Molecular screening of vector-borne pathogens in ectoparasites from bats in Ukraine

Abstract

Bats (Mammalia: Chiroptera) represent the second-most diverse order of mammals after rodents, being described as hosts for a wide range of bloodsucking arthropods that are able to circulate bacterial, protozoal and viral agents. Bats join other mammals in facilitating the spread of pathogens and are natural reservoir hosts of a large variety of zoonotic diseases with the ability to cross species barriers. In addition, due to their migratory habits, bats can facilitate the long-distance dispersal of pathogens.
The aim of this study was to perform a molecular screening for selected vector-borne pathogens in ectoparasites collected from bats in Ukraine. Bats from a mine system and from urban (Kharkiv city) and rural areas (Kharkiv region) in Ukraine were captured and checked for ectoparasites. The mines (Liptsy mines) are used by bats for swarming and hibernation. In Kharkiv city area bats were examined during hibernation season, and in countryside areas breeding colonies from buildings were checked. From the total number of collected ectoparasites, so far 143 samples were morphologically identified as Nycteridopsylla eusarca fleas (n=100) and Carios vespertilionis soft ticks (n=43) then screened for pathogens. Nycteridopsylla eusarca fleas were collected only from Nyctalus noctula (urban area) bat species while C. vespertilionis ticks were collected from three different bat species from the mines: Myotis dasycneme (n=28), Myotis daubentonii (n=2) and Plecotus auritus (n=1), and two bat species from rural sites: Pipistrellus kuhlii (n=1) and Pipistrellus pygmaeus (n=11). All ectoparasites were tested individually by PCR for Rickettsia spp., Bartonella spp., Ehlichia/Anaplasma spp. and Babesia spp. while only C. vespertilionis samples were tested by nested PCR for Borrelia spp. The amplified PCR products were sequenced for species identification.
Only C. vespertilionis samples were positive for Rickettsia spp. and following sequencing 16.3% (7/43) of samples showed 100% identity to Rickettsia parkeri (GenBank accession number: CP040325.1). Six of the positive ticks were found on Pipistrellus pygmaeus bat species while one sample was found on Pipistrellus kuhlii. Bartonella spp. was detected only in N. eusarca (7%; 7/100) and after sequencing, three samples showed 99 to 99.4% identity to uncultured Bartonella sp. (access. no.: MK140218.1) previously found in C. vespertilionis while the other four samples had 100% identity to uncultured Bartonella sp. (access. no.: AJ871615.1) previously reported in the blood of Nyctalus noctula. Ehrlichia/Anaplasma spp. PCR showed 58 positive samples: 56% (56/100) of N. eusarca and 4.7% (2/43) of C. vespertilionis. Selected Ehlichia/Anaplasma spp. positive samples were sequences and the obtained sequences from N. eusarca were identical to Wolbachia endosymbiont (access. nos.: MH618381.1 and EU315781.1) and an uncultured bacterium clone layman_j06 (access. no.: DQ980970.1). The sequences from C. vespertilionis matched Candidatus Ehrlichia shimanensis (AB074459.1) and uncultured Anaplasma sp. clone Erz1600 (access. no.: MT601947.1). The results for Ehrlichia/Anaplasma spp. might suggest that the used PCR reaction is not specific for the identification of the pathogenic species. All ectoparasite samples were negative for Babesia spp., while Borrelia spp. was detected in 4.7% (2/43) C. vespertilionis samples without identifying the Borrelia species after sequencing.
In this study we report for the first time in Ukraine the molecular detection of several bacterial agents in two species of ectoparasites found on six species of bats. The data presented extend the knowledge on the distribution of ectoparasite species on bats and their involvement to potentially circulate pathogenic agents.

