Screening of protozoan and microsporidian parasites in feces of great cormorant (Phalacrocorax carbo)
Screening of protozoan and microsporidian parasites in feces of great cormorant (Phalacrocorax carbo)
Piotr Rzymski 0 1 2 3
Anna Słodkowicz-Kowalska 0 1 2 3
Piotr Klimaszyk 0 1 2 3
Piotr Solarczyk 0 1 2 3
Barbara Poniedziałek 0 1 2 3
0 Department of Biology and Medical Parasitology, Faculty of Medicine I, Poznan University of Medical Sciences , Poznań , Poland
1 Department of Environmental Medicine, Faculty of Health Sciences, Poznan University of Medical Sciences , Poznań , Poland
2 Responsible editor: Robert Duran
3 Department of Water Protection, Faculty of Biology, Adam Mickiewicz University , Poznań , Poland
The global population of great cormorants (Phalacrocorax carbo L.) is on the rise. These birds, characterized by rapid metabolism, can deposit large quantities of feces, and because they breed on the land but forage on water, both terrestrial and aquatic environments can be simultaneously affected by their activities. The contribution of great cormorants in the dispersal of bacterial and viral pathogens has been immensely studied; whereas, the occurrence of eukaryotic parasites such as protozoans and microsporidians in these birds is little known. The present study investigated the presence of dispersive stages of potentially zoonotic protozoans belonging to the genera Blastocystis, Giardia and Cryptosporidium, and Microsporidia spores in feces collected from birds inhabiting the breeding colony established at one lake island in Poland, Europe. The feces were examined by coprological techniques (staining with iron hematoxylin, Ziehl-Neelsen, and modified Weber's chromotrope 2R-based trichrome), and with immunofluorescence antibody MERIFLUOR Cryptosporidium/Giardia assay. As found, the Cryptosporidium oocysts were identified rarely in 8% of samples (2/25; 3-5 × 103/g) and no cysts of Giardia and Blastocystis were detected. Microsporidian spores were detected in 4% of samples (1/25) but at very high frequency (4.3 × 104/g). No dispersive stages of parasites were identified in water samples collected from the littoral area near the colony. Despite the profuse defecation of cormorants, their role in the dispersion of the investigated parasites may not be as high as hypothesized.
Cormorants; Bird feces; Cryptosporidium; Blastocystis; Microsporidia; Microbial dispersion
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The global population of great cormorant (Phalacrocorax
carbo L.) is on the systematic rise, particularly in some parts
of Europe where the number of breeding pairs is estimated to
exceed 400,000 (Bregnballe et al. 2014; Klimaszyk and
Rzymski 2016). This bird species, exterminated for decades
by humans, have become numerous not only due to
international and national law enforcements but also as a result of its
high ecological adaptation, ability to forage on marine and
freshwater environments, lack of regular predators, and
increase in fish biomass due to the eutrophication and climate
changes (Cramp and Simmons 1997; White et al. 2011; Skov
2011; Klimaszyk and Rzymski 2016). As these birds are
generally gregarious, appear collectively, gather in flocks, nest in
colonies on land, can feed on a relatively large area (up to
30 km from the colony), and simultaneously on various water
systems, they may represent a significant biological factor that
could trigger environmental modifications (Klimaszyk et al.
2015a; Klimaszyk and Rzymski 2016).
The effect of P. carbo on nutrient cycling, soil chemistry,
terrestrial and aquatic vegetation, excessive algae growth
(Ligęza and Smal 2003; Klimaszyk et al. 2015a, b),
invertebrate communities (Kolb et al. 2010), and fish populations
(Ostman et al. 2013) has been studied extensively (for
review see Klimaszyk and Rzymski 2016). Some studies also
addressed the role of these birds in dispersion of bacterial
pathogens such as Escherichia coli (Klimaszyk 2012;
Klimaszyk and Rzymski 2013a, 2016), avian influenza virus
(Albini et al. 2014), avian paramyxovirus (Schelling et al.
1999), and West Nile virus (Iashkulov et al. 2008; Table 1).
Recent studies also investigated the presence of gastric
nematodes in these birds (Dziekońska-Rynko and Rokicki 2008;
El-Dakhly et al. 2012).
The occurrence of dispersive stages of intestinal protozoan
parasites such as Giardia cysts and Cryptosporidium oocysts
in great cormorants is, however, largely unknown and, so far,
reported only in two studies examining the bird feces
(Medema 1999; Plutzer and Tomor 2009). The parasites were
detected in cormorant droppings, but due to low number of
samples in both studies, the definite conclusions on the role of
cormorants in dispersion of these potential pathogens cannot
be drawn. The presence of microsporidian spores in great
cormorant, on the other hand, was so far a subject to only
one study conducted recently in Slovakia. The spores,
identified molecularly as Encephalitozoon cuniculi were detected
using PCR in several fecal samples (Malčeková et al. 2013).
