During the summer of my sophomore going into my Junior year, I participated in a research experience for undergraduates (REU) program at The Institute of Coastal Plain Science at Georgia Southern University.
Current progress of the reintroduction of Grizzly bears (Ursus arctos horribilis) into the North Cascades of Washington has been hindered due to an unknown pathogen. The culprit was eventually determined to be a bacterial pathogen transmitted by a parasitic worm, i.e. Neorickettsia SF agent. This bacterial pathogen is endosymbiotic within the digenean Nanophyetus salmincola, which use fishes as their second intermediate host. Grizzly bears were becoming infected with the parasite and subsequently the bacteria after ingesting infected salmon. Unfortunately, little is known on the strain diversity of this bacteria, the distribution or prevalence of the parasite, N. salmincola, or the distribution or prevalence of Neorickettsia SF agent. Therefore, this project had two major aims:
AIM 1: What is the prevalence of N. salmincola metacercariae in chinook salmon from two rivers in the North Cascades of Washington?
AIM 2: What is the prevalence of Neorickettsia SF agent within the collected N. salmincolametacercariae?
In the multi-stage lifecycle of the Neorickettsia SF agent, the Metacercariae stage was focused on. This life cycle included the egg stage, followed by the first intermediate host in a water snail (Rediae), then the second intermediate host in the Chinook salmon (Metacercariae), then infecting the definitive host in the grizzly bear.
Collection: Chinook salmon were collected from the Metatchee and Wenatchee rivers in the North Cascades Washington by the State Fish and Game and dissected to isolate kidney and muscle tissue.
Isolation of MTC: Kidneys and muscle tissues were crushed to increase surface area and dissolved in a Pepsin HCl solution, filtered through a sieve and metacercariae (MTC) removed.
DNA Extractions: MTC were pooled in groups of 10 (by fish), crushed using a pestle, sonicated, and DNA extracted following a salt precipitation method.
Real time PCR: DNA sequences of the GroESL operon were used to design a TaqMan probe and primers. MTC DNA extractions were tested for the presence of SF agent using the newly developed probe and primers.
PCR and Gel: Samples testing positive by real-time PCR were verified using Nested PCR and Sequencing of the 16S rRNA gene.
In the Metachee River samples, 94.66% of the 131 Chinook Salmon samples had N. salmincola metacercariae prevalence. In those 131 Chinook Salmon samples from the Metachee River, there was a 2.29% prevalence of the Neorickettsia SF agent Bacteria.
In the Wenatchee River samples, 88.10% of the 84 Chinook Salmon samples had N. salmincola metacercariae prevalence. In those 84 Chinook Salmon samples from the Wenatchee River, there was a 1.19% prevalence of the Neorickettsia SF agent Bacteria.
The overall prevalence of N. salmincolamtc was high at 88%-95%. While the overall prevalence of Neorickettsia SF agent was at 1.2%-2.3%. This low Neorickettsia SF agent is not to be taken lightly because the Grizzly Bear, which are the ones being affected by these bacteria, are known to eat large amounts of salmon. This means that any amount of prevalence, no matter how small, is causing bears to die at a higher than normal rate.The newly developed TaqMan real-time PCR assay was successful in amplifying SF agent DNA.
The data collected will be relayed to colleagues at the Washington State University Bear Center, which asked the lab to do this prevalence assay. The Center works directly with the reintroduction of the Grizzly Bears in the Northern Cascades in Washington State. The lab will do larger scale screening of more salmon from a greater geographic area. The lab also plans to look into future screening of snail first intermediate hosts shedding N. salmincola cercariae, which could provide insight into the transmission dynamics of SF agent. Also, nested PCR and sequencing of a 1,900 bp fragment of the GroESL gene will be completed on all real-time PCR positive samples for final verification of SF agent infection.