Chitosan-alginate/R8 ternary polyelectrolyte complex as an oral protein-based vaccine candidate induce effective mucosal immune responses.

Vaccination is the most effective method for preventing infectious diseases. Oral vaccinations have attracted much attention due to the ability to boost intestinal and systemic immunity. The focus of this study was to develop a poly (lactide-co-glycolide) acid (PLGA)-based ternary polyelectrolyte complex (PEC) with chitosan, sodium alginate, and transmembrane peptides R8 for the delivery of antigen proteins. In this study, the antigen protein (HBf), consisting of the Mycobacterium avium subspecies paratuberculosis (MAP) antigens HBHA, Ag85B, and Bfra, was combined with R8 to generate self-assembled conjugates. The results showed that PEC presented a cross-linked reticular structure to protect the encapsulated proteins in the simulated gastric fluid. Then, the nanocomposite separated into individual nanoparticles after entering the simulated intestinal fluid. The ternary PEC with R8 promoted the in vivo uptake of antigens by intestinal lymphoid tissue. Moreover, the ternary PEC administered orally to mice promoted the secretion of specific antibodies and intestinal mucosal IgA. In addition, in the mouse models of MAP infection, the ternary PEC enhanced splenic T cell responses, thus reducing bacterial load and liver pathology score. These results suggested that this ternary electrolyte complex could be a promising delivery platform for oral subunit vaccine candidates, not limited to MAP infection.

Selenium nanoparticles promotes intestinal development in broilers by inhibiting intestinal inflammation and NLRP3 signaling pathway compared with other selenium sources.

This study aimed to investigate how various selenium sources affect the intestinal health of broiler chickens. A total of 384, one-day-old Arbor Acres broilers were weighed and randomly allocated to four treatment groups. The control diet was a basal diet added with: 0.2 mg/kg Sodium Selenite (SS-control), 0.2 mg/kg Selenium nano-particles (Nano-Se), 0.2 mg/kg Selenomethionine (SeMet), and 0.2 mg/kg Selenocysteine (Sec) as the treatments. The results indicated that Nano-Se and SeMet were effective in enhancing the villus height (VH) and the villus height/crypt depth ratio (VH/CD) in the jejunum compared to (SS) (P < 0.05). The inclusion of Nano-Se into the diets increased the mRNA levels of zonula occluden-1 (ZO-1), ZO-2, Occludin, Claudin-1, and Claudin-3 compared to the SS diet (P < 0.05). The SeMet increased the levels of ZO-1 and Claudin-3 compared to the SS (P < 0.05). Moreover, SeMet upregulated the marker genes of intestinal enteroendocrine cells, stem cells, and epithelial cells compared to the SS diet (P < 0.05). However, supplementation of Nano-Se reduced the mRNA levels of interleukin 1ß (IL-1ß), and IL-8 and the concentration of reactive oxygen species (ROS) in the jejunum compared to the SS (P < 0.05). The Nano-Se and SeMet also increased the protein levels of CAT and SOD compared to the SS and Sec diet (P < 0.05). The number of the goblet cells and Mucin-2 (Muc2) levels were the highest in the Nano-Se group (P < 0.05). The protein expression levels of goblet cell differentiation regulator (v-myc avian myelocytomatosis viral oncogene homolog, c-Myc) were highest in the Nano-Se compared to the SS diet (P < 0.05). The Nano-Se decreased the mRNA and protein levels of NLRP3 signaling pathway-related genes compared to the SS diet (P < 0.05). In conclusion, our study demonstrated that Nano-Se and SeMet are better at improving the intestinal health of 21-day-old broilers. Additionally, Nano-Se demonstrated superior antioxidant and anti-inflammatory effects, promoting the development of intestinal goblet cells by modifying the NLRP3 signaling pathway.

Fabrication of red-emitting perovskite LEDs by stabilizing their octahedral structure.

