Gene Fragments Research Articles

DNA Barcoding Using 18S rRNA Gene Fragments for Identification of Tick-Borne Protists in Ticks in the Republic of Korea

The objective of this study was to evaluate the diversity and prevalence of tick-borne protists in the Republic of Korea via DNA barcoding using 18S rRNA gene fragments and PCR. Between 2021 and 2022, questing ticks were collected using the flagging method, with a total of 13,375 ticks collected and pooled into 1003 samples. Of these, 50 tick pools were selected for DNA barcoding targeting the V4 and V9 regions of 18S rRNA using the MiSeq platform. A taxonomic analysis of the amplicon sequence variants identified three genera of protozoa, namely Hepatozoon canis, Theileria luwenshuni, and Gregarine sp. However, the number and abundance of protists detected were different depending on the primer sets, and T. gondii was not identified in DNA barcoding. Furthermore, conventional PCR confirmed the presence of H. canis, Toxoplasma gondii, T. luwenshuni, and Theileria sp. in the collected ticks. This study identified H. canis and T. gondii in Ixodes nipponensis for the first time. It demonstrated that the results of DNA barcoding using 18S rRNA gene fragments can vary depending on the primer sets and further optimization is required for library construction to identify tick-borne protists in ticks. Despite these limitations, the findings highlight the potential of DNA barcoding using 18S rRNA gene fragments for screening the diversity of tick-borne protists in ticks.

Regional microbial content of fermented traditional and industrial East Mediterranean sausages from the islands of Cyprus and Mytilini

Objective: To characterize the microbial biodiversity of fermented sausages from the East Mediterranean islands of Cyprus and Mytilini, and to identify differences between the microbial diversity of traditionally and industrially produced Cypriot sausages. Method: The microbial diversity of thirty sausages from Cyprus and Mytilini (traditionally and industrially produced) was analyzed using high throughput sequencing (HTS) (amplicon sequencing) of 16S rRNA gene and ITS loci fragments. By applying microbial signature detection and machine learning algorithms, we identified key microbes that distinguish traditionally produced sausages from those produced industrially. Focusing on selected microbial taxa and using interaction network analysis, we identified associations among the sausages’ microbiota that may affect the shaping of the sausages’ microbial consortia. Results: Cypriot and Mytilini sausages indicated increased relative representation of Lactobacillus and Leuconostoc . Cypriot sausages were distinguished by the presence of the fungi Debaryomyces hansenii and Candida spp. , whereas Mytilini sausages by the bacteria Lactococcus and Streptococcus . Traditionally produced sausages from the Pitsilia region of Cyprus were differentiated by the presence of the species Lactobacillus helveticus , whereas industrially produced sausages were differentiated by the species Leuconostoc mesenteroides . Focusing mainly on Lactobacillus and Leuconostoc , negative associations with pathogenic bacteria, such as Salmonella , and spoilage fungi, such as Fusarium poae , were revealed. Conclusion: The present metataxonomic analysis provided insights into the microbial communities that characterize Cypriot and Mytilini sausages. The findings provide an indication that the microbial diversity might be applied as an additional marker of Cypriot sausages’ authenticity.

New insights into the systematics of Cyclocoelidae (Trematoda: Echinostomatoidea) based on novel morphological and molecular data, with description of a new species and a new genus.

In light of the morphological and molecular data for cyclocoelids observed from the air sacs of Mareca strepera (Linnaeus) (Anatidae, Anseriformes) caught in the southern region of the Russian Far East, we suggest new insights into the systematics of the family Cyclocoelidae. A comparative morphological and phylogenetic analyzes revealed that new cyclocoelids represented the new genus and species Paracyclocoelum lobatum. Based on the 28S rRNA gene fragment we showed the significant genetic divergence of P. lobatum from the type species of the type genus for the family, Cyclocoelum mutabile (Zeder, 1800) Brandes, 1892 and along with the confusing morphological similarity by the prepharyngeal position of the genital pore it most likely indicate homologous development of the reproductive system of Paracyclocoelum and Cyclocoelum. Here, we provide a new dichotomous key for five cyclocoelid genera from the subfamily Cyclocoelinae including Paracyclocoelum n. g. The new genus Paracyclocoelum had sister relationship to the cyclocoelin genus Circumvitellatrema. Based on the polyphyletic interrelationships of Cyclocoelum and Circumvitellatrema the Cyclocoelinae were assigned with the status sensu lato.

