Identification Of Molecular Species Research Articles

Candidatus Midichloria mitochondrii can be vertically transmitted in Hyalomma anatolicum

In this study, a tick intracellular symbiont, Candidatus Midichloria mitochondrii, was detected in Hyalomma anatolicum from Xinjiang, China. Morphological identification and cytochrome oxidase subunit I sequence alignment were used for molecular identification of the tick species. PCR detection further revealed the presence of endosymbiont C. M. mitochondrii in the tick. Specific primers were designed for Groel and 16S rRNA genes of C. M. mitochondrii for PCR amplification and phylogenetic analysis. To further investigate the vertical transmission characteristics of C. M. mitochondrii, specific primers were designed based on the FabⅠ gene fragment to detect C. M. mitochondrii in different developmental stages and organs of the tick using qPCR. Of the 336 tick specimens collected from the field, 266 samples were identified as H. anatolicum on the basis of morphological characteristics. The gene fragment alignment results of COI confirmed that these ticks were H. anatolicum. The phylogenetic analysis showed that Groel gene of C. M. mitochondrii clustered with Midichloria strains detected in Ixodes ricinus ticks from Italy and Ixodes holocyclus ticks from Australia, with 100% sequence similarity. Furthermore, the 16S rRNA gene of C. M. mitochondrii clusters with the strains isolated from Hyalomma rufipes ticks in Italy, exhibiting the highest degree of homology. qPCR results showed that C. M. mitochondrii was present at all developmental stages of H. anatolicum, with the highest relative abundance in eggs, and lower relative abundance in nymphs and unfed males. With female tick blood feeding, the relative abundance of C. M. mitochondrii increased, and a particularly high relative abundance was detected in the ovaries of engorged female ticks. This study provides information for studying the survival adaptability of H. anatolicum, and provides data for further investigation of the mechanisms regulating tick endosymbionts in ticks, enriching the reference materials for comprehensive prevention and control of tick-borne diseases.

Measurement of Li-Ion Self-Diffusion in Laser Structured Electrodes

It is generally stated that a limiting factor in fast charging and high-power discharging of lithium-ion batteries (LiB) stems from the diffusion kinetics of Li+ in the electrode pore network. Numerous approaches in enabling fast charging of LiBs include active material development, electrolytes with high ionic conductivity and the management of the charging and discharging temperature. Another promising method is laser micro structuring to modify the electrode architecture regarding an enhanced lithium-ion diffusion kinetics. An increased high-rate capability and boost in cell lifetime have been demonstrated with such 3D electrodes [1, 2] but the related mechanisms leading to a substantial impact to diffusion kinetics are still poorly understood. Furthermore, it is imperative to find an optimal ablation pattern with regard to the desired application scenario that minimizes active mass loss in order to create the economic basis for efficient upscaling of the process.Nuclear Magnetic Resonance (NMR) measurements of longitudinal (T1) and transverse relaxation times (T2), as well as the T2/T2 exchange, are routinely used for the identification of different molecular species. The exchange NMR experiment is a well-known technique for studying local environments of nuclei such as liquids in pores and approximating their shapes and sizes [3]. The evolution of the transverse relaxation rate (T2) of a probed nuclei allows to determine if the specie is confined (shorter T2) or in liquid bulk (longer T2). In this work, so-called Exchange Nuclear Magnetic Resonance (T2/T2 exchange or EXSY), is conceptualized to study the impact of laser generated 3D patterns in pore self-diffusion of electrolyte species.Exchange NMR and T1 and T2 measurements were realized at various soaking times and electrolyte amounts for the following electrolyte species: Li+, PF6 -, ethyl carbonate (EC), and ethyl methyl carbonate (EMC), using laser structured, graphite-based electrodes casted on non-metallic substrates (to reduce RF interference). 2D exchange maps show the presence of confined species as well as the approximation of the diffusion properties. Using isotope exchange experiments with 6Li enriched electrolyte, concentration maps revealed the rate of 6LiPF6 intrusion into 7LiPF6 rich pre-soaked electrodes. The impact of laser generated 3D patterns in pore diffusion will be discussed in detail.[1]. Zheng, Y. et al. 3D silicon/graphite composite electrodes for high-energy lithium-ion batteries. Electrochim. Acta 317, 502–508 (2019).[2]. Smyrek, P., Pröll, J., Rakebrandt, J.-H., Seifert, H. J. & Pfleging, W. Manufacturing of advanced Li(NiMnCo)O2 electrodes for lithium-ion batteries . Laser-based Micro- Nanoprocessing IX 9351, 93511D (2015).[3]. Mitchell, J. et al. Validation of NMR relaxation exchange time measurements in porous media. J. Chem. Phys. 127, (2007).

