Distribution of Fascioloides magna in Elk in Southwestern Alberta
Drs. L.H. Laméris
Abstract
Fascioloides magna is a trematode that occurs in the liver of infected wild and domestic
ruminants mainly in North America. Previous research has shown a high occurrence of F.
Magna in Southwestern Alberta. The following study was performed to produce an overview
of the distribution of F. magna in Southwestern Alberta, because concerns were raised of this
parasite circling within the wildlife population and possible transmission to cattle. To acquire
the necessary data nine herds were selected for having contact with cattle (Beauvais Lake,
Crowsnest Pass, Waterton, Castle Carbondale, Livingstone, Porcupine and Whaleback) or
having no interaction with cattle (Jasper, Yaha Tinda). Fecal samples were obtained from
March-May 2010. The fecal samples were processed using the flukefinder (Visual Difference,
Moscow, Idaho, USA). Animals shedding F. Magna were demonstrated in five of the nine
herds with percentages between 1,4 % and 76.7 %. Several factors that can affect the
differences in occurrence of F.magna between the herds have been considered and options for
further research are given.
Introduction
F. magna, also named the giant liver fluke, the large American liver fluke or deer fluke, is a
trematode that occurs in the liver of infected wild and domestic ruminants mainly in North
America. In Southwestern Alberta there have been reports of high prevalence’s of F. magna
amongst elk. (Kingscote et. al 1987, Pybus 1990). Not in all ruminants however, will the
fluke fully develop into an egg producing adult fluke. Bovids are dead-end hosts, which
means that the flukes successfully reach the liver, but rarely mature into egg producing flukes
(Pybus 2001). Nevertheless it is possible that the flukes reach adulthood and produce eggs,
but because the fluke is encysted in the
liver, or sometimes in other tissues, the
eggs will not be released in the
environment (Craig 2007). F. magna
has several definitive hosts, including
elk/Wapiti (Cervis canadensis). Adult
flukes, that occupy the liver of infected
definitive hosts, produce eggs which are
released with the feces into the
environment. In the proper surroundings
(aerated water) eggs hatch and release
miracidia, that need a suitable
intermediate host for their further
development. These intermediate hosts
are aquatic snail species. The miracidia
enter the intermediate host and develop
Figuur 1 Life Cycle of F. magna (Pybus 2001)
further into rediae. In the liver of the
intermediate host these rediae produce cercariae that leave the snail, encyst on the vegetation
and are then called metacercariae. Metacercariae are ingested by grazing ruminants and
eventually, in a suitable definitive host like elk, migrate to the liver and mature in adult liver
flukes. They become encapsulated in the liver parenchyma, where they start producing eggs.
(Pybus 2001). It can take an average of five months to finish the lifecycle. The dispersal of F.
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magna is determined by the presence of a suitable intermediate host and a reservoir of
definitive hosts to allow the cycle occur (Kennedy et. al.1990).
An infection with flukes is transmitted seasonally. Transmission of the miracidia to the
intermediate host occurs during the latter half of the snail activity season. In colder climates
snails hibernate during winter and stay underground. (Craig 2007) Conditions that are suitable
for snails include footprint pools created by livestock, mud flats bordering slow moving rivers
or streams, river backwater, permanent or seasonal runoff streams, irrigation ditches, dugouts
and marshy areas (Dunkel et. al. 1996, Kennedy et. al. 1990) Also the hatching of the
miracidia is influenced by the surrounding temperature. Temperatures below around 20 °C
slow down the development and also the level of moisture has an important effect. There are
two periods where transmission to a final host is most common, namely in late summer and
fall when great numbers of the infectious state of the fluke are present in in the environment
and in spring when final hosts seek lush wetlands to feed (Pybus 2001).
There are several factors that can influence the prevalence within a herd, for example higher
density of animals and favorable environmental factors tend to increase the prevalence.
Furthermore prevalence increases in definitive hosts with age and stabilizes in the adult age
groups (Pybus 2001). Also the amount of time spent in one location, where the intermediate
host is present, plays an important role. The longer an infected herd resides in one place, the
more the parasite pressure will rise and with it the prevalence within the herd.
To diagnose an infection with F. magna in live definitive hosts a fecal sample is taken and
examined to find eggs of this liver fluke. The eggs of F. magna (~160-96 μm) are
recognizable through their size, oval shape, thin shell and operculum. This method is not
usable to diagnose an infection with F. magna in a dead-end host, because no eggs will be
shed in the feces. A diagnosis of an infection with F. magna in a dead-end host is usually
obtained post-mortem from inspection of the liver. To diagnose an infection in a live dead-end
host it would be necessary to develop an Elisa. For F. Magna this has not yet been done, but
for the diagnosis of Fasciola hepatica enzyme-linked immunoassays for blood and milk are
available.
