Flotation techniques (FLOTAC and mini-FLOTAC) for detecting gastrointestinal parasites in howler monkeys
Alvarado-Villalobos et al. Parasites & Vectors
Flotation techniques (FLOTAC and mini-FLOTAC) for detecting gastrointestinal parasites in howler monkeys
Mayra Alejandra Alvarado-Villalobos 0 3
Giuseppe Cringoli 2
Maria Paola Maurelli 2
Aurelie Cambou 1
Laura Rinaldi 2
Colin A. Chapman
Juan Carlos Serio-Silva 0 3
0 Red de Biología y Conservación de Vertebrados, Instituto de Ecología A.C , Xalapa, 91070 Veracruz , Mexico
1 ENSAIA (Ecole National e Supérieure d'Ágronomie et des Industries Alimentaires) , Vandoeuvre-lés-Nancy , France
2 Unit of Parasitology and Parasitic Diseases, Department of Veterinary Medicine and Animal Productions, University of Naples Federico II , Naples , Italy
3 Red de Biología y Conservación de Vertebrados, Instituto de Ecología A.C , Xalapa, 91070 Veracruz , Mexico
Background: Analyses of environmental correlates of the composition of gastrointestinal parasite communities in black howler monkeys (Alouatta pigra) have been hindered by inadequate calibration techniques of detection and quantification methods of the parasites. Here we calibrate samples and compare the likelihood of parasite detection using two flotation techniques, FLOTAC and Mini-FLOTAC, and compare flotation solution, preservation method and dilution ratio for egg detection and counts of the most common parasites (Controrchis spp. and Trypanoxyuris spp.) in howler monkeys. Results: For samples preserved in 5% formalin, the Mini-FLOTAC technique was the best option for qualitative and quantitative copro-microscopic analysis. This technique displays an 83.3% and 100% detection of Controrchis spp. and Trypanoxyuris spp. infections, respectively. For the trematode Controrchis spp., more eggs per gram of feces (EPG) were recorded with the flotation solution (FS) #7 (zinc sulfate; specific gravity SG = 1.35) at 1:20 and 1:25 dilution than other methods. By contrast, for the nematode Trypanoxyuris spp., the best results were recorded with FS1 (sucrose and formaldehyde; SG = 1.20) at 1:10 dilution. Conclusions: We recommend the Mini-FLOTAC technique for general use with parasite analysis on frugivore/ folivores like the howler monkey, especially if many samples are analyzed. The technique has a high detection rate and the best EPG counts, allowing the qualitative and quantitative analysis of parasite load among the species or populations without the need for specialized equipment.
Quantitative flotation; Gastrointestinal parasites; Nematodes; Trematodes; Howler monkeys; Alouatta
The accurate detection of the prevalence and intensity
of gastrointestinal parasite infections is key to
understanding the effect of parasites on the biology, behavior,
and the conservation of hosts. Gastrointestinal parasites
are most often surveyed in the feces [
] of wild host
populations using light microscopy [
], which is
particularly effective where the feces of host populations
can be identified and collected in the wild, as this
eliminates the need to capture or handle host individuals.
An increasing number of studies have focused on the
gastrointestinal parasites of howler monkeys (Alouatta
spp.), in which polyparasitism is common, including
helminths, protozoans and acanthocephalans [
these parasites, trematodes (Controrchis spp.) and
nematodes (Trypanoxyuris spp.) are the most common
gastrointestinal parasites of howler monkeys [
Controrchis spp. are common in most studies [
may influence the ecology of the host [
]. By contrast,
Trypanoxyuris spp. appear to be indicators of ecosystem
health with low prevalence in human dominated
landscapes and high prevalence in more natural landscapes
]. Most studies report low levels of Controrchis spp.
and Trypanoxyuris spp. parasitism in the black howler
monkey (Alouatta pigra) [
], possibly due to
unsuitable sampling or analytical methods.