Quantification using a HPLC-FLD-based Detector and Molecular Identifi-cation of Ochratoxin-A Producing Aspergillus Species Isolated from Stored Grain Samples.

Quantification using an HPLC-FLD based detector and Molecular identification of Ochratoxin-A producing Aspergillus Species isolated from stored grain samples. Fungi are cosmopolitan in origin and are known to grow in any suitable substra-tum. In the present investigation, Aspergillus species isolated from stored grain samples were analyzed for ochratoxin-A production. The objective of this study is the quantification of Ochratoxin and identification of ochratoxigenic fungi. A total of n=34 black Aspergilli and n=1 Ochre Aspergilli were isolated from grain samples of Bihar, India and it was tested for OTA production. The limit of detection (LOD) is found to be 0.33μg/Kg and the limit of quantification (LOQ) is found to be 1μg/Kg for OTA in HPLC-FLD. In the present study, out of all the fungal isolates, only TiB fungal isolate was able to produce the ochratoxin-A above the level of LOQ. The positive isolate TiB obtained from stored sesame seed samples was able to produce 25.54 μg/Kg of OTA. ITS sequence analysis of TiB isolate was able to matche 100% with Aspergillus welwitschiae and Aspergillus foetidus. This is the initial report of ochratoxigenic Aspergillus fungal isolate isolated from stored sesame seed samples of flood-prone areas of Bihar, India.

Applicability and perspectives for DNA barcoding of soil invertebrates

Belowground invertebrate communities are dominated by species-rich and very small microarthropods that require long handling times and high taxonomic expertise for species determination. Molecular based methods like metabarcoding circumvent the morphological determination process by assigning taxa bioinformatically based on sequence information. The potential to analyse diverse and cryptic communities in short time at high taxonomic resolution is promising. However, metabarcoding studies revealed that taxonomic assignment below family-level in Collembola (Hexapoda) and Oribatida (Acariformes) is difficult and often fails. These are the most abundant and species-rich soil-living microarthropods, and the application of molecular-based, automated species determination would be most beneficial in these taxa. In this study, we analysed the presence of a barcoding gap in the standard barcoding gene cytochrome oxidase I (COI) in Collembola and Oribatida. The barcoding gap describes a significant difference between intra- and interspecific genetic distances among taxa and is essential for bioinformatic taxa assignment. We collected COI sequences of Collembola and Oribatida from BOLD and NCBI and focused on species with a wide geographic sampling to capture the range of their intraspecific variance. Our results show that intra- and interspecific genetic distances in COI overlapped in most species, impeding accurate assignment. When a barcoding gap was present, it exceeded the standard threshold of 3% intraspecific distances and also differed between species. Automatic specimen assignments also showed that most species comprised of multiple genetic lineages that caused ambiguous taxon assignments in distance-based methods. Character-based taxonomic assignment using phylogenetic trees and monophyletic clades as criteria worked for some species of Oribatida but failed completely for Collembola. Notably, parthenogenetic species showed lower genetic variance in COI and more accurate species assignment than sexual species. The different patterns in genetic diversity among species suggest that the different degrees of genetic variance result from deep evolutionary distances. This indicates that a single genetic threshold, or a single standard gene, will probably not be sufficient for the molecular species identification of many Collembola and Oribatida taxa. Our results also show that haplotype diversity in some of the investigated taxa was not even nearly covered, but coverage was better for Collembola than for Oribatida. Additional use of secondary barcoding genes and long-read sequencing of marker genes can improve metabarcoding studies. We also recommend the construction of pan-genomes and pan-barcodes of species lacking a barcoding gap. This will allow both to identify species boundaries, and to cover the full range of variability in the marker genes, making molecular identification also possible for species with highly diverse barcode sequences.