Light-emitting diodes (LEDs) based on metal halide perovskites (PeLEDs) with high colour quality and facile solution processing are promising candidates for full-colour and high-definition displays1-4. Despite the great success achieved in green PeLEDs with lead bromide perovskites5, it is still challenging to realize pure-red (620-650 nm) LEDs using iodine-based counterparts, as they are constrained by the low intrinsic bandgap6. Here we report efficient and colour-stable PeLEDs across the entire pure-red region, with a peak external quantum efficiency reaching 28.7% at 638 nm, enabled by incorporating a double-end anchored ligand molecule into pure-iodine perovskites. We demonstrate that a key function of the organic intercalating cation is to stabilize the lead iodine octahedron through coordination with exposed lead ions and enhanced hydrogen bonding with iodine. The molecule synergistically facilitates spectral modulation, promotes charge transfer between perovskite quantum wells and reduces iodine migration under electrical bias. We realize continuously tunable emission wavelengths for iodine-based perovskite films with suppressed energy loss due to the decrease in bond energy of lead iodine in ionic perovskites as the bandgap increases. Importantly, the resultant devices show outstanding spectral stability and a half-lifetime of more than 7,600 min at an initial luminance of 100 cd m-2.

First identification of Cytauxzoon manul in Eurasian lynx (Lynx lynx) in northwestern China.

BACKGROUND: Multiple species of the genera Cytauxzoon and Hepatozoon can infect wild felines, but the diversity of these and other apicomplexan parasites in Eurasian lynx is scarcely known. The aim of this study was to detect Cytauxzoon and Hepatozoon species with molecular methods in Eurasian lynxes and their ticks in northwestern China. METHODS: DNA was extracted from the heart, liver, spleen, lung, and kidney samples of three Eurasian lynxes as well as from their five ixodid ticks. These DNA samples were screened with polymerase chain reactions (PCRs) for Cytauxzoon with the partial cytochrome b gene (CytB), cytochrome c oxidase subunit I gene (COI), and small subunit ribosomal RNA gene (18S rRNA), and Hepatozoon with three different fragments of small subunit ribosomal RNA gene (18S rRNA). PCR products were sequenced, aligned, and phylogenetically analyzed. RESULTS: One adult female of Eurasian lynx (#1, adult female) was co-infected with Cytauxzoon manul and Hepatozoon felis genotype I, while an adult male lynx (#2) was infected with C. manul. Interestingly, H. felis genotype I was both detected in a male cub (#3) and two out of five infesting Hyalomma asiaticum ticks. CONCLUSIONS: For the first time, Cytauxzoon manul is reported here from Eurasian lynx. In addition, H. felis has not been known to occur in this host species in China and Central Asia. Thus, the findings of this study extend our knowledge on the geographical distribution and host range of these haemoprotozoan parasites. Moreover, this is also the first evidence of C. manul and H. felis co-infection in Eurasian lynx.

Impact of Hypoxia-Inducible Factor-1α on Host Immune Metabolism and Tissue Damage During Mycobacterium bovis Infection.

HIF-1α is a pivotal regulator of metabolic and inflammatory responses. This study investigated the role of HIF-1α in M. bovis infection and its effects on host immune metabolism and tissue damage. We evaluated the expression of immunometabolism markers and MMPs infected with M. bovis, and following HIF-1α inhibition in vitro. To understand the implications of HIF-1α inhibition on disease progression, mice at different infection stages were treated with the HIF-1α inhibitor, YC-1. Our results revealed an upregulation of the HIF-1α in macrophages post-M. bovis infection, facilitating enhanced M1 macrophage polarization. The blockade of HIF-1α moderated these responses but escalated MMP activity, hindering bacterial control. Consistent with our in vitro results, early-stage treatment of mice with YC-1 aggravated pathological alterations and tissue damage, while late-stage HIF-1α inhibition proved beneficial in managing the disease. Overall, our findings underscored the nuanced role of HIF-1α across varying phases of M. bovis infection.

Clinical and historical infection of Tacheng tick virus 2: A retrospective investigation.

