The preparation of hybrid silicon quantum dots by one-step synthesis for tetracycline detection and antibacterial applications.

In this study, we prepared three different silicon quantum dots (SiQDs-1, SiQDs-2 and SiQDs-3) by hydrothermal synthesis with rose Bengal as the reducing agent and triacetoxy(methyl)silane and allyloxytrimethylsilane as silicon sources. The as-prepared SiQDs not only exhibited potent antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) but also showed specific responses to tetracycline (TC). The minimum inhibitory concentrations (MICs) of SiQDs-1, SiQDs-2 and SiQDs-3 were 0.55 mg mL-1, 0.47 mg mL-1 and 0.39 mg mL-1 against E. coli, respectively, and 0.45 mg mL-1, 0.34 mg mL-1 and 0.34 mg mL-1 against S. aureus, respectively. By examining the morphologies of bacteria and generation of reactive oxygen species (ROS), we speculated that these SiQDs shrink the bacteria and even directly destroy the bacterial structural integrity through the production of singlet oxygen. In addition, the fluorescence quenching effectiveness of SiQDs-3 also showed a strong linear relationship with TC concentration in the range of 0-1.2 µM with a detection limit of 0.318 µM, as a result of the internal filtering effect. Together, SiQDs not only can be a candidate to treat resistant bacterial infections, but also may be applied in practical detection of TC.

Enhanced antibacterial activity with increasing P doping ratio in CQDs.

It is an urgent challenge to develop efficient antibacterial agents against resistant bacteria in the treatment of infectious diseases. Carbon quantum dots (CQDs) have attracted much attention owing to their good stability, low toxicity and excellent biocompatibility. In this work, CQDs doped with different contents of the element phosphorus (P) were prepared by a simple hydrothermal method using valine as a carbon source, triethylamine as a nitrogen source and different volumes of phosphoric acid as a phosphorus source. The average diameter and the surface charge could be regulated from 2.89 nm to 1.56 nm and +2.58 mV to +5.47 mV by increasing the content of the element P in these CQDs. Importantly, these CQDs showed effective bacterial inhibition against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The minimal inhibitory concentration (MIC) decreased from 0.71, to 0.51 to 0.18 mg mL-1 on E. coli and S. aureus with the increase of P element content. Furthermore, the morphologies of E. coli cells and S. aureus were damaged and became irregular upon treatment with these CQDs. The results of singlet oxygen (1O2) detection demonstrated that intracellular 1O2 was generated during the antibacterial process. We speculated that bacterial inhibition induced by these CQDs was accompanied by disruption of permeability and structural integrity, owing to strong electrostatic interactions between negatively charged bacteria and positively charged CQDs and production of singlet oxygen of CQDs. Together, this study indicates that the CQDs can be a candidate to treat resistant bacterial infections and may improve the understanding of killing pathogens by antibacterial CQD drugs.

Theoretical and Experimental Studies of Gallate Melilite Electrides from Topotactic Reduction of Interstitial Oxide Ion Conductors.

A nonstoichiometric La1.5Sr0.5Ga3O7.25 melilite oxide ion conductor features active interstitial oxygen defects in its pentagonal rings with high mobility. In this study, electron localization function calculated by density functional theory indicated that the interstitial oxide ions located in the pentagonal rings of gallate melilites may be removed and replaced by electron anions that are confined within the pentagonal rings, which would therefore convert the melilite interstitial oxide ion conductor into a zero-dimensional (0D) electride. The more active interstitial oxide ions, compared to the framework oxide ions, make the La1.5Sr0.5Ga3O7.25 melilite structure more reducible by CaH2 using topotactic reduction, in contrast to the hardly reducible nature of parent LaSrGa3O7. The topotactic reduction enhances the bulk electronic conduction (σ ∼ 0.003 S/cm at 400 °C) by ∼ 1 order of magnitude for La1.5Sr0.5Ga3O7.25. The oxygen loss in the melilite structure was verified and most likely took place on the active interstitial oxide ions. The identified confinement space for electronic anions in melilite interstitial oxide ion conductors presented here provides a strategy to access inorganic electrides from interstitial oxide ion conductor electrolytes.

Progranulin regulates the development and function of NKT2 cells through EZH2 and PLZF.

