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Molecular detection of Trypanosoma spp. in Lipoptena cervi and Lipoptena fortisetosa (Diptera: Hippoboscidae) and their potential role in the transmission of pathogens

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The family Hippoboscidae is a less known group of blood-sucking flies. Deer ked are particularly important for animal health; they may act as potential vectors of disease to ungulates, and may transmit pathogens to animals and humans. The aim of this study was to investigate the presence of Trypanosoma (Megatrypanum) DNA in deer keds using molecular methods. Results prove the presence of Megatrypanum trypanosome DNA in the studied winged adult deer keds and this is the first detection of this pathogen in Lipoptena fortisetosa. In addition, this paper evidences the occurrence of L. fortisetosa in two new locations: one in the Białowieża Primeval Forest, and another in the Strzałowo Forest Inspectorate (Piska Forest), both in north-eastern Poland.
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Parasitology
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Review
Cite this article: Werszko J, Steiner-
Bogdaszewska Ż,Jeżewski W, Szewczyk T,
KuryłoG,Wołkowycki M, Wróblewski P,
Karbowiak G (2020). Molecular detection of
Trypanosoma spp. in Lipoptena cervi and
Lipoptena fortisetosa (Diptera: Hippoboscidae)
and their potential role in the transmission of
pathogens. Parasitology 147, 16291635.
https://doi.org/10.1017/S0031182020001584
Received: 7 May 2020
Revised: 24 August 2020
Accepted: 24 August 2020
First published online: 1 September 2020
Key words:
Hippoboscidae;Lipoptena cervi;Lipoptena
fortisetosa;Megatrypanum;Trypanosoma
Author for correspondence:
Joanna Werszko,
E-mail: joanna.werszko@gmail.com
© The Author(s), 2020. Published by
Cambridge University Press
Molecular detection of Trypanosoma spp.
in Lipoptena cervi and Lipoptena fortisetosa
(Diptera: Hippoboscidae) and their potential
role in the transmission of pathogens
Joanna Werszko1,Żaneta Steiner-Bogdaszewska1, Witold Jeżewski1,
Tomasz Szewczyk1, Grzegorz Kuryło2, Marek Wołkowycki2, Piotr Wróblewski1
and Grzegorz Karbowiak1
1
Witold Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818, Warsaw, Poland and
2
Institute of Forest Sciences, Faculty of Civil Engineering and Environmental Sciences, Białystok University of
Technology, Wiejska 45e, 15-351, Białystok, Poland
Abstract
The family Hippoboscidae is a less known group of blood-sucking flies. Deer ked are particu-
larly important for animal health; they may act as potential vectors of disease to ungulates,
and may transmit pathogens to animals and humans. The aim of this study was to investigate
the presence of Trypanosoma (Megatrypanum) DNA in deer keds using molecular methods.
Results prove the presence of Megatrypanum trypanosome DNA in the studied winged adult
deer keds and this is the first detection of this pathogen in Lipoptena fortisetosa. In addition,
this paper evidences the occurrence of L. fortisetosa in two new locations: one in the
Białowieża Primeval Forest, and another in the Strzałowo Forest Inspectorate (Piska
Forest), both in north-eastern Poland.
Introduction
Flies in the family Hippoboscidae (Diptera), known as louse fliesor kedsare a group of obli-
gate parasites of mammals and birds (Rahola et al., 2011). A recent checklist of Hippoboscidae
across the world retains three subfamilies (Ornithomyinae, Hippoboscinae and Lipopteninae)
more than 213 species and 21 genera (Dick, 2006; Petersen, 2013). It has been shown that the
two subfamilies Hippoboscinae and Lipopteninae are monophyletic groups (Petersen et al.,
2007). From Europe, 30 species of Hippoboscidae are known (Petersen, 2013). In Poland,
the hippoboscid fauna is relatively not well known. About 10 species are present in Poland
including four which parasitize mammals: the forest fly Hippobosca equina L., 1758, the
sheep ked Melophagus ovinus L., 1758 and two species of the Lipoptena Lipoptena cervi
L., 1758 and Lipoptena fortisetosa Maa, 1965 (Borowiec, 1984; Borowiec and Zatwarnicki,
1989;Kowalet al., 2016).
