sharing of its publications and research. Forward, reprint, and quote SHIC material freely. For more information, visit http://www.swinehealth.org or contact Dr. Megan Niederwerder at [email protected] or Dr. Lisa Becton at [email protected].