T helper 2 (Th2) cytokine production by invariant natural killer T (iNKT) cells is involved in the development of asthma, but the regulation of Th2 cytokines in iNKT cells remains unknown. Although it is known that progranulin (PGRN) induces the production of Th2 cytokines in iNKT cells in vivo, the underlying mechanism is not clear. This study aims to investigate the role of PGRN in iNKT cells. The effects of PGRN on the differentiation of iNKT cells was detected by flow cytometry. Then stimulation of iNKT cells and airway resistance were carried out to evaluate the function of PGRN on iNKT cells. Furthermore, the mechanisms of PGRN in regulating iNKT cells was investigated by RT-PCR, WB, confocal and luciferase reporter assays. The absolute number of iNKT cells decreased in PGRN KO mice despite an increase in the percentage of iNKT cells. Furthermore, analyzing the subsets of iNKT cells, we found that NKT2 cells and their IL-4 production were reduced. Mechanistically, the decrease in NKT2 cells in the PGRN KO mice was caused by increased expression of enhancer of zeste homolog 2 (EZH2), that in turn caused increased degradation and altered nuclear localization of PLZF. Interestingly, PGRN signaling decreased expression of EZH2 and treatment of the PGRN KO mice with the EZH2 specific inhibitor GSK343 rescued the defect in NKT2 differentiation, IL-4 generation, and PLZF expression. Altogether, We have revealed a new pathway (PGRN-EZH2-PLZF), which regulates the Th2 responses of iNKT cells and provides a potentially new target for asthma treatment.

Germinal peptide eye drop promotes corneal epithelial and stromal defect healing in rabbit model.

OBJECTIVE: Corneal defect is a common disease in ophthalmology caused by trauma, inflammation, drug toxicity, or surgery. To investigate the effect of germinal peptide eye drop on corneal epithelial and stromal defects after lamellar keratectomy in rabbit model. METHODS: Eighty-five male New Zealand white rabbits were divided into five groups: Germinal Peptide eye drop at three different concentration groups, normal saline (negative control group), recombinant human epidermal growth factor (rh-EGF) eye drop (positive control group). Corneal epithelial and stromal defects of around 150-200 µm in depth were created with an 8 mm diameter trephine in the center of the right eyes of all animals. RESULTS: Germinal peptide eye drop with the concentration of 0.001%, 0.002%, and 0.004% and rh-EGF eye drop were more effective in promoting healing, reducing opacity, and edema during the process of corneal epithelial and stromal defect regeneration compared with 0.9% normal saline. No significant difference was observed among the three different doses of germinal peptide eye drop. Compared with the saline control group, the structures of the regenerated corneas were more orderly and less inflammatory cell infiltration was observed in the germinal peptide eye drop groups and the rh-EGF eye drop group. CONCLUSION: Germinal peptide eye drop (0.001%, 0.002%, and 0.004%) can significantly stimulate the regeneration of corneal epithelia and stroma and reduce corneal opacity and edema. Dose dependency was not observed in the current study.

P-Doped Carbon Quantum Dots with Antibacterial Activity.

It is a major challenge to effectively inhibit microbial pathogens in the treatment of infectious diseases. Research on the application of nanomaterials as antibacterial agents has evidenced their great potential for the remedy of infectious disease. Among these nanomaterials, carbon quantum dots (CQDs) have attracted much attention owing to their unique optical properties and high biosafety. In this work, P-doped CQDs were prepared by simple hydrothermal treatment of m-aminophenol and phosphoric acid with fluorescence emission at 501 nm when excited at 429 nm. The P-doped CQDs showed effective antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The minimal inhibitory concentrations (MICs) of P-doped CQD were 1.23 mg/mL for E. coli and 1.44 mg/mL for S. aureus. Furthermore, the morphologies of E. coli cells were damaged and S. aureus became irregular when treated with the P-doped CQDs. The results of zeta potential analysis demonstrated that the P-doped CQDs inhibit antibacterial activity and destroy the structure of bacteria by electronic interaction. In combination, the results of this study indicate that the as-prepared P-doped CQDs can be a promising candidate for the treatment of bacterial infections.

Mechanistically understanding adsorption of methyl orange, indigo carmine, and methylene blue onto ionic/nonionic polystyrene adsorbents.

