Anthony Domma

Animal Science 3900

Spring 2015

Comparison of Eggs Per Gram Counting Techniques for Haemonchus contortus infecting Ewes
and Goats
Abstract
The fecal egg count (FEC) of an animal can provide insight into how sick an animal can be. Having an
accurate test is important in determining the eggs per gram (EPG). Stat Spin and Fecalyzer tests have been
created to make the process of determining the EPG easier, particularly for small animal testing. However, this
amelioration might deteriorate the accuracy of these tests. In this experiment, using the McMasters tests as the
standard, the accuracy of Fecalyzer, double centrifugation (sugar) technique and Stat Spin was tested. Each test
was performed on samples from LSU ewes. A statistical analysis was performed on the results to see the
difference of the tests. Stat Spin and Fecalyzer tests were found to be statistically different from the McMasters,
while the double centrifugation (sugar) technique was shown to be statistically similar. This indicates that Stat
Spin and Fecalyzer are not accurate in determining an actual EPG. However, all tests showed floatation of eggs,
so all could be used in a qualitative analysis of a sample.
Introduction
Tests that determine the amount of eggs in an animals gastrointestinal system are important because an
animal can get very sick from these parasites. Different animals can be affected by parasites in different ways.
Parasitic worms like Haemonchus contortus , can cause anemia, edema around the neck, weight loss and
weakness. Detecting how infected an animal is can lead to the proper course of action needed to prevent the
effects of these worms. In most large animals, like ewes, there are always some worms in the intestine but they
cause inconsequential damage to the animal. However, if the FEC gets to high levels, control of the parasites is
vital to the animals health. In small animals any detection of worms can cause worry in pet owners so
veterinarians typically treat any detection of worms.
To determine how infected an animal is, tests can be run that count the eggs per gram (EPG) of
stool.One of the most commonly used tests is the Modified McMaster.. This test gives a EPG without having to
count all the eggs in a given sample. Other tests like double centrifugation technique, Stat Spin and Fecalyzer
aim to get the total count in a sample of feces by physically counting all eggs. Trichostrongyle type eggs are
denser than water, so they sink the bottom of a tube in a water solution. These tests work by changing the
specific gravity of a solution to one higher than the eggs. This will cause the eggs to float up to the top rather
than sink. Centrifugation can speed up the rate that these eggs float up by providing additional forces than
gravity.
With Modified McMasters and double centrifugation tests, the microscope slide often have debris that
might impede the counting process, and these tests require a labor intensive process to get the eggs on the slide.
The purpose of the Stat Spin and Fecalyzer tests were to provide a simpler technique and cleaner way to view
slides for determination of the EPG. However, the accuracy of these tests is questionable in determining how
many eggs were actually in the sample.

Materials and Methods

Collection:
Fecal samples were collected directly from the rectum from the LSU teaching herd of ewes. Most of the
ewes that the samples were taken from were taken a week after pregnancy.
Tests:
A comparison of quantitative eggs per gram (EPG) was performed to test the accuracy of the EPG. The
tests being compared were the Fecalyzer, double centrifugation (sugar) technique, Stat Spin and Modifiied
McMasters.
For the Modified McMasters preparation, 2g of sample feces was weighed out and homogenized with
15mL of non-Iodized Sodium Chloride (NaCl) salt solution (specific gravity of 1.20). After homogenization, 15
additional mL of salt solution were added and lightly blended with a mixer. The homogenate was carefully
transferred with a pipette to a McMaster slide ensuring that as few bubbles from the solution as possible are
created. The slides were viewed under a microscope and eggs counted using the 10x objective. The counted egg
number was then multiplied by 50 to get the EPG. The Modified McMasters test is one of the most accepted
tests to determine EPG.
For double centrifugation (sugar) technique, 2g of sample was homogenized in 15mL of water. The
homogenate was transferred to 15mL tubes. The tubes were centrifuged at 1500 rotations per minute (rpm) for
10 minutes. The supernatant was discarded and the pellet kept. 15mL of sugar solution (specific gravity of 1.251.27) was then added and the pellet broken up and mixed with the added solution. Additional sugar solution was
added to the tube until a positive meniscus was formed over the tube. A cover slip was placed on top of the
tubes and the tubes were centrifuged again at 1500 rpm for 10 minutes. After centrifugation, the coverslip was
removed and transferred to a microscope slide to be counted.
For the Stat Spin, the procedure was followed as the manufactures instructed. The manufacturer has a pipette
with a collection chamber that when filled assumes that 1g of sample is collected. This sample was broken up
and dissolved in a Sodium Nitrate (NaNO3) salt solution (specific gravity of 1.2). The tube was then centrifuged
at 3500 rpm for 5 minutes in a fixed angle centrifuge. Finding a
centrifuge that fit the tall tubes was problematic. Eventually, we
had to resort to wrapping the tubes in paper towels and putting
the tube-towel complex in a 50mL test tube and using a 50mL
centrifuge.
After centrifugation a cover slip was placed on top of the tube
and the solution level was brought up until the cover slip was
layered with homogenate, creating a seal. The cover slip sat for 5 minutes and the cover slip was removed and
placed on a slide for viewing using the 10x objective.
The Stat Spin was done twice for each sample. Once was done exactly as the manufacture instructed
(described above). The other test (Stat Spin EPG #2) was performed but the weight of the sample was
determined and recorded.
For the Fecalyzer the procedure was followed as the manufactures instructed. The instrument has a
compartment that when filled assumes 1g of sample is being used. The tube was homogenized with a Sodium
Nitrate (NaNO3) salt solution (specific gravity of 1.2). More solution was added to the tube until a positive

