Molecular pathogen screening of louse flies (Diptera: Hippoboscidae) from domestic and wild ruminants in Austria

Abstract

Background: Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria.

Methods: Louse flies were collected from naturally infested cattle (n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses.

Results: A total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida.

Conclusions: Molecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.

Keywords: Barcoding; Bartonella; Hippobosca equina; Hippoboscidae; Keds; Lipoptena cervi; Louse flies; Melophagus ovinus; Ruminants; Vector-borne pathogens.

Fig. 1

Origin of hippoboscid flies from Austria. Collection sites of hippoboscid flies from domestic and wild ruminants in Schwaz (A), Kufstein (B), Bludenz (C), Leobersdorf (D), Saalfelden (E) and Eisenstadt (F), in Austria. See the main text for further information

Fig. 2

Hippoboscid flies for molecular pathogen screening. Representative individuals of Hippobosca equina (A), Melophagus ovinus (B) and Lipoptena cervi (C) collected from domestic and wild ruminants in Austria

Fig. 3

Single and co-infections in pathogen-positive hippoboscid flies. Percentage of hippoboscid flies positive to one, two or three pathogens in the same individual of Hippobosca equina, Melophagus ovinus and Lipoptena cervi collected from domestic and wild ruminants in Austria

Fig. 4

Genetic diversity of Bartonella detected in hippoboscid flies. Median-joining haplotype network of the gltA sequences (338 bp) of selected Bartonella spp. from the present and previous studies showing their geographical distribution (A) and the reported hosts (B). Circles represent haplotypes, and numbers within the circles represent the number of individuals. If no number is shown, then only one individual is represented. Labels next to circles specify representative GenBank accession numbers of the haplotypes; white circles represent intermediate nodes; bars on branches interconnecting haplotypes represent the number of substitutions. Asterisks mark haplotypes detected in the present study

Fig. 5

Genetic diversity of Trypanosoma detected in hippoboscid flies. Median-joining haplotype network of the 18S rRNA sequences (779 bp) of selected Trypanosoma spp. from the present and previous studies showing their geographical distribution (A) and the reported hosts (B). Circles represent haplotypes and numbers within the circles represent the number of individuals. If no number is shown, then only one individual is represented. Labels next to circles specify representative GenBank accession numbers of the haplotypes; white circles represent intermediate nodes; bars on branches interconnecting haplotypes represent the number of substitutions. Asterisks mark haplotypes detected in the present study