FINAL REPORT FOR PIGEON RIVER FISH KILL

 

Michigan State University: Water Quality and Environmental Microbiology Laboratory

Marc P. Verhougstraete, Sangeetha Srinivasan, Rebecca Ives, and Joan B. Rose

August 18, 2008

 

In June 2008, a discharge from dam was reported to increase flow and sediment on the Pigeon River. This event is thought to be the culprit for a large ensuing fish kill occurring down river. A number of dead fish were collected and frozen within twenty-four hours of discovery for future analysis. The Water Quality and Environmental Microbiology Laboratory at Michigan State University was contacted to test these samples for bacterium and molecular source markers from the gill arches and gastrointestinal tract (GIT) of the collected fish. Initial results indicate elevated levels of total coliforms, E. coli, and enterococci in several of the assayed fish. This demonstrates that the fish were exposed to waters with fecal pollution which could arise from either animals or humans. Neither the human enterococcal surface protein (esp) or the Bacteroides human marker were detected in any of the fish samples. One specimen, the Northern Redbelly dace, tested positive for the presence of the bovine Bacteroides but all other samples were negative for the same indicator. Table 1 summarizes the results of tests performed on each sample.

 

Methods

The Aquatic Animal Health Lab of Michigan State University extracted the gill arches and GIT from eight species of preserved fish (stickleback, fathead, blacknose dace, northern redbelly dace, brook trout, creek chub, white sucker, and brown trout) and placed them into individual Whirl-Pak® bags. The samples were stored on ice, transported to the water quality laboratory and processed on the same day.

 

Contents of each bag were placed into sterile 250 ml centrifuge bottles. Each Whirl-Pak® was rinsed using phosphate buffer water (PBW) and poured into the centrifuge bottles. The bags were rinsed until the resulting volume in the centrifuge bottle equaled 250 ml and the Whirl-Pak® was clean. The only exception to this process was the brown trout GIT because of the large size of this organ. The brown trout GIT was placed into a sterile blender and blended for approximately one minute until homogenized. Twenty-five ml of the homogenized sample was added to a sterile centrifuge bottle containing 225 ml of PBW.

 

All samples were then placed into an automatic wrist shaker and vigorously shaken for five minutes. Immediately after shaking, 100 ml of each sample was processed for total coliforms and Escherichia coli (E. coli) using IDEXX Colilert and Enterococci using IDEXX Enterolert. Each of these methods is Environmental Protection Agency (EPA) approved.

 

DNA was extracted from each specimen (gill arches and GIT from each species) using the QIAmp® DNA Mini kit. The final DNA volume was 200 μl. Human and bovine Bacteroides were tested using the appropriate markers. Enterococci esp gene was assayed using the forward primer 5’-TAT GAA AGC AAC AGC ACA AGT-3’ and the conserved reverse primer 5’ –ACG TCG AAA GTT CGA TTT CC-3’ for all reactions. The Bacteroides and esp were assayed using triplicate samples for PCR. Each sample was run through a previously tested PCR program.

 

After each sample completed the PCR program, the amplified product was evaluated in agarose gel electrophoresis. The PCR product was run in 1.2% w/v agarose and 1.0X Tris-Acetate-EDTA buffer at 100V for 30-60 minutes. After the run, the gel was removed and viewed using a UV transilluminator to check for bands at the appropriate size (680 bp).

 

 

Results

Fish were collected and delivered to the Aquatic Animal Health Lab of Michigan State University on July 21st, 2008 processed for the bacteria on July 24th, 2008, and for the source tracking markers on August 14th, 2008. 

 

The initial results from the bacterial tests indicate that increased levels of total coliforms were present in the brook trout GIT and gill arches, white sucker gill arches, brown trout GIT and gill arches, and the stickle back GIT. The resulting E. coli levels were found in 6 of 12 samples ranging in concentration from 1.4 to 1996.4 (MPN) per gram of organ.  Enterococci levels were elevated in the brook trout GIT, creek chub GIT, white sucker GIT, and brown trout GIT and gill arches. The enterococci bacteria were present in 10 of 12 samples at estimated levels ranging from .5 to 454.1 (MPN) per gram of organ.

 

The enterococcal surface protein (esp) indicating human sewage was not detected in any of the samples. The Bacteroides human marker was not detected in any of the fish samples. The only sample that tested positive for the bovine Bacteroides was the Northern Redbelly dace; all other samples were negative for the bovine indicator.

 

Thus there is evidence that the fish were exposed to waters with extensive fecal pollution and some of the origin of this was identified as bovine.

 

Table 1. Summary of bacteria, esp, and Bacteroides results from Pigeon River fish kill

Fish ID number

Fish species

Organ

Total coliforms (Est. conc./g organ)

E. coli        (Est. conc./g organ)

Enterococci  (Est. conc./g organ)

esp

Human

Bacteroides Human

Bacteroides Bovine

080721-1A

Stickleback

Stomach

279.98

2.50

0.50

-

-

-

080721-1B

Stickleback

Gill arches

8.55

0.00

9.75

-

-

-

080721-2A

Fathead

Stomach

0.00

0.00

0.00

-

-

-

080721-2B

Fathead

Gill arches

0.00

0.00

0.00

-

-

-

080721-3A

Northern Redbelly dace

Stomach

ND

ND

ND

-

-

+

080721-3B

Northern Redbelly dace

Gill arches

ND

ND

ND

-

-

-

080721-4A

Brook trout

Stomach

>201.63

0.00

>201.63

-

-

-

080721-4B

Brook trout

Gill arches

>302.45

0.00

32.66

-

-

-

080721-5A

Blacknose Dace

Stomach

ND

ND

ND

-

-

-

080721-5B

Blacknose Dace

Gill arches

ND

ND

ND

-

-

-

080721-6A

Creek chub

Stomach

ND

4.55

>383.58

-

-

-

080721-6B

Creek chub

Gill arches

101.09

60.66

243.85

-

-

-

080721-7A

White sucker

Stomach

1.37

1.37

>454.13

-

-

-

080721-7B

White sucker

Gill arches

>1996.37

31.02

343.23

-

-

-

080721-8A

Brown trout

Stomach

>302.45

0.00

248.29

-

-

-

080721-8B

Brown trout

Gill arches

>604.90

10.05

162.20

-

-

-

ND: Not tested due to lack of sufficient organ size

* > means greater than and that samples were not diluted enough during analysis and high levels of bacteria exceeded the limits of the test.