The involved interaction information concerning adsorbate-adsorbate and adsorbate-adsorbent is indispensable for developing and optimizing adsorption treatment of dye wastewater. Single and bi-solute adsorption of methyl orange (MO), indigo carmine (IC), and methylene blue (MB) on polystyrene anion exchanger (PsAX), defunctionalized version of PsAX (DF-PsAX), and hyper-cross-linked polystyrene adsorbent (MN200) were investigated to obtain a mechanistic understanding. Under acidic condition, higher adsorption efficiencies of PsAX due to extra intermolecular interactions of MO between the protonated tertiary amine group and the sulfate groups were observed, while strong alkaline condition is favorable for the adsorption of IC and MB by PsAX. MN200 exhibited much larger adsorption capacity toward MB than that toward MO or IC, because the fused-rings structure of MB is more polarizable and can form stronger nonionic intermolecular attractions with the matrix structure of polystyrene adsorbents. Bi-solute adsorption reveals that MO has obvious competitive effect toward IC adsorption at low concentrations, but it is not the case for the adsorption at high concentrations, where IC molecules can form intermolecular H-bonding interactions to defend the competition. the thermodynamic parameters confirm the endothermic and spontaneous nature of MO adsorption by PsAX, and ≈ 48 KJ mol-1 of the enthalpy change (∆H) imply the adsorption is not just physical absorption. Additionally, water/ethanol mixture solution of NaCl can almost thoroughly regenerate the exhausted PsAX, whereas only aqueous solution without ethanol is invalid.

Germinal peptide eye drops promote corneal wound healing and decrease inflammation after alkali injury.

Germinal peptide is being developed to treat corneal injuries. The purpose of this study was to investigate its effect on corneal epithelial cells in vitro and its ability to promote healing in an alkali injury model in vivo. Cultured rabbit corneal epithelial cells were treated with germinal peptide at three concentrations. Cell proliferation and migration were assessed and compared with the effect of recombinant human epidermal growth factor (rh-EGF). In vivo, the corneas of New Zealand albino rabbits were chemically burned with 1 mol/l NaOH for 30 s. The injured eyes were topically treated with germinal peptide (10, 20, and 40 µg/ml), rh-EGF, or phosphate-buffered saline thrice daily. At fixed time points post injury, the healing of the cornea and its histopathology were evaluated. There was no difference in the effect of germinal peptide on cultured cell proliferation. However, cell migration was significantly higher than that in the control groups, with germinal peptide at concentrations of 20 and 40 µg/ml being the most efficacious. In vivo, 20 and 40 µg/ml germinal peptide significantly alleviated corneal opacity and edema. By day 21, the areas of corneal neovascularization in the germinal peptide-treated groups were smaller than those in the rh-EGF and control groups. The repaired corneas in the germinal peptide- and rh-EGF-treated groups also had more corneal epithelial layers and fewer inflammatory cells than the controls. Germinal peptide may be developed as a novel topical treatment agent for corneal wound healing in clinical settings.

Development of Melilite-Type Oxide Ion Conductors.

Lowering the operating temperature of solid oxide fuel cells (SOFCs) requires high performance oxide ion conductor electrolytes. Recently tetrahedra-based structures have been attracting considerable attention for oxide ion conductor development, among which the layered tetrahedral network melilite structure appears particularly interesting owing to its remarkable capability to accommodate and transport interstitial oxide ions, compared with isolated tetrahedral anion structures. Stabilization and migration mechanisms of interstitial oxide ions in melilites have been systematically investigated using local structural relaxation from both electrostatic Coulomb interaction and chemical bonding aspects based on atomic and electronic structures respectively using experimental and theoretical approaches. These reveal cationic size and chemical bonding effects on stabilization and migration mechanisms of interstitial oxide ions. Lately, full crystallization from glass, an innovative synthesis method, was employed to produce new metastable melilite oxide ion conductors which are inaccessible using classic solid state reaction owing to cationic size effect. Finally, the thermal and chemical stability at low temperature and the high oxide ion conductivity of the best melilite oxide ion conductors based on LaSrGa3 O7 are likely to provide real possibilities of applications of melilite-type electrolytes in SOFCs and other related devices.

Trigonal-Planar Low-Spin Co2+ in a Layered Mixed-Polyhedral Network from Topotactic Reduction.