Molecular detection and identification of Enteromonas species in human and animal hosts using polymerase chain reaction and DNA sequencing

Enteromonas hominis, a human intestinal protozoan parasite of the diplomonad group, has been overlooked because of its commensal features; therefore, molecular studies on this parasite are limited. To address this gap, we designed a molecular screening protocol using polymerase chain reaction (PCR) and DNA sequencing targeting the 18S small subunit ribosomal RNA gene and applied this screening method to the molecular epidemiological analysis of Enteromonas spp. in humans and various livestock. We validated our methodology using stool samples collected from 215 humans and 270 animal hosts (buffaloes, pigs, dogs, goats, horses, rodents, chickens, and ducks) during an annual epidemiological investigation conducted from 2013 to 2016 on Sumba Island, Indonesia. The overall prevalences of Enteromonas spp. were 33.9 % (n = 73/215) in humans and 25.2 % (n = 68/270) in mammals and avians. The positive predictive value of this PCR method for Enteromonas spp., as evaluated through sequencing, was 90.1 % in human samples and 58.1 % in non-human samples (particularly low, 11.4 % in rodents). Although the specificity of the PCR approach may not be perfect, in combination with DNA sequencing, it was effective in detecting and identifying a partial sequence (1458 bp) of the target gene region in Enteromonas species.

Development of a DNA macroarray for the molecular biological identification of trade-relevant tropical CITES timber species and their look-alikes

Abstract The control authorities of timber trade lack quick and easy methods to verify the legality of the traded product. This includes the clear identification of the timber species. In this study, a simple genetic method, the macroarray technique, was modified and optimised for this purpose. The internal transcribed spacer (ITS) was used as barcode region. Specific probes were designed and tested using this barcode of trade-relevant, CITES protected wood species and their look-alikes. The results reveal that 61 of these probes are suitable for identification purposes. Hence, 32 of the 50 investigated species can be clearly identified using the developed macroarray. Additional 15 species are represented by probes in groups of two to three species, which enables a delimitation of the species present. For four species, no identification was possible applying the probes created. The findings indicate the potential of time and cost savings compared to currently established methods in species control in the timber trade.

Comparison of morphological and molecular Strongylus spp. identification in equine larval cultures and first report of a patent Strongylus asini infection in a horse.

Surveillance of Strongylus vulgaris and other Strongylus spp. in equids is important for targeted intervention in parasite control, requiring reliable routine diagnostic methods. Comparing morphological examination and PCR analyses of larval cultures to identify Strongylus spp. species based on German diagnostic samples from 2018. Method comparison. During the routine diagnostic investigations, in total 712 strongyle-egg positive equine faecal samples were cultured. Third-stage larvae (L3) were morphologically differentiated. For molecular validation, samples were examined using S. vulgaris real-time PCR and Strongylus edentatus/Strongylus equinus/Strongylus asini high-resolution melting PCRs. Based on 28S rRNA PCR, 594 samples positive for nematode DNA were included in the study. The inter-rater reliability to compare morphological and molecular species identification was fair for Strongylus spp. without species identification and for S. edentatus, slight for S. equinus and poor for S. vulgaris. The frequency based on morphological and molecular data in this study were for S. vulgaris 0% and 0.8%, respectively, for S. edentatus 0.3% and 1.5%, respectively, and for S. equinus 2.0% and 0.2%, respectively. Based on molecular analyses, one sample obtained from a domestic horse contained S. asini DNA, which was confirmed by sequencing. For many samples, no or only incomplete data regarding clinical history, the exact geographical location and whether samples were obtained on individual or farm level, were available. Results of morphological and molecular examination methods of strongyle L3 from equine samples can differ substantially. Further evaluation of these methods is required to provide reliable and cost-effective methods of screening equine parasites. Further studies using approaches suitable to detect S. asini are needed to evaluate its clinical and epidemiological relevance.