Consequently, standardization of the copro-microscopic
techniques is essential for the diagnosis of gastrointestinal
In wild primates, including howler monkeys, parasitic
infections are typically detected by identifying eggs,
larvae, oocysts, or cysts in the feces of the host by
flotation procedures [
] or sedimentation
8, 10, 12
]. Sodium nitrate (NaNO3) is a common
flotation solution (FS) for fecal samples from wild
]. However, studies of black howler
monkeys (Alouatta pigra) have also used sodium chloride
], zinc sulfate (ZnSO4) [
sucrose (C12H22O11) for samples preserved in 10% formalin
8, 12, 14, 15
]. In folivore-frugivore primates, such as
Alouatta pigra [
], identification of parasites in fecal
samples is often complicated by the high fiber content of
their diet [
], as well as the common presence of pollen,
plant tissue, flowers, and invertebrate fragments
(accidentally ingested with the plants) [
19, 21, 22
], all of which can
be misclassified as parasitic structures.
We suggest that the development and calibration of
parasite copro-microscopic techniques and the standardization
of existing methods have not received sufficient attention,
particularly as calibration is the foundation of a good
diagnosis. The accuracy of fecal egg count (FEC) techniques
depends on the analytic sensitivity of the technique, the
choice of the flotation solution, and dilution as well as fecal
preservation method [
1, 4, 23
The aim of this study is to evaluate the efficiency of
detection and counts of the eggs of the most common
parasites (Controrchis spp. and Trypanoxyuris spp.) of
howler monkeys, using two novel techniques, FLOTAC
and Mini-FLOTAC, six flotation solutions, three
different dilutions, and two preservation methods. FLOTAC
and Mini-FLOTAC are innovative multivalent
quantitative diagnostic techniques sufficiently accurate to
estimate the number of parasites in fecal samples [
of 1 g (volume = 10 ml; analytical sensitivity = 1 egg,
larvae, oocyst, cyst per gram of feces, EPG/LPG/OPG/
CPG) with FLOTAC [
] and up to 0.2 g (volume =
2 ml; analytical sensitivity = 5 EPG/LPG/OPG/CPG) with
An additional file shows the “Guide to the
recommended quali-quantitative flotation method” which
describes the procedure for performing the
recommended Mini-FLOTAC basic and dual technique
(Additional file 1). A composite from 13 individual fecal
samples of wild A. pigra individuals (360 g) was
performed and divided into sub-samples depending on
calibration schemes (Additional file 2: Table S1). Individuals
were naturally infected with the trematode Controrchis
spp. (Fig. 1a) and nematode Trypanoxyuris spp. (Fig. 1b).
Two fecal preservation methods were used: (i) anaerobic
storage by vacuum packing samples (VPF) in the fridge
at 4 °C; and (ii) a 5% formalin solution.The VPF samples
were analyzed 10 days after collection.
As is typical for howler monkeys, the fecal samples
had a high fiber content that made the identification of
parasite difficult. Therefore, three different dilution
ratios: 1:10, 1:20 and 1:25 (g of feces/ml of water or water
plus fixative depending on the calibration scheme) were
used to calibrate the samples. The following six FSs were
]: FS1 (sucrose and formaldehyde, SG = 1.2); FS2
(sodium chloride, SG = 1.2); FS3 (zinc sulphate, SG =
1.2); FS4 (sodium nitrate, SG = 1.2); FS6 (magnesium
sulphate, SG = 1.28); and FS7 (zinc sulphate, SG = 1.35).
The FS5 established in the protocol of Cringoli et al. [
was not implemented because the reagent is corrosive
and expensive for routine use. All FSs were prepared at
room temperature, and their SG was checked with a
hydrometer. For each FS, 6 replicates were performed.
