Characterization of Polish feline B. henselae isolates by multiple-locus tandem repeat analysis and pulse-field gel electrophoresis.

Knowledge about molecular epidemiology of B. henselae is important for recognizing the geographical distribution of strains and identification of isolates virulent for humans. Eleven Polish feline B. henselae isolates were typed, using 2 different techniques: pulse-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA). PFGE analysis distinguished 6 different PFGE types, with subtypes within 3 of them, whereas 10 MLVA types were assigned. Global diversity index (D.I.) for MLVA equaled 0.93. For 7 isolates, the results of MLVA confirmed cluster assignments based on PFGE. Both PFGE and MLVA results were in accordance with epidemiological data. Although PFGE has been previously demonstrated to be a suitable method for the differentiation of B. henselae isolates/strains, our results show the superiority of MLVA over PFGE with respect to higher discriminatory power, distinguishing genotypes I and II isolates, easier analysis of results, and possibility to compare the numerical data obtained by different laboratories. With MLVA, 7 new profiles were observed, compared to previous results from around the world; whereas 3 known profiles were previously described mainly in European B. henselae isolates. Our results confirm that some VNTR profiles can be used as specific geographical markers.

A new typing technique for the Rickettsiales Ehrlichia ruminantium: multiple-locus variable number tandem repeat analysis.

Ehrlichia ruminantium (ER) is a member of the order Rickettsiales transmitted by Amblyomma ticks. This obligatory intracellular bacterium is the causative agent of a fatal disease in ruminants, named heartwater. It represents a constraint on breeding development in sub-Saharan Africa and in the Caribbean. The genetic diversity of the strains of ER, which could be a limiting factor to obtain effective vaccines, needs to be better characterized. For this purpose, we developed a molecular typing technique based on the polymorphism of variable number tandem repeat (VNTR) sequences, MLVA (multiple locus VNTR analysis). Eight (out of 21) VNTR candidates were validated using 17 samples representing a panel of ER strains from different geographical origins from West, South Africa, and Caribbean areas and in ER infected ticks and goat tissues. This result demonstrated the ability of these VNTRs to type a wide range of strains. The stability of the selected VNTR markers was very good, at the time scale needed for epidemiological purposes: in particular, no difference in the VNTR profiles was observed between virulent and attenuated strains (for Gardel and Senegal strains) and between strains (Gardel and Blonde strains) isolated in the same area 19years apart. We validated the strong discriminatory power of MLVA for ER and found a high level of polymorphism between the available strains, with 10 different profiles out of 13 ER strains. The MLVA scheme described in this study is a rapid and efficient molecular typing tool for ER, which allows rapid and direct typing of this intracellular pathogen without preliminary culture and gives reliable results that can be used for further epidemiological studies.

Differential effects of Bartonella henselae on human and feline macro- and micro-vascular endothelial cells.

Bartonella henselae, a zoonotic agent, induces tumors of endothelial cells (ECs), namely bacillary angiomatosis and peliosis in immunosuppressed humans but not in cats. In vitro studies on ECs represent to date the only way to explore the interactions between Bartonella henselae and vascular endothelium. However, no comparative study of the interactions between Bartonella henselae and human (incidental host) ECs vs feline (reservoir host) ECs has been carried out because of the absence of any available feline endothelial cell lines.To this purpose, we have developed nine feline EC lines which allowed comparing the effects of Bartonella strains on human and feline micro-vascular ECs representative of the infection development sites such as skin, versus macro-vascular ECs, such as umbilical vein.Our model revealed intrinsic differences between human (Human Skin Microvascular ECs -HSkMEC and Human Umbilical Vein ECs - iHUVEC) and feline ECs susceptibility to Bartonella henselae infection.While no effect was observed on the feline ECs upon Bartonella henselae infection, the human ones displayed accelerated angiogenesis and wound healing.Noticeable differences were demonstrated between human micro- and macro-vasculature derived ECs both in terms of pseudo-tube formation and healing. Interestingly, Bartonella henselae effects on human ECs were also elicited by soluble factors.Neither Bartonella henselae-infected Human Skin Microvascular ECs clinically involved in bacillary angiomatosis, nor feline ECs increased cAMP production, as opposed to HUVEC.Bartonella henselae could stimulate the activation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) in homologous cellular systems and trigger VEGF production by HSkMECs only, but not iHUVEC or any feline ECs tested.These results may explain the decreased pathogenic potential of Bartonella henselae infection for cats as compared to humans and strongly suggest that an autocrine secretion of VEGF by human skin endothelial cells might induce their growth and ultimately lead to bacillary angiomatosis formation.

Molecular epidemiology of feline and human Bartonella henselae isolates.

Multiple locus variable number tandem repeat analysis was performed on 178 Bartonella henselae isolates from 9 countries; 99 profiles were distributed into 2 groups. Human isolates/strains were placed into the second group. Genotype I and II isolates shared no common profile. All genotype I isolates clustered within group B. The evolutive implications are discussed.