An infection with F. magna usually has little clinical consequences, for bovids as well as for
elk, but can sometimes cause significant health problems and even death. Moreover, infection
in cattle can bring about considerable economic losses, because the livers have to be
condemned at slaughter. (Wobeser et al. 1985, Foreyt et al. 1976). Thereby are the possible
subclinical effects, for example higher susceptibility for other pathogens, lower nutrition
efficiency or loss of body condition, caused by F. magna never assessed. Aberrant hosts
(figure 1), mostly Ovine species, are however very sensitive to infection with F. Magna. In
these aberrant hosts the parasite is not able to finish the migration within the animal, but does
cause significant tissue damage, which often results in death. (Pybus 2001)
F. magna spreads in different hosts through natural and artificial means. Translocation of wild
animals by humans to for example reestablish a population in an area, creates a risk of
spreading disease carried by the translocated animals to naïve regions. In the case of F. magna
it creates a risk for not only the definitive hosts, but also for the aberrant hosts and the deadend hosts. For this reason it is important to get an overview of the distribution of F. magna in
southwestern Alberta. (Pybus 1990) Moreover, if there is (spatial) interaction between
wildlife and livestock, which is the case in the southwestern Alberta region, there is a risk of
transmitting diseases. Natural infections with F. magna occur in a variety of domestic
livestock in North America. Wild definitive hosts act as reservoirs of infection for domestic
species in enzootic areas. (Pybus 2001) Therefore F. magna is commonly found in livestock
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that shares pastures with infected wild definitive hosts like elk. (Foreyt et al. 1976). In Alberta
cow-calf ranches near Waterton National Park were confronted with massive infections of
their livestock with F. Magna and even loss of calves and heifers. Concerns were raised by
veterinarians and ranchers about F. magna circulating in the wildlife populations and the
transmission to cattle. This gave ground for performing this survey of the distribution of F.
Magna within elk herds residing in southwestern Alberta.
Materials and Methods
Study design
Fecal samples have been collected from elk herds in southwestern Alberta, which have been
selected based on either high or low (spatial) interaction with livestock. In collaboration with
the Elk Montane research project the information about the interaction was obtained through
140 elk, which have been equipped with a GPS collar at several occasions and since 2007 the
equipped elk are followed. These collars provide information about the location of the elk
and are also used to assess the elk home ranges. These home ranges are also assessed by
performing winter surveys (counts of elk from airplane or helicopter by governmental wildlife
agencies) and local knowledge from, for example wildlife officers, ranchers and scientists.
Based on all the information collected, 7 herds (Beauvais Lake, Porcupine, Livingstone,
Crowsnest Pass,Whaleback, Castle Carbondale and Waterton) were selected for having
interaction with livestock. Two control herds (Jasper and Yaha Tinda) were included for
having no interaction with livestock. Information about herd size and herd location were also
obtained through the collaboration with the Elk Montane research project.
Elk fecal samples were obtained between March 3rd and May 15th 2010 starting with the herds
in the National Parks and concluded with the herds in the ranch land areas. As mentioned
before the home ranges of the different elk herd were determined by information obtained
from the GPS collars and from local knowledge. The sampling strategy used consisted of
three parts. On location the sampling strategy commenced with the observation of the herd,
which includes identification of movement patterns, subgroup identification and dynamic of
the subgroups and takes a couple of days. The following step is identification of the optimal
herd situation for sampling and the final step is the actual collection of the samples. The main
objective of this strategy is to collect fresh samples from distinct individuals. Important for
determining the percentage of shedding animals as close as possible in a study like this, it is
essential to have representable samples of the whole population being sampled. Wildlife
sampling can sometimes give rise to several difficulties to overcome. One of these difficulties
is convenience or subjective sampling, which means the sampling is not random and is not a
correct presentation of the population sampled (Anderson 2001). In the case of this study the
elk herds were composed of unstable subgroups that besides this travel large areas, which
made randomized sample collection not feasible. Also identifying individuals can be difficult,
but was tried to overcome by making careful notes of the location of feces and in certain areas
the bedding places of individual animals were easily distinguished. Another reason that made
randomized sampling a challenge was the sometimes poor accessibility to the elk herds. From
the seven herds having either low or high interaction with livestock, six of them were not
encountered as a whole, comparing the numbers to the counts of the herds by the Elk Montane
research project, and therefore some parts of the herd couldn’t be sampled. The risk of
resampling was minimized by the large spatial distribution of the individual animals and by
selecting the right herd situation (static/resting).
