Urban emergence of Dermanyssus gallinae lineage L1 and Ornithonyssus sylviarum in Hungary: phylogenetic differentiation between the roles of migrating vs transported synanthropic birds.

BACKGROUND: Among Dermanyssoidea, the chicken red mite (Dermanyssus gallinae) and the northern fowl mite (Ornithonyssus sylviarum) are considered to be the cause of high economic losses endured by the poultry industry in the Holarctic region, with O. sylviarum predominating in North America and D. gallinae in Europe. Both species have a short life-cycle (thereby allowing a rapid build-up of massive infestations), a wide range of hosts, synanthropic presence and the ability to bite humans. The aim of this study was to analyze dermanyssoid mite specimens, collected in two human dwellings and two racing pigeon premises in different urban areas in Hungary, with molecular-phylogenetic methods. METHODS: Mite species were identified morphologically. This was followed by DNA extraction and molecular-phylogenetic analyses of selected mites, based on the cytochrome c oxidase subunit I (cox1) and 28S rRNA (28S) genes. RESULTS: Mites that had invaded a home from a pigeon nest and were linked to human dermatitis were morphologically and molecularly identified as D. gallinae special lineage L1. Specimens collected at all other sampling sites were identified as O. sylviarum, including mites that had invaded a home from a house martin (Delichon urbicum) nest, as well as those which were collected from racing pigeons. House martin- or pigeon-associated O. sylviarum specimens showed the highest sequence identity and closest phylogenetic relationship with conspecific mites reported in GenBank from Israel or Canada, respectively. CONCLUSIONS: Detailed morphological and molecular-phylogenetic analyses of D. gallinae lineage L1 confirmed its status as a cryptic species within D. gallinae (s.l.). Taking into account the well-documented latitudinal migratory routes of house martins between Hungary and Africa, O. sylviarum associated with this bird species most likely arrived on its host from the eastern Mediterranean region. On the other hand, mites collected from pigeons in Hungary showed cox1 genetic homogeneity with North American O. sylviarum, which can only be explained by a long-distance (west-to-east intercontinental) connection of birds and their mites as part of human activity (e.g. transportation to exhibitions or trading). In summary, this is the first molecularly confirmed and phylogenetically analyzed case of O. sylviarum infestation of birds in Hungary, implicating urban environment and involving distant parts of the country. This is also the first report of D. gallinae lineage L1 in central Europe.

First records and molecular-phylogenetic analyses of three tick species (Ixodes kaiseri, Hyalomma lusitanicum and Ornithodoros coniceps) from Malta.

The Maltese islands are situated south of mainland Europe and north of Africa, therefore are expected to share tick species and tick-borne pathogens with both continents. This situation highlights the importance of studying ticks in this country. Nevertheless, the tick fauna of Malta appears to be a seldom investigated issue, with hitherto only five tick species reported in the country. Here, as part of a tick collection campaign continuing since 2016 in Malta, three tick species new to the country are reported and analyzed in comparison with GenBank data. Ixodes kaiseri (collected from North African hedgehog in Malta) had unique cytochrome c oxidase subunit I (cox1) and 16S rRNA gene haplotypes (with 98.1-99.3 % sequence identity to I. kaiseri from Europe and China). Phylogenetically, these haplotypes from Malta clustered separately from other, mainland-associated haplotypes, with high support. On the other hand, Ornithodoros coniceps (collected from domestic chicken in Malta) had identical or nearly identical cox1 and 16S rRNA gene haplotypes with soft ticks reported from France, northern Africa and western African islands, similarly to Hyalomma lusitanicum (collected from rabbit and cat in Malta) in comparison with conspecific ticks in Spain and Portugal. These results are most likely related to differences in host associations and corresponding translocality of these three tick species. Taken together, results of the present study add three new tick species to those five already known to be present in Malta.

High mitochondrial sequence divergence in synanthropic flea species (Insecta: Siphonaptera) from Europe and the Mediterranean.