Molecular characterization and phylogenetic analysis of infectious laryngotracheitis virus isolates from commercial chicken flocks in Turkey.

Infectious laryngotracheitis virus (ILTV) causes an acute and highly contagious respiratory disease in poultry. Live-attenuated vaccines are generally used to control and prevent infectious laryngotracheitis (ILT). However, these vaccines can revert to a virulent form due to multiple passages and thereby become an ILT source. Hence, monitoring of ILTV in the field through molecular characterization is critically important for controlling infection and differentiating circulating isolates. In this study, we genotypically characterized and phylogenetically analyzed eight ILTV isolates from chicken flocks located in four different cities of Turkey between 2019 and 2022. For all isolates, we analyzed two regions of the infected cell protein 4 gene (ICP4-1 and ICP4-2) and the thymidine kinase (TK) gene. The isolates were 100%, 100%, and 99.8-100% identical to each other in the ICP4-1 and ICP4-2 gene fragments and the TK gene, respectively. None of the ICP4 sequences had a deletion at nt 272-283, confirming that they were field isolates. None of the isolates were predicted to have a T252M mutation in the thymidine kinase, suggesting that they have low virulence. The isolates were 100%, 99.36%, and 99.91% identical to Turkish ILTV isolates in their ICP4-1, ICP4-2, and TK gene region, respectively. Phylogenetic analysis based on the ICP4-1 and TK genes confirmed that the ILTV isolates are closely related to Turkish ILTV isolates. This suggests that these ILTVs were endemic isolates, which in turn suggests that the ILTV isolates circulating in Turkey were evolutionarily close, originated from the field, and had low virulence.

Elucidating the Mitogenomic Blueprint of Pomadasys perotaei from the Eastern Atlantic: Characterization and Matrilineal Phylogenetic Insights into Haemulid Grunts (Teleostei: Lutjaniformes).

The parrot grunt fish, Pomadasys perotaei, has a limited distribution in the Eastern Atlantic Ocean and is an important species in marine capture fisheries across several West African countries. Despite its ecological and economic significance, the mitogenomic information for this species is lacking. This study utilized next-generation sequencing to generate the de novo mitogenome of P. perotaei from Eastern Atlantic. The resulting mitogenome is 16,691 base pairs and includes 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNAs, and an AT-rich control region (CR). Most of the PCGs exhibit nonsynonymous (Ka) and synonymous (Ks) substitution rates of less than '1', indicating strong negative selection across haemulid fishes. The control region of Pomadasys species contains four conserved domains, as seen in other teleost's, with polymorphic nucleotides that can be used to study population structures through the amplification of short mitochondrial gene fragments. Additionally, Bayesian phylogenetic analysis based on PCGs revealed a non-monophyletic clustering pattern of Pomadasys within the haemulid matrilineal tree. Overall, the structural characterization and phylogenetic analysis enhance our understanding of the genetic composition and evolutionary history of Pomadasys species from the Indo-West Pacific and Eastern Atlantic Oceans.

Morpho-molecular characterization and phylogenetic relationships of Encotyllabe percussa n. sp. (Monogenea: Capsalidae) from the spangled emperor Lethrinus nebulosus (Teleostei, Lethrinidae).