Topotactic reduction of the perovskite oxide TbBaCo2O5.5 with CaH2 leads to a new crystalline phase TbBaCo2O4.5, adopting a 2 × 2 × 1 superstructure compared to TbBaCo2O5.5. The structure consists of a corner-shared network of square pyramidal CoO5 and trigonal planar CoO3 units. Magnetic susceptibility and variable temperature neutron diffraction data reveal that TbBaCo2O4.5 adopts a G-type antiferromagnetically ordered structure (TN ∼ 322 K). The ordered moments are consistent with the presence of low-spin Co2+ (S = 1/2) in trigonal-planar coordination and high-spin Co2+ centers in square pyramidal coordination. TbBaCo2O4.5 shows lower conductivity than TbBaCo2O5.5, which is consistent with the p-type conduction behavior. The unique anion vacancy arrangements in TbBaCo2O4.5 further complement the role of A-cations in controlling the oxygen vacancy distribution in LnBaCo2O5+δ series and demonstrate more opportunity to tune the structural and physical properties based on cationic and anionic lattice coupling.

Substitution-Induced Structure Evolution and Zn2+/Ga3+ Ordering in "114" Oxides MAZn2Ga2O7 ( M = Ca2+, Sr2+; A = Sr2+, Ba2+).

The "114" oxides LnBa(Co/Fe)4O7+δ represent a new family of materials that exhibits intriguing physical properties, including geometrically frustrated magnetism, oxygen storage, and magnetoelectric couplings. Various chemical substitutions have been conducted to modify their crystal and magnetic structures as well as physical properties. However, the principles beneath the substitution-induced structural evolution and charge/cationic ordering have not yet been understood. Thus, in this contribution, two complete solid solutions of MAZn2Ga2O7 ( M = Ca2+, Sr2+; A = Sr2+, Ba2+) were designed, synthesized, and characterized by Rietveld refinements based on high-resolution X-ray diffraction (XRD) and neutron diffraction (ND) data. The structure symmetry of MAZn2Ga2O7 is determined by the cationic size mismatch between M and A cations that can be defined by the tolerance factor t, i.e., symmetry transitions from P63 mc ( t > 0.87) to P31 c (0.87 > t > 0.75) and to Pna21 ( t < 0.75) were observed for MAZn2Ga2O7, associated with the rotation of T1O4 tetrahedra in the triangular layers. The Zn2+/Ga3+ ordering at T sites is also a consequence of the increase or decrease of the average sizes of M and A cations. A small concentration of interstitial oxygen ions can be obtained in Sr2Zn2- xGa2+ xO7+ x/2 ( x = 0.1, 0.2); however, no oxygen ionic conduction was observed at high temperatures, indicating the migration ability of the interstitial oxygen was very limited.

Observation of optic disc neovascularization using OCT angiography in proliferative diabetic retinopathy after intravitreal conbercept injections.

This study reports the short-term efficacy and safety of intravitreal conbercept injections for neovascularization at the disc (NVD) in patients with proliferative diabetic retinopathy (PDR). Conbercept is a recombinant fusion protein with a high affinity for all isoforms of vascular endothelial growth factor (VEGF)-A, placental growth factor and VEGF-B. A prospective case series study was conducted in 15 patients (15 eyes). Patients had complete ocular examinations and received a 0.5 mg intravitreal conbercept injection followed by supplemental pan-retinal photocoagulation (PRP). Optical coherence tomography angiography (OCTA) was performed before and after treatment. Before treatment, the mean NVD area was 1.05 ± 0.33 mm2, and it decreased to 0.56 ± 0.17 mm2 after an interval of 7.5 d (p = 0.000). One eye required vitrectomy during follow-up. Recurrent NVD was observed in 2 eyes, which resolved after repeated injections. The remaining 12 eyes were stable over a mean follow-up period of 8.3 months. The mean area of the NVD in 14 patients without vitrectomy was 0.22 ± 0.11 mm2 (p = 0.000) at the last visit. Intravitreal conbercept injections combined with intensive PRP are an effective and safe treatment for PDR with NVD. Quantitative information on NVD can be obtained with OCTA, which may be clinically useful in evaluating the therapeutic effect.

[Mechanism of chloroquine in promoting sensitivity of chemotherapeutics in oral squamous cell carcinoma CAL-27 cell line to cisplatin].