BACKGROUND: Tacheng tick virus 2 (TcTV-2) is an emerging tick-borne virus belonging to the genus Uukuvirus in the family Phenuiviridae. Initially isolated in 2019 from a patient in Xinjiang Uygur Autonomous Region (XUAR), northwestern China, who developed fever and headache after a tick bite, TcTV-2 was concurrently molecularly detected in hard ticks across various countries, including China, Kazakhstan, Romania, and Turkey. This study conducted a retrospective epidemiological investigation of TcTV-2 infection. METHODOLOGY: In this retrospective cohort study, we collected samples from 47 tick-bitten patients, 984 herdsmen, 7 Asian badgers, 13 red foxes, and 168 Hyalomma asiaticum tick egg batches. Patients' samples were primarily analyzed by using high-throughput sequencing, targeting the V3-V4 region of the bacterial 16S rRNA gene and viral cDNA libraries. Typical tick-borne pathogens were further confirmed using RT-PCR and detected in Asian badgers, red foxes and Hy. asiaticum tick egg batches. We also conducted enzyme-linked immunosorbent assay (ELISA) to detected specific IgM and IgG antibodies against TcTV-2 in herdsmen. Phylogenetic analysis was performed to genetically characterize TcTV-2 detected in this study. PRINCIPAL FINDINGS: TcTV-2 was detected in various samples, including blood, urine, and throat swabs from 12.77% (6/47) tick-bitten patients. It was found in blood samples of 14.29% (1/7) of wild badgers, 7.69% (1/13) of red foxes, and 13.69% (23/168) of Hy. asiaticum egg batches. Furthermore, ELISA results revealed that 9.55% (94/984) of the serum samples (34 from males and 60 from females) were tested positive for TcTV-2-specific IgG, while 2.95% (29/984, 7 males and 22 females) showed positivity for TcTV-2-specific IgM. Additionally, 1.02% (10/984, 4 males and 6 females) of the sera tested positive for both TcTV-2-specific IgM and IgG. Phylogenetic analysis indicated that the TcTV-2 strains detected in this study were genetically similar, regardless of their origin and host species. CONCLUSIONS: Clinical symptoms of TcTV-2 infection in patients are nonspecific, with common symptoms including headache, fever, asthenia, vomiting, myalgia, rash, and meningitis-like signs. TcTV-2 can be detected in blood, urine, and throat swab samples of infected patients. Among local herdsmen, 9.55% tested positive for TcTV-2-specific IgG and 2.95% for TcTV-2-specific IgM. Importantly, TcTV-2 can be transovarially transmitted in Hy. asiaticum ticks, and the Asian badgers and red foxes are potential reservoirs of TcTV-2.

Selenium nanoparticles enhance mucosal immunity against Mycobacterium bovis infection.

Selenium nanoparticles (SeNPs) enhance the immune response as adjuvants, increasing the efficacy of viral vaccines, including those for COVID-19. However, the efficiency of mucosal SeNPs in boosting vaccine-induced protective immunity against tuberculosis remains unclear. Therefore, this study aims to investigate whether the combination of SeNPs with the AH antigen (Ag85A-HspX) can boost respiratory mucosal immunity and thereby enhance the protective effects against tuberculosis. We synthesized SeNPs and assessed their impact on the immune response and protection against Mycobacterium bovis (M. bovis) as a mucosal adjuvant in mice, administered intranasally at a dose of 20 µg. SeNPs outperformed polyinosinic-polycytidylic acid (Poly IC) in stimulating the maturation of bone marrow-derived dendritic cells (BMDCs), which enhanced antigen presentation. SeNPs significantly activated and proliferated tissue-resident memory T cells (TRMs) and effector CD4+ T cells in the lungs. The vaccines elicited specific antibody responses in the respiratory tract and stimulated systemic Th1 and Th17 immune responses. Immunization with AH and SeNPs led to higher levels of mucosal secretory IgA in bronchoalveolar lavage fluid (BALF) and secretory IL-17 in splenocytes. Moreover, SeNPs immunized mice showed reduced M. bovis infection loads and inflammatory lesions in the lungs post-challenge. Notably, immunization with AH and SeNPs significantly reduced bacterial load in the lungs, achieving the lowest levels compared to all other tested groups. This study calls for pre-clinical investigation of AHB-SeNPs as an anti-bovine tuberculosis vaccine and for exploring its human vaccine potential, which is anticipated to aid in the development of innovative vaccines or adjuvants.