Worldwide, 30 Lipoptena species were recorded, five of them were described from Europe
(Dick, 2006; Petersen, 2013). Lipoptena cervi and L. fortisetosa have a more northern range
while the other three species have a restricted distribution in Southern Europe, including
the Mediterranean islands (Petersen, 2013; Kurina et al., 2019). In Europe, the L. fortisetosa
was first recorded from Czech Republic where it was initially described as a new species
L. parvula Theodore, 1967, but was later changed to L. fortisetosa by Grunin (1970)
(Kurina et al., 2019).
Its appearance in Europe is probably associated with the introduction of sika and Siberian
deer in 1839 (Bartoš,2009). The species is also able to colonize the European roe deer popu-
lation as a result of natural spread following contact with Siberian roe deer (Kowal et al.,
2016). Lipoptena fortisetosa was reported for a few European countries, including Germany,
Belarus, Moscow district in Russia, Austria, Lithuania, Moldova, Poland, the Czech
Republic, Romania, Slovakia and Switzerland (Kurina et al., 2019;Oboňaet al., 2019).
More recently the species has been also recorded in Italy and Estonia (Andreani et al.,
2019;Kurinaet al., 2019). The most important hosts of deer keds in Europe are mainly:
red deer Cervus elaphus L., 1758, roe deer Capreolus capreolus (L., 1758), Eurasian elk
Alces alces (L., 1758), sika deer Cervus nippon Temminck, 1838 and fallow deer Dama
dama (L., 1758). In North America, the parasite is common on wapiti Cervus canadensis
Erxleben, 1777 and white-tailed deer Odocoileus virginianus (Zimmermann, 1780). A wide
range of animals can also be accidental hosts of L. cervi: horses, cattle, European bison,
sheep, domestic dogs, red foxes, badgers and suids (Hermosilla et al., 2006; Karbowiak
et al., 2014;Kowalet al., 2016). Deer keds occasionally bite also humans (Kortet et al.,
2010). In Poland, while L. cervi has been observed throughout the country, L. fortisetosa
has been found only in the Dolnośląskie, Małopolskie and Warmińsko-Mazurskie
Voivodeships (Borowiec and Zatwarnicki, 1989;Kowalet al., 2009). Sokółand Gałęcki
(2017) reported the presence of L. fortisetosa in dogs in cities in central Poland. Both
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L. fortisetosa and L. cervi have a specific development cycle. Upon
settling on a suitable mammal host, deer keds shed their wings,
remaining in a wingless form for the rest of their life. These flies
are viviparous species: they generate fully-grown larvae that fall
to the ground and pupate. Although the keds occur throughout
the year, the winged adults only appear in high numbers from sum-
mer to early autumn (Haarløv, 1964; Borowiec, 1984).
Deer keds have an economic impact on the hunting
economy. Their infestation can induce scratching and itching
in free-ranging cervids, and this may impair the condition of
the host following secondary bacterial infection (Dehio et al.,
2004). Besides, although deer keds do not reproduce on humans
and accidental hosts, people who work in forestry, or visitors,
are still particularly vulnerable to deer ked infestation. As a
matter of fact, deer ked bites can result in the occurrence of
severe dermatitis in humans (Härkönen et al., 2009).