The single composite fecal sample was completely
homogenized, divided into two sub-samples of 180 g
each for analysis by the FLOTAC and Mini-FLOTAC
methods. The sub-samples were further subdivided into
two samples of 90 g each, which were either preserved
in VPF or 5% formalin at dilution ratio 1:4 (one part of
feces and three of fixative). After storage, each 90 g
sample was equally divided into three 30 g samples that were
diluted at the following ratios 1:10, 1:20, 1:25 (g of feces/
ml of water or water plus 5% formalin). Each dilution
was sieved (pore size = 250 μm), the waste was discarded
and the remaining sample was thoroughly homogenized.
For the FLOTAC method, 108 aliquots (in tubes)
preserved by VPF and 108 aliquots preserved in 5%
formalin (36 aliquots per dilution) were used. Each aliquot
contained 6 ml of fecal suspension. A total of 216
aliquots were centrifuged for three minutes at 1500× rpm
(170 RCF), and the supernatant discarded, leaving only a
pellet in the tube. Each pellet was randomly assigned to
one of the six FSs and re-suspended in 6 ml of each
solution in a tube. With a pipette, 5 ml (0.5 g) of the
resulting fecal suspension were transferred to each
centrifuge chamber (n = 216 chambers) for analysis by the
FLOTAC method, and the samples were centrifuged at
1000× rpm (120 RCF) for five minutes. Flotation in a
centrifuge causes the debris to sink to the bottom of the
chambers and the parasite elements to float to the top
under the two ruled grids [
]. After being
centrifuged, each 5 ml sample suspension in a chamber was
examined with a light microscope at 100× and 400×
For the Mini-FLOTAC method 216 tubes were
analyzed (180 g). Samples preserved by VPF (90 g) did not
need to be centrifuged and 5 g of feces sample were
weighed for every six replicates and homogenized them
in their respective FS and dilution. For samples
preserved in 5% formalin, 108 tubes (90 g) were centrifuged
at 1500× rpm for three minutes. The supernatant was
discarded, and each tube was filled with 6 ml of the
respective FS and dilution. The Mini-FLOTAC chambers
were filled with 1 ml (0.1 g) of the homogenized
suspension. Finally, the 216 replicates (n = 216 chambers) were
examined for parasites using a light microscope 100×
and 400× magnifications.
A total of 432 tubes (2 flotation methods × 2
preservation methods × 3 dilutions × 6 FSs × 6 replicates per
solution) were examined.
Infections are often described as the number of parasitic
elements per gram of feces: eggs (EPG), larvae (LPG),
oocysts (OPG), or cysts (CPG) per gram of feces. To
obtain these values, the analytic sensitivity of the technique
must be known. The analytic sensitivity is the ability to
detect the smallest number of parasitic elements
assessed by a technique, small values mean that the
technique has a high analytic sensitivity and is capable
of detecting parasitic infections even though the
excreted parasites eggs are low. This value is used as a
multiplication factor used to express the results in gram
of feces. Both techniques used in this paper present a
high analytical sensitivity compared to the most used
parasitological techniques [
For our work using FLOTAC, the multiplication factor
is 2 when the dilution ratio is 1:10, 4 using a 1:20
dilution ratio and 5 for 1:25 dilution ratio to obtain our
results in EPG. For the Mini-FLOTAC technique the
multiplication factors are 10 using 1:10, 20 with 1:20
dilution, and 25 when a 1:25 dilution ratio was used.
The efficiency of the methods was estimated from six
replicates (n = 6 chambers) as the number of EPG in the
flotation solution. The mean EPG, standard deviation
(SD) and the coefficient of variation expressed as a
percentage [CV (%) = (standard deviation/mean of EPG) ×
100] were estimated for each combination of flotation
method, fecal preservation method, dilution and FS
(Additional file 3: Table S2, Additional file 4: Table S3).