Forensic species identification: practical guide for animal and plant DNA analysis.

The importance of non-human DNA in the forensic field has increased greatly in recent years, together with the type of applications. The molecular species identification of animal and botanical material may be crucial both for wildlife trafficking and crime scene investigation. However, especially for forensic botany, several challenges slow down the implementation of the discipline in the routine.Although the importance of molecular analysis of animal origin samples is widely recognized and the same value is acknowledged to the botanical counterpart, the latter does not find the same degree of application.The availability of molecular methods, especially useful in cases where the material is fragmented, scarce or spoiled preventing the morphological identification, is not well known. This work is intended to reaffirm the relevance of non-human forensic genetics (NHFG), highlighting differences, benefits and pitfalls of the current most common molecular analysis workflow for animal and botanical samples, giving a practical guide. A flowchart describing the analysis paths, divided in three major working areas (inspection and sampling, molecular analysis, data processing and interpretation), is provided. More real casework examples of the utility of non-human evidence in forensic investigations should be shared by the scientific community, especially for plants. Moreover, concrete efforts to encourage initiatives in order to promote quality and standardization in the NHFG field are also needed.

DNA barcoding is currently unreliable for species identification in most crayfishes.

DNA barcoding is commonly used for species identification. Despite this, there has not been a comprehensive assessment of the utility of DNA barcoding in crayfishes (Decapoda: Astacidea). Here we examined the extent to which local barcoding gaps (used for species identification) and global barcoding gaps (used for species discovery) exist among crayfishes, and whether global gaps met a previously suggested 10× threshold (mean interspecific difference being 10× larger than mean intra specific difference). We examined barcoding gaps using publicly available mitochondrial COI sequence data from the National Center for Biotechnology Information's nucleotide database. We created two versions of the COI datasets used for downstream analyses: one focused on the number of unique haplotypes (N H) per species, and another that focused on total number of sequences (N S; i.e., including redundant haplotypes) per species. A total of 81 species were included, with 58 species and five genera from the family Cambaridae and 23 species from three genera from the family Parastacidae. Local barcoding gaps were present in only 30 species (20 Cambaridae and 10 Parastacidae species). We detected global barcoding gaps in only four genera (Cambarus, Cherax, Euastacus, and Tenuibranchiurus), which were all below (4.2× to 5.2×) the previously suggested 10× threshold. We propose that a ~5× threshold would be a more appropriate working hypothesis for species discovery. While the N H and N S datasets yielded largely similar results, there were some discrepant inferences. To understand why some species lacked a local barcoding gap, we performed species delimitation analyses for each genus using the N H dataset. These results suggest that current taxonomy in crayfishes may be inadequate for the majority of examined species, and that even species with local barcoding gaps present may be in need of taxonomic revisions. Currently, the utility of DNA barcoding for species identification and discovery in crayfish is quite limited, and caution should be exercised when mitochondrial-based approaches are used in place of taxonomic expertise. Assessment of the evidence for local and global barcoding gaps is important for understanding the reliability of molecular species identification and discovery, but outcomes are dependent on the current state of taxonomy. As this improves (e.g., via resolving species complexes, possibly elevating some subspecies to the species-level status, and redressing specimen misidentifications in natural history and other collections), so too will the utility of DNA barcoding.