Efficient and stable hybrid perovskite-organic light-emitting diodes with external quantum efficiency exceeding 40 per cent.

Light-emitting diodes (LEDs) based on perovskite semiconductor materials with tunable emission wavelength in visible light range as well as narrow linewidth are potential competitors among current light-emitting display technologies, but still suffer from severe instability driven by electric field. Here, we develop a stable, efficient and high-color purity hybrid LED with a tandem structure by combining the perovskite LED and the commercial organic LED technologies to accelerate the practical application of perovskites. Perovskite LED and organic LED with close photoluminescence peak are selected to maximize photon emission without photon reabsorption and to achieve the narrowed emission spectra. By designing an efficient interconnecting layer with p-type interface doping that provides good opto-electric coupling and reduces Joule heating, the resulting green emitting hybrid LED shows a narrow linewidth of around 30 nm, a peak luminance of over 176,000 cd m-2, a maximum external quantum efficiency of over 40%, and an operational half-lifetime of over 42,000 h.

A novel regulator of wheat tillering LT1 identified by using an upgraded BSA method, uni-BSA.

Branching/tillering is a critical process for plant architecture and grain yield. However, Branching is intricately controlled by both endogenous and environmental factors. The underlying mechanisms of tillering in wheat remain poorly understood. In this study, we identified Less Tiller 1 (LT1) as a novel regulator of wheat tillering using an enhanced bulked segregant analysis (BSA) method, uni-BSA. This method effectively reduces alignment noise caused by the high repetitive sequence content in the wheat genome. Loss-of-function of LT1 results in fewer tillers due to defects in axillary meristem initiation and bud outgrowth. We mapped LT1 to a 6 Mb region on the chromosome 2D short arm and validated a nucleotide-binding (NB) domain encoding gene as LT1 using CRISPR/Cas9. Furthermore, the lower sucrose concentration in the shoot bases of lt1 might result in inadequate bud outgrowth due to disturbances in the sucrose biosynthesis pathways. Co-expression analysis suggests that LT1 controls tillering by regulating TaROX/TaLAX1, the ortholog of the Arabidopsis tiller regulator REGULATOR OF AXILLARY MERISTEM FORMATION (ROX) or the rice axillary meristem regulator LAX PANICLE1 (LAX1). This study not only offers a novel genetic resource for cultivating optimal plant architecture but also underscores the importance of our innovative BSA method. This uni-BSA method enables the swift and precise identification of pivotal genes associated with significant agronomic traits, thereby hastening gene cloning and crop breeding processes in wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01484-7.

The bacterial patterns suggesting the dynamic features of tick-associated microorganisms in hard ticks.

BACKGROUND: Ticks are blood-feeding significant arthropods that can harbour various microorganisms, including pathogens that pose health risks to humans and animals. Tick-symbiont microorganisms are believed to influence tick development, but the intricate interactions between these microbes and the relationships between different tick-borne microorganisms remain largely unexplored. RESULTS: Based on 111 tick pool samples presenting questing and engorged statuses including 752 questing tick and 1083 engorged tick from cattle and goats, which were collected in two types of geographic landscape (semi-desert and alpine meadow). We observed significant variations in the composition of tick-borne microorganisms across different environments and blood-engorgement statuses, with a pronounced divergence in symbionts compared to environmental bacteria. Metabolic predictions revealed over 90 differential pathways for tick-borne microorganisms in distinct environments and more than 80 metabolic variations in response to varying blood engorgement statuses. Interestingly, nine pathways were identified, particularly related to chorismate synthesis and carbohydrate metabolism. Moreover, microbial network relationships within tick-borne microorganism groups were highly distinct across different environments and blood-engorgement statuses. The microbial network relationships of symbionts involve some pathogenic and environmental microorganisms. Regression modelling highlighted positive correlations between the Coxiella symbiont and related pathogens, while some environmental bacteria showed strong negative correlations with Coxiella abundance. We also identified commensal bacteria/pathogens in bacterial cooccurrence patterns. Furthermore, we tested pathogenic microorganisms of each tick sample analysis revealed that 86.36% (1601/1855) of the tick samples carried one or more pathogenic microorganisms, The total carrier rate of bacterial pathogens was 43.77% ((812/1855). Most blood samples carried at least one pathogenic microorganism. The pathogens carried by the ticks have both genus and species diversity, and Rickettsia species are the most abundant pathogens among all pathogens. CONCLUSION: Our findings underscore that the bacterial pattern of ticks is dynamic and unstable, which is influenced by the environment factors and tick developmental characteristics.