Encotyllabe percussa n. sp. is proposed based on morphology and DNA sequences analysis of ribosomal (18S, 28S) and mitochondrial (COI) gene fragments. Encotyllabe percussa n. sp. was found infecting the spangled emperor Lethrinus nebulosus (n = 75) with higher prevalence from Dibba, Musandam (Gulf of Oman) than in Dhofar Salalah (Indian Ocean), Oman (p = 0.03). The general morphology of E. percussa n. sp. resembles E. caballeroi, E. chironemi and E. spari, which exhibit pre-equatorial testes. However, E. percussa n. sp. shows unique morphological characteristics distinguishing from congeneric species: the large hamuli bear notch allocated externally between the first half proximal of the root, and the small hamuli exhibit semicircular shape with undivided roots. Phylogenetic relationships within the Encotyllabe genus remain unresolved. However, the tree topology with the 28S showed overall consistency with the principal component analysis arrangement (PCA) derived from the morphological analysis. Which showed that the large and small hamuli, marginal hooks, ovary, testes (length and width) and peduncle are currently the most important morphological traits within the genus. Cytochrome c oxidase subunit I (COI) gene fragment showed high interspecific genetic divergence adding unambiguous resolution to discriminate/designate species identity. Interrelations within the genus support the identity of Encotyllabe percussa as a new species. This is the first species characterized with three gene fragments, the second congeneric species described in L. nebulosus and the first recorded in Oman.

Description of larvae of three Opatrini species (Coleoptera: Tenebrionidae: Blaptinae) from China, with molecular species delimitation and diagnoses

Opatrini Brullé, 1832 is the most species-rich tribe in the subfamily Blaptinae Leach, 1815. In this study, a preliminary phylogenetic relationship among 14 species of five genera of Opatrini is hypothesized based on mitochondrial gene (COI) fragment. Based on the phylogenetic topology and the results of three molecular species delimitation analyses, three larval specimens are assessed for the adults. Additionally, the larvae of these three species are described and illustrated: Gonocephalum bilineatum (Walker, 1858), Gonocephalum gracile (Bates, 1879), and Mesomorphus latiusculus Chatanay, 1917.

The characteristics of T-cell receptor repertoire in relation to systemic immune response of patients with ischemic stroke.

T lymphocytes play a vital role in the immune-inflammatory response following a stroke. However, the specific mechanisms behind the contrasting functions of T cells in the brain and peripheral tissues after a stroke remain unclear and require further investigation. T-cell receptors (TCRs) are essential in controlling how T lymphocytes develop and become active. This study aims to gain a deeper understanding of the biological function of T lymphocytes by analyzing the TCR repertoire in patients who have experienced an acute ischemic stroke (AIS). High-throughput TCR sequencing was conducted on peripheral blood samples from 25 AIS patients and 10 healthy controls. We compared the percentage of T cells and the characteristics of the TCR repertoire, specifically focusing on the recombination of V(D)J gene fragments and the diversity of the complementarity determining region 3 (CDR3) of the Vβ gene. Additionally, this study analyzed the potential biological significance of the skewed TCR repertoire in AIS patients. In patients with AIS, the proportion of circulating lymphocytes (LY%) decreased while the systemic immune-inflammatory index (SII) increased compared to healthy controls. The average number of TCR read pairs decreased, corresponding with the presence of lymphopenia. However, the recombination of V(D)J gene fragments, the number of CDR3 clonotypes, and the diversity of CDR3 was elevated in the peripheral blood of AIS patients. Furthermore, the increased number of CDR3 amino acid or nucleotide clonotypes was negatively correlated with neurologic deficits but positively correlated with AIS patients' systemic immune condition and functional outcomes. Our findings suggest that both immunosuppression and enhanced antigen-specific T-cell response may exist in the periphery of the AIS patients. Further investigation into the mechanisms underlying these opposing changes may lead to the discovery of novel targets to reverse immunosuppression or mitigate the detrimental effects of T cells in the lesioned brain of AIS patients.

Restoration of the spike architectonics in ancient barley excavated at the twelfth-century settlement of Usvyaty

Background. The data are presented on the architectonics of ancient barley spikes from the 12th century, excavated in 2019 at Usvyaty Settlement. Modern molecular genetics approaches were used to study domestication genes (Btr1, Btr2, and Vrs) in ancient and contemporary barleys (germplasm accessions preserved at VIR).Materials and methods. The carbonized kernels found by archaeologists during the excavations at Usvyaty were analyzed. Primers for domestication genes were designed, and PCR was performed on contemporary and ancient barley grains. Ancient kernels were studied in accordance with the rules established for organizing a paleogenetics laboratory, which excluded any contamination with contemporary DNA. Fragments of domestication genes from contemporary and ancient barley grain samples underwent Sanger sequencing. Results. Ancient DNA was isolated and enriched. The analysis of domestication gene sequences made it possible to reconstruct the ancient barley spike’s features. Conclusion. The ancient cereal crop architectonics was restored to ascertain a brittle two-row spike of ancient barley

