Metabolic characterization of serum from mice challenged with Orientia tsutsugamushi-infected mites.

Scrub typhus is an acute zoonosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. To better understand the host response elicited by natural infection by chigger feeding, ICR mice were infected by Leptotrombidium chiangraiensis (Lc1) chiggers, and the metabolic profiles of their serum were examined over several time points after initiation of feeding. ICR mice were infected by either naive Lc1 chiggers (i.e. not infected by O. tsutsugamushi, NLc1) or O. tsutsugamushi-infected Lc1 chiggers (OLc1). Serum was collected from both groups of mice at 6 hours and 10 days after initiation of feeding. Metabolites were extracted from the serum and analysed by ultra performance liquid chromatography-tandem mass spectrometry. The resulting ion/chromatographic features were matched to a library of chemical standards for identification and quantification. Biochemicals that differed significantly between the experimental groups were identified using Welch's two-sample t tests; p ≤ 0.05 was considered statistically significant. A number of biochemicals linked to immune function were found to be significantly altered between mice infected by the NLc1 and OLc1 chiggers, including itaconate, kynurenine and histamine. Several metabolites linked to energy production were also found to be altered in the animals. In addition lipid and carbohydrate metabolism, bile acid and phospholipid homeostasis, and nucleotide metabolism were also found to be different in these two groups of mice. Markers of stress and food intake were also significantly altered. Global untargeted metabolomic characterization revealed significant differences in the biochemical profiles of mice infected by the NLc1 versus OLc1 chiggers. These findings provide an important platform for further investigation of the host responses associated with chigger-borne O. tsutsugamushi infections.

Rapid detection and strain identification of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR.

AIMS: To develop and validate assays based on real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for rapid detection and strain identification (European and North American strains) of porcine reproductive and respiratory syndrome virus (PRRSV) by using SYBR Green I and TaqMan probe chemistries. METHODS AND RESULTS: This study describes two alternative assays based on real-time RT-PCR for rapid detection and strain identification of PRRSV in comparison with conventional RT-PCR. The first assay utilized SYBR Green I with melting curve analysis; another assay was performed using strain-specific TaqMan probes. Primers were selected from the conserved regions within ORF7 (N) of both strains whereas two TaqMan probes labelled with different fluorescent dyes were specifically designed for each strain. The result of strain identification was confirmed by direct sequencing. Both assays can be used for rapid detection and strain identification of PRRSV with a sensitivity of 10(4) and 10(3) copies microl(-1) for SYBR Green and TaqMan probe, respectively. CONCLUSIONS: Real-time RT-PCR is a powerful method combining rapidity, specificity and efficiency for large-scale screening and strain identification of PRRSV. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that the methods developed are invaluable for detecting low levels of PRRSV infection in swine.