As some microsporidian and protozoan parasites are
potentially infectious in mammals including human (Ehsan et al.
2015), it is of high priority to conduct further studies
elucidating the role of great cormorants in their dissemination. As
these birds represent a very important intermediate link in
some food webs (Gwiazda et al. 2010, Skov et al. 2014) and
a factor facilitating the dislocation of matter between
terrestrial and aquatic ecosystems (Marion et al. 1994), it can be rather
anticipated that they could also be responsible for high
dispersion of parasites because they consume relatively large fish
biomass, estimated at 350 g per day (Carss 1997). Various fish
species were, in turn, identified as potential reservoirs of
protozoan intestinal parasites such as Giardia sp. (Yang et al.
2010; Ghoneim et al. 2012) or Cryptosporidium sp.
(Barugahare et al. 2011; Gabor et al. 2011) as well as
microsporidian parasites (Lom and Nilsen 2003). Moreover,
cormorants are characterized by rapid metabolism and the
birds defecate on average 30 g dry weight of droppings per
day (Marion et al. 1994). Deposited on relatively small area of
colony, their chemical and microbial content can be
subsequently transported with surface runoff and/or groundwater
to the nearby lake (Klimaszyk and Rzymski 2011, 2013b;
Klimaszyk et al. 2015a, b). Therefore, it is of great interest
to evaluate the importance of great cormorants as vectors of
dispersive stages of intestinal protozoan parasites in terrestrial
and aquatic environments.
The current state of knowledge on the human pathogens dispersed by great cormorant (Phalacrocorax carbo)
Bacteria
Escherichia coli Poland, Czech Republic (as a
intestinal commensal it is
spread anywhere the
cormorant is present)
O25b-ST131 clone was isolated. The
increased E. coli counts were observed
in lake littoral and groundwater within
the colony area
Predominantly serious
urinary tract infections
(O25b-ST131)
Highly pathogenic avian
influenza transmitted
between birds and to
mammals resulting in death
Newcastle disease in poultry
and wild birds. Clinical
symptoms in human
West Nile fever. Rarely
neurological symptoms
Encephalitozoon cuniculi was identified Intestinal parasitosis, diarrhea Malčeková et al.
(Slovakia). 2013; This study
The present study aimed to investigate the presence of
Cryptosporidium oocysts, cysts of Giardia and Blastocystis,
and microsporidian spores in fecal samples collected from the
colony of P. carbo during the breeding season. The colony,
constituted of 170 breeding pairs, was located on the island of
recreationally used, eutrophic Lake Chrzypsko (Northern
Poland, Europe). To the best of our knowledge, this is the first
study not only to survey such number of these birds in this
regard but also to highlight that the role of great cormorants in
dispersion of human intestinal protozoan and microsporidian
parasites may not be as significant as expected.
Material and methods
The cormorant colony
The studied colony inhabits the Lake Chrzypsko (Poland,
Europe) which is in the state of moderate eutrophy
(Klimaszyk 2012). Owing to its location, the lake is intensely
used for recreation. Numerous holiday resorts and bathing
places are located on its shores. In the west bay, there is also
a rowing training center, the racetrack of which stretches near
the cormorant colony. The colony has been existing since the
beginning of the twenty-first century (Klimaszyk 2012). It
occupies the most northward island of the lake (Fig. 1) at the
latitude and longitude of 52o 36′ 57″ N and 16o13′ 23″ E,
respectively. An island has an area of 0.9 ha and slight
elevation, up to 40 cm above the lake level. The counting of birds
Fig. 1 The studied island on
Lake Chrzypsko (Poland)
inhabited by cormorants and
sampling points
was performed by two independent observers during
dawndusk prior to collection of fecal samples. During the
investigated period (June 2013), 170 breeding pairs (approx. 600
individuals including adults and rearing chicks) were
recorded. The foraging area of cormorants during this period is in the
radius of 50 km from the colony, but outside the breeding
season, adult birds and younglings may spread across the
Europe (Bregnballe et al. 2014).
Samples of cormorant feces were collected from the colony
area in June 2013 using 10 trays (60 × 60 cm) located directly
beneath bird nests at various points within the island (Fig. 1).
The birds’ behavior was observed from the boat using
binoculars and feces were systematically collected from each tray. A
special care was taken to avoid collecting the fecal samples
from the same nest. Plant detritus (leaves, branches) was
removed from trays to minimize contamination of samples.
