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Molecular characterization of Lipoptena cervi from environmental samples collected in Poland

ABSTRACT: The activity of Lipoptena cervi has intensified in Poland in recent years. The population genetics of this ectoparasite in Poland has never been described in the literature. The objectives of this study were to investigate the population genetics of L. cervi in selected regions of Poland, to evaluate molecular differences between L. cervi populations, and to determine phylogenetic relationships with other L. cervi sequences obtained in previous studies. In 2019, louse flies were sampled in natural mixed forests in five Polish voivodeships. Seven samples of L. cervi were collected from each voivodeship, and a total of 35 insects were analyzed molecularly. In the first step, Lipoptena spp. were identified to species level under a stereoscopic microscope. A fragment of the rRNA 16S gene was used as a marker to identify L. cervi by the PCR assay. The sequences were assigned accession numbers MT337409 to MT337416. A total of eight haplotypes were identified, two of which were dominant. In the obtained sequences, intraspecific pairwise genetic distances varied between 0.000 and 0.0496 (m = 0.0135; SD = 0.0149; SE = 0.0006; V = 110.11). Mean interpopulation diversity was d = 0.0135 (SE = 0.0027). The acquired nucleotide sequences were highly similar to the sequences from the Czech Republic (MF495940, AF322437), Lithuania (MN889542-MN889544) and Poland (MF541726–MF541729). The similarity with GenBank sequences ranged from 97.24% to 100%. This study revealed two dominant haplotypes of L. cervi in Poland, MT337410 and MT337413. Fragments of the analyzed sequences were detected in only one voivodeship. These findings suggest that the two dominant sequences are the oldest sequences that gave rise to the locally identified haplotypes. The lack of significant correlations with the sequences obtained in regions situated west of the research sites suggests the presence of other genetic populations in Europe. Graphical abstract Image 1 Highlights • Eight new haplotypes with 0.000–0.0496 intraspecific pairwise genetic distance.• Similarity with sequences from GenBank ranged from 97.24% to 100%.• Two dominant haplotypes of L.cervi were observed in Central Europe.• Part of the sequences have been discovered in single studied location.• The lack of significant connections with sequences obtained from West Europe.

SUBMITTER: Ga?ecki R

PROVIDER: S-EPMC7776119 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Project description:The bacteria of the genus Bartonella are obligate parasites of vertebrates. Their distribution range covers almost the entire world from America, Europe, Asia to Africa and Australia. Some species of Bartonella are pathogenic for humans. Their main vectors are blood-sucking arthropods such as fleas, ticks and blood-feeding flies. One such dipteran able to transfer vector-borne pathogens is the deer ked (Lipoptena cervi) of the family Hippoboscidae. This species acts as a transmitter of Bartonella spp. in cervid hosts in Europe.In the present study, 217 specimens of deer ked (Lipoptena cervi) were collected from 26 red deer (Cervus elaphus) hunted in January 2014. A short fragment (333 bp) of the rpoB gene was used as a marker to identify Bartonella spp. in deer ked tissue by PCR test. A longer fragment (850 bp) of the rpoB gene was amplified from 21 of the positive samples, sequenced and used for phylogenetic analysis.The overall prevalence of Lipoptena cervi infection with Bartonella spp. was 75.12% (163/217); 86.67% (104/120) of females and 60.82% (59/97) of males collected from red deer hunted in the Strza?owo Forest District in Poland (53°45'57.03?N, 21°25'17.79?E) were infected. The nucleotide sequences from 14 isolates (Bartonella sp. 1) showed close similarity to Bartonella schoenbuchensis isolated from moose blood from Sweden (GenBank: KB915628) and human blood from France (GenBank: HG977196); Bartonella sp. 2 (5 isolates) and Bartonella sp. 3 (one isolate) were similar to Bartonella sp. from Japanese sika deer (GenBank: AB703149), and Bartonella sp. 4 (one isolate) was almost identical to Bartonella sp. isolated from Japanese sika deer from Japan (GenBank: AB703146).To the best of our knowledge, this is the first report to confirm the presence of Bartonella spp. in deer keds (Lipoptena cervi) in Poland by molecular methods. Bartonella sp. 1 isolates were most closely related to B. schoenbuchensis isolated from moose from Sweden and human blood from France. The rest of our isolates (Bartonella spp. 2-4) were similar to Bartonella spp. isolated from Japanese sika deer from Japan.

Project description:BackgroundCoxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes.ResultsTo assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples.ConclusionsThis newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

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Molecular characterization of Lipoptena cervi from environmental samples collected in Poland

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