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A sample size between 55 and 70 per herd (depending on the herd size) was chosen in order
to detect a 5% prevalence with 95% confidence. Overall, there are 9 herds of which fecal
samples (720 samples) have been collected.
Herd descriptions
For this study nine herds in southwestern Alberta have been sampled. The Waterton-herd
consists of about 800-1000 animals. The Castle Carbondale herd includes around 600
animals. During sampling there were one big group of around 120 animals and several smaller
subgroups of 20-30 animals present and sampled. In Beauvais Lake there are many subgroups
present and they are spread out over a large area. The part of the herd sampled in the
Livingstone area consists of about 110-130 elk of the total of around 300 animals. In the
Whaleback area there are 3 subgroups of different numbers (20,60,120) of elk sampled. The
whole herd in the Whaleback region consists of about a 1000 animals. A group of about 100
elk and a smaller subgroup were sampled in the Porcupine region from the total of 1200 elk
that are part of this herd. The Whaleback and Porcupine herd consist of so many animals that
reside in such large areas, from which only portions are part of the study area, which
consequently means that not the whole herds are part of the study. In Crowsnest Pass two
subgroups were sampled of respectively 20 and 40 animals. The rest of the herd, which in
total contains 200 animals, couldn’t be found. In the Yahatinda area one main herd was found
of about 200 animals and one smaller herd of 40 males. In Jasper there were several smaller
sub-groups of 10-40 elk found and mixing between groups occurred. The elk present here
were really habituated to people, which facilitated sampling of these groups.
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Laboratory methods
To detect eggs of F. magna in the samples taken from the elk herds a sedimentationflotation technique has been used. Two grams of feces of each sample have been
processed using the flukefinder (Visual Difference, Moscow, Idaho, USA). The flukefinder
consists of a two sieved system with different pore size. The first sieve has a larger pore
size, which makes sure the largest pieces of debris in the fecal samples are removed. The
eggs of F. magna will pass through this first sieve. The second sieve has a smaller pore
size than the first, which will retain the eggs of F. magna. Subsequently the second sieve
will be flushed with water and this solution is collected in a cup. The solution is than
poured into a tube and several sedimentation steps follow. The objective of these
sedimentation steps are the removal of more debris, as the eggs will sink. The sediment
obtained after following the protocol (addendum 1) is analyzed using a dissecting
microscope and any eggs found have been counted.
Results
Of all the samples taken 30 of each herd were already processed. Of the following herds,
Castle Carbondale (51), Whaleback (63), Crowsnest pass (9), Beauvais Lake (41), Porcupine
(39) and Livingstone (46) all the remainder of the samples (between the parenthesis) were
analyzed. From the other three herds, Waterton, Yaha Tinda and Jasper, only the 30 samples
processed previously have been analyzed.
In three of the herds which have contact with cattle, Beauvais Lake, Crowsnest Pass,
Waterton, animals shedding F. magna have been found. Besides these herds, the control
herds, Jasper and Yaha Tinda, show animals shedding F. magna eggs. In the other herds,
Castle Carbondale, Livingstone, Porcupine and Whaleback no positives have been found. The
result of the analysis are shown in Table 1.
Table 1 Result of sample analysis
Discussion
Before it is possible to conclude anything from the results it is important to put the samples in
perspective. As mentioned in the introduction, the prepatent period for F. magna takes around
five months. The samples that were analyzed during the last two months have been collected
in the period March until May of the year 2010. This would mean the samples are taken in the
right period, because the transmission to the final host takes place in late summer-early fall.
But taken into account the fact that in Alberta winter can be very rigorous and temperatures
can stay low until May, the samples are possibly taken too early in the year. Because, as
5
mentioned before, temperature has a great retarding influence on the developing eggs, as well
as on the miracidia. This can cause a lower parasite pressure and less shedding animals.
Since the collection of the samples, they have been saved in a -80 °C freezer for this whole
period, which may also have an effect on the quality of the samples. Of each sample two
grams was taken. Because the samples were frozen and had to be remained frozen, it was not
possible to mix the entire sample. Mixing of the entire sample would assure a representative
part of the sample to be processed with the flukefinder. All these factors, concerning the
samples, named above, could result in an underestimation of the number of infected animals.