PURPOSE: To study the role and mechanism of autophagy in chemotherapy of oral squamous cell carcinoma, and provide theoretical evidence to improve chemotherapeutic efficacy of oral squamous cell carcinoma patients. METHODS: The cell survival rate changes induced by cisplatin (DDP) and chloroquine (CQ) in CAL-27 cells were assayed by methyl thiazolyl tetrazolium method(MTT). The LC3-II expression level was detected by laser scanning confocal microscope; The apoptotic rate was determined by flow cytometry. SPSS17.0 software package was used for statistical analysis. RESULTS: MTT results showed that compared with the control group, the cell survival rate reduced with the increasing time of DDP and CQ treatment; The optimal concentration of CAL-27 cells was 5 mg/L after treatment with CQ. IC50 of the CAL-27 cells was 5 mg/L after treatment with DDP; MTT results showed that the cell survival rate of CQ+DDP group was significantly lower than control group, CQ group and DDP group (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, immunofluorescence results showed that the average fluorescence intensity of DDP group was significantly higher than the other 3 groups (P<0.05), while it was significantly lower in CQ group than the other 3 groups (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, flow cytometry results showed that the cell apoptosis rate of DDP group and CQ+DDP group were significantly higher than control group and CQ group. The cell apoptosis rate of CQ+DDP group was significantly higher than DDP group (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, cells in G1 phase of DDP group and CQ+DDP group increased, indicating G1 phase blockage. The cell count in G1 phase of CQ+DDP group was significantly higher than DDP group (P<0.05). CONCLUSIONS: Inhibition of autophagy can enhance the chemotherapeutic sensitivity of DDP in CAL-27 cells. Autophagy in CAL-27 cells is an important mechanism for chemotherapy resistance of oral squamous cell carcinoma. Autophagy inhibitor may have significant potential to be a novel chemotherapeutic sensitizer for oral squamous cell carcinoma.

Protective effect of indomethacin in renal ischemia-reperfusion injury in mice.

OBJECTIVE: To evaluate the renoprotection effects of non-steroidal anti-inflammatory drugs (NSAIDs) in renal ischemia-reperfusion injury (IRI) and the cyclooxygenase (COX)-1/2 blockade association by indomethacin (IMT) in the mice model. METHODS: After the left renal pedicle of mice was clamped, IMT was administrated by intraperitoneal injection with four doses: 1, 3, 5, and 7 mg/kg. Blood and kidney samples were collected 24 h after IRI. The renal functions were assayed by the cytokines and serum creatinine (SCr) using enzyme-linked immunosorbent assay (ELISA) kits. Kidney samples were analyzed by hematoxylin and eosin (H&E) and immunohistochemistry stainings. RESULTS: The mice administered with 5 mg/kg IMT had a marked reduction in SCr and significantly less tubular damage. The tumor necrosis factor α (TNF-α) activity in renal homogenates and interleukin 6 (IL-6) activity in serum had a marked reduction at doses of 5 and 7 mg/kg IMT. The administration of 3 and 5 mg/kg IMT had a marked reduction in the ratio of thromboxane B2 to 6-keto-prostaglandin F1α. COX-1 and COX-2 stainings were weaker in 5 mg/kg IMT groups than that in the other groups. CONCLUSIONS: There was a dose response in the IMT function of renal IRI in mice, and IMT had a protective effect in a certain dose range. The effect of IMT on mice IRI was related to COX-1/2 blockades.

Urinary serum- and glucocorticoid-inducible kinase SGK1 reflects renal injury in patients with immunoglobulin A nephropathy.

AIM: Serum- and glucocorticoid-inducible kinase SGK1 functions as an important regulator of transepithelial sodium transport by activating epithelial sodium channel in renal tubules. Considerable evidence demonstrated that SGK1 was associated with hypertension and fibrosing diseases, such as diabetic nephropathy and glomerulonephritis. The present study was performed to evaluate the role of SGK1 played in immunoglobulin A (IgA) nephropathy. METHODS: Seventy-six patients of biopsy-proven IgA nephropathy and 33 healthy volunteers were enrolled in this study. All patients and healthy volunteers' urinary and serum samples were tested for SGK1 expression by indirect enzyme-linked immunosorbent assay. Meanwhile all patients' renal tissues were semi-quantified for SGK1 expression by immunohistochemistry assay. The relationships between SGK1 expressions and clinical or pathological parameters were also assessed. RESULTS: SGK1 expression was upregulated in urine and renal tubules in patients of Oxford classification T1 and T2, whereas its expression in serum did not increase significantly. Relationship analysis indicated that urinary and tissue SGK1 expressions were associated with heavy proteinuria and renal insufficiency in patients with IgA nephropathy. On the other hand, RAS blockades would reduce the SGK1 levels both in urine and renal tissues. CONCLUSION: These results suggested that urinary SGK1 should be a good indicator of tubulointerstitial damage in patients of IgA nephropathy. SGK1 expressions in urine and renal tissues were associated with the activity of renin-angiotensin-aldosterone system.