According to the life cycle of insect vectors, Trypanosoma
species are divided into two sections: Stercoraria (subgenera:
Megatrypanum Hoare, 1964; Herpetosoma Doflein, 1901;
Schizotrypanum Hoare, 1972) and Salivaria (subgenera:
Duttonella Chalmers, 1918; Nannomonas Hoare, 1964;
Trypanozoon Luhe, 1906; Pycnomonas Hoare, 1964) (Hoare,
1972). Stercorarian trypanosomes develop only in the hindgut
(rectum) of the host thus allowing the metacyclic forms to leave
the vector organism with the feces. The infection takes place
through damaged skin or mucous membrane of the host
(Hoare, 1972). During their developmental cycle, Salivarian try-
panosomes enter the salivary glands of the insect vector so that
it can transmit the pathogen injecting saliva into vertebrate
hosts. The subgenus Megatrypanum comprises a group of large
trypanosomes that infect almost all mammalian orders (Hoare,
1972). Previous studies of these parasites have typically been
restricted to morphological and morphometric examinations of
samples from hostsblood and cultured forms. More recently,
complex phylogenetic analyses concerning trypanosomes of the
subgenus Megatrypanum revealed two main lineages of
Trypanosoma theileri (TthI and TthII) and 10 genotypes asso-
ciated with the host species: four genotypes from cattle, one
from water buffalo, one from deer, two from duikers and one
from sitatunga (Rodrigues et al., 2010a,2010b; Garcia et al.,
2011a,2011b). The species of Megatrypanum trypanosomes
described in Poland are presented in Table 1. Although
Megatrypanum infections in animals are typically subclinical,
some clinical cases are reported; such disease or deaths tend to
be associated with stressed cattle or with the presence of concomi-
tant infections such as bovine leucaemia virus (Matsumoto et al.,
2011). Trypanosoma theileri can result in leucocytosis, neonatal
death, anaemia, weight loss and a considerable drop in milking
capacity (Matsumoto et al., 2011). Several neurological manifesta-
tions associated with T. wrublewskii infection, including depres-
sive neurological signs, apathy and oedema have been observed
in European bison from the Białowieża Primeval Forest
(Wrublewski, 1912; Kingston et al., 1992). However, further inves-
tigation on infection with parasites of European bison did not
confirm these clinical signs (Kingston et al., 1992; Karbowiak
et al., 2014).
Megatrypanum trypanosomes are typically transferred to
the vertebrate hosts by contamination of the oral mucosa with
feces or the gut contents of the infected insects such as tabanid
flies (Böse et al., 1987). Hoare (1972) described that the entire
life cycle of Megatrypanum trypanosomes takes place in the
alimentary tract of the invertebrate host. Only a few reports
have described the occurrence of Megatrypanum trypanosomes
in deer keds; for example, Böse and Petersen (1991) documented
the presence of trypanosomatids in the midgut and hindgut of
L. cervi.
It is much more challenging to assess the effect of pathogen-
icity on arthropods as they tend to display higher tolerance to
parasites, and thus less pronounced signs and symptoms
(Lipa, 1968). Nonetheless, some studies highlighted the effect of
Trypanosoma infections on the condition and survivability
of the invertebrate host. Nelson (1956) reports high mortality of
sheep keds, caused by obstruction of the intestine with a massive
number of trypanosomes. However, Hoare (1972) did not notice
any changes in the appearance and behaviour of sheep ticks in his
research.
The Hippoboscidae, including deer keds, are potential vectors
of a number of pathogens, including bacteria such as Bartonella
spp., Anaplasma spp., Coxiella spp. and Ehrlichia spp., protozoa
such as Trypanosoma (Megatrypanum) spp. and apicomplexan
parasites such as Theileria spp. (Halos et al., 2004; Lee et al.,
2016; Szewczyk et al., 2017).
The occurrence of Trypanosoma melophagium in sheep ked
was confirmed by Martinkovićet al.(2012). Billeter et al.
(2008) identified Bartonella melophagi in sheep ked (M. ovinus)
and B. chomelii in forest flies (H. equina) in Algeria. Bartonella
spp. have also been identified in deer keds collected from
deer in Poland (Szewczyk et al., 2017), while the presence of
Anaplasma phagocytophilum was confirmed in deer keds col-
lected from deer in Slovakia (Víchová et al., 2011). These findings
underline the potential role of these blood-sucking hippobocids in
the mechanical transmission of pathogenic bacteria within the
population of wild animals. They also highlight the risk of trans-
mission of pathogens to humans and animals via the bite of
infected haematophagous ectoparasites.
Our previous study reported the presence of Megatrypanum
trypanosomes in some species of blood-sucking flies belonging
to the Tabanidae family (Werszko et al., 2020). We hypothesize
that the same trypanosomes could also be present in two species
of Lipoptena:L. cervi and L. fortisetosa.