Genetic Identification of Putative Hybrids Between Grey Wolf and Golden Jackal

We describe the results of genetic analysis of 11 phenotypically deviant individuals of grey wolf (Canis lupus Linnaeus, 1758) sensu lato collected in Voronezh State Nature Biosphere Reserve (Chernozem zone of European Russia) and Dagestan (Northern Caucasus, Russia) putatively identified morphologically as hybrids between grey wolf and golden jackal (Canis aureus Linnaeus, 1758). By means of maternally inherited mtDNA (sequences of cytochrome b gene fragment) and paternal lineage markers ZfY no F1 wolf-jackal hybrids were identified. As well, possibility of classification of the studied individuals to next generation hybrids from crosses between different wolf-jackal F1s. However, attribution of these animals to complex hybrids such as various backcrosses cannot be rejected. From the results of analysis by a set of autosomal microsatellite loci we putatively diagnosed a single F2 hybrid. As well, we obtained data that can be considered as traces of hybridization between wolf and jackal in southern regions of European Russa, albeit direct indications of introgression between these species not found. At the same time, the results of both genetic and craniological studies could be interpreted as indication to hybridization between wolves and domestic dogs on the same territories.

Molecular Detection of Anaplasma marginale in Capybara (Hydrochoerus hydrochaeris) from Corrientes, Argentina.

Monitoring wildlife health is essential for understanding global disease patterns, particularly as vector-borne infections extend the geographic ranges and thereby hosts due to environmental shifts. Anaplasma marginale, primarily impacting cattle, has economic implications and has been found in diverse hosts, yet its presence in capybaras (Hydrochoerus hydrochaeris), influential in tick-borne pathogen spread, lacks comprehensive understanding. From 2015 to 2022, 14 capybaras were surveyed across two different areas of northeastern Argentina. In 1 of 14 (7%) capybaras, the presence of A. marginale was confirmed through the amplification of specific genes, msp5 and msp1β. In addition, A. marginale DNA was detected in the capybara's blood sample through quantitative PCR, with a cycle threshold value of 30.81 (800 copies per reaction). Amplification of a fragment of the msp1α gene revealed PCR products of three different sizes, suggesting the presence of at least three coinfecting A. marginale variants in the capybara host. This study suggests that capybaras are wild hosts for A. marginale in the Ibera Wetlands in Argentina, potentially influencing the infection dynamics of both domestic and wild species. This finding highlights the necessity for thorough studies on the role of capybaras in disease dynamics, crucial for understanding wildlife health and the spread of disease.

Evidence of the involvement of Xylella fastidiosa in the occurrence of walnut leaf scorch in Xinjiang, China.

