Veterinary Parasitology 260 (2018) 45–48

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Research paper

Determination of the specific gravity of eggs of equine strongylids, Parascaris T

spp., and Anoplocephala perfoliata
⁎
Jamie K. Norrisa, , Ashley E. Steuera, Holli S. Gravattea, Paul Slusarewiczb, Jennifer L. Bellawa,
Jessica A. Scarea, Martin K. Nielsena
a
M.H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA
b
MEP Equine Solutions, 3905 English Oak Circle, Lexington, KY, 40514, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Given the ever-increasing levels of anthelmintic resistance in livestock parasites globally, it is recommended to
Specific gravity use parasite fecal egg counts to make treatment decisions and to evaluate treatment efficacy. The consensus in
Flotation equine parasitology is to use a flotation medium with a specific gravity (SG) of ≥ 1.20 to float the main parasite
Ascarid egg types of interest in egg counting techniques. However, the density of common equine endoparasite eggs has
Strongylid
been sparsely investigated. Equine tapeworm eggs are known to be particularly difficult to determine and count
Anoplocephalid
in fecal samples. It is unknown whether this could be because of differences in egg density. The aim of this study
was to provide estimates of relative densities for equine ascarid, strongyle, and tapeworm eggs. Six aqueous
glucose-salt solutions with specific gravities ranging from 1.06 to 1.16 were made and placed from most to least
dense into thirteen 15 mL centrifuge tubes. Concentrated aqueous suspensions of the three types of endoparasite
eggs were placed on top of each tube. These tubes were then centrifuged at 800 g for 20 min and each layer of
flotation solution was carefully pipetted and transferred to a McMaster egg counting slide. Egg type and count
were recorded for each specific gravity layer. Each egg was assigned a specific gravity based on the specific
gravity layer it was observed in. In a second trial of this study, five similar flotation media were made ranging
from 1.02 to 1.10 and were used in four subsequent replicates. In total between the two trials, the mean egg SGs
of Anoplocephala perfoliata (n = 3811), Parascaris spp. (n = 3478), and strongylid type eggs (n = 9291) were
1.0636 (95% confidence interval (CI): 1.0629–1.0642), 1.0903 (95% CI: 1.0897–1.0909), and 1.0453 (95% CI:
1.0448–1.0458), respectively. The three egg types were statistically different from each other (p < 0.0001).
This is the first time that the specific gravity of equine strongylid and Anoplocephala perfoliata eggs has been
determined. With a tapeworm egg density demonstrated to be between that of strongylids and Parascaris spp.,
the poor recovery of tapeworm eggs in equine fecal samples must have other explanations.

1. Introduction
Due to ever-increasing levels of anthelmintic resistance in livestock
parasites (Kaplan, 2004; Kaplan and Vidyashankar, 2012), re-
commendations for sustainable parasite control involve systematic
parasite surveillance. Determination of parasite fecal egg counts is used
to identify treatment needs (Kenyon et al., 2009), to evaluate anthel-
mintic treatment efficacy (Coles et al., 1992); specifically, in modern
equine parasite control, fecal egg counts are recommended to identify
consistently high strongylid egg shedders and differentiate between
strongylid and ascarid positive adolescents (Nielsen et al., 2013).
The use of a flotation solution to identify and count parasite eggs
was first described over 100 years ago (Bass, 1909), and the principle
has been used widely since. Despite a multitude of techniques available
today, all protocols rely on the difference in densities between parasite
eggs and that of the surrounding feces. Most protocols are composed of
steps involving some variation of homogenizing the fecal sample,
adding a flotation solution, and allowing the eggs (sometimes aided by
centrifugation) to float to the surface of the solution where they can be
identified and counted. The amount of feces, flotation solution, and use
of counting slide vary immensely between techniques. Typical flotation
media used include zinc sulphate (specific gravity (SG) = 1.18–1.20),
sodium nitrate (SG = 1.18–1.20), saturated salt (1.18–1.20), Sheather’s
sugar solution (SG = 1.27) (Dryden et al., 2005), and saturated glucose-
salt (SG = 1.25–1.27) (Roepstorff and Nansen, 1998). The difference in
specific gravity among helminth eggs and that of feces must be

