Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples.

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

Declining diversity of egg-associated bacteria during development of naturally spawned whitefish embryos (Coregonus spp.)

Fish eggs are associated with microbes, whose roles range from mutualism to parasitism. Recent laboratory experiments have shown that the taxonomic composition of associated microbial communities on the egg influences embryonic development. Host genetics also plays an important role in determining the consequences for embryonic growth and survival in this interaction. Moreover, it has been found that the importance of host genetics increases during embryogenesis. These findings suggest that during embryogenesis, the host increasingly influences the composition of its associated microbial community. However, little is known about the composition of microbial communities associated with naturally spawned eggs in the wild. We sampled fertilized whitefish eggs (Coregonus spp.) of different developmental stages from six sub-Alpine lakes and used a universal primer pair and 454 pyrosequencing in order to describe the taxonomic composition of egg-associated bacterial communities. We found bacterial communities on early embryos to be very diverse and to resemble the bacterial composition of the surrounding water environment. The bacterial communities on late embryos were significantly less diverse than on early embryos and displayed a clear shift in taxonomic composition that corresponded poorly with the bacterial composition of the surrounding water environment. The main bacterial components on whitefish eggs in this study were Proteobacteria, Actinobacteria, and Firmicutes, while the five most common families were Leuconostocaceae, Streptococcaceae, Comamonadaceae, Oxalobacteraceae and Moraxellaceae. Their putative relationships with the host are discussed. We conclude that natural symbiotic bacterial communities become more specialized during embryogenesis because of specific interactions with their embryo host.

Chlamydiaceae family, Parachlamydia spp., and Waddlia spp. in porcine abortion.

At present, despite extensive laboratory investigations, most cases of porcine abortion remain without an etiological diagnosis. Due to a lack of recent data on the abortigenic effect of order Chlamydiales, 286 fetuses and their placentae of 113 abortion cases (1-5 fetuses per abortion case) were investigated by polymerase chain reaction (PCR) methods for family Chlamydiaceae and selected Chlamydia-like organisms such as Parachlamydia acanthamoebae and Waddlia chondrophila. In 0.35% of the cases (1/286 fetuses), the Chlamydiaceae real-time PCR was positive. In the Chlamydiaceae-positive fetus, Chlamydia abortus was detected by a commercial microarray and 16S ribosomal RNA PCR followed by sequencing. The positive fetus had a Porcine circovirus-2 coinfection. By the Parachlamydia real-time PCR, 3.5% (10/286 fetuses of 9 abortion cases) were questionable positive (threshold cycle values: 35.0-45.0). In 2 of these 10 cases, a confirmation by Chlamydiales-specific real-time PCR was possible. All samples tested negative by the Waddlia real-time PCR. It seems unlikely that Chlamydiaceae, Parachlamydia, and Waddlia play an important role as abortigenic agents in Swiss sows.

Parachlamydia spp. and related Chlamydia-like organisms and bovine abortion.

Chlamydophila abortus and Waddlia chondrophila cause abortion in ruminants. We investigated the role of Parachlamydia acanthamoebae in bovine abortion. Results of immunohistochemical analyses were positive in 30 (70%) of 43 placentas from which Chlamydia-like DNA was amplified, which supports the role of Parachlamydia spp. in bovine abortion.

Different rates of defense evolution and niche preferences in clonal and nonclonal milkweeds (Asclepias spp.).

Given the dual role of many plant traits to tolerate both herbivore attack and abiotic stress, the climatic niche of a species should be integrated into the study of plant defense strategies. Here we investigate the impact of plant reproductive strategy and components of species' climatic niche on the rate of chemical defense evolution in the milkweeds using a common garden experiment of 49 species. We found that across Asclepias species, clonal reproduction repeatedly evolved in lower temperature conditions, in species generally producing low concentrations of a toxic defense (cardenolides). Additionally, we found that rates of cardenolide evolution were lower for clonal than for nonclonal species. We thus conclude that because the clonal strategy is based on survival, long generation times, and is associated with tolerance of herbivory, it may be an alternative to toxicity in colder ecosystems. Taken together, these results indicate that the rate of chemical defense evolution is influenced by the intersection of life-history strategy and climatic niches into which plants radiate.