BACKGROUND: Adult fleas are haematophagous ectoparasites of warm-blooded vertebrates, particularly mammals. Among them, the cat flea (Ctenocephalides felis) and the human flea (Pulex irritans) have high veterinary-medical significance, owing to their cosmopolitan distribution and role in the transmission of important vector-borne pathogens. While the taxonomy of Ct. felis has been investigated on a morphological basis during the past decades, its molecular-phylogenetic analyses have been only recently conducted. This study expands the knowledge on Ct. felis from hitherto less studied geographical regions, and includes representatives from additional flea families, less investigated with molecular approaches. METHODS: Fleas were collected in four countries of the Mediterranean Basin (Croatia, Italy, Malta and Israel), as well as in Hungary, from domestic and wild carnivores, rodents and humans. The DNA extracts of representative fleas (n = 148), belonging to ten species of eight genera, were used for PCR amplification of part of their cytochrome c oxidase subunits 1, 2 (cox1, cox2) and 18S rRNA genes, followed by sequencing and phylogenetic analyses. RESULTS: The majority (65.6%) of Ct. felis felis cox2 sequences showed 99.4-100% similarity to each other (haplogroup A), whereas those from Malta and Israel had 98.1-98.7% sequence similarity (haplogroup B), and a third sequence from Israel (haplotype C) had as low as 96.3% sequence similarity in comparison with a reference sequence from group "A". Except for the shape of the head, no consistent morphological differences (e.g. in chaetotaxy) were found between haplogroups "A" and "C". Haplotypes of Ct. canis were genetically more homogenous, with 99.6-100% sequence similarity to each other. However, when P. irritans collected from humans was compared to those from three species of wild carnivores, these only had 96.6% cox2 similarity. The mouse flea, Leptopsylla segnis and the northern rat flea, Nosopsyllus fasciatus were both shown to have haplotypes with low intraspecific cox2 similarities (96.2 and 94.4%, respectively). Taken together, differences between mitochondrial lineages within four flea species exceeded that observed between two Chaetopsylla spp. (which had 97.3% cox2 similarity). The topologies of cox1 and cox2 phylogenetic trees were in line with relevant sequence comparisons. Conversely, 18S rRNA gene analyses only resolved differences above the species level. CONCLUSIONS: Ctenocephalides felis felis, P. irritans, L. segnis and N. fasciatus were shown to have such a high level of mitochondrial gene heterogeneity, that the uniformity of these flea taxa should be reconsidered. Although the present results are limited (especially in the case of L. segnis and N. fasciatus), there appears to be no geographical or host restriction, which could explain the divergence of these genetic lineages.

Infestation of Rhipicephalus sanguineus sensu lato on cats in Malta.

Studies on cats as hosts of Rhipicephalus sanguineus sensu lato (s.l.) are scarce. Cats are regarded as infrequent hosts of this species complex, and usually only when dogs are also present. In order to compare the occurrence of developmental stages and mitochondrial DNA haplotypes of R. sanguineus s.l. on cats and other domestic or synanthropic mammalian species, 540 ticks were collected from cats, dogs, hedgehogs and one goat. Collections were made from April to September in 2016 and 2017, from 20 locations in Malta in southern Europe. The sampling sites included six cat colonies, where no dogs were present. Compared to adults, significantly more immatures of R. sanguineus s.l. were found on cats (123 larvae and nymphs versus 10 adults) than on dogs (190 larvae and nymphs versus 173 adults). Furthermore, compared to nymphs, significantly more larvae of R. sanguineus s.l. were found on cats (50 larvae versus 73 nymphs) than on dogs (11 larvae versus 179 nymphs). Adult ticks predominated on male dogs (42 adults versus 28 larvae or nymphs), whereas immatures were significantly more abundant compared to adult ticks on female dogs (142 larvae or nymphs versus 80 adults). Similarly, immature as compared with adult ticks were significantly more likely to occur on female cats (72 immature ticks versus 1 adult) in comparison with male cats (46 immature ticks versus 8 adults). Moreover, R. sanguineus s.l. larvae were found significantly more frequently as compared with nymphs on female cats (38 larvae versus 34 nymphs) than on male cats (12 larvae versus 34 nymphs). To confirm morphological identification and to compare mitochondrial markers (cytochrome c oxidase subunit 1 [cox1] and 16S rRNA genes) of ticks across hosts, 57 ticks were analysed. The amplified parts of the cox1 and 16S rRNA genes of R. sanguineus s.l. collected from various hosts showed 100% sequence identity with each other and with those in GenBank from the middle to western Mediterranean Basin. In conclusion, the present study highlights that cats can be important hosts of the immature life stages of R. sanguineus s.l., even in the absence of dogs. This finding has veterinary-medical significance, because stray cats and free-roaming cats may transport immature stages of R. sanguineus s.l. into gardens, i.e. near dogs and humans.