A generalized linear logistic model (binomial error
distribution) was used to compare the likelihood of
parasite detection (prevalence) among the combinations of
flotation methods, fecal preservation methods, dilutions
and FSs. Differences in the number of EPG as intensity
value (or the capacity of the methods to float parasite
elements) were analyzed with generalized linear models
using a Poisson distribution and adjusting interactions at
fourth level [glm (EPG ~ (method + fecal preservation
method + dilution + FS) ^ 4, Poisson)]. Models were
checked for homoscedasticity and normality of the
residuals. The presence/absence of the parasite and the
number of EPG counted (quantitative technique) were the
respective response variables. The flotation methods,
fecal preservation methods, dilutions, and FSs were
explanatory variables. Initially fitted the saturated model
and then followed a model simplification procedure to
eliminate the explanatory variables that did not improve
the model fit to the data. To find the best model we used
the Akaike’s information criterion (AIC) [
]. Also, when
necessary, levels were conflated within a given factor to
construct the simplest model. GLMs with the R package
] were performed within the statistical program
R version 3.2.0 (R Development Core Team, 2015) [
Eggs of Controrchis spp. floated only using FS2, FS3, FS6
and FS7. The values of FS2, FS3 and FS6 were grouped
into a single category, due to the low numbers of eggs
detected in these solutions. Only in the FS7, eggs floated
with both techniques at all dilution ratios. The likelihood
of detection based on presence/absence vary significantly
in relation to apparatus (χ2 = 5.5, df = 1, P = 0.019),
preservation methods (χ2 = 7.4, df = 1, P = 0.007) and
FSs (χ2 = 4.3, df = 1, P = 0.037). The samples preserved
in 5% formalin and analyzed with FLOTAC with FS7
showed the best results with a probability of detection
of 83.3% (n = 15/18) for Controrchis spp. infections.
The evaluation based on counts of EPG in flotation
differ in FSs (χ2 = 445.4, df = 1, P < 0.001), preservation
methods (χ2 = 167.2, df = 1, P < 0.001) and dilution ratios
(χ2 = 85.2, df = 2, P < 0.001). All significant GLM results
are shown in Additional file 5: Table S4. FS7 exhibited
significantly larger counts than those obtained from the
groups FS1-FS6 (Fig. 2). The highest values of EPG were
FLOTAC / FS 1 6
Preservation method Preservation method
Fig. 2 Controrchis spp. total egg counts. Comparison between calibration variables: methods, FSs, preservation methods and dilutions. a Total number
of EPG using the FLOTAC technique with groups FS1-FS6. b Total number of EPG using the Mini-FLOTAC technique with groups FS1-FS6. c Total
number of EPG using the FLOTAC technique with FS7. d Total number of EPG using the Mini-FLOTAC technique with FS7. Letters above the bars
indicate the homogeneous groups based on the contrasts done. Differences for bars with the same letter were not statistically significant while those
with different letters were statistically different. In all the bars the standard error is represented as a measure of dispersion around the mean
Mini FLOTAC / FS 1 6
Mini FLOTAC / FS7
obtained by 1:20 and 1:25 dilution, and 5% formalin also
proved their efficiency in counts of EPG in flotation.
The values of EPG in flotation was better using 5%
formalin compared with VPF (n = 454 EPG, mean = 2.1 and
n = 140 EPG, mean = 0.65, respectively).
Although there were no differences in relation to
apparatus (χ2 = 1.1, df = 1, P = 0.296), Mini-FLOTAC was
marginally better than FLOTAC based on counts of EPG
in flotation (Fig. 2). The most appropriate combination
of elements was (i) fecal samples preserved in 5%
formalin and analyzed with FS7 at 1:20; and (ii) fecal samples
preserved in 5% formalin and analyzed with FS7 at 1:25
dilution ratio. Using Mini-FLOTAC with the both
previous combination an 83.3% of detection value is obtained
for Controrchis spp. infections.
The eggs of Trypanoxyuris spp. floated with all the six
FSs (Fig. 3) and there were differences in apparatus in
detectability (χ2 = 17.6, df = 1, P < 0.001). The FLOTAC
method was, however, better than the Mini-FLOTAC
method (50.4 and 13.8%, respectively) (Table 1).
Detection based on counts of EPG in flotation
varied significantly among FSs (χ2 = 286.8, df = 5,
P < 0.001), preservation methods (χ2 = 123.8, df = 1,
P < 0.001), and dilution (χ2 = 33.7, df = 2, P < 0.001).
