Be Practical, Be Proactive - UC Davis School of Veterinary Medicine

Donkey Parasites
Hands-On Laboratory:
Be Practical, Be Proactive
The Second Donkey Welfare Symposium
UC Davis, California
November 7-9, 2014
Authors:
Rosina (Tammi) C Krecek FRSSAf, BS, MS, PhD, MAP, MBA
Visiting Professor, Department of Veterinary Pathobiology
College of Veterinary Medicine & Biomedical Sciences (CVM)
Texas A&M University (TAMU), College Station, Texas 77843, USA
Email: [email protected]
Eddy Krecek
908 Ladove Drive, College Station, Texas 77845, USA
Email: [email protected]
Table of Contents
Introduction.................................................................................................3
What test is best for detecting...................................................................4
parasites in my donkey?
What are the common................................................................................5
worm eggs?
How do I test for parasite...........................................................................6
anthelmintic resistance
in my donkey?
What flotation solution..............................................................................7
can I use for my fecal
egg counts?
Acknowledgements.....................................................................................8
References....................................................................................................8
2
Introduction
Parasite egg-counting techniques are recommended for estimating the
extent of parasite egg contamination on pastures grazed by infected animals,
determining the efficacy of drug treatment and measuring the impact of other
interventions (e.g. removal of feces) (Zajac and Conboy, 2012; Matthee et al.,
2002: Krecek et al., 1994). Egg counts are of limited value in making judgments
about the clinical condition of individual animals because many factors affect
parasite egg production, including parasite species, individual host species,
host immunity and stage of infection. Also, counts performed on combined
samples from a number of animals may not accurately reflect parasites within
that herd. Individual animals produce higher egg counts (“high shedders”)
than others (“low shedders”) (Krecek et al., 1994).
A commonly used quantitative test for counting parasite eggs in equids is the
modified McMaster test. The test requires the use of special reusable slides
(Figure 1) available from several suppliers (e.g., TE Krecek, www.mcmaster.
co.za; [email protected]; [email protected]). The capacity of the counting
chamber and the number of chambers counted per sample affect the sensitivity of the test. Saturated salt and sugar solutions are used as flotation solution
for this test (Reinecke, 1983). In the US, salt is commonly used (Zajac and
Conboy, 2012).
As it is most commonly used currently in the US, the modified McMaster test
has a sensitivity of 25 or 50 eggs per gram (epg) of feces. This level of sensitivity is acceptable since monitoring of parasite eggs for parasite control programs do not usually require detection of lower epgs (Zajac and Conboy, 2012;
Krecek et al., 1994).
Figure 1. McMaster slide used for counting parasite eggs
in fecal examination (T.E. Krecek).
Regardless of the procedure used to count parasites, the most important
element is consistency. Each step of the procedure should be performed in the
same way for every sample.
3
What test is best for detecting parasites
in my donkey?
Quantitative Fecal Flotation Modified McMaster Test for Equids
Equipment you will need to perform the test:
• Fresh fecal sample from donkey
• Scale to weigh the fecal sample
• Cheesecloth or a small, fine mesh sieve
• 2 chamber McMaster slide*
• Broad tipped disposable pipette, with diameter of 1/8 inch (3 mm)
• Compound microscope, preferably with ocular micrometer in one eye
piece for measuring eggs
• Flotation solution
*Several suppliers are available: e.g., TE Krecek, www.mcmaster.co.za
The method follows:
1. Combine 4 g of fecal material with 56 ml of flotation solution (a method
for making a sodium chloride solution is below).
2. Mix well and strain through cheesecloth or sieve.
3. Mix the solution thoroughly and draw fluid into the pipette. Fill each
chamber of the slide completely.
4. Let the slide sit for at least 5 minutes to allow the eggs to rise to the
surface.
5. Examine microscopically with low power (10 X objective) and maximum
light.
6. At this magnification the engraved lines of the grid will also be in focus.
Count the eggs, oocysts and any other parasite stages for each individual
lane of each chamber. Identify eggs to categories of parasites (e.g.,
strongyle eggs), and to genus or species level (e.g., ascarid eggs) where
possible.
7. The EPG is calculated as follows:
Total number of eggs X 50
= EPG
2 chambers
4
What are the common worm eggs?
The table below is useful because if gives the common parasite eggs and a scale
to show their approximate size, shape and characteristics. It is important if you
wish to measure them to have an ocular micrometer in your microscope.
(Zajac and Conboy, 2012)
5
How do I test for parasite
anthelmintic resistance in my donkey?
Use the Fecal Egg Count Reduction Test (FECRT)
One of the principal uses of the Fecal Egg Count Reduction Test (FECRT)
is the evaluation of drug efficacy. Anthelmintic resistance in strongyle
nematodes of equids including donkeys is rapidly increasing worldwide, and
drug resistance is being reported in the literature. The gold standard today to
determine if anthelmintic resistance has developed is the FECRT.