Walnut (Juglans regia Linn.) leaf scorch (WLS) was first reported in 2012 in Hotan, Xinjiang, China (Zhang et al., 2012). Initially, brown spots appear at the apex of the leaflets with slight shrinking. These spots spread inward along the leaf margins in a flame-like pattern. The scorched areas curl inward with a yellow halo. No powdery or bacterial signs were observed on the leaf surface. In severe cases, the leaves dried up and shrank, affecting the entire tree. However, new leaves did not show any signs of scorching. We collected 300 symptomatic leaf samples from 10-12 year-old trees of the susceptible WLS species Wen185, located in Daryaboyi (40°72'N, 80°49'E), Xakal (40°69'N, 80°52'E), and Karatal (40°73'N, 80°39'E) for X. fastidiosa PCR detection analysis. X. fastidiosa was detected in asymptomatic leaves of trees with severe WLS, as well as in asymptomatic leaves of trees exhibiting mild WLS symptoms, and it was even found in asymptomatic leaves of trees without any WLS symptoms.To isolate X. fastidiosa, walnut leaves with petioles were disinfected with 3% bleach for 10 minutes, followed by four washes in autoclaved deionized water. The midrib and petiole of the leaf were cut off with a sterile blade, and a 2 mm to 3 mm section was excised. Then, the cut ends were squeezed with a pair of pliers, and the sap was blotted onto Periwinkle Wilt (PW) plates (Davis et al. 1983). The plates were sealed with parafilm and incubated at 28°C in the dark, with daily observations for the development of individual colonies. The sap collected from 5 out of 20 leaf samples with scorch symptoms and from 20 leaves without symptoms never showed bacterial growth in PW.. Six colonies were tested and confirmed positive for X. fastidiosa using the RST31 and RST33 primer pair (Minsavage et al., 1994). The uploaded sequence accession numbers are PP871340-PP871342. Blast analysis of Multilocus Sequence Typing (MLST) sequences from the isolated strain using the X. fastidiosa MLST Database (http://pubmlst.org/xfastidiosa) revealed a perfect match with sequences of alleles leuA_3, petC_3, malF_, cysG_3, holC_4, nuoL_3, and gltT_3 (PP871343-PP871349). Using MEGA software to concatenate the MLST single gene fragments and construct a ML multi-gene fragment phylogenetic tree through Maximum Likelihood (ML) analysis, the bacterium was identified as X. fastidiosa subsp. multiplex To fulfill Koch's postulates, a purified colony from the PW plate was suspended in 500 μL of deionized water at 1×10^8 CFU·mL-1. A sterilized 5 ml medical needle was used to prick a small hole on the main branch, 5 cm from the base, of three-year-old walnut seedlings, and the bacterial solution was injected into the small hole. Three biological replicates were performed for each treatment (5 μL and 10 μL of bacterial solution, deionized water), and the experiment was repeated three times. Scorching symptoms were observed three months after inoculation in 11 out of the 15 seedlings inoculated with a 10 μL bacterial solution, and isolation of X. fastidiosa occurred in leaf samples from 5 of these seedlings, completing Koch's postulates. Walnut seedlings with water stayed asymptomatic, PCR negative. To our knowledge, X. fastidiosa has been found infecting grapes (Chu, 2001) in Shaanxi, China and pears (Su et al., 2016) in Taiwan, China. This is the first report of X. fastidiosa involvement in WLS in China. Further research on the occurrence of the disease will help prevent the spread of the disease.

ONT sequencing identifies a high prevalence of crt sensitive, triple mutant dhfr and single mutant dhps parasites within an ANC population in Nigeria

BackgroundMalaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxine-pyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated Plasmodium falciparum malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACT-resistant P. falciparum, maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria.MethodsDried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. P. falciparum genomic DNA was extracted by the Chelex® method and Pf18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of Pfdhps, Pfdhfr, Pfmdr1, Pfcrt, Pfk13 and Pfama1 genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology.ResultIn total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. P. falciparum genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. Pfama1, pfk13, Pfdhps, Pfdhfr, Pfmdr1 and Pfcrt were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of Pfdhfr and Pfdhps SP resistance haplotypes, IRN for pyrimethamine resistance and ISGKA/IAGKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the Pfcrt CVIET resistant haplotype was at approximately 22% (7/32).Conclusion and recommendationsHere a multiplexed amplicon-based ONT assay established that triple mutant Pfdfhr-IRN, double mutant Pfdhps-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.

Clinical Manifestations and Molecular Identification of Giardia duodenalis in Pediatric and Adolescent Cancer Patients in Southwestern Iran.

This study aimed to investigate the clinical and molecular characteristics of Giardia duodenalis (G. duodenalis) infection and identify potential risk factors in children and teenagers with malignancies in Shiraz, southwestern Iran. A total of 200 fresh fecal samples were collected from children and adolescents suffering from 32 different cancer types at Amir, Nemazee, and Saadi hospitals affiliated with Shiraz University of Medical Sciences between October 2021 and May 2022. Direct microscopy using saline and iodine wet mount was conducted, and all fecal samples were rechecked by SSU-PCR. Subsequently, a specific fragment of the tpi gene was amplified on all samples for prevalence, sequencing, and assemblage identification. Our study found a 4% (8/200) prevalence of G. duodenalis using microscopy and PCR. The molecular findings were consistent with the microscopic results. All eight positive samples with SSU-rRNA gene were also detected as positive with tpi gene and were correctly sequenced. Among the examined cancer patients, two assemblages were identified: A [sub-assemblage AI (2/8, 25%) and sub-assemblage AII (3/8, 37.5%)] and B [sub-assemblage BIV (3/8, 37.5%)]. Notably, patients were more vulnerable to G. duodenalis infection after receiving at least 8 treatment episodes (p < 0.05) and displaying gastrointestinal symptoms (p > 0.05). The demographic characteristics of cancer patients with G. duodenalis infection and the statistical conclusions were separately detailed. The small sample size and low prevalence rate in this study hindered precise epidemiological conclusions. Nonetheless, the results suggest that G. duodenalis infection among cancer patients in Shiraz city originates from humans, without any specific animal groups (C-H) involved.