Materials and methods
Fly collection and taxonomical study
Hippoboscid specimens were collected manually from the fur
of red deer during the autumn hunting seasons in the years
2018/2019, and from vegetation in autumn, using an entomo-
logical net. The flies were collected from the Strzałowo Forest
Inspectorate (Piska Forest) (53°46N, 21°27E) and three localities
in the vicinity of the Białowieża Primeval Forest (Białowieża
52°42N, 23°52E, Hajnówka 52°44N, 23°35E and Smolany
Sadek 52°48N, 23°36E) (Fig. 1). All the locations where deer
keds were collected before 2019 as well as those identified during
the current study are marked on the map in Fig. 1, according to
the UTM (the Universal Transverse Mercator) geographical
grid. This method is commonly used for plotting the ranges of
animals on a regional scale in faunistic research. Before the
taxonomical identification, hippoboscid specimens were rinsed
in ultrapure water (Direct-Pure® adept Ultrapure Lab Water
Systems, RephiLe Bioscience, Ltd., China) and then air-dried
and prepared for optical observations. Sex determination and
species identification were carried out using taxonomic keys,
according to Borowiec (1984) and Salvetti et al.(2020) under
an OPTA-TECH microscope (Warsaw, Poland). In the current
study, two dimensions were measured: the total length of the
body and the largest width of the abdomen.
PCR and sequence analyses
DNA from each fly was extracted using a Genomic Mini AX
Tissue kit (A&A Biotechnology, Gdynia, Poland), according to
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the manufacturers instructions and stored at i °C until molecu-
lar analysis. Flies were individually screened for the presence of
trypanosomes using polymerase chain reaction (PCR). The fol-
lowing 18S rDNA oligonucleotides TrypF 150 (5-GAA ACA
CGG GAG CGG TTC CTT-3) and TrypR 800 (5-ACC TCA
AAG CTT TCG CGT GAA G-3) were used as previously
described (Werszko et al., 2020). These primers amplified a
650 bp fragment of the 18S rRNA gene of Trypanosoma spp.
PCR reactions were conducted in a 50 μL reaction mixture
containing 36 μL of deionized water, 3 μLofa25μMsolution of
MgCl
2
, 0.5 μL of Allegro Taq DNA polymerase (5 U μL
1
)
(Novazym, Poznań, Poland), 0.5 μL of dNTP-mix (10 mM), 5 μL
of 10 × Taq DNA polymerase buffer (with 25 mMMgCl
2
),
0.5 μL of each primer (20 pmol μL
1
) and 4 μL of template
DNA. DNA from a Trypanosoma sp. (GenBank acc. no.:
MK088728) isolated from Haematopota pluvialis (Tabanidae),
was used as a positive control. As a negative control, nuclease-free
water was added to the PCR mix instead of the DNA sample.
The amplification conditions include initial denaturation at
94°C for 2 min, followed by 35 cycles of denaturation at 94°C
for 30 s, annealing at 55°C for 30 s, primer extension at 72°C
for 30 s and final extension at 72°C for 3 min. The final phase
of PCR reaction included cooling the samples to 10°C.
PCR products were visualized on a 1% agarose gel stained with
ethidium bromide. Visualization was performed using ChemiDoc,
MP Lab software (Imagine, BioRad, Hercules, USA). The result-
ing product was compared using the Nova 100 bp DNA Ladder
Novazym (Poznań, Poland). PCR products were purified using
Table 1. Trypanosoma (Megatrypanum) species and their respective hosts, described in Poland, on basis of morphological and morphometric data
Trypanosoma species Host References
Trypanosoma wrublewskii European bison (Bison bonasus) Wrublewski (1912)
Trypanosoma theileri Cattle (Bos taurus) Demiaszkiewicz and Lachowicz (1991)
Trypanosoma stefanskii Roe deer (Capreolus capreolus) Kingston et al.(1992)
Trypanosoma cervi Red deer (Cervus elaphus) Wita and Kingston (1999)
Trypanosoma ornata Water shrew (Neomys fodiens) Karbowiak et al.(2005)
Fig. 1. The documented occurrence of Lipoptena fortisetosa in Poland. (1) Localities where L. fortisetosa was collected before 2019; (2) New record-sites described
during the study.
Parasitology 1631
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the QIAEX II Gel extraction kit (Qiagen, Hilden, Germany) and
sequenced by Genomed (Warsaw, Poland). The purified PCR
products were assembled into contigs using ContigExpress,
Vector NTI Advance 11.0 (Invitrogen Life Technologies,
New York, USA). The obtained sequences were compared by
using BLAST (BasicLocal Alignment Search Tool) with sequences
available in GenBank.