For convenience, a standard period of 14 days before collection of post
treatment samples is often recommended. To compare the Fecal Egg Count of
your donkey before or pre treatment (Day 0) and after or post treatment (Day
14) use the following formula.
% FECR =
[
Pre treatment FEC- Post treatment FEC
Pre treatment FEC
]
X 100
To interpret the FECR percentages, we follow the American Assocation of
Equine Practitioners (AAEP) Guidelines. Fully effective modern anthelmintics
in equids can reliably decrease fecal egg counts by > 95%. However, under
typical field conditions, it may be more practical to suspect that some portion
of the worm population is resistant if the percentage reduction is less than
90%. For example, if the percentage reduction is 10%, a large proportion
of worms in the animals are probably resistant. If the reduction is 80%, the
proportion of resistant worms is much smaller, but still significant. For equids,
minimum cutoff values for drug efficacy in FECRT have been suggested as 95%
for macrolides (e.g., moxidectin, ivermectin, etc.) and 90% for benzimidazoles
and pyrantel.
To further evaluate which worm genera are resistant in ruminants, feces can
be cultured at a diagnostic lab. In equids, drug resistance is present in small
strongyles (cyathostomins), which cannot be differentiated to species by larval
identification.
6
What flotation solution can I use
for my fecal egg counts?
A commonly used flotation solution is the sodium chloride (NaCl) solution
(Luksovsky, 2014). This is used for the diagnosis of equine nematode eggs. It
can be used for eggs per gram (EPG) tests. This solution can also be purchased
commercially.
If you prefer to prepare this yourself, the method follows:
1. Boil 8000 ml of water in a stainless steel pot.
2. Measure out 3500 ml of salt (e.g., common kitchen salt).
3. Add salt to boiling water. *
*Note: When adding salt to water, it is best to add in increments. Add
approximately 500 ml of salt into water at the time, stirring until mostly
dissolved. Continue to add salt in approximately 500 ml increments until all
salt is used.
4. Since this is a supersaturated solution, you should expect to see a “salt plate”
on the surface of the solution. A salt plate looks like a thick plate of salt on the
top of the water. When the salt plate begins forming, place 90 ml of solution
into a 100 ml graduated cylinder. Allow cool to room temperature. Placing the
graduated cylinder in an ice bath can speed up this process.
Warning: Make sure the solution is cooled before checking specific gravity.
If the temperature is too hot or too cool, it will be an incorrect reading.
Again, make sure to cool to room temperature.
5. Drop hydrometer into graduated cylinder once solution is cool and check
specific gravity. The specific gravity should be 1.2.
• If specific gravity is lower than 1.2, add more salt.
• If specific gravity is higher than 1.2, add more water.
Note: This part is really more of a guessing game. Make sure to add the salt or
the water slowly and check the specific gravity often until is it 1.2.
6. Pour into containers labeled “NaCl Solution” once completely cooled.
7
Acknowledgements
The authors thank Robin Houston (UC Davis), Thomas Craig (TAMU), Joe Luck
(TAMU), Karen Snowden (TAMU), and Eric Davis (UC Davis) for providing biological
material and assistance; Araceli Lucio-Forster for helminth egg photos; and the staff of
Creative Technologies (TAMU-CVM), specifically Jennie L. Lamb and Sydney Sund,
for illustrations and design.
References
American Association of Equine Practitioners, 2013. http://www.aaep.org/custdocs/
ParasiteControlGuidelinesFinal.pdf
Krecek RC, Guthrie AJ, Van Nieuwenhuizen LC and Booth LM (1994) A comparison
between the effects of conventional and selective antiparasitic treatments on
nematode parasites of horses from two management schemes. Journal of the South
African Veterinary Association 65: 97–100.
Levecke B, Behnke JM, Ajjampur SSR, Albonico M, Ame SM, et al. (2011) A
Comparison of the Sensitivity and Fecal Egg Counts of the McMaster Egg Counting
and Kato-Katz Thick Smear Methods for Soil-Transmitted Helminths. PLoS Neglected
Tropical Diseases 5(6): e1201. doi:10.1371/journal.pntd.0001201
Luksovsky, J. (2014). Sodium Chloride Flotation Solution Method. Parasitology,
College of Veterinary Medicine, Texas A&M University, College Station, Texas, 77843,
USA.
Matthee S, Krecek RC, Milne S, Boshoff M and Guthrie A (2002) Impact of
management interventions on helminth levels, and body and blood measurements in
working donkeys in South Africa. Veterinary Parasitology 107: 103–113.
Reinecke RK (1983) Veterinary Helminthology. Butterworths, Durban, South Africa.
392 pp.
The RVC/FAO Guide to Diagnostic Veterinary Parasitology. http://www.rvc.ac.uk/
review/parasitology/EggCount/Principle.htm
University of Pennsylvania http://cal.vet.upenn.edu/projects/parasit06/website/
mcmaster.htm
Zajac AM and Conboy GA (2012) Veterinary Clinical Parasitology. 8th Edition.
Wiley-Blackwell. 354 pp.
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