Functional characterization of chalcone reductase gene CHR3 through RNAi

In this experiment, the soybean genome "Jilin 30" was used as a template to clone the target gene CHR3 using specific primers. The sense fragment, antisense fragment, and intron of the target gene were ligated into pCAMBIA3300 through double enzyme digestion to form a stem loop structure. We then transferred the constructed RNAi vector pCAMBIA3301-CHR3 RNAi into the recipient soybean genome via Agrobacterium mediated method, specifically reducing the expression of the target gene CHR3. By genetic transformation, positive plants were obtained, and molecular biology methods such as Southern blotting, real-time quantitative PCR, isoliquiritigenin analysis, and identification of resistance to Phytophthora root rot were used to detect the expression level of the CHR3 gene in the positive plants and study its function.The RNAi expression vector pCAMBIA3300-CHR3-RNAi was successfully constructed using the sense and antisense fragments of 428 bp CHR3 gene and an intron of 110 bp was inserted to form the stem-loop structure of the RNAi vector. PCR was used to detect 6 lines of the T1 generation and 15 lines of the T2 generation. The results of Southern analysis confirmed the integration of CHR3 and marker genes into the soybean genome. The expression CHR3 gene in the T1 genotypes decreased by 13.1 to 58.3%; the expression in the root decreased by 0.9 to 47.4%; the expression in the leaf decreased by 20 to 44.2%; and the expression in the stem decreased by 7.6 to 23%. The content of isoliquiritigenin decreased by 26.7% as transgenic shoots had a lower content of isoliquiritigenin and reduced resistance to Phytophthora sojae. Bangladesh J. Bot. 53(3): 627-633, 2024 (September) Special

Genetic characterization of Neisseria meningitidis isolates recovered from patients with invasive meningococcal disease in Lithuania.

Neisseria meningitidis is a gram-negative bacterium responsible for life-threatening invasive infections known as invasive meningococcal disease and is associated with high fatality rates and serious lifelong disabilities among survivors. This study aimed to characterize N. meningitidis isolates cultured from blood and cerebrospinal fluid collected between 2009 and 2021 in Lithuania, assess their genomic relationships with European strains, and evaluate the possibility of using a cost-effective method for strain characterization, thus improving the national molecular surveillance of invasive meningococcal disease. In total, 321 N. meningitidis isolates were collected and analyzed using multilocus restriction typing (MLRT). Amplification of the penA gene and restriction fragment length polymorphism analysis were performed to identify the modified penA genes. Based on the MLRT genotyping results, we selected 10 strains for additional analysis using whole-genome sequencing. The sequenced genomes were incorporated into a dataset of publicly available N. meningitidis genomes to evaluate genomic diversity and establish phylogenetic relationships within the Lithuanian and European circulating strains. We identified 83 different strains using MLRT genotyping. Genomic diversity of N. meningitidis genomes analysed revealed 21 different sequence types (STs) circulating in Lithuania. Among these, ST34 was the most prevalent. Notably, three isolates displayed unique combinations of seven housekeeping genes and were identified as novel STs: ST16969, ST16901, and ST16959. The analyzed strains were found to possess virulence factors not commonly found in N. meningitidis. Six distinct penA profiles were identified, each with different frequencies. In the present study, we also identified N. meningitidis strains with new penA, NEIS0123, NEIS1320, NEIS1525, NEIS1600, and NEIS1753 loci variants. In our study, using the cgMLST scheme, Minimum Spanning Tree (MST) analysis did not identify significant geographic relationships between Lithuanian N.meningitidis isolates and strains from Europe. Discussion: To our knowledge, this is the first study to employ whole genome sequencing (WGS) method for a comprehensive genetic characterization of invasive N. meningitidis isolates from Lithuania. This approach provides a more detailed and precise analysis of genomic relationships and diversity compared to prior studies relying on traditional molecular typing methods and antigen analysis.