Deposited feces were collected to sterile polypropylene tubes
by pooling droppings from five different birds as one sample.
All samples were immediately preserved with 20 mL
potassium dichromate and transported to the laboratory in a
lightproof insulated box containing a cooling factor. A total
number of 25 pooled samples, and estimated droppings from 125
cormorants, were collected for subsequent parasitological
examination. Additionally, samples of lake water (10 L each)
were collected from four sampling sites at littoral area near
the colony (shore zone) into sterile vessels (Fig. 1). These sites
were selected because the shore lake zone was previously
shown to be characterized by a high density of fecal bacteria
originating from cormorant species (Klimaszyk and Rzymski
2013a; Klimaszyk et al. 2015b).
Parastiological examination of feces
All fecal samples were examined using coprological methods.
From each pooled fecal sample, four smears were made. One
direct wet smear was immediately microscopically examined
under high dry power (total magnification ×400). The
remaining three smears were stained with either: (i) modified
Weber ’s chromotrope 2R–based trichrome stain for
Microsporidia spores (Weber et al. 1992), (ii) Ziehl-Neelsen
stain for Cryptosporidium oocysts, or (iii) iron hematoxylin
stain for cysts of Giardia and Blastocystis (Garcia 2001).
Stained smears were microscopically screened using an
oilimmersion objective (total magnification ×1000).
Additionally, to confirm identification of Cryptosporidium
oocysts and/or Giardia cysts, all positive specimens were
tested using a direct immunofluorescence antibody (IFA) test kit,
M E R I F L U O R C r y p t os p o r i d i u m / G i a rd i a ( M e r i di a n
Diagnostic, Cincinnati, Ohio, USA), was used according to
the manufacturer’s instructions.
Parastiological examination of water samples
All water samples were examined using modified U.S.
Environmental Protection Agency Method 1623 (U.S.
Environmental Protection Agency 1999). The sediment was
obtained by filtration using SM 16274 filter chamber
(Sartorius, Germany) on cellulose acetate membranes with a
nominal pore size of 0.8 μm (Merck Millipore, Ireland). The
filters were then dissolved in acetone according to Graczyk
et al. 1997. Each sample concentrate was analyzed using the
Ziehl-Neelsen (Cryptosporidium oocysts), modified Weber’s
chromotrope 2R-based trichrome stain (Microsporidia spores)
and iron hematoxylin (Giardia and Blastocystis cysts)
methods, and immunofluorescent assay (IFA).
Fig. 2 Spores of Microsporidia
stained with modified Weber’s
chromotrope 2R-based trichrome
(a) and oocysts of
Cryptosporidium stained with
Ziehl-Neelsen (b), identified in
Phalacrocorax carbo feces
Cryptosporidium oocysts were identified in 2/25 (8%) of
pooled fecal samples of great cormorant (Fig. 2a). All samples
detected as positive by Ziehl-Neelsen staining were also
positive by the immunofluorescence technique. In both
Cryptosporidium-positive samples, a small number of
oocysts, i.e., five to ten per slide, were detected, at frequency
of 3 × 103/g and 5 × 103/g of feces. The mean length (±SD)
and width (±SD) of identified oocysts was 5.0 (±0.0) and 5.4
(±0.5) μm, respectively. Spores of Microsporidia (Fig. 2b)
were identified only in 1/25 (4%) of pooled fecal samples
but at high concentration of 4.3 × 104/g of feces. The mean
length (±SD) and width (±SD) of these spores was 1.8 (±0.4)
and 1.2 (±0.2) μm, respectively. None of the investigated
pooled fecal samples contained detectable cysts of Giardia
and Blastocystis.
None of investigated water sample was identified to
contain microsporidian spores and dispersive stages of
Cryptosporidium, Blastocystis and Giardia.
The birds represent an important factor harboring and dispersing
the microorganisms, including pathogens (Graczyk et al. 1998;
Okulewicz 2014). The bird microbiota has been demonstrated to
be affected by many different factors, such as infections and
general health status, diet, and local microbial communities in
environment (Palmgren et al. 1997; Lu et al. 2003; Santos et al.
2012). A main route through which the birds can take part in the
dispersion of various microorganisms, including protozoa and
microsporidia, is fecal excretion. Despite that great cormorants
were previously reported to deposit large amounts of feces
within the colonized areas (Marion et al. 1994; Klimaszyk and
Rzymski 2016), the present study indicates that their role in
dispersion of intestinal protozoan parasites may be largely
limited and decidedly lower than theoretically expected. Some
colonies of this bird may, however, still represent a source of
dispersion of other human pathogens (Table 1).
It is important to fully elucidate the biological vectors of
dispersive stages of protozoan and microsporidian parasites.