Results
In total, 155 flies (118 L. cervi and 37 L. fortisetosa) were collected,
including 27 adult winged deer keds (L. cervi) from vegetation
caught in one single location (Smolany Sadek) through sweeping
net (Table 2). In the current study L. fortisetosa was recorded in
two localities in Poland: one in the Białowieża Primeval Forest,
and another in the Strzałowo Forest Inspectorate (Puszcza Piska
Forest), both in north-eastern Poland (Fig. 1).
Morphological identification
The most significant difference between the two species is the
body size: L. fortisetosa is smaller than L. cervi. In both species,
females are larger than males. The mean length of L. fortisetosa
is 4.43 ± 0.0963 mm for females and 3.75 ± 0.0172 mm for
males. In L. cervi, the mean body length is 5.90 ± 0.070 mm for
females and 5.55 ± 0.050 mm for males. The mean abdomen
width of L. fortisetosa is 2.31 ± 0.099 mm for females and
2.079 ± 0.022 mm for males while in L. cervi,theabdomenwidth
is 2.86 ± 0.052 mm for females and 2.96 ± 0.050 mm for males.
The sutural pattern and the distribution of bristles in the thor-
acic region reveal significant morphological features to distinguish
the two species. Lipoptena cervi is hairier than L. fortisetosa and
the dimensions of its bristles vary, whereas all bristles in L. forti-
setosa are of equal dimensions (Fig. 2).
Female terminalia show other important taxonomical differ-
ences: the number of bristles on the genital opening, and the fea-
tures of the pregenital sclerites and plates. Lipoptena cervi shows
three pregenital aligned sclerites, with the central triangular scler-
ite bearing four or six bristles, and each oval external sclerites with
three or four bristles. In contrast, L. fortisetosa has only one cen-
tral pregenital sclerite with two long, strong bristles in the middle
and one on both sides (Fig. 2).
PCR and sequence analyses
The overall positivity to Trypanosoma spp. DNA in deer keds was
27.09% (42/155). The presence of Trypanosoma spp. was detected
in 24 out of 118 (20%) L. cervi. In the tested group of winged
L. cervi 5 out of 27 (18.51%) were infected. The presence of try-
panosomes was detected in 18 out of 37 (48.64%) L. fortisetosa.
The males and females of both species of Lipoptena demonstrated
similar prevalence of trypanosome infection. The positivity
to trypanosome DNA among females and males L. cervi and
L. fortisetosa from different locations is given in Table 2.
Four partial 18S rDNA nucleotide sequences were obtained
from L. fortisetosa and three from L. cervi, including one sequence
obtained from winged deer ked. Two isolates of Trypanosoma
spp. obtained from L. cervi (isolates Lc99KG and Lc106KG)
and one obtained from winged L. cervi (isolates Lc6SM) were
identical each other, and all shared 100% similarity to T. theileri
from a tse-tse fly Glossina fuscipes fuscipes Newstead, 1910 from
Central Africa (KR024688) and Trypanosoma cf. cervi from white-
tailed deer (Odocoileus virginianus Zimmermann, 1780) from the
USA (JX178193).
Sequences of Trypanosoma spp. obtained from two L. fortise-
tosa (isolates Lf4KG and Lf15KG) were identical to each other
and showed 100% similarity to T. theileri from European bison
Bison bonasus from Poland (KF765799). These isolates also
showed 100% identity with T. theileri from cattle from Poland
(KF924257). In addition, they also shared a high identity
(100%) with trypanosomes isolated in the USA and Brazil
(JX853185, JX178162, JX178185, JX178188 and AY773679) and
water buffalo Bubalus bubalis (L., 1758) from Brazil (AY773674).
One nucleotide sequence from L. fortisetosa (isolate Lf10KG)
infected with Trypanosoma spp. was 100% identical to T. melopha-
gium from sheep ked (M. ovinus)fromCroatiaandtheUK
(HQ664912 and FN666409). One sequence derived from L. fortise-
tosa (isolate 1LfHka) infected with Trypanosoma spp. demon-
strated high similarity (99.6%) to T. theileri from sitatunga
Tragelaphus spekii (Sclater, 1863) from Cameroon (FM202489)
and Trypanosoma sp. from horse fly Hybomitra tarandina (L.,
1758) from Russia (MK156791). The derived sequences of
Trypanosoma spp. were submitted to the GenBank database
under the accession numbers: MT394044, MT393974, MT393977,
MT393982, MT393983, MT393984 and MT393991.