Rapid Identification of Brucella Genus and Species In Silico and On-Site Using Novel Probes with CRISPR/Cas12a.

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis.

Anaplasma bovis and Bartonella spp. in Libyan Jirds (Meriones libycus) from China.

Anaplasma bovis (1), Bartonella krasnovii (3), and Bartonella sp. (17) were detected in 80 Libyan jirds (Meriones libycus) from China. These findings extend the known host and geographic ranges of these pathogens, with neither A. bovis nor B. krasnovii previously confirmed in Libyan jirds.

Multicomponent Pathogen-Mimicking Nanoparticles Induce Intestinal Immune Responses against Paratuberculosis.

Given the worldwide problem posed by enteric pathogens, the discovery of safe and efficient intestinal adjuvants combined with novel antigen delivery techniques is essential to the design of mucosal vaccines. In this work, we designed poly (lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) to codeliver all-trans retinoic acid (atRA), novel antigens, and CpG. To address the insolubility of the intestinal adjuvant atRA, we utilized PLGA to encapsulate atRA and form a "nanocapsid" with polydopamine. By leveraging polydopamine, we adsorbed the water-soluble antigens and the TLR9 agonist CpG onto the NPs' surface, resulting in the pathogen-mimicking PLPCa NPs. In this study, the novel fusion protein (HBf), consisting of the Mycobacterium avium subspecies paratuberculosis antigens HBHA, Ag85B, and Bfra, was coloaded onto the NPs. In vitro, PLPCa NPs were shown to promote the activation and maturation of bone marrow-derived dendritic cells. Additionally, we found that PLPCa NPs created an immune-rich microenvironment at the injection site following intramuscular administration. From the results, the PLPCa NPs induced strong IgA levels in the gut in addition to enhancing powerful systemic immune responses. Consequently, significant declines in the bacterial burden and inflammatory score were noted in PLPCa NPs-treated mice. In summary, PLPCa can serve as a novel and safe vaccine delivery platform against gut pathogens, such as paratuberculosis, capable of activating both systemic and intestinal immunity.

Molecular detection of Bartonella rochalimae and Hepatozoon canis in red foxes (Vulpes vulpes) from China.

Red foxes (Vulpes vulpes) have been recognized as natural reservoirs for multiple pathogens and a source of infection for domestic animals, wildlife and humans. To date, no reports are available on the Bartonella rochalimae and Hepatozoon canis infection in red foxes from China. In 2018-2022, a total of 16 red foxes were sampled in two counties and a city in Xinjiang Uygur Autonomous Region (XUAR) in northwest China. Subsequently analyzed by DNA extraction amplified by polymerase chain reaction (PCR). In the present study, based on nucleotide sequence and phylogenetic tree analyses, B. rochalimae and H. canis were molecularly identified in red foxes. Our findings provide the first molecular evidence of B. rochalimae and H. canis in red foxes from China.

Discussing the safety and effectiveness of transcatheter arterial embolization combined with intravenous chemotherapy in treating locally advanced breast cancer.