FS1 performed best with a significantly larger EPG count
than FS2 (z = 3.1, P = 0.002), FS3 (z = 3.1, P = 0.002)
and FS4 (z = 2.0, P = 0.044). Fecal samples preserved
in 5% formalin detected more Trypanoxyuris spp.
infections with a better EPG count in flotation than
VPF. All significant GLM results are shown in
(Additional file 6: Table S5).
Although there were no differences related to
apparatus (χ2 = 2.0, df = 1, P = 0.159) based on counts of EPG
in flotation, the combination Mini-FLOTAC method
using FS1 at 1:10 dilution (80 EPG, mean = 13.3 EPG,
SD = 5.2 EPG, CV = 38.7%) with samples preserved in
5% formalin performed the best EPG counts. This
combination detected 100% of Trypanoxyuris spp. infections
FLOTAC / Formalin 5%
FLOTAC / VPF
b b b
Mini FLOTAC/ Formalin 5%
a a a
Mini FLOTAC/ VPF
b b b
a a a
a a a
a a a a a
Fig. 3 Trypanoxyuris spp. total egg counts. Comparison between calibration variables: methods, FSs, preservation methods and dilutions. a Total
number of EPG using the FLOTAC technique with the six FS and 5% formalin as the preservation method. b Total number of EPG using the
FLOTAC technique with the six FS and VPF as the preservation method. c Total number of EPG using the Mini-FLOTAC technique with the six FS
and 5% formalin as the preservation method. d Total number of EPG using the Mini-FLOTAC technique with the six FS and VPF as the
preservation method. Letters above the bars indicate the homogeneous groups based on the contrasts done. Differences for bars with the same letter
were not statistically significant while those with different letters were statistically different. In all the bars the standard error is represented as a
measure of dispersion around the mean
with less variation among samples compared to the same
elements at 1:20 dilution (100 EPG, mean = 16.6 EPG,
SD = 19.6 EPG, CV = 118.0%).
Although significant advances have been made in the
parasitology and epidemiology of Alouatta pigra in the
], evaluation of the best methods to use
were not available until now. We demonstrated that the
likelihood of detecting a parasite and the egg count are
dependent on the choice of preservation method,
flotation technique, dilution ratio, and FS. In our study,
many of the combinations of these elements were not
adequate to detect the parasites and in a few specific
combinations, the EPG counts were as much as 100
times higher, especially for Controrchis spp. These
findings emphasize the need for standardization and
calibration of the copro-microscopic techniques for an
accurate detection and counting of parasites.
We do not know the real value of EPG of Controrchis
spp. and Trypanoxyuris spp. infections although our
study still provides useful guidelines. Infection levels
reported in other studies of howler monkeys are
generally low, the mean EPG of Controrchis spp. values
reported using the sugar-flotation technique (SG = 1.2) by
Kowalzik et al. [
] was 2.3 ± 1.9 (mean ± SD) and by
Behie et al. [
] was 3.2 ± 1.4 eggs per gram of feces.
Similarly, Behie et al. [
] reported an EPG value of 2.0 ±
1.4 for Trypanoxyuris spp. using sucrose solution (SG =
1.26). The general infection levels found in the present
study using the best combination of elements for each
parasite were 16.6 ± 8.1 EPG (with Mini-FLOTAC, FS7 at
1:20 dilution), and 25 ± 15.8 EPG (with Mini-FLOTAC,
FS7 at 1:25 dilution) for Controrchis spp. and 13.3 ± 5.2
EPG for Trypanoxyuris spp. (with Mini-FLOTAC, FS1 at
1:10 dilution). For all combinations, fecal samples
preserved in 5% formalin show significantly better results.