An essential condition for an infection of fascioloides to establish in a herd of elk is the
presence of the intermediate host. The intermediate host in an aquatic snail species, Lymnaeid
species and in the introduction favorable environmental conditions for survival of this species
are described. The presence of the intermediate host is highly dependable on the
environmental conditions, which includes at least slow running water and a temperature that
is not too low. The differences in numbers of infected animals between the herds could be
explained by differences in vegetation, elevation and favorable habitats for the intermediate
host of the study areas. Each research area probably also differs in food and water availability,
which also has an influence on the need for travelling of the herd to find other food/water
sources if these are not sufficient. If this is not the case, there is no need to move and the herd
will stay in one place for a longer amount of time. This predilicts for a rise in the incidence of
F. magna, because of a rise in infection pressure.
There are several herd characteristics that could play part in the distribution of F. magna. For
example, prevalence can rise if the density of elk in the area increases. Density of animals is
usually higher if there are food and water sources readily available. The different sampling
area´s should be compared on these aspects to assess differences in food and water
availability. The Waterton herd shows a high prevalence for F. Magna in which density can
play a part. In Waterton national park wild life management entails the protection of elk from
hunting and this has had an increase in density as consequence.
Not only elk can serve as a definitive host for F. magna, but also other cervids, like whitetailed deer and caribou, can act as a definitive host and contribute to sustaining the
contamination of the surroundings with infectious stages of F. magna. To put the results in
perspective it is important to know the number of other definitive hosts present in the sampled
areas. If in certain areas where elk reside, the number of definitive hosts other than elk are
considerably higher, the incidence of F. magna is probably also higher. More definitive hosts
means more chance for the completion of the lifecycle of F. magna and there are more
susceptible animals to be infected.
The herds, Jasper, Yaha Tinda, Crowsnest Pass and Wateron, were selected for having no or
low contact with cattle. Interestingly, the results show the highest prevalence in these herds.
As mentioned before cattle are a dead end host for F. magna. Consequently if cattle gets
infected no eggs will be shed by the infected animal, which means this animal will not
contribute to the contamination of the environment and no transmission to other animals will
take place. Another factor to take into account is that the intake of infectious stages of the
liver fluke by cattle can cause a lower infection pressure for the definitive hosts, such as elk.
This hypothesis could explain the results from this study, which show higher incidence of F.
magna in herds that have no or low interaction with cattle.
Protocol evaluation
6
The method used for detecting the F. magna eggs has been evaluated. Eggs were collected
from a positive fecal sample, using a pipet (10µl). The known number of eggs were then
added during step 3 of the protocol before pouring the suspension of feces into the
flukefinder and the rest of the protocol was finished as usual. (see addendum 1) The
analyzing of the sample was also done as usual and in the same time (20 minutes). The
results of the test evaluation are shown in table 2&3.
Table 2 results of test evaluation
Table 2 shows the results of the evaluation when just one egg was added each time to the
mixture before using the flukefinder. The 10µl pipet used for carrying out this experiment
contained too much volume and this in combination with the high number of eggs in the
positive sample used to get the eggs for this experiment, is probably the cause for finding the
two eggs in the first try.
Table 3 results of test evaluation
Table 3 shows the results of the evaluation when 30 eggs were picked and added to the
mixture before using the fluke finder. The processing of the samples has been done with the
flukefinder. The flukefinder method has been used in many different studies to determine
liver fluke ova in fecal samples (Foreyt 1992, Knapp et. al.1992), but the test qualities of the
fluke finder have never been undoubtedly established. The protocol evaluation done in this
study is not elaborate and accurate enough to draw conclusions about the fluke finder
technique on this matter, but it would be good to determine the test qualities of the method
used to process the samples.
Possibilities for further research
First it is important to finish the processing of the rest of the samples. There are some
questions about the period in which the samples are taken. The first opportunity could be to
take the samples in spring/early summer (starts in Alberta around May) and compare the
results of the analysis of the new samples to the results obtained from this study. For some of
the herds, like for example Crowsnest Pass, it is necessary to take more samples to get a more
reliable result of the prevalence. The analyzing method used in this study is the fluke finder.