⁎
Corresponding author.
E-mail address: jamie.norris@uky.edu (J.K. Norris).

https://doi.org/10.1016/j.vetpar.2018.08.004
Received 12 July 2018; Received in revised form 6 August 2018; Accepted 8 August 2018
0304-4017/ © 2018 Elsevier B.V. All rights reserved.
J.K. Norris et al.
substantial enough to separate the eggs from other particles present in
the feces and allow easy visualization under the microscope.
A few studies have estimated the SG of various endoparasite eggs
(David and Lindquist, 1982; Harnnoi et al., 1998). Many egg types, such
as those of the ascarid parasites Toxascaris leonina, Toxocara canis, and
T. cati as well as the hookworm eggs of Ancylostoma caninum all have SG
at or below 1.10 (David and Lindquist, 1982), which means they should
float well in flotation media above this threshold. Eggs of the porcine
ascarid, Ascaris suum, and the whipworm species, Trichuris vulpis and T.
suis, were somewhat denser in the 1.13–1.14 range, and the highest
specific gravities were measured for eggs of Physaloptera spp. and
Taenia spp., which both were above 1.20 (David and Lindquist, 1982).
Thus, flotation media with higher SG are required to effectively float
these egg types. In one study, for example, significantly higher counts of
T. vulpis and T. canis eggs were obtained when the SG of the flotation
solution was elevated above 1.20 (Dryden et al., 2005).
Very little is known about relative densities of commonly occurring
equine parasite eggs. One study estimated SG for Parascaris equorum to
be 1.0969 (David and Lindquist, 1982) but no published studies have
attempted to determine such estimates for equine strongylids or the
commonly occurring tapeworm Anoplocephala perfoliata. The latter is of
particular interest as Anoplocephala eggs are notoriously difficult to
recover in flotation-based egg counting techniques, and standard
McMaster methods perform with diagnostic sensitivities below 10% for
this parasite (Nielsen, 2016). This is often assumed to be because of an
uneven distribution of tapeworm eggs in the fecal matter, and mod-
ifications with larger amounts of feces processed do perform sub-
stantially better (Nielsen, 2016). However, it is unknown if egg flota-
tion capability may also play a role.
The aim of this study was to determine SG for eggs of equine
strongylids, Parascaris spp., and Anoplocephala perfoliata.
2. Materials and methods
This study was carried out with a first trial (13 replicates) completed
during January-March and a second trial (four replicates) done during
April-August 2017.
2.1. Egg isolation
Eggs were either retrieved from fecal samples collected from natu-
rally infected horses, or from dissection of adult gravid worms, as de-
tailed in the following section.
2.1.1. Strongyle type and ascarid eggs
The source of strongyle and ascarid egg types was fecal samples
collected from naturally infected horses. Feces was collected from an
equine parasitology research herd, which has been kept without an-
thelmintic intervention since 1979 (Lyons et al., 1990). This herd has
been shown to harbor mixed species equine strongylid infection (Lyons
et al., 1990) as well as Parascaris univalens (Nielsen et al., 2014). Since
karyotyping was not carried out for the present study, an ascarid spe-
cies determination was not carried out, and, hence, this manuscript
refers to the genus, Parascaris spp., throughout. Parasite fecal egg
counts were performed using the Mini-FLOTAC method (Cringoli et al.,
2017) to identify samples from high strongylid shedding horses (> 500
eggs per gram of feces (EPG)). Similarly, foals with ascarid egg
counts > 80 EPG were used as ascarid egg donors for this project.
In the first trial, 15 g of feces were homogenized with 45 mL of
water, filtered through cheese cloth, and 2 mL of the filtrate was mixed
with 10 mL of a glucose-salt flotation solution (SG = 1.25) into a 15 mL
conical centrifugation tube. For the second trial, larger amounts of
equine feces (∼60 g) were used along with 50 mL centrifugation tubes
to facilitate egg retrieval. All other steps remained the same. This sus-