First report of lily mottle virus naturally infecting lily (Lilium asiatica) in Texas, USA.

Lily (Lilium asiatica) is an important plant grown for its range of flower colors and heavy scent. In March 2024, potyvirus-like symptoms consisting of light-yellow mottling and mosaic were noticed on 12/20 lily plants in a private property in Weslaco, Hidalgo County, Texas. Two symptomatic plants (WTX1 and WTX2) were sampled randomly for virus diagnosis. The leaf extracts of both samples were negative for the potyvirus group using Agdia's Poty ImmunoStrip® (Agdia, Inc., Elkhart, IN, USA). However, rub-inoculation of the extracts onto healthy Nicotiana benthamiana and Vigna unguiculata plants (n=4, each) induced mild mottle symptoms on the systemic leaves of both herbaceous test plants 20 to 28 days post inoculation, indicating the presence of a mechanically transmissible agent in the samples. No virus-like symptoms were observed on the mock-inoculated plants (n=1, each) of both species. To test for suspected potyvirus infection, 2-µg of total nucleic acid extracts (Dellaporta et al. 1983) from WTX1 and WTX2 were used for complimentary DNA (cDNA) synthesis with Oligo(dT) primer and the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, USA). One microliter aliquot of each cDNA served as template in 12.5-µl conventional PCR reaction volumes with 5 U Taq polymerase (Roche Diagnostics, Indianapolis, IN), and two pairs of degenerate primers targeting the partial cylindrical inclusion (CI) gene and the helper component protease (HC-Pro) of potyviruses (Ha et al. 2008). The expected ~700-bp DNA product of each gene target was amplified from both samples. The amplicons were excised, gel eluted (Zymoclean™ Gel DNA Recovery kit) and cloned individually into the pJET1.2/Blunt vector (Life Technologies). Recombinant plasmids from two transformed Escherichia coli cells per cloned DNA insert were Sanger sequenced. BLASTn analysis of each gene-specific nucleotide (nt) sequence fragment revealed their identities (83 to 92 % nt) as lily mottle virus (LMoV; genus Potyvirus) at 93 to 98% query coverage. In pairwise comparison, the obtained sequences of LMoV isolates from TX specific to the CI (GenBank accession nos. PQ037260-61) and the HC-Pro (PQ037262-63) shared ~99% nt and 100% amino acid (aa) identities with each other and ~91-92% nt and 98-100% aa identities with the closest isolate, Bate5 (JN127341) from Australia. Based on these results, a pair of LMoV-specific primers (LM_v109-F: 5'-GGCCAGTAATGTGCACAAGC-3' and LM_c527-R: 5'-TCGCTGTAGCTAGCGACGTAC-3') was newly designed to target the partial CI gene, internal to the 700-bp fragment obtained above with the generic primers. The primer pair was used to screen each of the mechanically inoculated test plants by RT-PCR as previously described above. The expected 438-bp fragment of the LMoV CI gene was amplified from all the inoculated plants of both N. benthamiana and V. unguiculata; the results were confirmed by Sanger sequencing as described above. The mock-inoculated plant of each experimental host plant tested negative for LMoV. LMoV is a quarantine pest and was previously reported in the U.S. (Brierley and Smith, 1944) and other lily-producing parts of the world, such as the Netherlands, China, Korea, and Brazil. To our knowledge, this is the first confirmed report of the virus in Texas, thus expanding its geographical range. Testing of lily foundation stocks before propagation is essential to prevent further spread of LMoV via planting material.

Wildlife Infection of Peste des Petits Ruminants Detected in China, 2024.

