“The Analysis of Filth Flies as potential Vectors for Disease from Septic Tanks and Latrines at Mpala Research Center” *Part of* Life at the Watering Hole””A One Health Approach to Disease Surveillance in Laikipia County, Kenya

Flies are major vectors of disease that mechanically transmit different pathogens to human populations. The Filth Fly Study aims to further evaluate how flies function as mechanical vectors of disease by collecting and analyzing the genetic makeup of flies from latrines and septic tanks to humans at the Mpala Research Center. Identifying what pathogens are present in local flies would be vital to understanding and potentially improving the health of individuals at the Mpala Research Center. A three-day collection period of flies was undertaken at the Mpala Research Center around the latrines and septic tanks through the placement of flytraps. After a four-hour collection period, the captured flies were homogenized and had their DNA extracted for a Polymerase Chain Reaction (PCR) test. As of right now, results can not be discussed as requested by the Mpala Research Center, but it can be stated that pathogens have been noted from testing done. Overall, further testing should be done on local flies to better expand upon knowledge of flies as vectors for disease.

When you think of African wildlife, what comes to mind? Elephants? Lions? Welp, how about arthropods? Insects, bugs, arachnids; they are everywhere. Usually, they are manifested in nightmares, like an inherent, primal fear. These creatures are usually regarded as pests. At best, they annoy their hosts by swarming around them, or with their loud noises. But at worst, they cause epidemics that can kill millions. This is where the GMU STIP team comes in handy.

Led by Dr. Michael Von Fricken, a former professor of community health at GMU and the current director of the One Health Center of Excellence at the University of Florida, the George Mason team efficiently tackled our mission by dividing into three groups: ticks, flies (which we belonged to), and water. Our fly group consisted of Sonny, Gabriel, and James. While we assisted all three groups to some extent, our primary focus for the STIP project was evaluating flies as mechanical vectors of disease. This involved collecting flies from latrines and septic tanks at the Mpala Research Center in Laikipia County, Kenya, and analyzing their genetic makeup. Mpala, located in a secluded rural area, provided pristine wildlife testing grounds. We gathered around 5 flies from each site, homogenized the samples, and utilized a Biomeme machine using a CAVE B panel test for real-time PCR amplification of pathogen DNA carried by the flies. The panel tests included detecting pathogens such as Salmonella, Listeria, Legionella, Giardia, Campylobacter, E. coli, human fecal matter, and Cryptosporidium.

The first step was gathering the flies. This was done by placing a sheet of 3×5 inch fly paper at our six chosen locations and using sugar/fruit to lure them onto it. Over a 4-hour period from 10:00 am to 2 pm, the traps would be checked at 1-hour intervals on a daily basis for the three days of collection. The pool size representing each site was 5 flies, with 1 pool being collected from each site. The tested sites were a public men’s latrine, two separate septic tanks near housing, and a staff latrine. It should be noted that we originally also included areas around the dining facility, however, due to staff concerns, we were unable to complete testing there.
The second step was homogenizing our samples. After we collected the flies from the traps with tweezers, we put them into labeled test tubes. To begin, 400 microliters of lysis buffer was taken from the Sample prep Biomeme cartridge and put into the sample test tube. After 2 minutes the flies are crushed up using a pestle to fully lyse the cell membrane so as to obtain the nucleic materials. Afterward, the Biomeme cartridge is put to use, and the different formulated buffers inside each of the cartridge’s sections are pumped to the recommended set numbers with a syringe. Through this process, the subsequent buffers allow for the nucleic acids to be cleaned and ready for analysis. Once these steps are completed, 800 microliters are extracted and put in another clean test tube for it to be analyzed later.
The third and final step is simply putting it into the Biomeme machine, which is a portable thermocycler, so it can process and analyze each sample for the presence or absence of the searched for target pathogens. How we put it in the machine is by transferring 3 sets of 20 Microliter samples from the test tube into a Go-strip, a set of 3 small vials containing our master mix, and pump the solution using a pipet to ensure there are no bubbles. Finally, we carefully put it into the Biomeme machine, and wait for it to finish reading the fly samples.

So, to preface this aspect, we did find pathogens at all the tested sites. Although our results cannot currently be discussed due to varying factors, the research completed certainly allowed for a variety of new experiences and learning opportunities. In addition, this experience gave us a better scope of the logistics behind research and our limitations. For example, having an increased budget and a longer allotted collection period could potentially allow for more definite results. In addition, the collection of multiple and larger pools for sampling could allow for more accurate results given the increased time window. The use of other means of collection is another topic to consider. Using an apparatus such as a high-powered aspirator could allow for differing results due to the live, whole nature of the flies that the flypaper didn’t always produce. Overall, the opportunity to take part in this research project was an eye-opening experience, and certainly a good start into the field of research.

Although the GMU STIP team worked diligently on vector-borne illnesses, there was also some time for fun and exploring Kenya. We went to an animal habilitation sanctuary. This place helps rescue and rehabilitate troubled wild animals. Another cool thing we got to do was hike to a small waterfall. It was spectacular, to say the least. And of course, we also got to see the local wildlife on a game drive.

We would like to thank the STIP and overall OSCAR program for allowing us to take part in such an exciting program. Without our funding, this project never would have gotten off the ground. In addition, we would like to thank Dr. Von Fricken, the Mpala Research Center staff, and our partner project Wastewater.