Complete mitochondrial genome of the worm snail Thylacodes adamsii (Littorinimorpha: Vermetidae) from South Korea

The worm snail Thylacodes adamsii (Mörch, 1859) (Littorinimorpha: Vermetidae) is a sessile gastropod that mainly inhabits rocky shores along the warm temperate to tropical ocean. Herein, the complete mitochondrial genome (mitogenome) of T. adamsii from South Korea was characterized. The genome is 14,913 bp in length and contains 13 protein-coding genes (PCGs), 22 tRNA genes, and 2 rRNA genes. The genome organization and base composition of T. adamsii are similar to those of other vermetids. A phylogenetic tree was reconstructed using maximum likelihood based on the nucleotide sequences of the 13 PCGs; this tree supported the monophyly of Vermetidae. The complete mitogenome of T. adamsii can assist with molecular species identification and vermetid phylogenetic research in the future.

The complete mitochondrial genome of a ground beetle Synuchus nitidus (Carabidae: Harpalinae: Sphodrini) from South Korea

The ground beetle Synuchus nitidus (Motschulsky, 1861) (Carabidae: Harpalinae: Sphodrini) is one of the most common species in the forests of South Korea, which has the potential to be utilized as an environmental indicator. Here, we characterized the complete mitochondrial genome (mitogenome) of S. nitidus, which is the first in the harpaline tribe Sphodrini. Its genome is 16,392 bp in length and composed of 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an A + T rich region. In addition, we reconstructed a maximum likelihood tree to elucidate the phylogenetic position of Sphodrini among the seven harpaline tribes using nucleotide sequences of the 13 PCGs. The ML tree supported a monophyletic clade of the subfamily Harpalinae and showed a close relationship between Sphodrini and Lebinii with a low bootstrap value. The complete mitogenome of S. nitidus could be helpful for molecular species identification and exploring phylogenetic relationships among carabids.

Application of pulsed-field gel electrophoresis for molecular identification of pathogenic Leptospira species in Iran: a rapid and reliable method.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira serovars. The genus Leptospira cannot differentiated by conventional techniques. However, identity determination of pathogenic serovar is precious of public health problems and epidemiological studies. Pulsed-field gel electrophoresis facilitates rapid identification of Leptospires to the serovar levels. In this study, we employed PFGE to evaluate 28 Leptospira isolates, with animal, human and environmental origin, obtained from Razi Vaccine and Serum Research Institute of Karaj, Iran. PFGE patterns of 28 Leptospira serovars were generated using the Not I restriction enzyme in comparison with the lambda ladder. Out of 28 serovars evaluated, we identified 22 different pulsed types, designated P1-P22. Out of 22 pulse groups, 3 were found to be a common type, but others were a single Type. Groups consisting of the common type were P3, P9, P14, and P16. The results showed that the discriminatory index of PFGE by Not I enzyme was 0.99, demonstrating heterogeneous differentiation among serovar members. The PFGE methodology used in this study showed excellent interlaboratory report usability, rapid, reliable, enabling standardization and data sharing between laboratories.

The development of multiplex PCR assays for the rapid identification of multiple Saccostrea species, and their practical applications in restoration and aquaculture