Full text

Cristian Răileanu1, Oliver Tauchmann1, Anton Vlaschenko2,3, Denys Muzyka3,4, Valeria Bohodist5, Serhii Filatov4,
Cornelia Silaghi1,6
1 Institute of Infectology, Friedrich-Loeffler-Institut, Suedufer 10, 17492, Greifswald-Insel Riems, Germany
2 Bat Rehabilitation Center of Feldman Ecopark, 62340 Lesnoye, Kharkiv Region, Ukraine
3 Institute of Natural Sciences, Department of Zoology, H.S. Skovoroda Kharkiv National Pedagogical University, Valentynivska st., 2, Kharkiv, 61168, Ukraine
4 National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, Pushkinska St., 83, Kharkiv, 61023, Ukraine.
5 Bila Tserkva National Agrarian University, Veterinary Medicine department, Stavishchanskaya st., 126, 09111, Bila Tserkva, Ukraine
6 Department of Biology, University of Greifswald, Domstraße 11, 17489, Greifswald, Germany
Molecular screening of vector-borne pathogens in ectoparasites
from bats in Ukraine
Conclusions
• This study reports for the first time in Ukraine the molecular detection of several bacterial agents in two species of
ectoparasites found on bats.
• Detection of Rickettsia parkeri in Carios vespertilionis corroborated with previous reports indicating that this soft
tick can bite humans, suggests the role of this species as a potential source of pathogens for humans.
• The data presented extend the knowledge on the distribution of ectoparasite species on bats and their involvement
to potentially circulate pathogenic agents.
References
1. Calisher, C. H., Childs, J. E., Field, H. E., Holmes, K. V. & Schountz, T. Bats: im-
portant reservoir hosts of emerging viruses. Clin Microbiol Rev. 19, 531–545 (2006).
2. Hornok, S. et al. DNA of piroplasms of ruminants and dogs in Ixodid bat ticks. PLoS
One 11, e0167735 (2016).
Bats (Mammalia: Chiroptera) represent the second-most diverse order of mammals after rodents, being described as hosts for a wide range of
bloodsucking arthropods that are able to circulate bacterial, protozoal and viral agents. Bats join other mammals in facilitating the spread of
pathogens and are natural reservoir hosts of a large variety of zoonotic diseases with the ability to cross species barriers [1,2]. In addition, due to
their migratory habits, bats can facilitate the long-distance dispersal of pathogens.
The aim of this study was to perform a molecular screening for selected vector-borne pathogens in ectoparasites collected from bats in Ukraine.
Context and Objective
Bats were captured and checked for ectoparasites at three different locations in
Ukraine:
• A mine system (Liptsy mines) used by bats for swarming and hibernation.
• Urban area (Kharkiv city): bats were examined during hibernation season.
• Rural areas (Kharkiv region): the breeding colonies from buildings were checked.
From the total number of collected ectoparasites, so far 143 samples were mor-
phologically identified and screened for pathogens: 100 Carios vespertilionis (short-
legged bat tick) and 43 Nycteridopsylla eusarca (noctule bat flea) (Table below).
All ectoparasites were tested individually by:
• PCR for Rickettsia spp., Bartonella spp., Ehlichia/Anaplasma spp. and Babesia spp.
• Carios vespertilionis ticks were tested additionally by nested PCR for Borrelia spp.
The amplified PCR products were sequenced for species identification.
Sequencing of Carios vespertilionis samples
Sequencing of Nycteridopsylla eusarca samples
1. COLLECTION OF ECTOPARASITES FROM BATS
2. SCREENING OF ECTOPARASITES FOR PATHOGENS
Bat host species Carios
vespertilionis
Nycteridopsylla
eusarca
Collection area
Myotis dasycneme 28 0 Mine system
Myotis daubentonii 2 0 Mine system
Nyctalus noctula 0 100 Urban area
Pipistrellus kuhlii 1 0 Rural area
Pipistrellus pygmaeus 11 0 Rural area
Plecotus auritus 1 0 Mine system
Total 43 100
Pathogen Target
gene Positive
samples Sequenced
samples Identified species
Rickettsia spp. ompA 7 7 Rickettsia parkeri
Anaplasma/
Ehrlichia spp.
16S rRNA
2
1 Candidatus E. shimanensis
1 Uncultured Anaplasma sp.
Borrelia spp. 16S-23S
IGS 2 2 -
Pathogen Target
gene Positive
samples Sequenced
samples Identified species
Bartonella spp. gltA 10 7 Uncultured Bartonella sp.
Anaplasma/
Ehrlichia spp.
16S rRNA
56 2 Uncultured bacterium
6 Wolbachia endosymbiont
Methodology and Results
R. parkeri ompA - C. vespertilionis Uncultured Anaplasma sp. 16S rRNA C. Ehrlichia shimanensis 16S rRNA Uncultured Bartonella sp. gltA
C. vespertilionis C. vespertilionis N. eusarca
ML + Tamura 3-parameter ML + Hasegawa-Kishino-Yano ML + Kimura 2-parameter using a discrete Gamma distribution
ML + Tamura 3-parameter using a discrete Gamma distribution
The phylogenetic tree based on the ompA partial sequence demonstrates that all
the Rickettsia sequences detected in Carios vespertilionis in this study belong to
Rickettsia parkeri, clustering with species within the spotted fever group (SFG).
The phylogenetic tree based on the 16s rRNA partial sequence indicates
that the Anaplasma sequence detected in Carios vespertilionis in this study
clusters in a clade that includes Uncultured Anaplasma and Anaplasma ovis.
The phylogenetic analysis based on the 16s rRNA partial sequence indica-
tes that the Ehrlichia sequence detected in Carios vespertilionis clusters
in a clade that includes Candidatus Ehrlichia shimanensis, Uncultured Ehr-
lichia, Ehrlichia minasensis or Ehrlichia canis.
The phylogenetic analysis based on the gltA partial sequence
shows that three Bartonella sequences detected in Nycteri-
dopsylla eusarca cluster in a clade that includes Uncultured Bar-
tonella, Bartonella washoensis and Bartonella sp. The additional
four obtained sequences cluster in a separate clade.
Nycteridopsylla eusarca
http://britishfleas.myspecies.info
Carios vespertilionis
https://biology.stackexchange.com
Myotis dasycneme
https://batslife.eu
Nyctalus noctula
https://batslife.eu
Myotis daubentonii
https://batslife.eu
Pipistrellus kuhlii
https://batslife.eu
Pipistrellus pygmaeus
https://batslife.eu
Plecotus auritus
https://batslife.eu
3. PHYLOGENETIC ANALYSIS