To investigate the efficacy and safety of drug-eluting bead-transarterial chemoembolization (DEB-TACE) combined with systemic chemotherapy in HR+/Her2- locally advanced breast cancer (LABC) patients. A controlled study was conducted on LABC patients treated at Jianyang People's Hospital and the First Affiliated Hospital of Chengdu Medical College from December 2020 to June 2022. The patients were randomly divided into the experimental group and the control group. The experimental group received DEB-TACE combined with the TAC regimen (175 mg/m2 paclitaxel-loaded albumin, 50 mg/m2 Doxorubicin, and 500 mg/m2 cyclophosphamide), while the control group received the TAC regimen intravenously. The therapeutic efficacy was evaluated using the mRECIST criteria. Statistical analysis was performed using SPSS 22.0 software, and baseline characteristics, overall response rate (ORR), pathological complete response (PCR), adverse reactions, and complications were compared between the two groups using paired t-test and chi-square test. A total of 60 patients were included, with 30 patients in the experimental group (50%) and 30 patients in the control group (50%). After the first treatment, the ORR was 90% in the experimental group and 60% in the control group (P < 0.05). The overall ORR was 100% in the experimental group and 83% in the control group (P < 0.05). PCR was achieved in 14 patients (47%) in the experimental group and 4 patients (13%) in the control group. The main adverse reactions in the experimental group were skin blistering, pigmentation, and pain. There was no statistically significant difference in vomiting and grade II or above bone marrow suppression between the two groups. No grade III or above adverse events occurred in either group. The comparison of tumor shrinkage between the two groups was P = 0.051, and axillary lymph node shrinkage was P < 0.05. The use of drug-eluting beads in combination with neoadjuvant chemotherapy is a feasible and safe treatment option for locally advanced breast cancer patients.

Novel trypanosomatid species detected in Mongolian pikas (Ochotona pallasi) and their fleas in northwestern China.

BACKGROUND: In the family Trypanosomatidae, the genus Trypanosoma contains protozoan parasites that infect a diverse range of hosts, including humans, domestic animals, and wildlife. Wild rodents, as natural reservoir hosts of various pathogens, play an important role in the evolution and emergence of Trypanosomatidae. To date, no reports are available on the trypanosomatid infection of pikas (Lagomorpha: Ochotonidae). METHODS: In this study, Mongolian pikas and their fleas were sampled at the China-Mongolia border, northwestern China. The samples were analyzed with polymerase chain reaction (PCR) and sequencing for the presence of Trypanosomatidae on the basis of both the 18S ribosomal RNA (18S rRNA) gene and the glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. The morphology of trypomastigotes was also observed in peripheral blood smears by microscopy. RESULTS: Molecular and phylogenetic analyses revealed a new genotype of the Trypanosoma lewisi clade that was found both in pika blood and flea samples. This genotype, which probably represents a new species, was provisionally designated as "Trypanosoma sp. pika". In addition, a novel genotype belonging to the genus Blechomonas of Trypanosomatidae was detected in fleas. On the basis of its molecular and phylogenetic properties, this genotype was named Blechomonas luni-like, because it was shown to be the closest related to B. luni compared with other flea-associated trypanosomatids. CONCLUSIONS: To the best of our knowledge, this is the first study to report any trypanosomatid species in Mongolian pikas and their fleas. Further studies are needed to investigate the epidemiology of these protozoan parasites, as well as to evaluate their pathogenicity for humans or domestic animals.

Detection of Sarcocystis albifronsi, Eimeria alpacae, and Cystoisospora felis in Eurasian lynx (Lynx lynx) in northwestern China.