BackgroundThe ecology and biology of oysters (Ostreidae) across the tropics is poorly understood. Morphological plasticity and shared characteristics among oysters have resulted in the misidentification of species, creating challenges for understanding basic species-specific biological information that is required for restoration and aquaculture. Genetic barcoding has proven essential for accurate species identification and understanding species geographic ranges. To reduce the costs of molecular species identification we developed multiplex assays using the cytochrome c oxidase subunit I (COI or cox1) barcoding gene for the rapid identification of five species of oysters within the genus Saccostrea that are commonly found in Queensland, Australia: Saccostrea glomerata, Saccostrea lineage B, Saccostrea lineage F, Saccostrea lineage G, and Saccostrea spathulata (lineage J).ResultsMultiplex assays were successful in species-specific amplification of targeted species. The practical application of these primers was tested on wild spat collected from a pilot restoration project in Moreton Bay, Queensland, with identified species (S. glomerata, lineage B and lineage G) validated by Sanger sequencing. DNA sampling by extraction of oyster pallial fluid was also tested on adult oysters collected from the Noosa estuary in Queensland to assess whether oysters were able to be identified non-destructively. DNA concentrations as low as 1 ng/ μL still amplified in most cases, allowing for identification, and mortality at 6 weeks post pallial fluid collection was low (3 out of 104 sampled oysters).ConclusionThese multiplex assays will be essential tools for species identification in future studies, and we successfully demonstrate their practical application in both restoration and aquaculture contexts in Queensland. The multiplex assays developed in this study outline easily replicable methods for the development of additional species-specific primer sets for the rapid identification of other species of Saccostrea found across the Indo-Pacific, which will be instrumental in unravelling the taxonomic ambiguities within this genus in tropical regions.

Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland

BackgroundBovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk.ResultsThe results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland.ConclusionsThe results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Basic and Advanced Logical Concept Derived from Surface Enhanced Infrared Spectroscopy (SEIRS) as Sensing Probe for Analysis of Chemical Species: A Brief Review

The worldwide concern for environmental pollution, climate change and health hazards caused by various pollutants has significantly increased in the recent past. Various techniques have so far been employed for sensing applications of such organic as well as inorganic pollutants. Amongst the different techniques, surface enhanced infrared spectroscopy (SEIRS) is a powerful tool which is utilized for label-free and unambiguous identification of molecular species. SEIRS overcomes the limitations of the conventional infrared spectroscopy and has emerged as a potential technique with high surface sensitivity by enhancing the signals by many folds and also facilitates new studies from the fundamental aspect to applied sciences. The current review is dedicated to a comprehension of the SEIRS technique to provide a critical overview of its application as sensing probe for analysis of chemical species. The major features of Fourier Transform Infrared spectroscopy and SEIRS have been critically discussed.

Molecular Identification of a New Species of Absidia (Cunninghamellaceae, Mucorales) Isolated from Soil in Korea

The species within the family Cunninghamellaceae during an investigation of soil microfungi in Korea, in which three strains were isolated from Gangwon, Chungbuk, and Gyeongbuk provinces, designated as KNUF-22-121A, KNUF-22-126A, and KNUF-22-316, respectively. Because the morphological and molecular analyses of these three strains were identical, KNUF-22-316 underwent further detailed study. Phylogenetic analyses based on the concatenated nucleotide sequences of the internal transcribed spacer region and the large subunit 28S rRNA gene revealed that the strain belonged to the genus Absidia, but occupied a distinct phylogenetic position. The strain KNUF-22-316 was compared with closely related species Absidia radiata CGMCC 3.16257T and Absidia yunnanensis CGMCC 3.16259T, morphologically different with shorter sporangiophores, smaller sporangia and columellae, and the consistent presence of collars. Here, we provide a detailed description and images of this proposed new species, which we have named Absidia microsporangia sp. nov.

Comparative analysis the chloroplast genomes of Celastrus (Celastraceae) species: Provide insights into molecular evolution, species identification and phylogenetic relationships

