Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated Neorickettsia sp. in Europe

A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi, fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was >/=4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.

Using PCR and an automated laser fluorescent DNA sequencer, we amplified and sequenced a 1,234-bp fragment of the citrate synthase-encoding gene (gltA) of 28 bacteria belonging to the genus Rickettsia. Comparative sequence analysis showed that most of the spotted fever group (SFG) rickettsiae belonged to one of two subgroups. The first subgroup included Rickettsia massiliae, strain Bar 29, Rickettsia rhipicephali, "Rickettsia aeschlimanni," and Rickettsia montana, which have been isolated only from ticks. The second subgroup was larger and included the majority of the human pathogens and also rickettsiae isolated only from ticks; the members of this subgroup were strain S, Rickettsia africae, "Rickettsia monglotimonae," Rickettsia sibirica, Rickettsia parkeri, Rickettsia conorii, Rickettsia rickettsii, the Thai tick typhus rickettsia, the Israeli tick typhus rickettsia, the Astrakhan fever rickettsia, "Rickettsia slovaca," and Rickettsia japonica. The sequence analysis also showed that the tick-borne organisms Rickettsia helvetica and Rickettsia australis and the mite-borne organism Rickettsia akari were associated with the SFG cluster, that Rickettsia prowazekii and Rickettsia typhi, two representatives of the typhus group, clustered together, and that Rickettsia canada; Rickettsia bellii, and the AB bacterium probably represent three new groups. We compared the phylogenetic trees inferred from citrate synthase gene sequences and from 16S ribosomal DNA (rDNA) sequences. For rickettsial phylogeny, the citrate synthase approach was more suitable, as demonstrated by significant bootstrap values for all of the nodes except those in the larger subgroup defined above. We also compared phylogenetic analysis results obtained in a comparison of the sequences of both genes for all of the representatives of the domain Bacteria for which the gltA sequence was determined. We believe that comparison of gltA sequences could be a complementary approach to 16S rDNA sequencing for inferring bacterial evolution, especially when unstable phylogenetic models are obtained from ribosomal sequences because of high levels of sequence similarity between the bacteria studied.

A highly specific real-time polymerase chain reaction (PCR) assay was developed to detect spotted fever and typhus group rickettsiae using the citrate synthase gene as the target. The assay amplified rickettsial members of the spotted fever and typhus group including Rickettsia akari, R. australis, R. conorii, R. honei, "R. marmionii," R. sibirica, R. rickettsii, R. typhi, and R. prowazekii. The ancestral group rickettsia, R. bellii, did not produce a positive reaction, nor did other members of the order Rickettsiales or any non-rickettsial bacteria. The assay had a sensitivity of one target copy number per reaction as determined by serial dilutions of a plasmid containing a spotted fever group target sequence. This quantitative assay is useful for the enumeration of rickettsiae in clinical specimens and the diagnosis of rickettsial illnesses, when rickettsial numbers are very low.