MicroRNA-275 and its target vitellogenin-2 are crucial in ovary development and blood digestion of Haemaphysalis longicornis

Background: The hard tick Haemaphysalis longicornis is widely distributed in eastern Asia, New Zealand and Australia and is considered the major vector of Theileria and Babesia, harmful parasites to humans and animals. Female ticks need successful blood meals to complete the life-cycle. Therefore, elucidation of the underlying molecular mechanisms of H. longicornis development and reproduction is considered important for developing control strategies against the tick and tick-borne pathogens. Methods: Luciferase assays were used to identify the targets of micro RNA miR-275 in vitro. RNAi of Vitellogenin (Vg) was used in phenotype rescue experiments of ticks with miR-275 inhibition, and these analyses were used to identify the authentic target of miR-275 in vivo. The expression of miR-275 in different tissues and developmental stages of ticks was assessed by real-time PCR. To elucidate the functions of miR-275 in female ticks, we injected a miR-275 antagomir into female ticks and observed the phenotypic changes. Statistical analyses were performed with GraphPad5 using Student’s t-test. Results: In this study, we identified Vg-2 as an authentic target of miR-275 both in vitro and in vivo by luciferase assays and phenotype rescue experiments. miR-275 plays the regulatory role in a tissue-specific manner and differentially in developmental stages. Silencing of miR-275 resulted in blood digestion problems, substantially impaired ovary development and significantly reduced egg mass (P < 0.0001). Furthermore, RNAi silencing of Vg-2 not only impacted the blood meal uptake (P < 0.05) but also the egg mass (P < 0.05). Significant rescue was observed in miR-275 knockout ticks when RNAi was applied to Vg-2. Conclusion: To our knowledge, this study is the first demonstration that miR-275 targets Vg-2 in H. longicornis and regulates the functions of blood digestion and ovary development. These findings improve the molecular understanding of tick development and reproduction

Detection and differentiation of Borrelia burgdorferi sensu lato in ticks collected from sheep and cattle in China

<p>Abstract</p> <p>Background</p> <p>Lyme disease caused by <it>Borrelia burgdorferi </it>sensu lato complex is an important endemic zoonosis whose distribution is closely related to the main ixodid tick vectors. In China, isolated cases of Lyme disease infection of humans have been reported in 29 provinces. Ticks, especially ixodid ticks are abundant and a wide arrange of <it>Borrelia </it>natural reservoirs are present. In this study, we developed a reverse line blot (RLB) to identify <it>Borrelia </it>spp. in ticks collected from sheep and cattle in 7 Provinces covering the main extensive livestock regions in China.</p> <p>Results</p> <p>Four species-specific RLB oligonucleotide probes were deduced from the spacer region between the 5S-23S rRNA gene, along with an oligonucleotide probe which was common to all. The species specific probes were shown to discriminate between four genomic groups of <it>B. burgdorferi </it>sensu lato i.e. <it>B. burgdorferi </it>sensu stricto, <it>B. garinii, B. afzelii</it>, and <it>B. valaisiana</it>, and to bind only to their respective target sequences, with no cross reaction to non target DNA. Furthermore, the RLB could detect between 0.1 pg and 1 pg of <it>Borrelia </it>DNA.</p> <p>A total of 723 tick samples (<it>Haemaphysalis, Boophilus, Rhipicephalus </it>and <it>Dermacentor</it>) from sheep and cattle were examined with RLB, and a subset of 667 corresponding samples were examined with PCR as a comparison. The overall infection rate detected with RLB was higher than that of the PCR test.</p> <p>The infection rate of <it>B. burgdoreri </it>sensu stricto was 40% in south areas; while the <it>B. garinii infection rate </it>was 40% in north areas. The highest detection rates of <it>B. afzelii </it>and <it>B. valaisiana </it>were 28% and 22%, respectively. Mixed infections were also found in 7% of the ticks analyzed, mainly in the North. The proportion of <it>B. garinii </it>genotype in ticks was overall highest at 34% in the whole investigation area.</p> <p>Conclusion</p> <p>In this study, the RLB assay was used to detect <it>B. burgdorferi </it>sensu lato in ticks collected from sheep and cattle in China. The results showed that <it>B. burdorferi senso stricto </it>and <it>B. afzelii </it>were mainly distributed in the South; while <it>B. garinii </it>and <it>B. valaisiana </it>were dominant in the North. <it>Borrelia </it>spirochaetes were detected in <it>Rhipicephalus </it>spp for the first time. It is suggested that the <it>Rhipicephalus </it>spps might play a role in transmitting <it>Borrelia </it>spirochaetes.</p