This method has never been tested for specificity and sensitivity qualities. Here also lies an
opportunity and this knowledge would give more clarity how to interpret the results obtained
in this survey. To get more certainty about which factors play a part in the differences in
occurrence of F. Magna between the herds it would be necessary to do a survey of the
sampled areas for as well the intermediate host as other final hosts (white tailed deer, moose
7
etc.) and take a closer look at other environmental factors. Interesting to know would also be
the prevalence of F. Magna within populations of other definitive hosts and compare this to
the prevalence found in elk. This part of the research focuses on the infection in elk
populations, but it would also be interesting to see the exposure in cattle. To diagnose the
infection in live dead-end host the development of a serologic test is essential. At this moment
in Calgary an western blot is being developed to obtain a serological diagnosis of F. magna in
cattle and the results are promising.
8
Literature
ANDERSON D.R., (2001) The need to get the basics right in wildlife field studies Wildlife
Society Bulletin 29(4) 1294-1297
CRAIG T.M. (2007) Beef Cattle; Epidemiology and Control of Internal Parasites Department
Veterinary Pathobiology, Texas A&M University College Station, TX, USA, 79th Western
Veterinary Conference
DUNKEL A.M., ROGNLIE M.C., JOHNSON G.R., KNAPP S.E. (1996) Distribution of
potential intermediate hosts for Fasciola hepatica and Fascioloides magna in Montana, USA
Veterinary Parasitology 62 63-70
FOREYT W.J. (1992) Experimental fascioloides fagna infections of mule deer (odocoileus
hemionus hemionus) Journal of Wildlife Diseases 28(2) 183-187
FOREYT W.J., TODD A.C. (1976) Effect of six fasciolicides against F. magna in whitetailed deer Journal of Wildlife Diseases 12 361-366
KENNEDY J.M., ACORN R.C., MORAIKO D.T. (1990) Survey of F. magna in farmed
wapiti in Alberta Canadian Veterinary Journal 40 252-254
KINGSCOTE B.F., YATES W.D.G. TIFFIN G.B. (1987) Diseases of Wapiti utilizing cattle
range in Southwestern Alberta Journal of Wildlife Diseases 23(1) 86-91
KNAPP S.E., DUNKEL A.M., HAN K., ZIMMERMAN L.A. (1992) Epizootiology of
fascioliasis in Montana Veterinary Parasitology 42 241-246
PYBUS M.J. (1990) Survey of hepatic and pulmonary helminths of wild cervids in Alberta
Canada Journal of Wildlife Diseases 26(4) 453-459
PYBUS M.J. (2001) Liver Flukes, In: SAMUEL W.M., PYBUS M. J., KOCAN A.A.,
Parasitic diseases of wild mammals 2nd ed. Iowa State University Press, Ames 121-136
WOBESER G., GAJADHAR A.A., HUNT H.M. (1985) F. magna: Occurrence in
Saskatchewan and Distribution in Canada Canadian Veterinary Journal 26 241-244
Acknowledgements
This study was conducted under the guidance of Dr. Karin Orsel and Mathieu Pruvot. Dr.
Susan Kutz provided the laboratory and supplies for processing the samples. Thanks to all.
9
Addendum 1
“Flukefinder®” Procedures
1. Fit the 2 units together with the label upright and at the top.
2. At a slight angle with vent hole facing upward, wet the screens with water running
through the top.
3. Mix known volume of feces (2 grams) with 30ml of water in a plastic cup and pour
into the top section of the column. Make sure the feces are dissolved completely.
4. Tap flukefinder against the side of the sink to expedite the passing of water through
the screens.
5. Briefly hold the column under a cold tap running the stream at half force.
6. Repeat steps 5, than 4, 3 times. Then separate the top unit from the bottom. Invert top
section and backwash debris into the sink until clean.
7. Invert bottom unit over a cup and backwash the eggs and debris from the screen into
the cup with a strong stream from a squirt bottle of water. Make sure the screen is
completely clean.
8. Swirl suspension in the cup and pour into a 16x125 test tube and allow to settle (about
4 minutes for a completely full test tube, 2 minutes for half full test tube).
9. Slowly, in 1 fluid movement, pour supernatant from the tube without disturbing the
sediment and refill tube with 3 inches of water, making sure sediment is well
dispersed.
10. Let stand for 2 minutes only and pour supernatant from the tube.
11. Repeat steps 9 and 10 two or three times (until no more debris remains suspended after
a two minute sedimentation).
12. Pour off supernatant, swirl to resuspend sediment and pour quickly into the shallowest
concentric striped petridish. Rinse test tube and pour into dish. Look for eggs using a
dissecting microscope at 25x or compound scope at 4x magnification. A drop of
methylene blue dye will greatly enhance viewing.
13. Thoroughly rinse flukefinder after use to ensure accuracy of the next essay.
10