These microorganisms are resistant to various environmental
conditions, can lead to serious, acute gastrointestinal
infections in human, and are usually characterized by the low
infectious dose (Szumowski and Troemel 2015; Messner and
Berger 2016). The presence of Cryptosporidium oocysts and
Giardia cysts in source waters have already caused numerous
documented outbreaks related in both drinking and
recreational waters (Karanis et al. 2007). Various birds have already
been demonstrated to contribute to contamination of surface
waters with dispersive stages of these parasites, including
species and genotypes representing a threat to human health
(Smith et al. 1993; Graczyk et al. 1998; Majewska et al.
2009). In a Hungarian survey investigating feces of different
bird species, one Giardia sp. cyst was identified
microscopically and the presence of Cryptosporidium sp. was confirmed
with PCR but the study examined only a single fecal sample
collected from cormorant (Plutzer and Tomor 2009).
Cryptosporidium oocysts (but not Giardia cysts) were also
detected in feces collected from ten P. carbo individuals in
the Netherlands inhabiting areas nearby man-made human
reservoirs. The prevalence amounted to 20% and mean
concentration in positive samples was estimated at 64 oocysts per
gram—high enough to significantly contribute to the
contamination of water reservoir (Medema 1999). Contrary to these
findings, the present study, which employed fecal samples
collected from significantly greater number of birds (125
individuals) found that the prevalence of Cryptosporidum
oocysts was very low and no Blastocystis or Giardia cysts were
present. This indicate that this species, at some inhabited sites,
may not represent a significant source of dispersive stages of
human protozoan parasites—particularly if one considers that
none of investigated protozoans were identified in lake water
near the colony.
The infection of the gastrointestinal tract by Microsporidia
can also lead to severe, persistent diarrhea (Didier 2005). As
shown, microsporidian species known to infect humans such
as E. hellem are present in aquatic bird species including Anas
platyrhynchos, Anser anser, Balearic pavonina, Cygnus
atratus, C. melanocoryphus, C. olor and Coscoroba
coscoroba (Słodkowicz-Kowalska et al. 2006). The
E. cuniculi (but not E. hellem, E. intestinalis or E. bieneusi)
was found in the Slovakian pilot study examining 40 samples
of great cormorant feces at a relatively high prevalence of
42.5% (Malčeková et al. 2013). The present study showed
decidedly lower frequency of spores in investigated
population of 125 birds indicating that the role of great cormorants in
Microsporidia dispersion may be highly site specific.
However a relatively high number of spores were identified
in one pooled sample (4.3 × 104/g); no dispersive stages of
Microsporidia were identified in lake water near the colony. It
should be highlighted that however the most widely used
staining method to detect spores (chromotrope 2R modified
trichome) was employed in the present study, it does not allow
t o d i s t i n g u i s h p a r t i c u l a r s pe c i e s o r g e n o t y p e s of
microsporidia. This is important if one considers that only
some species (at least 15 from over 1200 identified so far)
are known to be pathogenic for humans (Ramanan and Pritt
2014). These, in turn, can be identified by means of
immunofluorescence assays using polyclonal or monoclonal
antibodies and/or PCR (Ramanan and Pritt 2014). Further
investigations are required to fully elucidate the environmental
conditions contributing to the presence of microsporidian dispersive
stages in cormorant feces, and to estimate risks for human
health.
The present study was limited only to one great cormorant
colony situated at the lake of low human pressure; therefore,
the results should be treated cautiously upon any
extrapolation. Sewage discharge can lead to increased contamination of
water with dispersive stages of parasites and their presence in
biota including waterfowls (Słodkowicz-Kowalska et al.
2015); thus, the prevalence of studied parasites may be
different at sites varying in human pressure. It should be, however,
highlighted that great cormorants usually nest within areas of
negligible human impact (Klimaszyk and Rzymski 2016).
The present study investigated the presence of dispersive
stages of potentially zoonotic protozoans belonging to the
genera Blastocystis, Cryptosporidium and Giardia, and
Microsporidia spores in feces of great cormorant. It was
hypothesized that due to specific behavior and metabolism, these
birds may represent an important vector for these parasites.
Contrary to this, the prevalence of Cryptosporidium oocysts
and microsporidian spores was very low, and no cysts of
Giardia and Blastocystis were identified. The study indicates
that this species may not play, at least at certain locations, a
profound role in the dissemination of investigated parasites.
Further research employing immunological and molecular
methods is necessary to elucidate exact species of
Microsporidia, and evaluate whether cormorants may
disseminate those associated with human infection.
Acknowledgments Piotr Rzymski is supported by the Foundation for
Polish Science (FNP) (START 091.2016).
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