Babesia spp., Theileria spp. and Anaplasma ovis are important intracellular agents that are transmitted by tick bites. However, Babesia spp., Theileria spp. and A. ovis in ticks have not been systematically reported along the border of northwestern China. In this study, a total of 1,084 adult ticks, including 134 Haemaphysalis punctata, 337 Hyalomma asiaticum, 233 Dermacentor nuttalli, 69 Rhipicephalus turanicus and 265 Dermacentor marginatus were collected from 11 counties or cities of Xinjiang Uygur Autonomous Region. The ticks were identified from morphological and molecular characteristics. Two fragments of 18S rRNA gene were used to determine the species level of Babesia and Theileria. Msp4 gene encoding major surface protein 4 was used to determine A. ovis. Of the 1,084 samples, five species of Babesia (B. occultans, B. caballi, B. motasi, B. major and Babesia sp. detected in this study), two kinds of Theileria (Theileria ovis and Theileria sp. detected in this study) and A. ovis with six phylogenic branches were detected in the border of northwestern China. Babesia occultans, first found in China, was first molecularly detected in D. nuttalli. Babesia caballi and Babesia sp. detected in this study were first molecularly detected in Hy. asiaticum. Genotype III of A. ovis was predominant in the border regions of northwestern China.

The aims of this study were to establish optimal doses of doxycycline (dox) against Haemophilus parasuis on the basis of pharmacokinetic-pharmacodynamic (PK-PD) integration modeling. The infected model was established by intranasal inoculation of organism in pigs and confirmed by clinical signs, blood biochemistry, and microscopic examinations. The recommended dose (20 mg/kg b.w.) was administered in pigs through intramuscular routes for PK studies. The area under the concentration 0- to 24-hr curve (AUC ), elimination half-life (T ), and mean residence time (MRT) of dox in healthy and H. parasuis-infected pigs were 55.51 ± 5.72 versus 57.10 ± 4.89 μg·hr/ml, 8.28 ± 0.91 versus 9.80 ± 2.38 hr, and 8.43 ± 0.27 versus 8.79 ± 0.18 hr, respectively. The minimal inhibitory concentration (MIC) of dox against 40 H. parasuis isolates was conducted through broth microdilution method, the corresponding MIC and MIC were 0.25 and 1 μg/ml, respectively. The Ex vivo growth inhibition data suggested that dox exhibited a concentration-dependent killing mechanism. Based on the observed AUC /MIC values by modeling PK-PD data in H. parasuis-infected pigs, the doses predicted to obtain bacteriostatic, bactericidal, and elimination effects for H. parasuis over 24 hr were 5.25, 8.55, and 10.37 mg/kg for the 50% target attainment rate (TAR), and 7.26, 13.82, and 18.17 mg/kg for 90% TAR, respectively. This study provided a more optimized alternative for clinical use and demonstrated that the dosage 20 mg/kg of dox by intramuscular administration could have an effective bactericidal activity against H. parasuis.

The mechanisms of invasion and intracellular survival of Brucella are still poorly understood. Previous studies showed that the two-component regulatory systems (TCSs) play an important role in the intracellular survival of Brucella. To investigate if TCSs involve in the virulence and cytotoxicity of Brucella melitensis, we introduced a mutation into one of the TCSs in chromosome II in Br. melitensis 16M strain, and generated 16MΔTceSR, a mutant of Br. melitensis 16M strain. In vitro infection experiments using murine macrophage cell line (RAW 264.7) showed that the survival of 16MΔTceSR mutant in macrophages decreased 0·91-log compared with that of wild type Br. melitensis 16M strain at 2 h postinfection, replication of 16MΔTceSR mutant in macrophages was 5·65-log, which was much lower than that wild type strain. Results of lactate dehydrogenase cytotoxicity assays in macrophages demonstrated high dose infection with wide type strain produced high level cytotoxicity to macrophages, but 16MΔTceSR mutant had very low level cytotoxicity, indicating mutation of TCSs impaired the cytotoxicity of Br. melitensis to macrophages. Animal experiments showed that the spleen colonization of 16MΔTceSR was significantly reduced compared with its wild type strains. The lower levels of survival of 16MΔTceSR in various stress conditions suggested that the mutation of the TCSs of Br. melitensis was the causative factor of its reduced resistance to stress conditions. Taken together, our results demonstrated TCS TceSR involves in the intracellular survival, virulence and cytotoxicity of Br. melitensis during its infection. Significance and impact of the study: Two-component systems (TCSs) are predominant bacterial signal transduction mechanisms. The pathogenicity of Brucella is due to its ability to adapt to the intracellular environment including low levels of acidic pH, high-salt and heat shock. TCSs are designed to sense diverse stimuli, transfer signals and enact an appropriate adaptive physiological response. Here, we show that Br. meilitensis TCS TceSR is not only involved in regulation of Br. meilitensis virulence and adaptation of environmental stresses, but also can regulate cytotoxicity in macrophages.