Table 2. Specimens of Lipoptena cervi and Lipoptena fortisetosa collected from different localities in Poland, analysed for Trypanosoma spp. infection (number and
percentage of infection)
Processed deer ked (species and sex)
Location
Overall
Nadleśnictwo Strzałowo
(Puszcza Piska Forest) Białowieża Hajnówka
Smolany Sadek
(winged deer ked)
L. cervi Analysed 27 6 9 10 52
Infected 5 0 2 3 10
Analysed 38 3 8 17 66
Infected 7 2 3 2 14
Total Analysed 65 9 17 27 118
Infected 12 2 5 5 24 (20.33%)
L. fortisetosa Analysed 16 8 –– 24
Infected 10 3 –– 13
Analysed 11 2 –– 13
Infected 4 1 –– 5
Total Analysed 27 10 –– 37
Infected 14 4 ––18 (48.64%)
1632 Joanna Werszko et al.
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Discussion
First records of L. fortisetosa in Poland date back to the 1980s
(Borowiec and Zatwarnicki, 1989). However, due to its dynamic
spread observed in recent years, it can be considered an invasive
species in the country; furthermore, owing to the possibility that it
could be introduced to other European countries with its host, it
could also be considered as an alien invasive species (Kowal et al.,
2016). In addition, the presence of these blood-sucking flies may
play a significant role in the circulation and maintenance of
vector-borne pathogens; they may also demonstrate significant
vector competence for infectious agents, despite not being well
recognized. Indeed, as the deer keds shed their wings when they
find a suitable host and settle for the rest of their lives, the possi-
bility of transmitting a pathogen from one host to another is
believed relatively low. Nevertheless, deer keds are recognized as
an important group of haematophagous insects for both veterin-
ary parasitology and medical reasons since they have been proved
able to transmit Bartonella spp., Borrelia spp. and Trypanosoma
(Megatrypanum) spp. (Böse and Petersen, 1991; Dehio et al.,
2001; Vichová et al., 2011; Szewczyk et al., 2017), they are recog-
nized as an important group of haematophagous insects for both
veterinary parasitology and medical reasons. It is important to
note that most part of the studies performed so far have con-
cerned L. cervi (Halos et al., 2004), while much less is known
about the competence of L. fortisetosa as a pathogen vector,
and less data have been acquired on trypanosomes in other
Lipoptena species.
In the current study, the overall prevalence of infection with
Trypanosoma spp. was 27.09% (42/155) among deer keds. The
highest positivity was reported in the case of L. fortisetosa
(48.64%, 18/37). The presence of Trypanosoma spp. was detected
in 24 of the 118 (20%) L. cervi. Including the tested group of
winged L. cervi, five out of 27 (18.51%) were infected.
Böse and Petersen (1991) report the identification of
Megatrypanum trypanosomes in the midgut and hindgut of 9/37
L. cervi collected from deer. High levels of infection with other
pathogens have also been observed in L. cervi; the presence of
Bartonella spp. was observed in 75.12% of L. cervi collected
from deer in Poland (Szewczyk et al., 2017). Víchová et al.
(2011) detected A. phagocytophilum infection in only two out
of 19 tested deer keds collected from deer in Slovakia.
Pathogens present in L.fortisetosa have not been well studied.
Lee et al.(2016) report the presence of Coxiella spp., Theileria
luwenshuni and Theileria ovis in L. fortisetosa in inland regions
of South Korea.
The studies mentioned above were based on wingless flies col-
lected from hosts. In this case it was not known whether the fly
was infected or the pathogen was present only in the host blood
withdrawn from the vertebrate. We provide the first molecular
evidence for Trypanosoma spp. DNA in five out of 27 investigated
winged deer keds (L. cervi) collected from vegetation. The winged
Fig. 2. Features on the thorax and female terminalia of Lipoptena cervi (A, B) and L. fortisetosa (C, D).