pension was centrifuged at 1000 g and 4 °C for 10 min. The top 1 mL
was collected, placed in a 15 mL tube, and 12 mL of distilled water were
46
Veterinary Parasitology 260 (2018) 45–48
added. The tube was then centrifuged for 10 min at 1000 g, and su-
pernatant was pipetted off, leaving the pellet at the bottom. The pellet
was then resuspended in 12 mL of glucose-salt solution via vortex
homogenizer and centrifuged at 1000 g for 10 min. The entire volume
of flotation medium containing the eggs, was used in the subsequent
filtering steps described below.
Both strongylid and ascarid eggs were concentrated via selective
filtration through a series of pluriStrainer® cell strainers (pluriSelect Life
Science, Saxony, Leipzig, Germany) arranged, in decreasing pore size,
from 400, 200, 100, to 27 μm. The fecal debris suspension mentioned
above was poured onto the topmost 400 μm filter, drawn through with
suction, rinsed with distilled water, and collected from the 100 μm and
27 μm filters. The eggs were collected from these filters by rinsing with
a p1000 micropipette and placed in a 15 mL plastic conical tube. These
tubes were stored at 4 °C for at least 24 h before use and were kept for
no more than three months. Counting of a homogenized 0.5 mL of egg
suspension to ensure an adequate number of identifiably mature and
representative eggs was undertaken using a McMaster egg counting
chamber. Trial 1 utilized a concentrated egg suspension containing 742
eggs per mL of strongyle type eggs and 286 eggs per mL of ascarid eggs.
The second trial used suspensions of 16,052 strongyle type and 2412
ascarid eggs per mL.
2.1.2. Anoplocephala perfoliata eggs
In the case of Anoplocephala perfoliata, adult worm specimens were
collected at equine necropsy performed with horses from the above-
mentioned university research herd. Worm specimens were obtained
from locations near the ileocecal junction and preserved in phosphate
buffered saline solution until dissection. Worm specimens were mor-
phologically identified to be A. perfoliata (Skrjabin and Spasskii, 1951)
and gravid proglottids were removed and bifurcated to release eggs.
These eggs were collected via P20 single channel pipette and added to a
15 mL conical tube with distilled water. Counted similarly to the
aforementioned strongyle type and ascarid eggs, suspensions containing
1054 and 2340 anoplocephalid eggs per mL were used in the first and
second trials, respectively.
2.2. Flotation solution specific gravity determination
As specific gravity is defined as the density of a sample divided by
the density of the same amount of water, 1 mL of distilled water was
pipetted using a calibrated p1000 pipette and weighed in a plastic
weigh boat to within 0.1 mg accuracy utilizing a scale (Mettler Toledo®
MS3001S Precision Balance, Columbia, Maryland, USA) which was set
to zero. The weight of five 1 mL replicates of flotation solution were
then compared to 1 mL of water and specific gravity was determined.
In the second trial, an optical spectrophotometer was included for a
second measure of specific gravity. Spectrophotometer readings were
converted from the Brix scale to specific gravity using the following
⎛ ⎞
Brix
equation: = ⎜ ⎟ + 1.
⎛258.6 − ( Brix ) x 227.1⎞
⎝ ⎝ 258.2 ⎠ ⎠
2.3. Determination of specific gravity of eggs
A range of specific gravities were obtained via a series of dilutions of
a stock saturated glucose-salt flotation solution (SG = 1.25, measured
at 4 °C) with distilled water. 2 mL of each were stacked one atop the
other in 15 mL conical centrifuge tubes (Fig. 1). For the first trial the
following specific gravity layers were included; 1.00, 1.06, 1.08, 1.10,
1.12, and 1.14. For the second trial, the same general format was used,
but the specific gravity layers were changed slightly; 1.00, 1.02, 1.04,
1.06, 1.08, and 1.10.
Minute amounts of food grade dye (Market Pantry™, Target
Corporation, Minneapolis, Minnesota, USA) were added to each specific
gravity solution to better visualize separation of layers. The solutions
were chilled to 4 °C for 48 h before use and pipetted slowly, heaviest to
J.K. Norris et al. Veterinary Parasitology 260 (2018) 45–48