BackgroundThe genus Celastrus is an important medicinal plant resource. The similarity of morphology and the lack of complete chloroplast genome analysis have significantly impeded the exploration of species identification, molecular evolution and phylogeny of Celastrus. PurposeIn order to resolve the phylogenic controversy of Celastrus species, the chloroplast genome comparative analysis was performed to provide genetic evidence. MethodsIn this study, we collected and sequenced ten chloroplast genomes of Celastrus species from China and downloaded three chloroplast genomes from the databases. The chloroplast genomes were compared and analyzed to explore their characteristics and evolution. Furthermore, the phylogenetic relationships of Celastrus species were inferred based on the whole chloroplast genomes and protein-coding genes. ResultsAll the 13 Celastrus species chloroplast genomes showed a typical quadripartite structure with genome sizes ranging from 155,113 to 157,366 bp. The intron loss of the rps16 gene occurred in all the 13 Celastrus species. The GC content, gene sequence, repeat types and codon bias pattern were highly conserved. Ten highly variation regions were identified, which can be used as potential DNA markers in molecular identification of Celastrus species. Eight genes, including accD, atp4, ndhB, rpoC1, rbcL, rpl2, rpl20 and ycf1, were detected to experience positive selection. Phylogenetic analysis showed that Celastrus was a monophyletic group and Tripterygium was the closest sister-group. Noteworthy, C. gemmatus Loes. and C. orbiculatus Thunb. can be discriminated using the chloroplast genome as a super barcode. The comparative and phylogenetic analysis results proposed that C. tonkinensis Pitard. was the synonym of C. hindsii Benth. ConclusionThe comparative analysis of the Celastrus chloroplast genomes can provide comprehensive genetic evidence for molecular evolution, species identification and phylogenetic relationships.

Epidemiology of rumen fluke infection in selected buffalo farms in perak, malaysia: prevalence, molecular species identification, and associated risk factors.

Rumen flukes cause heavy economic losses in the ruminant industry worldwide, especially in tropical and subtropical countries. This study estimated the prevalence of rumen flukes in buffaloes, identified the species diversity, and determined risk factors associated with rumen fluke prevalence in Perak, Peninsular Malaysia. A cross-sectional study was conducted, and 321 faecal samples were collected from six buffalo farms. A structured questionnaire was developed, and farmers were interviewed to obtain information regarding risk factors associated with rumen fluke infection. The faecal samples were examined using sedimentation and Flukefinder® techniques. Genomic DNA was extracted from the fluke eggs recovered using the Flukefinder® method, and the internal transcribed spacer 2 (ITS2) fragment was amplified and sequenced to facilitate species identification. The results showed that the overall prevalence of rumen fluke across the sampled farms was 40.2% (129/321). Three rumen fluke species were identified, namely, Fischoederius elongatus, F. cobboldi, and Orthocoelium streptocoelium. Several management factors had a significant association (P < 0.05) with rumen fluke prevalence, including production type, cleaning of the stable, drinking water system, flooding around the farm, grazing system, pasture sharing with other livestock, and deworming program. This work constitutes the first attempt to understand the epidemiology of rumen fluke infection in the region and suggests that good farm management, pasture management, choosing appropriate drugs, and proper husbandry practices may improve buffalo health and production in areas where rumen flukes are prevalent.

Molecular identification of Sarcocystis species in wild boar (Sus scrofa) and pigs (Sus scrofa domesticus) in Brazil

Sarcocystis spp. are protozoan parasites that form cysts in the organs and musculature of various animal species. The species Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs and wild boars (Sus scrofa), acting as intermediate hosts, while humans are the definitive host for S. suihominis. To date, there have been no reports of the identification of these coccidian species in Sus scrofa in Brazil. Therefore, in this study, we conducted the first molecular identification of Sarcocystis species using PCR-RFLP and sequencing. A total of 210 samples were analyzed, of this total, 67 tested positive for Sarcocystis spp., representing 31.9% of the total samples assessed. Out of the total positive samples, 55 (82.1%) were identified as S. miescheriana and 8 (11.9%) as S. suihominis, a zoonotic species. Additionally, other species related to bovines, such as S. cruzi and zoonotic S. hominis, were detected in 3.0% of the samples, serving as contaminants in the pork products. The presence of S. suihominis in swine and wild boar samples is concerning due to the zoonotic risk and potential environmental contamination, as humans act as definitive hosts, also for the presence of S. hominis as a bovine contaminant in pork sausages. Furthermore, we confirmed the efficacy of the PCR-RFLP technique as a reliable tool for the identification of Sarcocystis species, demonstrating its potential use in laboratories for molecular diagnosis and rapid identification of these parasites, aiming to protect public health and ensure food safety.