Here FLOTAC obtained the best results for detecting
the presence of Controrchis spp. and Trypanoxyuris spp.
infections. FLOTAC is a cylindrical device with two 5 ml
flotation chambers, which allows up to 1 g of stool to be
prepared for microscopic analysis [
]. However, species
of the genus Alouatta are folivore-frugivore primates
], and their feces have a high fiber content [
with pollen, plant tissue, flowers, and other elements
being abundant [
19, 21, 22
] and all this debris
accumulates in the large chambers (5 ml) of FLOTAC hindering
the quantitative analysis. As a result, we suggest the
quantitative analysis of EPG using a Mini-FLOTAC as a
better option. Mini-FLOTAC is considered in other
studies, as the most sensitive method for detecting
helminth infections compared with the formol-ether
concentration and direct fecal smear methods for the
diagnosis in humans [
24, 29, 30
]. It is also more sensitive
than the McMaster (FEC technique) for the diagnosis of
Eimeria in goats [
]. Barda et al. [
] reported a
detection of 90% in helminth infections and 68% of protozoan
infections. Also in the veterinary field Maurelli et al.
], report a 100% of detection of the three most
common intestinal nematodes in dogs (Toxocara canis,
Trichuris vulpis and hookworm) with this method. In
our study, with the appropriate combination of
elements, Mini-FLOTAC performed good detection based
on presence and EPG counts of both Controrchis spp.
and Trypanoxyuris spp. avoiding the large amount of
debris in the 5 ml of FLOTAC chambers.
Mini-FLOTAC is a simple technique (FEC technique)
with two 1 ml flotation chambers, which are designed
for the optimal examination of faecal sample
suspensions (total volume = 2 ml) [
]. In our study, the
probability of detection is higher (83.3% for detection of
Controrchis spp. and 100% of Trypanoxyuris spp.
infections) than in previous studies [
The use of 5% formalin is recommended as this was
associated with significantly higher egg detection and
EPG counts for Controrchis spp. and Trypanoxyuris spp.
infections compared with VPF. These findings are
encouraging because in field conditions, high temperatures
and humidity do not allow the preservation of fresh
samples for later analysis and is effective to avoid
degradation and loss of parasitic forms [
We suggest the use of zinc sulfate (SG = 1.35) for the
detection of trematode eggs is the best option. For the
detection of trematodes such as Controrchis spp., some
authors use sedimentation techniques [
our results and those of previous studies [
that the flotation of this group of parasites can be
achieved with FSs that have a high specific gravity. Also,
Barda et al. [
] found that the FS7 is the most sensitive
solution to detect Ascaris lumbricoides infections using
the Mini-FLOTAC method, which is of interest because
species of this parasite genus has also been found in
howler monkeys [
For nematodes such as Trypanoxyuris spp., FS1
(sucrose and formaldehyde; SG = 1.20) is recommended.
These FSs have been used before on howler monkey
fecal samples with different specific gravity [
]. It is
relevant to mention that, in this study, a popular
solution used for copro-microscopic analysis of Alouatta
pigra, FS2 [
], provided a low detection of the
parasites; Trypanoxyuris spp. were detected in 18.1% (n = 13/
72) of samples and Controrchis spp. in 2.8% (n = 2/72) of
infections. Similarly, our results showed that FS4, which
is considered optimal for samples from wild primates
], was not suitable for the analysis of feces of
howler monkeys, because it did not detect the presence
of trematodes, and Trypanoxyuris spp. were detected
only in 29.2% (n = 21/72) of samples.
If a qualitative or quantitative analysis of parasites of
Alouattais needed, we suggest Mini-FLOTAC with the
different combination of elements shown (depending on
the parasite group to be detected) is an appropriate
method (Additional file 1). Our recommendations are
made based on two species of parasites belonging to two
parasitic groups (trematodes and nematodes), but the
richness of parasites reported in Alouatta spp. is higher,
thus similar studies evaluating different species are
]. Finally, it was not possible to calibrate with
fresh samples because FLOTAC technique needed
specialized equipment (large volume or microtiter
] that was not possible to use in the field.