Comparative analysis of microRNA profiles between wild and cultured Haemaphysalis longicornis (Acari, Ixodidae) ticks

The miRNA profiles of a Haemaphysalis longicornis wild-type (HLWS) and of a Haemaphysalis longicornis cultured population (HLCS) were sequenced using the Illumina Hiseq 4000 platform combined with bioinformatics analysis and real-time polymerase chain reaction (RT-PCR). A total of 15.63 and 15.48 million raw reads were acquired for HLWS and HLCS, respectively. The data identified 1517 and 1327 known conserved miRNAs, respectively, of which 342 were differentially expressed between the two libraries. Thirty-six novel candidate miRNAs were predicted. To explain the functions of these novel miRNAs, Gene Ontology (GO) analysis was performed. Target gene function prediction identified a significant set of genes related to salivary gland development, pathogen-host interaction and regulation of the defence response to pathogens expressed by wild H. longicornis ticks. Cellular component biogenesis, the immune system process, and responses to stimuli were represented at high percentages in the two tick libraries. GO enrichment analysis showed that the percentages of most predicted functions of the target genes of miRNA were similar, as were certain specific categories of functional enhancements, and that these genes had different numbers and specific functions (e.g., auxiliary transport protein and electron carrier functions). This study provides novel findings showing that miRNA regulation affects the expression of immune genes, indicating a considerable influence of environment-induced stressful stimulation on immune homeostasis. Differences in the living environments of ticks can lead to differences in miRNAs between ticks and provide a basis and a convenient means to screen for genes encoding immune factors in ticks

Molecular Evidence of Bartonella melophagi in Ticks in Border Areas of Xinjiang, China

Bartonella are gram-negative intracellular bacteria; certain species of Bartonella can cause diseases in mammals and humans. Ticks play a major role in the transmission of Bartonella. Xinjiang is the largest province in China according to land area and has one-third of the tick species in China; the infection rate of Bartonella in ticks in the Xinjiang border areas has not been studied in detail. Therefore, this study investigated tick infections by Bartonella in Xinjiang border areas, and the purpose of the study was to fill in gaps in information regarding the genetic diversity of tick infections by Bartonella in Xinjiang. We tested 1,549 tick samples from domestic animals (sheep and cattle) for Bartonella using ribC-PCR. Positive samples from the ribC-PCR assay for Bartonella spp. were further subjected to PCR assays targeting the ITS, rpoB and gltA genes followed by phylogenetic analyses. Bartonella DNA was detected in 2.19% (34/1,549) of tick samples, and the ITS, rpoB and gltA genes of ribC gene-positive samples were amplified to identify nine samples of Bartonella melophagi. In this study, molecular analysis was used to assess the presence and genetic diversity of B. melophagi in ticks collected from sheep and cattle from Xinjiang, China. This study provides new information on the presence and identity of B. melophagi in ticks from sheep and cattle

Genetic Variation of Promoter Sequence Modulates XBP1 Expression and Genetic Risk for Vitiligo

