Marco Valerio Bernasconi

Real-Time PCR Investigation of Potential Vectors, Reservoirs, and Shedding Patterns of Feline Hemotropic Mycoplasmas

ABSTRACT Three hemotropic mycoplasmas have been identified in pet cats: Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum,” and “Candidatus Mycoplasma turicensis.” The way in which these agents are transmitted is largely unknown. Thus, this study aimed to investigate fleas, ticks, and rodents as well as saliva and feces from infected cats for the presence of hemotropic mycoplasmas, to gain insight into potential transmission routes for these agents. DNA was extracted from arthropods and from rodent blood or tissue samples from Switzerland and from salivary and fecal swabs from two experimentally infected and six naturally infected cats. All samples were analyzed with real-time PCR, and some positive samples were confirmed by sequencing. Feline hemotropic mycoplasmas were detected in cat fleas and in a few Ixodes sp. and Rhipicephalus sp. ticks collected from animals but not in ticks collected from vegetation or from rodent samples, although the latter were frequently Mycoplasma coccoides PCR positive. When shedding patterns of feline hemotropic mycoplasmas were investigated, “Ca. Mycoplasma turicensis” DNA was detected in saliva and feces at the early but not at the late phase of infection. M. haemofelis and “Ca. Mycoplasma haemominutum” DNA was not amplified from saliva and feces of naturally infected cats, despite high hemotropic mycoplasma blood loads. Our results suggest that besides an ostensibly indirect transmission by fleas, direct transmission through saliva and feces at the early phase of infection could play a role in the epizootiology of feline hemotropic mycoplasmas. Neither the investigated tick nor the rodent population seems to represent a major reservoir for feline hemotropic mycoplasmas in Switzerland.

Phylogenetic relationships among muscoidea (Diptera: calyptratae) based on mitochondrial DNA sequences.

The utility of a mitochondrial DNA (mtDNA) fragment of about 1100 bp (including partial COI and COII sequences and tRNALeu) for evolutionary studies in Muscoidea is discussed. The species investigated are Scathophaga stercoraria, Microprosopa pallidicauda and Trichopalpus fraterna (family Scathophagidae), Musca domestica (Muscidae), Lasiomma seminitidum (Anthomyiidae) and Fannia armata (Fanniidae). Comparisons were made with published mtDNA sequences of Drosophila, Anopheles and three Calliphoridae species. The molecular phylogeny obtained here matches the classical morphological taxonomy reasonably well. This varies considerably, however, at different taxonomical levels. At a high taxonomic level, there is a clear separation between the Nematocera and the Brachycera, but the Calyptratae–Acalyptratae division is not always supported. At a lower taxonomic level, all species belonging to the same family are well grouped, but at an intermediate level, within the Calyptratae, it is impossible to clearly separate the Muscoidea and Calliphoridae, preventing a firm conclusion on the phylogenetic relationships among Muscoidea families. The entire COI sequence of S. stercoraria, as well as other mtDNA sequences (including the proximal portions of the COI gene, tRNATrp, tRNACys and tRNATyr genes) in Muscoidea species, are also presented and discussed.

Assessment of intraspecific mtDNA variability of European Ixodes ricinus sensu stricto (Acari: Ixodidae).

The Ixodes ricinus complex is composed of 14 species distributed worldwide. Some members of this complex are involved in the transmission of a number of diseases to animals and humans, in particular Lyme borreliosis, tick-borne encephalitis, ehrlichiosis and babesiosis. While the phylogenetic relationships between species of the I. ricinus complex have been investigated in the past, still little is known about the genetic structure within the species I. ricinus sensu stricto. We have investigated the intraspecific variability among 26 I. ricinus s.s. ticks collected in various European countries, including Switzerland, Italy, Austria, Denmark, Sweden, and Finland by using five mitochondrial gene fragments corresponding to the control region, 12S rDNA, cytb, COI, and COII. The five genes considered here showed a low genetic variability (1.6-5%). Our results based on both statistical parsimony (applied to the COI + COII + cytb + 12S + CR data set, for a total of 3423 bp) and maximum parsimony (applied to the COI + COII + cytb + 12S data set, for a total of 2980 bp) did not provide any evidence for a correlation between the identified haplotypes and their geographic origin. Thus, the European I. ricinus s.s. ticks do not seem to show any phylogeography structure.

Population Genetics of Vibrio vulnificus: Identification of Two Divisions and a Distinct Eel-Pathogenic Clone

ABSTRACT Genetic relationships among 62 Vibrio vulnificus strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the recA and glnA genes. Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic. Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the V. vulnificus population into two divisions (divisions Ι and ΙΙ). One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2). A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St. Peters fish (biotype 3). RAPD analysis of the 62 V. vulnificus isolates identified 26 different profiles separated into two divisions as well. In general, this subdivision was comparable (but not identical) to that observed by MLEE. Phylogenetic analysis of 543 bp of the recA gene and of 402 bp of the glnA gene also separated the V. vulnificus population into two major divisions in a manner similar to that by MLEE and RAPD. Sequence data again indicated the overall subdivision of the V. vulnificus population into different biotypes. In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees. None of the methods showed an association between distinct clones and human clinical manifestations. Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed. In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results. The significance of the two divisions (divisions Ι and ΙΙ) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.

