The main goal of our project has been the creation of a diagnostic tool for detecting the level of infestation of small strongyles (or cyathostomins) in horse feces. For this purpose, transcriptomics and phage display analysis have been performed. These techniques, however, require large amounts of clean sterilized strongyles.
For this reason, the first part of our project has been the recovery and the processing of the nematodes, to obtain samples usable for the following segments of the project. The co-culturing between nematodes and E. coli has then been performed with the obtained strongyles. This process requires, in fact, the presence of only strongyles and bacteria.
While dealing with this task, however, many times our group has faced one problem: the lack of information. Not much, in fact, has been published about strongyles and techniques to handle them. For this reason, this first part of the project often involved the adaptation of a pre-existing protocol or the creation of entirely new ones.
In order to establish the protocols for the recovery of the nematodes from the feces, a process of trial and error has proven necessary. This has lead to the process being divided in 4 main steps:
- Nematode isolation
- Nematode purification
- Nematode sterilization
- Nematode separation
The first two steps allow the retrieval of sterile small strongyles samples, ready for the following parts of our project, while the last step was necessary for obtaining large strongyles.
After obtaining sterile samples, some were used for co-culturing, and some for phage display. The co-culturing has been performed with varying amounts of sterilized nematode samples and E. coli MG1655.
Figure 1. Schematic picture of the process.
From the faecal samples provided by Vidilab we needed to extract the worms in order to perform experiments on them later. For each extraction, approximately 20 g of feces are used.
The first step is the incubation of the feces in a plastic cup in a 29 degree oven for one week. During this time the eggs hatch and the nematodes reach the third larval stage. This is the infectious stage of the strongyles and the end of its development in the feces/grass.
Figure 2. Measurement of feces for incubation.
Figure 3. Cups incubated at 29°C.
After the one week incubation period, the cup was filled almost to the brim with physiological saline solution and left upside down on a petri dish overnight. This allowed the worms to swim out of the feces and into the water — which is easy to recover in different falcon tubes.
Even though most of the fecal debris was left in the cup some smaller impurities were still present in some of the falcons. This debris would drastically reduce the efficiency of the sterilization process, since contaminant organisms would be able to survive inside the debris. In order to remove it, we set up a simple filter system using pasteur pipettes to which we added a small amount of cotton, loosely inserted.
The worm and debris solution is inserted in the pasteur pipette and is left above the cotton filter for a time between 30 minutes and 1 hour. During this time the nematodes get to swim through the filter, while larger impurities are stopped. The solution recovered was pure enough to proceed to the sterilization step.
Figure 4. Nematode cotton sterilization setup.
The purified worm solutions have undergone sterilization. This was done for the following phage display and co-culturing. In the co-culturing, if the sterilization step is not done or is insufficient, it could lead to upregulation of other genes than those that are under investigation, which in turn would lead to the accumulation of false positives. Therefore this step is fundamental.
The sterilization was done by applying a solution of bleach to the nematodes. The concentration of bleach was kept at 1% so that the nematodes survived and the bacteria died. After the sterilization washes was made with sterile physiological solution (saline). This led to the removal of the bleach and created a sterile environment in which the nematodes could survive.
In order to make sure that the sterilization was successful, a control was made by putting an aliquot of the sterilized worm solution into a small flask of LB. This was then put into incubation overnight. If nothing had started growing in the flask sufficient sterilization had been achieved. An example of this can be seen below in figure 4 where the sample that was put into the rightmost flask was shown to be sterile while the others was not.
Figure 5. The picture shows the result from a sterilization test. The aliquote that was put into the rightmost flask proved to be sterile
To investigate the possibility of adapting the workflow of the development of the diagnosis tool for small strongyles to large strongyles, samples containing uniquely large strongyles are needed.
Since small strongyles can be present in horses without large strongyles, but the opposite doesn’t occur, a system to separate the two types of strongyles was needed. The only pre-existing system employed by researchers that intend to collect nematodes of a certain class is the use of a pick under a microscope. This however is very time consuming and doesn’t allow obtaining a high concentration of nematodes and therefore a different procedure was needed. With this goal a microfluidic chip has been created by first 3D-printing a mold. The chip was then cast with PDMS in the mold. When the chip had solidified it was adhered to a glass slide.
Figure 6. Microfluidics chip.
The chip presented a single channel that splits in two. The solution containing both types of nematodes, and any kind of contaminant, can be pumped inside the single channel. Two valves, connected to the two split channels, can be used to redirect the flow from the main channel to one of the two. Positioning this chip under a microscope, and analysing a strongyle when reaching the bifurcation allows the determination of the nature of the nematode. The flow is then directed towards the collection tubes.
This system, even though it doesn’t allow an immediate separation of all the large strongyles from the sample, leads to a quicker separation compared to hand picking. A low flow pump, such as a pressure or a syringe pump, is required. These details are described in the protocol we developed.
So, having achieved sterilisation of the small strongyles, the question is what do we do with them? The next step was the co-culturing. In this process we grew bacteria in media containing the newly sterilised nematodes. The bacteria are cultured with the nematodes for around 4 h, enough time to allow the bacteria to develop a genetic response to the presence of the strongyles in the media, in order to detect the response on the transcriptomic level.
We chose to work with MG1665 as our E. coli strain as it closely resembles the K-12 wild-type, which is the strain closest to the E. coli that is naturally found in the gut of horses, having a higher probability of possessing a response system for the presence of nematodes. As growth media we chose M9, a minimal media, in order to minimise the noise from the expression of common metabolism related proteins.
The co-culture was started from an OD of around 0.05, and was allowed to grow until an OD of 0.8. Once the bacteria were done growing, the strongyles had to be removed, which was achieved by vacuum filtration through a Whatman n°1 filter, which allows the passage of bacteria but not of the nematodes. When the filtration was finished, the bacteria were ready to be handed over to the transcriptomics and phage display groups for further study.