Detection of Bartonella schoenbuchensis (sub)species DNA in different louse fly species in Saxony, Germany: The proof of multiple PCR analysis necessity in case of ruminant-associated bartonellae determination.

BACKGROUND: Hippoboscid flies are bloodsucking arthropods that can transmit pathogenic microorganisms and are therefore potential vectors for pathogens such as Bartonella spp. These Gram-negative bacteria can cause mild-to-severe clinical signs in humans and animals; therefore, monitoring Bartonella spp. prevalence in louse fly populations appears to be a useful prerequisite for zoonotic risk assessment. METHODS: Using convenience sampling, we collected 103 adult louse flies from four ked species (Lipoptena cervi, n = 22; Lipoptena fortisetosa, n = 61; Melophagus ovinus, n = 12; Hippobosca equina, n = 8) and the pupae of M. ovinus (n = 10) in the federal state of Saxony, Germany. All the samples were screened by polymerase chain reaction (PCR) for Bartonella spp. DNA, targeting the citrate synthase gene (gltA). Subsequently, PCRs targeting five more genes (16S, ftsZ, nuoG, ribC and rpoB) were performed for representatives of revealed gltA genotypes, and all the PCR products were sequenced to identify the Bartonella (sub)species accurately. RESULTS AND CONCLUSIONS: The overall detection rates for Bartonella spp. were 100.0%, 59.1%, 24.6% and 75.0% in M. ovinus, L. cervi, L. fortisetosa and H. equina, respectively. All the identified bartonellae belong to the Bartonella schoenbuchensis complex. Our data support the proposed reclassification of the (sub)species status of this group, and thus we conclude that several genotypes of B. schoenbuchensis were detected, including Bartonella schoenbuchensis subsp. melophagi and Bartonella schoenbuchensis subsp. schoenbuchensis, both of which have previously validated zoonotic potential. The extensive PCR analysis revealed the necessity of multiple PCR approach for proper identification of the ruminant-associated bartonellae.

Two novel Bartonella (sub)species isolated from edible dormice (Glis glis): hints of cultivation stress-induced genomic changes.

Bartonelloses are neglected emerging infectious diseases caused by facultatively intracellular bacteria transmitted between vertebrate hosts by various arthropod vectors. The highest diversity of Bartonella species has been identified in rodents. Within this study we focused on the edible dormouse (Glis glis), a rodent with unique life-history traits that often enters households and whose possible role in the epidemiology of Bartonella infections had been previously unknown. We identified and cultivated two distinct Bartonella sub(species) significantly diverging from previously described species, which were characterized using growth characteristics, biochemical tests, and various molecular techniques including also proteomics. Two novel (sub)species were described: Bartonella grahamii subsp. shimonis subsp. nov. and Bartonella gliris sp. nov. We sequenced two individual strains per each described (sub)species. During exploratory genomic analyses comparing two genotypes ultimately belonging to the same species, both factually and most importantly even spatiotemporally, we noticed unexpectedly significant structural variation between them. We found that most of the detected structural variants could be explained either by prophage excision or integration. Based on a detailed study of one such event, we argue that prophage deletion represents the most probable explanation of the observed phenomena. Moreover, in one strain of Bartonella grahamii subsp. shimonis subsp. nov. we identified a deletion related to Bartonella Adhesin A, a major pathogenicity factor that modulates bacteria-host interactions. Altogether, our results suggest that even a limited number of passages induced sufficient selective pressure to promote significant changes at the level of the genome.

Role of invasive carnivores (Procyon lotor and Nyctereutes procyonoides) in epidemiology of vector-borne pathogens: molecular survey from the Czech Republic.

BACKGROUND: Vector-borne pathogens (VBPs) are a major threat to humans, livestock and companion animals worldwide. The combined effect of climatic, socioeconomic and host composition changes favours the spread of the vectors, together with the expansion of invasive carnivores contributing to the spread of the pathogens. In Europe, the most widespread invasive species of carnivores are raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides). This study focused on the detection of four major groups of VBPs namely Babesia, Hepatozoon, Anaplasma phagocytophilum and Bartonella in invasive and native carnivores in the Czech Republic, with the emphasis on the role of invasive carnivores in the eco-epidemiology of said VBPs. METHODS: Spleen samples of 84 carnivores of eight species (Canis aureus, Canis lupus, Lynx lynx, P. lotor, Martes foina, Lutra lutra, Mustela erminea and N. procyonoides) were screened by combined nested PCR and sequencing for the above-mentioned VBPs targeting 18S rRNA and cytB in hemoprotozoa, groEL in A. phagocytophilum, and using multilocus genotyping in Bartonella spp. The species determination is supported by phylogenetic analysis inferred by the maximum likelihood method. RESULTS: Out of 84 samples, 44% tested positive for at least one pathogen. Five different species of VBPs were detected in P. lotor, namely Bartonella canis, Hepatozoon canis, Hepatozoon martis, A. phagocytophilum and Bartonella sp. related to Bartonella washoensis. All C. lupus tested positive for H. canis and one for B. canis. Three VBPs (Hepatozoon silvestris, A. phagocytophilum and Bartonella taylorii) were detected in L. lynx for the first time. Babesia vulpes and yet undescribed species of Babesia, not previously detected in Europe, were found in N. procyonoides. CONCLUSIONS: Wild carnivores in the Czech Republic are hosts of several VBPs with potential veterinary and public health risks. Among the studied carnivore species, the invasive raccoon is the most competent host. Raccoons are the only species in our study where all the major groups of studied pathogens were detected. None of the detected pathogen species were previously detected in these carnivores in North America, suggesting that raccoons adapted to local VBPs rather than introduced new ones. Babesia vulpes and one new, probably imported species of Babesia, were found in raccoon dogs.