Sensitivity analysis, molecular systematics and natural history evolution of Scathophagidae (Diptera: Cyclorrhapha: Calyptratae)

The 60 000 described species of Cyclorrhapha are characterized by an unusual diversity in larval life‐history traits, which range from saprophagy over phytophagy to parasitism and predation. However, the direction of evolutionary change between the different modes remains unclear. Here, we use the Scathophagidae (Diptera) for reconstructing the direction of change in this relatively small family (≈ 250 spp.) whose larval habits mirror the diversity in natural history found in Cyclorrhapha. We subjected a molecular data set for 63 species (22 genera) and DNA sequences from seven genes (12S, 16S, Cytb, COI, 28S, Ef1‐alfa, Pol II) to an extensive sensitivity analysis and compare the performance of three different alignment strategies (manual, Clustal, POY). We find that the default Clustal alignment performs worst as judged by character incongruence, topological congruence and branch support. For this alignment, scoring indels as a fifth character state worsens character incongruence and topological congruence. However, manual alignment and direct optimization perform similarly well and yield near‐identical trees, although branch support is lower for the direct‐optimization trees. All three alignment techniques favor the upweighting of transversion. We furthermore confirm the independence of the concepts “node support” and “node stability” by documenting several cases of poorly supported nodes being very stable and cases of well supported nodes being unstable. We confirm the monophyly of the Scathophagidae, its two constituent subfamilies, and most genera. We demonstrate that phytophagy in the form of leaf mining is the ancestral larval feeding habit for Scathophagidae. From phytophagy, two shifts to saprophagy and one shift to predation has occurred while a second origin of predation is from a saprophagous ancestor.

Mechanism of metronidazole resistance in Helicobacter pylori : Comparison of the rdxA gene sequences in 30 strains

ABSTRACT The rdxA gene of 30 independently isolatedHelicobacter pylori strains was sequenced. A comparison of the rdxA sequences revealed a higher percentage of amino acid substitutions in the corresponding protein than in other housekeeping genes. Out of 122 point mutations, 41 were missense and 4 were nonsense. A resistant strain with a nucleotide insertion in therdxA sequence was also found. With the exception of the point mutations and the insertion generating a stop signal, no particular nucleotide mutation or amino acid substitution could be associated to metronidazole resistance. Moreover, phylogenetic analysis of the 30 nucleotide sequences did not demonstrate specific clusters associated with the resistance phenotype.

Population genetics of Helicobacter pylori in the southern part of Switzerland analysed by sequencing of four housekeeping genes (atpD, glnA, scoB and recA), and by vacA, cagA, iceA and IS605 genotyping

The population biology of 78 Helicobacter pylori strains (71 from Swiss Italian, 4 from East Asian and 3 from South African patients) was investigated by sequence analysis of four housekeeping genes: atpD, scoB, glnA and recA. The vacA genotype, the presence of cagA and IS605, the iceA allelic type, and the resistance to metronidazole, clarithromycin and amoxycillin were determined. A high percentage of DNA polymorphic sites (19.8% for atpD, 21.3% for scoB, 23.7% for glnA and 20.3% for recA) was found. The phylogenetic trees based on the nucleotide sequences of the four gene fragments showed different topologies and were incongruent. The virulence-associated markers were distributed over the dendrograms and no association was found with phylogenetic clusters or clinical manifestations (chronic gastritis, gastric or duodenal ulcer, MALT lymphoma). Moreover, the H ratios (calculated with the homoplasy test) ranged from 0.742 to 0.799, depending on the gene fragment examined. All these observations suggest that H. pylori exists as a recombinant population. The clustering of the strains according to their geographical origin (USA/Europe, East Asia, South Africa) that has recently been demonstrated elsewhere could only be confirmed for the East Asian vacA s1c strains. In contrast, the South African strains clustered together only in the atpD tree. Presumably, recombination at the different loci has masked the evolutionary relationship among the strains.

Tick zoonoses in the southern part of Switzerland (Canton Ticino): occurrence of Borrelia burgdorferi sensu lato and Rickettsia sp.

