- Catherine Herzog
Investigating an Outbreak
My paired student, Andrew Chota, has started his field research. He received news of a suspected PPRV outbreak in the Ngorongoro area north of where we are staying in Arusha. He headed to the field on May 6 and returned May 10 with samples. As with many trips, the travel time took longer than expected and he had to take great care to transport his serum, blood, and nasal swab samples. Out in the field the serum was pipetted into cyrotubes so this saved a step in his future work in the lab. Andrew is working in respiratory diseases in small ruminants (sheep and goats), including my disease of interest PPRV as well as contagious caprine pleuropneumonia (CCPP) and Pneumonic pasteurellosis (PP). Here is a look at his many samples – over 200 in all – and that is just the serum samples!
Andrew arrived in Morogoro at Sokoine University of Agriculture (SUA) a few days after Megan, Fulgence, Elifuraha, and I – all fellow PEHPL program students – arrived. I was excited to see the samples processed and tested in the lab. A SUA PhD student named Tebogo Kgotele, was there to assist teaching Andrew how to run cELISA test. Additionally, the helpful and capable lab technician, Miriam, was available.
You are probably asking, what is a cELISA test? ELISA stands for enzyme-linked immunosorbent assay (that’s a mouthful, right?). There are several types of ELISAs and we were using the competition ELISA, aka cELISA, so I will focus on how this test works. This cELISA test works to detect the presence of antibodies to a particular pathogen (disease causing agent such as virus, bacteria, etc). More details can be found here. http://www.elisa-antibody.com/ELISA-Introduction/ELISA-types/competitive-elisa In our case, we had a kit tailored to PPRV and purchased it from a company called ID-VET.
The plates in the kit are already coated with an antigen (a molecule capable of inducing an immune response) that resembles an antigen on the outside of the PPRV virus. During infection, the immune system of sheep and goats create antibodies to this protein to try to attack and control the PPRV infection. So when we put the samples Andrew collected in the pre-coated cELISA plate, they should bind to these antigens. The more antibodies there are, the more binding will happen. After allowing this to happen (an “incubation period”) and a rinse of the plate, a conjugate is added – which consists of a PPRV antibody that has had a color attached that will allow for detection of color at the end of the test. This conjugate competes for attachment on the wall of the plate wells with the antibody from the sample. After incubating again, the plates are rinsed and a few more steps occur with different solutions that enable the color to develop.
Andrew, Miriam, and I all worked on a plate of samples – doing each of the steps required. Each plate has 96 wells, four of which are reserved for two positive and two negative controls that are used to validate the rest of the plate – that is, to make sure results are believable.
I was not expecting a hands-on experience that day but I am so glad that I was encouraged to be involved! It was exciting adding the final solution and watching the plate wells change from blue or clear to yellow or clear! The reading of the plate is inverse – such that the more color (from the conjugate) means there was less PPRV antibody in the sample. The plate is then taken to a plate reader machine and it reads from beneath, calculating the intensity of color. Following the printed guide, we were able to calculate cutoff values and determine which samples had been positive. In short, there was a lot of PPRV! So it was not a suspected outbreak – it was an actual PPRV outbreak!
Andrew stayed in Morogoro a few more days to process his other samples and run PCR and amplify the nucleic acids for eventual sequencing.
I wanted to share this process in depth to give an idea of some of the field and lab work PEHPL students are carrying out. It was great to be involved in this hands-on experience assisting my paired student Andrew on my first trip.
Photo 1: Serum samples in cryovials, collected by PEHPL Student Andrew Chota. Photo Credit: Catherine Herzog
Diagram 1: Image from: http://www.elisa-antibody.com/ELISA-Introduction/ELISA-types/competitive-elisa
Photo 2: PEHPL Student Andrew Chota performing a wash step. Photo Credit: Catherine Herzog