In 2013, the second outbreak of peste des petits ruminants occurred in China, leading to a spillover in more than 20 provinces and municipalities over the next few months. Thereafter, the epidemic situation was stable owing to strict prevention and control measures. In February 2024, several bharals and argali with suspected symptoms of PPR were discovered in Rutog country, Tibet Autonomous Region. Samples collected from these animals were delivered to our laboratory for diagnosis; the results of fluorescence quantitative reverse-transcription (RT) PCR indicated that all samples were positive for PPR viral RNA. The N and F gene fragments were amplified successfully via RT-PCR, and these results confirmed that these animals were infected with PPRV. A PPRV strain (subsequently named ChinaTibet2024) was sequenced, and its genome length was 15,954 nucleotides. A phylogenetic tree analysis using N and F genes and viral genomes showed that the ChinaTibet2024 genome was classified into lineage IV of the PRRV genotypes. The genome of the ChinaTibet2024 strain was found to be closely related to PPRVs isolated in China between 2013 and 2014. A base insertion and a base deletion were detected in the M gene 5' untranslated region. Results indicated that the prevalent PPRV strains in China did not show significant changes and that special attention should be paid to the surveillance of wild animals as an important part of PPR prevention and control.

Prevalence and genotyping of Toxoplasma gondii in questing Ixodes ricinus ticks from forest areas of Northern Poland.

Toxoplasma gondii occurs in a wide range of intermediate hosts, whose blood may be a meal for different tick species. A few studies have examined the role of ticks in the life cycle of T. gondii. This one includes the largest number and all stages of Ixodes ricinus collected from the widest area, covering seven recreational localities within a forest biotope in Northern Poland. This study aimed to determine the prevalence of T. gondii DNA in 2144 collected questing ticks to establish whether they may be involved in T. gondii life cycle. The additional goal was to genotype the detected T. gondii, as knowledge about its genotypes occurring in European ticks is insufficient. A further purpose was to detect coinfection with T. gondii and Borreliaceae in the collected ticks, as all of them have previously been tested for the presence of bacteria DNA. Nested PCR and sequencing of the obtained B1 gene fragment were conducted. T. gondii DNA was detected in 0.9% of all ticks (1.1% of nymphs and 0.7% of larvae). The presence of T. gondii in unfed larvae and nymphs may indicate the possibility of its vertical transmission. The prevalence of T. gondii DNA in ticks collected from individual sites was focal (0-4.3%) and seems to depend on local climatic conditions. Among all examined ticks, 0.3% were coinfected with T. gondii and Borreliella spp., vs. 0.6% of specimens with a single T. gondii infection. The obtained B1 sequences showed the greatest similarity (99.71-100%) to the sequence representing type III.

Blastocystis occurrence and subtype diversity in European wild boar (Sus scrofa) from the Iberian Peninsula.

The ongoing increase in wild boar populations across Europe has fostered human-wildlife conflicts, including the transmission of emerging pathogens with zoonotic importance. Blastocystis is a ubiquitous, faecal-oral transmitted protist that can cause gastrointestinal illnesses and is observed in humans and animals worldwide. The role of wildlife in the epidemiology of Blastocystis is insufficiently understood. Thus, we investigated the occurrence and subtype diversity of Blastocystis in free-ranging wild boars from the Iberian Peninsula using conventional PCR and next-generation amplicon sequencing of a fragment of the ssu RNA gene. A total of 459 wild boar faecal samples were collected across Spain (n = 360) and Portugal (n = 99) between 2014 and 2021. Blastocystis was present in 15.3% (70/459; 95% CI 12.1-18.9) of the wild boars analysed, and its occurrence was significantly higher in Portugal (34.3%, 34/99; 95% CI 25.1-44.6) than in Spain (10.0%, 36/360; 95% CI 7.1-13.6). Seven Blastocystis subtypes (ST5, ST10b, ST13-ST15, ST24b, and ST43) were detected among the surveyed wild boar populations, with greater variability detected in Portuguese samples. ST5 was identified in all the Blastocystis-positive animals, whereas 14.3% of them harboured ST mixed colonisations. Our results demonstrate that Blastocystis ST5 is particularly adapted to infect wild boars. The additional identification of zoonotic STs reinforces the role of wild boars as spreaders of zoonotic infections with public health significance.