The diversity and the distribution of tick species and their infection rates by the pathogenic micro-organism Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, and Rickettsia sp., were studied in Canton Ticino (the southern part of Switzerland). Ticks specimens collected from animals and humans were classified and analysed for the presence of both pathogens. In particular, PCR analysis was performed for the detection of Borrelia spirochetes in Ixodes ricinus and Ixodes hexagonus, and the hemolymph test was done on Rhipicephalus sanguineus for the detection of Rickettsia sp. PCR assays, performed on 424 of the 989 collected ticks, revealed a low rate of infection (around 2%) of both vectors I. ricinus and I. hexagonus by B. burgdorferi sensu lato. These results are in agreement with the modest number of Lyme borreliosis cases yearly recorded in Ticino. Further, through analysis of DNA sequences, the strains carried by the infected ticks were identified as belonging to the genomic group VS116. The widespread finding of the Mediterranean species Rhipicephalus sanguineus in different locations from July 1994 to October 1995 demonstrates its establishment in Ticino. Of the 210 specimens collected, 70 were analysed and one was infected by Rickettsia sp.

RecA and glnA sequences separate the bacteroides fragilis population into two genetic divisions associated with the antibiotic resistance genotypes cepA and cfiA.

The sequences of part of the glutamine synthetase-encoding gene (glnA) and of the RecA-encoding gene (recA) were determined and aligned for 45 Bacteroides fragilis isolates from different clinical and geographical origin. The patterns of sequence divergence of glnA and recA were very similar. The sequences of a 303-bp fraction of recA showed 45 nucleotide substitutions, 40 of which allowed the separation of B. fragilis into two major divisions, which were not found when the deduced amino acid sequences were considered. The 687-bp sequences analysed for the glnA gene showed 112 nucleotide substitutions, 96 of which separated the population into the same two divisions as those described for recA. In this case, the deduced amino acid sequences showed this subdivision as well: three of the six observed amino acid substitutions were division-specific. Within the two divisions, both genes presented a high degree of sequence conservation. Each B. fragilis division was associated with the presence of a different antibiotic resistance gene: cepA encoding a serine-beta-lactamase (division I) and cfiA encoding a metallo-beta-lactamase (division II). No particular clusters associated with geographical or clinical origin, or with the production of an enterotoxin were observed. Sequencing of the cfiA gene allowed identification of two different alleles in division II. However, no association of these different cfiA alleles with the expression of imipenem resistance was observed. In conclusion, the phylogenetic patterns observed by sequencing recA and glnA are in agreement with those obtained previously by MLEE (multilocus enzyme electrophoresis). Thus, it appears that the evolution of recA and glnA genes is similar to that of the whole chromosome of B. fragilis. Horizontal gene transfer between divisions I and II seems to be low, at best. However, the results of the present study could not clarify definitively whether divisions I and II should be considered as two different B. fragilis genospecies.

Molecular systematics of Dolichopodidae (Diptera) inferred from COI and 12S rDNA gene sequences based on European exemplars

With ~7000 species in ~220 genera, the Dolichopodidae is one of the most speciose families of Diptera. Though the family as such is well defined, knowledge on the internal phylogenetic relationships is generally poor and although authors of successive monographs and catalogues indifferently listed most genera in specific subfamilies, their decisions were rarely based on sound phylogenetic analyses and never on molecular data. In a first attempt to unravel the phylogeny of Dolichopodidae, a combined COI + 12S rDNA dataset (1199 characters) of 119 samples of 101 European species was used in Bayesian (BAY), neighbour joining (NJ) and weighted/unweighted maximum parsimony analyses (MP). At the subfamily level, our study supports the monophyly of Dolichopodinae, Sympycninae, and Hydrophorinae (including Machaerium Haliday, 1832). Ten (Campsicnemus Haliday, 1851, Chrysotus Meigen, 1824, Dolichopus Latreille, 1796, Gymnopternus Loew, 1857, Medetera Fischer von Waldheim, 1819, Poecilobothrus Mik, 1878, Rhaphium Meigen, 1803, Teuchophorus Loew, 1857, Sciapus Zeller, 1842, Syntormon Loew, 1857) of the 14 multispecies genera formed monophyletic assemblages in all analyses and relationships among Argyra Macquart, 1834 species were supported in most analyses. At the subgeneric level, seven of the nine stable species-groups in Dolichopus as established during previous research were supported in most analyses. The validity of the recent transfer of Hercostomus chrysozygos Wiedemann, 1817 to Poecilobothrus was clearly supported in all analyses. Within Argyra and Rhaphium, interspecific relationships reflected previously used subgeneric classifications (Lasiargyra Mik, 1878, Leucostola Loew, 1857 and Argyra s.s. in Argyra; Porphyrops Meigen, 1824, Rhaphium s.s. and Xiphandrium Loew, 1857 in Rhaphium). Further, subclades within Medetera corresponded to species-groups defined by other authors featuring a different morphology and ecology. Anepsiomyia flaviventris (Meigen, 1824) most probably does not belong to Sympycninae but its current position within Peloropeodinae could not be confirmed.