However, it is also important to compare different
preservation methods against fresh samples [
This is the first in-depth calibration of fecal egg count
flotation methods for analyzing samples from wild
howler monkeys and should be applicable for
comparisons of populations and species of howlers and other
similar frugivore/folivores. The Mini-FLOTAC method
is a promising technique for the qualificative and
quantitative analysis of nematodes and trematodes in howler
monkeys, and can be used in field without specialized
equipment. For the trematode Controrchis spp. the
highest EPG values were recorded with FS7 at 1:20 and
1:25 dilution; for the nematode Trypanoxyuris spp. the
highest EPG values were recorded with FS1 at 1:10
dilution, for samples preserved in 5% formalin. These
combinations achieved an 83.3% detection of
Controrchis spp. and 100% of Trypanoxyuris spp. infections.
Additional file 1: Guide to the recommended quali-quantitative
flotation method. (DOCX 14 kb)
Additional file 2: Table S1. Table showing in-depth calibration scheme.
(DOCX 19 kb)
Additional file 3: Table S2. Controrchis spp. egg counts, stratified by
flotation and preservation method, dilution and flotation solutions.
(DOCX 19 kb)
Additional file 4: Table S3. Trypanoxyuris spp. egg counts, stratified by
flotation and preservation method, dilution and flotation solutions.
(DOCX 25 kb)
Additional file 5: Table S4. Generalized linear model output for
Controrchis spp. egg counts. (DOCX 16 kb)
Additional file 6: Table S5. Generalized linear model output for
Trypanoxyuris spp. egg counts. (DOCX 16 kb)
CV: Coefficient of variation; EPG: Eggs per gram of feces; EPG/LPG/OPG/
CPG: Eggs/larvae/oocysts/cyst per gram of feces; FEC: Fecal egg count;
FS1: Sucrose and formaldehyde/specific gravity SG = 1.2; FS2: Sodium
chloride, SG = 1.2; FS3: Zinc sulfate, SG = 1.2; FS4: Sodium nitrate, SG = 1.2;
FS6: Magnesium sulfate, SG = 1.28; FS7: Zinc sulfate, SG = 1.35; G: Gram;
GLMs: Generalized linear models; SD: Standard deviation; VPF: Vacuum
packing in a fridge (4 °C)
We thank the Tejero family for their assistance during fieldwork, especially the
field assistant (Lolo, deceased). We thank landowners (Doña Chona, deceased,
Doña Tila and Don Polo) for allowing us to work on their private properties. We
thank my committee members Dr Luis Manuel Garcia Feria and Dr Martha Bonilla
Moheno (INECOL). We thank Julio García Hernández for their collaboration
providing the photographs of the eggs of the parasites. Finally, we appreciate the
invaluable help of the Medicine Conservation Laboratory of the Instituto
Politécnico Nacional, México (Don Mario, Omar, Dr Polo, Dr Nogueda).
JCSS supported most of the costs of this work including travel and lab
analysis expenses and MAAV was supported by a master degree grant
(CONACYT, scholarship number 280637). INECOL provided support through
an academic retreat program.
Availability of data and materials
The data used and/or analyzed during the current study are available from
the corresponding author upon reasonable request.
MAAV, GC, MPM, JCSS, LR and ABG conceived and designed the study.
MAAV and AC performed the experiments. MAAV identified specimens and
built the database. RG performed statistical analyses. MAAV, ABG, GC, MPM,
JCSS, RG and CAC wrote the manuscript; all authors provided relevant input
at different stages of manuscript preparation. All authors read and approved
the final manuscript.
Ethics approval and consent to participate
Sampling permit (number SEMARNAT:SGPA/DGVS/05938/08) was provided
by Secretaría de Medio Ambiente y Recursos Naturales (SEMARNAT).
Consent for publication
GC is the inventor and current patent holder of the FLOTAC and
MiniFLOTAC methods. The methods will be licensed free of charge to the WHO
and interested public non-commercial research centers. None of the other
authors declare any conflict of interest concerning the work reported in this
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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