High prevalence of spotted fever group rickettsiae in Amblyomma variegatum from Uganda and their identification using sizes of intergenic spacers.

The spotted fever group (SFG) rickettsiae are obligate intracellular bacteria transmitted by ticks that cause several tick-borne rickettsioses in humans worldwide. This study was intended to determine the prevalence of SFG rickettsiae in Amblyomma variegatum from 7 districts across Uganda. In addition to sequencing of gltA and ompA genes, identification of Rickettsia species based on the sizes of highly variable intergenic spacers, namely, dksA-xerC, mppA-purC, and rpmE-tRNA(fMet) was carried out. Application of multiplex PCR for simultaneous amplification of 3 spacers combined with capillary electrophoresis separation allowed simple, accurate, and high-throughput fragment sizing with considerable time and cost savings. Rickettsia genus-specific real-time PCR detected 136 positives out of 140 samples, giving an overall prevalence of 97.1%. Most samples (n=113) had a size combination of 225, 195, and 341 bp for dksA-xerC, mppA-purC, and rpmE-tRNA(fMet), respectively, which was identical to that of R. africae, a causative agent of African tick bite fever. In addition, several samples had size variants in either dksA-xerC or rpmE-tRNA(fMet). Nonetheless, the partial sequences of gltA and ompA genes of samples of all size combinations showed the greatest similarity to R. africae (99.3-100% for gltA and 98.1-100% for ompA). Given these results, it is highly possible that the tested ticks were infected with R. africae or closely related species. This is a first report on molecular genetic detection of R. africae and its high endemicity in Uganda. Clinicians in this country should be aware of this pathogen as a cause of non-malarial febrile illness. This study provided a starting point for the development of Rickettsia species identification based on the sizes of intergenic spacers. The procedure is simple, rapid, and cost-effective to perform; hence it might be particularly well suited for preliminary species identification in epidemiological investigations. The results may be more detailed and reliable when simultaneous sequencing analysis is performed.

Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping of Ehrlichia ruminantium and its application to infected Amblyomma variegatum collected in heartwater endemic areas in Uganda.

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost- effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.

Multi-locus sequence typing of Ehrlichia ruminantium strains from geographically diverse origins and collected in Amblyomma variegatum from Uganda.

BACKGROUND: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater in ruminants. A better understanding of the population genetics of its different strains is, however, needed for the development of novel diagnostic tools, therapeutics and prevention strategies. Specifically, the development of effective vaccination policies relies on the proper genotyping and characterisation of field isolates. Although multi-locus sequence typing (MLST) has been recently developed, only strains from geographically restricted collections have been analysed so far. The expansion of the MLST database to include global strains with different geographic origins is therefore essential. In this study, we used a panel of reference strains from geographically diverse origins and field samples of E. ruminantium detected from its vector, Amblyomma variegatum, in heartwater-endemic areas in Uganda. RESULTS: A total of 31 novel alleles (six, four, six, three, two, five, three, and two for gltA, groEL, lepA, lipA, lipB, secY, sodB, and sucA loci, respectively) and 19 novel sequence types (STs) were identified. Both neighbour-joining and minimum spanning tree analyses indicated a high degree of genetic heterogeneity among these strains. No association was observed between genotypes and geographic origins, except for four STs from West African countries. When we performed six different tests for recombination (GeneConv, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq) on concatenated sequences, four possible recombination events were identified in six different STs. All the recombination breakpoints were located near gene borders, indicating the occurrence of intergenic recombination. All four STs that localized to a distinct group in clustering analysis showed evidence of identical recombination events, suggesting that recombination may play a significant role in the diversification of E. ruminantium. CONCLUSIONS: The compilation of MLST data set across the African continent will be particularly valuable for the understanding of the existing genetic diversity of field isolates in African countries. Comprehensive information on the degree of cross-protection between strains and further understanding of possible relationships between genotypes and phenotypes such as vaccine efficacy are expected to lead to the development of region-specific vaccination strategies.