meniscus was formed. A cover slip was placed on the top. After 20 minutes of sitting, the cover slip was taken
off and placed on a slide to be read.
Results
The Trichostrongyle type egg count was determined under 10X magnification and the EPG was
calculated. Figure 1 shows the calculated EPG from each individual in the population of ewes from each test.

Eggs Per Gram (EPG)

Individual Ewe Results

40000

Sugar EPG

30000

Stat Spin EPG

Stat Spin EPG #2

20000

Fecalizer EPG

10000
6000

McMasters EPG

4000
2000
0

Ewe
Figure 1: This graph shows the individual eggs per gram (EPG) results from the samples of each animal using each test.

Eggs Per Gram (EPG)

Overall Average
20000
18000
16000
10000

**
**

Sugar EPG
Stat Spin EPG

8000

Stat Spin EPG #2

6000

Fecalizer EPG

4000

McMasters EPG

2000
0

Fecal Test
Figure 2: This graph shows the averages of the eggs per gram (EPG) of each test. The error bars show the deviation from that average. The (*) shows
statistical differences as performed by a T test while (**) shows a stronger significance.

The averages of the individual EPG was calculated. Figure 2 shows the averages plotted in a bar graph.
A T-test was performed to determine the statistical significance of the results. All tests were compared against
each other, there was no statistical difference between McMasters and the double centrifugation (sugar)
technique or the sugar vs any of the tests There was a statistical difference between the Stat Spin, Stat Spin #2
and Fecalyzer, when compared to the Modified McMaster. There was a smaller statistical difference with the

Stat Spin #2 than with the Stat Spin and Fecalyzer. So when the sample was actually weighed and the EPG
calculated from the weight rather than the assumed weight, the results were more closely related to the
McMasters test.
Discussion
From these results, the interpretation is that the double centrifugation (sugar) technique was closer to the
actual EPG than the other tests. The other tests do not show a statistical significance for providing an accurate
EPG for the animal. When the Stat Spin was weighed out and not assumed to be 1 gram the EPG was found to
be more accurate.
The Stat Spin and Fecalyzer tests both have cleaner microscope slides and the technique was less
complicated than the sugar and McMaster techniques. In general for sugar and McMaster, more material is
floated up. This means more eggs were on the slide but also more debris was on the slide. The cleaner Stat Spin
and Fecalyzer tests made it easier to count the eggs. However, the eggs that were counted did not provide an
accurate determination of the actual EPG relative to the other tests. The amount of debris in the Modified
McMaster and sugar may be reduced by including a filtration step using a tea strainer.
If a qualitative test is desired to determine if any eggs are in a sample, any of these test would suffice in
floating up eggs to be viewed under a microscope. This explains why these two tests are used to see if small
animals have any eggs in a sample. However, if an EPG is needed to test quantiatively the parasite burden of an
animal, there might not be a correlation of EPG results and pathogenicity when using the Stat Spin and
Fecalyzer tests.