Table 1
Mean specific gravity estimates with 95% confidence intervals (CI) for the three
equine egg types in the two trials of the study as well as overall.
Strongylid 95% CI Ascarid 95% CI Anoplocephalid 95% CI

Trial 1 1.0454 1.0438- 1.0891 1.0882- 1.0614 1.0606-

1.0470 1.0900 1.0622
Trial 2 1.0453 1.0448- 1.0914 1.0906- 1.0694 1.0685-
1.0458 1.0921 1.0704
Overall 1.0453 1.0448- 1.0903 1.0897- 1.0636 1.0629-
1.0458 1.0909 1.0642

Table 2
Total egg counts from each of the specific gravity (SG) layers in the two trials of
the study.
Trial 1

SG Strongylidaif Ascaridabch Anoplocephalidcf

1 460 1 206
1.06 873 210 1895
1.08 118 709 567
1.10 48 546 89
1.12 13 150 9
1.14 5 24 9
1.16 0 11 0
Fig. 1. Illustration of average resting place of the three egg types in relation to
specific gravity of glucose-salt flotation solution in trial 1. Strongyle type (a) Trial 2
came to rest in between 1.00 and 1.06 specific gravity (SG) solutions.
Anoplocephalid eggs (b) came to rest in the middle of the flotation solution SG Strongylidbdg Ascaridide Anoplocephalidheg
which measured 1.06 SG. Ascarid eggs (c) came to rest further down, between
the layers corresponding to 1.08 and 1.10 SG. While banding of eggs in solution 1 405 0 0
after centrifugation could be observed in some samples, it was not recorded for 1.02 818 1 12
1.04 4350 82 54
all samples and is included solely for summary.
1.06 1196 165 516
1.08 531 210 342
lightest to accomplish the required layering. Finally, 0.5 mL of an 1.10 474 1369 112
aqueous suspension of concentrated endoparasite eggs was carefully a, b, c, d, e, f, g - Superscripts indicate an overall statistically significant dif-
placed atop these gradient columns. The tubes were subsequently ference of (p ≤ 0.0001).
centrifuged at 800 g for 20 min (David and Lindquist, 1982). Each 2 mL h, i - Superscripts indicate an overall statistically significant difference of
layer was carefully removed 0.5 mL at a time with a p1000 micropipette (p < 0.05).
and transferred to a McMaster slide for counting at 100X and 400X
magnification. The counting chambers were carefully examined from between the layers corresponding to 1.00 and 1.06 specific gravity
top to bottom to ensure that all eggs present in the given layer were solution. In Trial 2, 71.69% of strongylid type eggs, 4.54% of Parascaris
counted. spp., and 6.37% of anoplocephalid eggs were found in these layers.

2.4. Statistical analysis 4. Discussion

The data were analyzed statistically using SAS, version 9.3 software
(Statistical Analysis Systems, Cary, North Carolina, USA). A mixed
linear model was constructed using the Mixed Procedure in SAS with
the specific gravity of the parasite eggs as response variable and egg
type (strongylid, ascarid, anoplocephalid) and trial (1 or 2) as covari-
ates. Replicate (1–17) was kept as a random effect. In case any of the
covariates were statistically significant, a pairwise comparison of Least
Squares Means w/ Tukey-Kramer adjustment was performed. All results
were interpreted at the 0.05 significance level.
3. Results
Mean densities of the three egg types considered are presented in
Table 1. Mean densities of all three eggs types were found to be sta-
tistically significantly different from one another (p < 0.0001). No
differences were observed between the two trials (p < 0.0001). Total
eggs counted, broken down by specific gravity in which they came to
rest after centrifugation and subsequent observation, are presented in
Table 2.
In trial 1, a proportion of eggs (30.32% of strongylid type eggs,
0.06% of Parascaris spp., 7.42% Anoplocephala perfoliata) were found
We are the first to determine the specific gravity of eggs of equine
strongylids and Anoplocephala perfoliata, and our results for Parascaris
spp. are in good agreement (99.9% similarity) with the only other
published measure (David and Lindquist, 1982). The results can be
useful in determining the optimum specific gravity of flotation media
used for determining equine fecal egg counts.
Parascaris spp. eggs were statistically denser than the other two egg
types (Table 2). This was expected given the typical appearance of the
egg with a thick proteinaceous capsule. A smaller proportion of equine
ascarid eggs appear partially or completely decorticated in routine fecal
samples (Donoghue et al., 2015), and could thus be expected to be
lighter. However, the appearance of decorticated ascarid eggs was not
accounted for in this study but could be interesting for future in-
vestigations. The results reported herein are in general agreement with
those reported for ascarid type eggs of T. canis and T. cati, while T.
leonina eggs are substantially lighter, and those of A. suum are markedly
denser (David and Lindquist, 1982). It should be noted that two Para-
scaris species have been described, P. equorum and P. univalens. The two
species are morphologically identical and can only be told apart
through the process of karyotyping and counting the chromosomes
(Nielsen et al., 2014). Karyotyping of eggs was not carried out in this

