Genetic characterization of Eucoleus aerophilus from different hosts and Countries

Abstract

The parasitic nematode Eucoleus aerophilus (syn. Capillaria aerophila) affects the respiratory tract of different carnivore species. Infected animals may display minimal respiratory signs to sneezing, wheezing, and chronic dry or moist cough; heavy parasite burdens lead to bronchopneumonia, respiratory failure and may be life-threatening (Traversa D et al, 2011, Parasitol Res, 109: S97-104). Occasionally E. aerophilus may cause lung damages in humans and can even mimick carcinoma-like masses (Lalosevic D et al, 2008, Am J Trop Med Hyg, 78: 14-16). Despite the importance of E. aerophilus and the recent evidence of an increasing trend of the infection in companion animals (Traversa D et al, 2010, Parasit Vectors, 3: 62), several aspects of lung capillariosis remains to be elucidated, also for the absence of genetic studies. AIM: this work based on molecular analysis of target region of the mitochondrial DNA of E. aerophilus isolates from different hosts and countries. The aim was to gain insights into the population structure, geographical distribution and possible affiliation of isolates of this nematode with wild and domestic animals. METHODS: single adult nematodes from 14 red foxes (4 from Romania, 3 from Portugal, 3 from Serbia, 3 from UK and 1 from Canada), from 3 beech marten from Portugal, and 44 egg batches from 33 dogs, 10 cats and 1 fox from Italy, were subjected to a PCR-coupled sequencing protocol to amplify a ~344 bp informative region within mitochondrial partial cytochrome c oxidase subunit 1 gene (pcox1) of E. aerophila. The open reading frames were confirmed by conceptual translation using the invertebrate mitochondrial code by MEGA5 (Tamura K et al, 2011, Mol Biol Evol, 28: 2731-2739). The evolutionary relationships of taxa belonging to Capillarinae available in GenBankTM with the herein generated sequences was inferred using the Neighbor-Joining method (Saitou N, Nei M, 1987, Mol Biol Evol 4: 406-425). RESULTS: Sixty-one amplicons of all E. aerophilus isolates were sequenced: fifteen sequence types (i.e. haplotypes I–XV) were found according to the different hosts and geographical origin. The most prevalent haplotypes from all collection sites were I (n = 36.59%), haplotype II and III (n = 8, 13.1%), followed by the other eleven haplotypes (n = 17, 27.8%). Haplotype I were represented by sequences of E. aerophilus found in dogs, cats and the fox from Italy, as well in foxes coming from Serbia and Romania. All 15 haplotypes were aligned over 299 positions. Sequences included 274 conserved and 25 variable sites, of which 21 were singletons and four parsimony-informative. The majority of the variable sites (n = 18; 72%) was at the third codon position, whereas the remainder (n = 7; 28%) at the first and second codon positions. Almost all the intraspecific nucleotide variations were synonymous. Amino acid sequences had an open reading frame in first position and did not contain a further stop codon. The mean intraspecific nucleotide differences among the haplotypes was of 1.5%, ranging from 0.4 to 5.1% in haplotype VIII (e.g., dogs, Italy) vs haplotype XIV (e.g., foxes, UK). All 15 haplotypes clustered all together, with strong statistical support (≥49%), and were separated from sequences representing other Capillarinae, inferring that all haplotypes, irrespective of their geographical and host origin, represented different haplotypes of E. aerophilus. CONCLUSIONS: This study identified significant genetic variation among sequences of E. aerophilus found in domestic and wild animals from different countries. Although 15 distinct haplotypes were identified, the sequence variation found in the cox1 region examined was lower than that among nematodes for which homologous sequence data are available in public databases. The existence of these population variants might be due to a higher mutation rate in this cox1 region to inbreeding within particular host populations and geographical regions. Some haplotypes were shared between domestic and wild animals in different areas, thus indicating that some E. aerophilus populations co-infect pets and wildlife. In summary, these results represent a foundation for further approaches to address epidemiology, phylo-geography and population-genetic make-up of this neglected lungworm in different hosts and countries.