Our previous genome-wide linkage analysis identified a susceptibility locus for generalized vitiligo on 22q12. To search for susceptibility genes within the locus, we investigated a biological candidate gene, X-box binding protein 1(XBP1). First, we sequenced all the exons, exon-intron boundaries as well as some 5′ and 3′ flanking sequences of XBP1 in 319 cases and 294 controls of Chinese Hans. Of the 8 common variants identified, the significant association was observed at rs2269577 (p_trend = 0.007, OR = 1.36, 95% CI = 1.09–1.71), a putative regulatory polymorphism within the promoter region of XBP1. We then sequenced the variant in an additional 365 cases and 404 controls and found supporting evidence for the association (p_trend = 0.008, OR = 1.31, 95% CI = 1.07–1.59). To further validate the association, we genotyped the variant in another independent sample of 1,402 cases and 1,288 controls, including 94 parent-child trios, and confirmed the association by both case-control analysis (p_trend = 0.003, OR = 1.18, 95% CI = 1.06–1.32) and the family-based transmission disequilibrium test (TDT, p = 0.005, OR = 1.93, 95% CI = 1.21–3.07). The analysis of the combined 2,086 cases and 1,986 controls provided highly significant evidence for the association (p_trend = 2.94×10−6, OR = 1.23, 95% CI = 1.13–1.35). Furthermore, we also found suggestive epistatic effect between rs2269577 and HLA-DRB1*07 allele on the development of vitiligo (p = 0.033). Our subsequent functional study showed that the risk-associated C allele of rs2269577 had a stronger promoter activity than the non-risk G allele, and there was an elevated expression of XBP1 in the lesional skins of patients carrying the risk-associated C allele. Therefore, our study has demonstrated that the transcriptional modulation of XBP1 expression by a germ-line regulatory polymorphism has an impact on the development of vitiligo

Biomimetic nanozyme-decorated hydrogels with H₂O₂-activated oxygenation for modulating immune microenvironment in diabetic wound

Diabetic foot ulcers (DFUs) remain a devastating threat to human health. While hydrogels are promising systems for DFU-based wound management, their effectiveness is often hindered by the immune response and hostile wound microenvironment associated with the uncontrollable accumulation of reactive oxygen species and hypoxia. Here, we develop a therapeutic wound dressing using a biomimetic hydrogel system with the decoration of catalase-mimic nanozyme, namely, MnCoO@PDA/CPH. The hydrogel can be designed to match the mechanical and electrical cues of skins simultaneously with H2O2-activated oxygenation ability. As a proof of concept, DFU-based rat models are created to validate the therapeutic efficacy of the MnCoO@PDA/CPH hydrogel in vivo. The results indicate that the developed hydrogel can promote DFU healing and improve the quality of the healed wound as featured by alleviated proinflammatory, increased re-epithelialization, highly ordered collagen deposition, and functional blood vessel growth.Ministry of Education (MOE)National Research Foundation (NRF)This research work was supported by the National Natural Science Foundation of China (Grant 22105131), the Guangdong Basic and Applied Basic Research Foundation (Grant 2022A1515011677), the International Postdoctoral Exchange Fellowship Program (Grant PC2021046), the National Research Foundation Singapore under Its Competitive Research Programme (Grant NRF-CRP26-2021-0002), and the Ministry of Education Singapore under the Research Centres of Excellence Scheme (Institute for Digital Molecular Analytics and Science)

Molecular detection and characterization of Anaplasma spp. in sheep and cattle from Xinjiang, northwest China

Abstract Background Anaplasmosis is caused by obligate intracellular bacteria in the genus Anaplasma. These bacterial pathogens are transmitted by ticks and impact both human and animal health. This study was conducted to determine the prevalence and molecular characterization of Anaplasma spp. in ruminants sampled in Xinjiang, northwest China. Methods A survey was performed in August 2012 in rural areas of six counties in Xinjiang province. A total of 250 blood samples from ruminants were collected and tested for the presence of Anaplasma spp. by PCR. Positive samples were genetically characterized based on the 16S rRNA and msp4 genes. Results The results showed a high prevalence of Anaplasma spp. in ruminants, with at least three different Anaplasma species detected (A. phagocytophilum, A. bovis and A. ovis). The mean prevalence of single infection with each species was 17.6% (A. phagocytophilum), 4.8% (A. bovis) and 40.5% (A. ovis). Coinfection occurred in 20 (8.0%) animals. Phylogenetic analysis of the 16S rRNA gene of A. bovis and A. phagocytophilum revealed a higher degree of genetic diversity for the latter. The results for A. ovis showed genotypic variation among geographic regions in China. In addition, a closely related isolate to the canine pathogen A. platys was identified in ruminants. Conclusions This survey revealed a high prevalence of Anaplasma sp. infections in sheep and cattle in the northwestern border regions of China, indicating the potential risk of transboundary disease