Parasitology 1633
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flies had not fed since they left the pupae; therefore, we suppose
the only explanation for the presence of trypanosome DNA is
transovarial transmission. The present findings support the
hypothesis of a transstadial transmission of Trypanosoma spp.
in these species of Lipoptena, but the topic remains open at this
stage of research. Korhonen et al.(2015) report the presence of
Bartonella DNA in L. cervi pupae and winged adults, thus sup-
porting the potential of deer ked for vector competence of
Bartonella spp. and indicating their transstadial transmission;
their findings also demonstrate that adult winged deer keds do
harbour bartonellae. Víchová et al.(2011) found winged deer
keds to be negative for the presence of A. phagocytophilum, indi-
cating that they do not serve as competent vectors of this patho-
gen. Similarly, all L. cervi collected in a field study by de la Fuente
et al.(2005) were found to be negative for the presence of A. pha-
gocytophilum and A. marginale. In contrast, Bartonella spp. was
found to be present in pools of winged unfed deer keds (Dehio
et al., 2004; Duodu et al., 2013) in midgut bacterial aggregates
of Bartonella schoenbuchensis. It is therefore possible that deer
keds support the replication of the pathogen and serve as poten-
tial biological vectors (Dehio et al., 2004).
Trypanosomes of the subgenus Megatrypanum do not show
high specificity for the insect as a host, and the same species of
trypanosomes may infect different flies (Böse et al., 1987). The
absence of A. phagocytophilum in winged deer keds collected
from vegetation and the presence of Bartonella pathogens may
indicate that these pathogens demonstrate host specificity for
the vector. Until now, deer keds have not been shown to transmit
any infectious agents to humans. The causes underlying the vari-
ation of prevalence and intensity of blood parasites are poorly
known (Sol et al., 2000). The prevalence of microparasite infec-
tion in an insect can arise both from factors intrinsic to the
host, such as genotype resistance, biochemical immunity pro-
cesses, behaviour or state of health and from extrinsic factors,
such as differences in exposure to vectors (Sol et al., 2000).
All isolates (except to isolate 1LfHka) obtained in this study
were identical to the Trypanosoma spp. sequences in the
GenBank database. Moreover, isolates Lf4KG and Lf15KG were
identical to those of T. theileri isolates obtained previously from
cattle and European bison inhabiting the same area.
It seems there is a growing range of arthropods that might
serve as potential vectors for transmissible pathogens. The blood-
sucking flies, including insects from the genus Lipoptena,are
important potential vectors that can disseminate a variety of
pathogens in natural foci of transmission disease.
In conclusion, the current study examines the presence of
Megatrypanum trypanosomes using molecular methods in L.
cervi and L. fortisetosa (Hippoboscidae). It describes the first
detection of Trypanosoma spp. in L. fortisetosa, and first recorded
presence of trypanosomes in an unfed winged L. cervi from vege-
tation. The study also identifies two new localities where L. forti-
setosa was recorded in Poland: one in the Białowieża Primeval
Forest, and another in the Strzałowo Forest Inspectorate
(Puszcza Piska Forest), both in north-eastern Poland.
Acknowledgements. The authors would like to express their gratitude to Marek
Bogdaszewski, head of the Witold Stefański Institute of Parasitology of the Polish
Academy of Sciences (Deer farm in Kosewo Górne), for enabling the samples to
be collected from red deer. We are grateful to Zbigniew Ciepluch, Forest
Inspector of the Strzałowo Forest Inspectorate for enabling sample collection.
Financial support. This study was supported by the MINIATURA 2 grant
no. 2018/02/X/NZ8/00037 Research Project, funded by the National Science
Centre, Poland.
Conflict of interest. The authors declare no conflicts of interest.
Ethical standards. Not applicable.
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... Lipoptena fortisetosa is a species typical of Asian deer: the Siberian roe deer (Capreolus pygargus) and the eastern deer (Cervus nippon) [11,12]. The first reports of its presence in Europe came in the 1960s [7], and its progressive range expansion has been observed over the past years [13,14]. In Poland, the first observations of L. fortisetosa were made in Lower Silesia at the end of the 1980s [15]. ...