Eurasian lynx (Lynx lynx) is widely distributed in various habitats in Asia and Europe, and it may harbor multiple pathogens. Currently, the information on protozoan infection in Eurasian lynx is scarce. In this study, we performed nested polymerase chain reaction (nPCR) analysis to detect intestinal protozoan infection in three dead Eurasian lynxes, in northwestern China. Three dead Eurasian lynxes, an adult female (#1), an adult male (#2), and a cub male (#3), were sampled in West Junggar Mountain, the northwestern region of Xinjiang Uyghur Autonomous Region. The intestine samples were analyzed using nPCR. We used primers targeting the cytochrome C oxidase subunit I gene (COI) for detection of Sarcocystis and Eimeria species and targeting the small subunit 18 S ribosomal RNA gene (18S rRNA) for detection of Cystoisospora species. The nPCR-positive products were sequenced, aligned, and phylogenetically analyzed. Three intestinal protozoa, Sarcocystis albifronsi, Eimeria alpacae, and Cystoisospora felis, were found in three Eurasian lynxes. The intestine sample of Eurasian lynx #2 was detected with S. albifronsi and E. alpacae. In addition, C. felis was only found in the intestine sample of Eurasian lynx #3. To the best of our knowledge, S. albifronsi and E. alpacae were detected in Eurasian lynx for the first time. In addition, C. felis was firstly found in Eurasian lynx in China. These findings extend our knowledge of the geographical distribution and host range of intestinal protozoa.

Application of DNA barcodes in the genetic diversity of hard ticks (Acari: Ixodidae) in Kazakhstan.

Forty-five tick species have been recorded in Kazakhstan. However, their genetic diversity and evolutionary relationships, particularly when compared to ticks in neighbouring countries, remain unclear. In the present study, 148 mitochondrial cytochrome c oxidase subunit I (COI) sequence data from our laboratory and NCBI (National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/ ) data were used to address this knowledge gap. Phylogenetic analyses showed that i) Hyalomma anatolicum anatolicum (Koch, 1844) ticks from Jambyl Oblast (southeastern Kazakhstan) and Gansu Province (northwestern China) constituted a newly deviated clade; and ii) Dermacentor reticulatus (Fabricius, 1974) ticks from South Kazakhstan Oblast were closer to those in Romania and Turkey. The network diagram of haplotypes showed that i) the H-1 and H-2 haplotypes of Dermacentor marginatus (Sulzer, 1776) ticks from Zhetisu and Almaty were all newly evolved; and ii) the H-3 haplotypes of Haemaphysalis erinacei (Pavesi, 1884) from Almaty Oblast and Xinjiang Uygur Autonomous Region (northwestern China) were evolved from the H-1 haplotype from Italy. In the future, more COI data from different tick species, especially from Kazakhstan and neighbouring countries, should be employed in the field of tick DNA barcoding.

Detection of Rickettsia raoultii in Vermipsylla alakurt-Like Fleas of Sheep in Northwestern China.

INTRODUCTION: To date, a total of 2574 validated flea species have been discovered. Vermipsyllidae is a family of fleas that comprises at least eight species. Vermipsylla is a genus of the family Vermipsyllidae within the order Siphonaptera of fleas. Here a novel Vermipsylla species was described, and rickettsial agent was also detected in it. METHODS: A total of 128 fleas were collected directly from 260 pastured sheep in China. Of these, eight representative fleas (four males and four females) were identified by key morphological features. Meanwhile, 120 flea DNAs, including six flea samples for molecular taxonomy, were subjected to Rickettsia spp. DNA detection. The molecular identity of fleas was determined by amplification and sequenmce analysis of four genetic markers (the 28S rDNA genes, the 18S rDNA genes, the mitochondrial cytochrome c oxidase subunit I and subunit II). In addition, five Rickettsia-specific gene fragments were used to identify the species of the rickettsial agents. The amplified products were sequenced and phylogenetically analyzed. RESULTS: The morphological characteristics of the flea species identified in this study were similar to Vermipsylla alakurt, but presented difference in hair number of the metepimeron, the third tergum, the genitals and the tibiae of hind leg. The 18S rDNA, 28S rDNA and COII genetic markers from fleas showed the highest identity to those of V. alakurt, shared 98.45% (954/969), 95.81% (892/931) and 85.86% (571/665) similarities, respectively. However, the COI sequence showed the highest identity to that of Dorcadia ioffi with 88.48% (576/651) similarity. Rickettsia raoutii tested positive in 14.17% (17/120) flea DNA samples. CONCLUSION: Our study reports the detection of R. raoultii in V. alakurt-like fleas infesting sheep in China.