47
J.K. Norris et al.
study, but the isolate has previously been identified to be P. univalens,
which appears to be the predominant equine ascarid species world-wide
(Nielsen et al., 2014). Thus, the ascarid eggs used in the present study
are unlikely to have been of mixed species origin.
The strongylid eggs evaluated here were of mixed species origin as
the donor herd has been maintained without anthelmintic intervention
since 1979 and, thus, harbors a relatively large strongylid species di-
versity, including the large strongyles Strongylus vulgaris and S. eden-
tatus (Lyons et al., 1990). This species diversity likely explains the wider
95% confidence interval for the strongylid eggs (Table 1). However, the
mean specific gravity determined is in good agreement with that of the
canine hookworm, A. caninum, which is also a strongylid type egg
(David and Lindquist, 1982). Thus, strongylid type eggs are likely to all
have similar specific gravities.
The results for the Anoplocephalid eggs are of particular interest
given the general difficulty in recovering and counting this egg type in
equine fecal samples (Nielsen, 2016). Two evaluations of simple
McMaster techniques found the diagnostic sensitivity to be below 10%
for anoplocephalid eggs (Meana et al., 1998; Tomczuk et al., 2014).
This is often assumed to be due to the eggs being released within pro-
glottids that are subsequently degraded within the intestinal tract to
release the eggs in an aggregated manner, and this is supported by the
observation that increasing the amount of feces processed can sub-
stantially improve the diagnostic sensitivity (Nielsen, 2016). However,
other cestode eggs (i.e., Taenia spp.) have been reported with a mean
specific gravity above 1.20 (David and Lindquist, 1982), which poses
diagnostic challenges with flotation-based techniques, so it cannot be
ruled out that flotation properties could play a role for A. perfoliata as
well. The results reported herein illustrate that eggs of A. perfoliata have
a specific gravity between that of strongylid and Parascaris spp. eggs
(Table 1). Thus, the challenges with coprologically diagnosing A. per-
foliata infections are unlikely to be explained by specific gravity.
It should be noted that the experiment reported herein was carried
out with one particular flotation medium, the glucose-salt solution, and
it is possible that results could differ if other flotation media had been
used. However, David and Lindquist (1982) used sucrose and as out-
lined above, their results are in good general agreement with ours.
Nonetheless, it could be of value to replicate this study with other
flotation media in the future. Another aspect worth investigating is the
degree of separation between the fecal debris and the objects of in-
terest, the eggs. As none of the three egg types evaluated herein had
mean specific gravities above 1.10, it is possible that the range of
1.20–1.30 for traditionally chosen flotation media for determining
equine fecal egg counts, might actually be suboptimal by allowing too
much fecal debris to float. This could potentially compromise the di-
agnostic performance, and would be a worthy topic of future in-
vestigation. Specific gravity estimates for other endoparasitic ova, such
as those of Eimeria leuckarti and Strongyloides westeri, would be useful to
determine as well.
In summary, we found eggs of Parascaris spp. to be significantly
denser than anoplocephalid eggs, which again were significantly denser
than the strongylid type eggs. All three egg types had mean specific
48
Veterinary Parasitology 260 (2018) 45–48
gravities below 1.10, meaning that they are unlikely to pose the diag-
nostic challenges known for denser egg types such as Trichuris spp.,
Ascaris spp., Physaloptera spp., and Taenia spp. Further investigation
into this may reveal an optimal flotation solution for equine fecal flo-
tation which would reduce non-egg fecal debris and preserve the ap-
pearance and structural integrity of eggs by not exposing them to un-
necessarily high osmotic forces.
Conflict of interest statement
The authors declare no conflict of interest.
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