Soil research aided by citizen scientists, boots and all

What lurks in the soil beneath your feet?

In the soil beneath us live billions of organisms, ranging in size from one-celled bacteria to gophers. These critters aerate the soil, break down decaying organic matter, enhance soil fertility, and eventually provide food for birds and small mammals. Some of them, including bacteria and viruses, are pathogens, which are organisms that cause disease. Soil bacteria may cause diseases such as tetanus, anthrax, and botulism, as well as gastrointestinal, skin, and respiratory illnesses.

Both beneficial and pathogenic bacteria rely on new food sources—such as dung, fertilizers, and other residues—to stay active. In addition to food, soil microbes need water and suitable temperatures to survive. As a result, both their presence and activity level vary from place to place.

A British research team combined low-tech and high-tech methods to examine the distribution and pathways used by bacteria in soil. They focused on Campylobacter, a global pathogen and the most common bacterial cause of gastroenteritis in the developed world. This genus of bacteria infects hundreds of thousands of people each year in Europe alone. These bacteria live primarily in the guts of birds and mammals, including livestock, but don’t typically make the animals sick. They are known to infect and sicken people through tainted food (e.g. milk, meat) or water, yet the transmission pathways of up to 50% of human cases are unknown. For example, the bacteria can also reside in soil tainted by animal feces and be transferred from soil to people, but the relative role of such environmental pathways in transmitting diseases is not well known.

Citizen scientists on the go

In the UK, incidence of Campylobacter infection is higher in summer, possibly because more people are active outdoors and could transfer bacteria from soil to their shoes, hands, and mouths. To investigate the potential for outdoor activity and presence of livestock to translate into greater soil-mouth contact and potential disease transmission, the researchers combined citizen scientist data collection with laboratory analysis.

The researchers wanted a sampling approach that mirrored human travel patterns, which they felt would be more representative than traditional sampling of small, discrete sites of water or soil over a large area. They also wanted one that sampled the whole foot of a person, to reflect typical contact with the soil.

The research team recruited a brigade of 60 citizen scientists to wear a fabric boot sock over one shoe and walk in the countryside at sites in two regions of England. The northwestern (NW) region supports large numbers of livestock, while the East Anglia (EA) region is drier and dominated by cropland.

In each region, the team selected three highly-traveled routes of up to 4 km, in areas with a median numbers of livestock for that region. Routes avoided newly plowed fields—which caused the socks to come off—as well as poultry farms and other likely bacteria hotspots—to avoid skewed data.

Volunteers walked the routes every three weeks throughout the year, more frequently in late spring and early summer, which are the peak season for Campylobacter infections, for a total of 40 walks on each route. They also carried a smartphone to record and submit environmental information (weather, trail conditions, and livestock seen). In addition to training, the research team sent reminders to walk leaders and created a newsletter to help keep participants engaged in the project.

The volunteers used gloves to remove the soiled socks and ship them to the scientists in bio-hazard bags to avoid contamination. With three volunteers on each walk, the citizen science team submitted 720 boot socks to the research lab.

The researchers then tested each boot sock for the presence of one of several Campylobacter species using higher-tech laboratory methods, including DNA and bacterial culture analysis. The culture process included filtering Campylobacter from other bacteria and incubating any Campylobacter colonies present in a sample.

For the DNA process, the team used a Campylobacter-specific polymerase chain reaction (PCR) assay to confirm the presence of these bacteria in each sample. PCR is a genetic analysis technique that amplifies a small number of copies of a DNA segment to generate thousands to millions of copies of that particular DNA sequence. Additional analyses helped them assign each sample to the probable infection source: cattle, sheep, pig, wild bird, or chicken.

Low-tech collection meets high-tech analysis

The citizen scientists successfully completed the walks, submitted their dirty, bacteria-laden boot socks, and recorded the livestock observed, the weather, and the underfoot conditions. They also took photos and discussed any data issues with the researchers.

The lab techniques enabled detection of Campylobacter on 156 (56%) of 240 total walks. The bacteria were either present or absent in the boot socks of all three volunteers in over 60% of the walks, suggesting the method is consistent. Altogether, the tests found Campylobacter bacteria on 47% of the 720 boot socks.

The PCR technique is more sensitive than the culture analysis and detected the Campylobacter in more cases, including those where the bacteria was present in low numbers, was dead, or was stressed. PCR was needed far more frequently to detect the bacteria in socks in the EA region, suggesting the bacteria there may be weakened or present in low numbers.

The Campylobacter came mostly from wild birds in the crop-dominated EA region and from a mix of sources (mainly sheep, chicken, wild birds) in the NW region, where livestock are more abundant. According to boot sock data, the bacteria were also more prevalent in the NW region.

The researchers found that environmental conditions for the seven days before each walk had the strongest association with presence of Campylobacter in the soil. Cooler temperatures and more rainfall were each related to higher likelihood of soil contamination, a result consistent with previous studies of this pathogen. The researchers suggest it is possible that the wet conditions also increased the probability of material sticking to the boot socks.

While the study found a peak in Campylobacter-laden boot socks in the winter months, there is no corresponding seasonal peak in infection in humans, likely because people visit the countryside less often in winter.

Understanding pathogen movement

The researchers state in their paper that successfully extracting the Campylobacter from a high proportion of boot socks worn by the volunteers “…highlights the potential of spatially distributed, citizen science based, environmental sampling for pathogens or potentially other particles in the environment.”

Working with the volunteers required substantial investment, including identifying and training them and providing them with support and feedback.

“However,” say the authors, “…if these are in place then citizen science can be used for successful, independent, long term, and systematic environmental sampling. All walks were successfully completed, every boot sock posted to the laboratory, and all additional observations submitted.”

Scientists have not yet identified all the ways pathogens transfer from the environment to humans. Beyond testing the citizen science approach, therefore, the research team also aimed to better understand the pathways taken by Campylobacter in the environment and across human populations, and ultimately to identify interventions that can reduce the risk of disease to humans.

The bacteria are also harbored in wild animals, often at low levels, which presents the potential of cross-contamination between domestic and wild species as humans and their animals expand across previously natural landscapes. Tracking the patterns and pathways of pathogens transmitted between humans and wildlife, including apes, could help scientists design interventions to reduce such transmissions to the benefit of both.

Citation

Natalia R Jones, Caroline Millman, Mike van der Es, Miroslava Hukelova, Ken J Forbes, Catherine Glover, Sam Haldenby, Paul R Hunter, Kathryn Jackson, Sarah J O’Brien, Dan Rigby, Norval J C Strachan, Nicola Williams, Iain R Lake. (2017). A novel sampling method for assessing human-pathogen interactions in the natural environment using boot socks and citizen scientists, with an application to the seasonality of Campylobacter. Applied and Environmental Microbiology; AEM.00162-17 DOI: 10.1128/AEM.00162-17