... Depending on the geographical region, there are differences among the main hosts of Lipoptena species. For example, moose is the main host for L. cervi in Scandinavia, and Cervus elaphus and Capreolus capreolus are indicated as important hosts of this species in different regions of Europe [2,14,16,[23][24][25] (own observations). In contrast, there are no clear data on the dominant host of L. fortisetosa, although it is suggested that this role is probably played by roe deer [7]. ...
... The greatest epidemiological importance is attributed to Bartonella schoenbuchensis, Borrelia burgdorferi, and Anaplasma phagocytophilum [29][30][31][32][33][34] as well as Trypanosoma (Megatrypanum) spp. [14,35]. People entering natural habitats of Lipoptena spp. ...
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... Moreover, they can play an important role as carriers of pathogens, mainly Anaplasma spp., Bartonella spp., Borrelia spp., Coxiella spp., Theileria spp., and Trypanosoma spp. [11][12][13][14][15][16][17]. Recently, the Asian species L. fortisetosa has colonized most European countries, including Italy, where it is competing with L. cervi for territories and host microniches [18]. ...
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... Lipoptena fortisetosa may be the potential biological vector for Trypanosoma spp. [35]. Currently, Bartonella spp. is the most comprehensively studied pathogen carried by Lipoptena spp., which appears to pose the greatest risk in infestation with these ectoparasites [40][41][42][43]. ...
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... Lipoptena fortisetosa has been found to mechanically carry pathogens, i.e., Coxiella-like bacteria (CLB), Theileria luwenshuni, and Theileria ovis [36]. In addition, very recently, both L. cervi and L. fortisetosa specimens from Poland were found positive to Trypanosoma DNA [37]. ...
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Background Deer keds are obligatory haematophagous parasites of large homeothermic animals, particularly cervids. Two of the five known species occurring in Europe—Lipoptena cervi (Linnaeus) and L. fortisetosa Maa—are known to have a relatively wide distribution. Lipoptena fortisetosa is considered to have been introduced into Europe with sika deer from the Eastern Palaearctic and is continuously expanding its range. Little is known about the medical importance of deer keds, but they can cause hair loss in cervids and are suspected to be vectors of several diseases. New information Details of the distribution of Lipoptena fortisetosa in Europe, including its northernmost record, are provided. This species has been shown to have a viable population in Southern Estonia. Furthermore, the differences from allied L. cervi are discussed, based on morphological and molecular characters.
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Hippoboscidae flies parasitize various animal species. Knowledge about these insects remains sparse, although they are known to cause stress and damage to their hosts, and can also accidentally infest humans, causing different sanitary risks. Research conducted in Tuscany assessing the biology and distribution of Lipoptena cervi (Linnaeus, 1758) (Diptera: Hippoboscidae), the most common ectoparasite of ungulates in Italy, revealed the presence of Lipoptena fortisetosa Maa, 1965 in Italy for the first time. This study includes a morphological comparative description of L. cervi and L. fortisetosa, emphasizing the peculiar differences between the two species to facilitate their accurate identification. The most pertinent morphological differences between the two species are highlighted, such as the external features of the antennae, distribution of bristles, and different features in the external genitalia. In both species, scanning electron microscopy of mouthparts revealed strong adaptive convergence in the feeding apparatus. Modified palps and a very thin proboscis are described in relation to feeding behaviour.
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The aim of this study was to identify the species of ked infesting dogs in the cities of central Poland. A total of 510 dogs were observed between June and September 2015. The presence of keds was noted in 182 (35.7%) animals. Keds were more prevalent in female (38.0%) than in male (33.2%) dogs, and were more frequently found in animals younger than 1 year (46.2%) and in long-haired dogs (36.6%). The body areas most heavily colonized by keds were the groin (35.4%) and neck (21.4%). A total of 904 keds were isolated from dogs, including Hippobosca equina (Diptera: Hippoboscidae) (17.2%), Lipoptena cervi (Diptera: Hippoboscidae) (32.0%), and two species not previously encountered in Poland: Hippobosca longipennis (45.0%) and Lipoptena fortisetosa (5.9%). Hippoboscidae may act as vectors of pathogens and any shifts in their geographic range may lead to the spread of new diseases affecting animals.