Joseph Harman
Biology 1615
Professor Lata E. Moses
10/25/12
Summary of Avian Botulism
Introduction
There have been large numbers of mortalities in fish-eating birds around the Great lakes since
1999 to present day (Specifically Lake Erie). Scientists have found a correlation of infection between
fish-eating birds of the Great Lakes and the local fish species. It is hypothesized that the fish-eating birds
are being exposed to the disease from their primary food source. Due to the lack of investigation into
this hypothesis, a study was conducted to gather more information on the particular species of fish
being preyed upon. The role of the fish as carriers or transport vectors of Clostridium Botulinum Type E
Neurotoxin (BoNT/E) was thoroughly investigated in the following experiment. By isolating four of the
primary species of fish found in the lakes (Rainbow Trout, Walleye, Round Goby, and yellow perch) and
exposing them to BoNT/E, studies were conducted to see how each species dealt with carrying the
strand of neurotoxin. As a result all the fish species exhibited change in pigmentation of the skin and
behavior before death from the exposure. Suggested from the results of this study is that the fish
species can be a main carrier/transport vector of BoNT/E to the affected bird species.
Methods
Before the study was conducted the group gathered four native species of fish. The species
were: Rainbow Trout, Walleye, Round Goby, and yellow perch. Once obtained, the fish were placed in
individual 350 1 (1.1m x 1.1m x 0.29 m) tanks. Each tank was carefully prepared and treated with fresh
flowing water and excellent water chemistry (in compliance with the University of Guelph’s Animal Care
Committee Guidelines).
Clostridium botulinum type E (BoNT/E) was grown in a lab over a period of four days in an anaerobic
chamber at 30 C. After the four day period the culture was centrifuged at 20,000 x G with RC5 Sorvall
centrifuge for 30 min at 4 C. Later, the supernatant was harvested. After creating the
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Clostridium botulinum type E neurotoxin that was to be used throughout the duration of the
experiment, the neurotoxin was then diluted into the concentrations that would be administered to
each of the four species of fish. Each dilution was tested by mouse bioassay (two mice x five dilutions =
10 mice total) by injection in the peritoneal of the fish. The concentration or the highest dilution that
was lethal to two mice tested became known as one Mouse Lethal Dose (MLD). The four concentrations
used were: 0, 800, 1,500, and 4000MLD of BoNT/E.
The BoNT/E was placed into No. 5 gelatin capsules with a volume of 100 µl. The gel capsules
were filled with 75% gelatinized corn starch and 100µl of trypsinized supernatant was later added.
Immediately, the capsules were placed in a freezer maintained at -20 C until they were ready to be
injected into the fish.
Once each capsule was concentrated in the respective MLD dosages, a negative control was
tested (0 MLD) or a placebo. The negative control was used to test the specific neutralized “C.
botulinum type E monovalent antiserum.” Of the fish that were tested using these capsules no fatalities
or signs of clinical illness were observed.
The fish had the capsules surgically implanted into the peritoneum. The fish were closely
watched and is important to note that no fish was reported to have regurgitated the capsule. After the
capsules were placed into the fish they were returned to their respective tanks for recovery and
observation. Once the fish began to recover to homeostasis they were closely observed to document
the effects that the BoNT/E would have on each species of fish. Each fish was fasted for six days prior to
the experiment in order to clear the digestive systems.
The Experimental design took place over four individual 10-day trials for each species of fish.
For each species tested, there were 12 fish used with 3 fish each intubated with each varying kind of
controls. To determine a fish a survivor it had to last the entire 10-day period. The “survivor” fish were
then humanely killed by a chemical overdose of tricaine methanesulfate (MS-222).
Results
In the rainbow trout, the mean time following the injection of the capsules to reach complete
loss of equilibrium and loss of motion were23+- to 47+- hrs. This was consistent with few differences
among the species. As for the remaining species there were various other results.
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The fish displayed changes in color prior to death. As they drew nearer to death, changes in
behavior increased. The random movement of the fish, an apparent loss of orientation, some were
even observed to have lied at the bottom of the tank on their sides. In the Gobies, the time to these
aggravated symptoms seemed to take longer to take effect, however after the symptoms did begin to
show the progression of time to death was rapid.
After exposure to BoNT/E there were some rainbow trout, walleye, and yellow perch that
exhibited a head up / tail down orientation. The following attempts to swim resulted in the breaching of
the water’s surface. The mortalities that occurred in the fish according to the dosage varied among
species. For example in the 4,000 MLD dosages the fish species with the highest mortality rate were the
round goby at 100%. In order of declining mortality rate went as follows: rainbow trout and walleye at
92% and the yellow perch at 67%. Additionally, not only did the round goby experience 100% mortality
rate but also died in the shortest period at 30 hours.
Discussion
The methods used, particularly the precise doses administered is what set this experiment apart
from other studies conducted. The experimental methods and controls used in this study made it easier
for it to be replicated again if additional data is ever needed. The study was consistent with the studies
and observations by Skulberg and Grande from (1967), but in contrast to the findings of Cann and Taylor
in 1982. In Cann and Taylor’s finding, they used 10,000 MLD of BoNT/E and observed no mortalities in
five rainbow trout. What was not recorded in Cann and Taylor’s experiment was whether or not
regurgitation was observed or even watched for. If regurgitation did occur it is highly possible that is the
reason the five trout survived.
The study has successfully made a strong correlation of fish-eating birds contracting the virus
from infected fish species. The experiment has shown that fish may very well be carriers or transport
vectors of Avian Botulism. The type of behavior and change in pigmentation noted in the study could
increase the chances of fish in a natural setting being more susceptible to being eaten by fish-eating
birds.
Conclusion
The experiment was unique by answering unanswered questions like: “Could avian botulism be
contracted due to the consumption of fish that are infected?”; “What species of fish could be successful
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carriers of BoNT/e?”; “How can these infected fish be identified?”; etc. The experiment was thorough
and followed each guideline that helped maintain control and good ethical practices (i.e. compliance
with the University of Guelph’s Animal Care Committee Guidelines). Thanks to the carefully recorded
data and well documented procedures, additional studies conducted will have a great reference to
compare to when attempting to answer similar and perhaps more specific questions.
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