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Recombinant adeno-associated virus (rAAV) is a viral vector commonly used in gene therapy. Residual host cell DNA is an impurity that has been associated with the risk of infection and oncogenicity. Thus, it needs to be monitored for quality control. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) method targeting 18S ribosomal RNA (rRNA) genes to quantitate residual host cell DNA. The copy number of the 18S rRNA gene was determined using two sets of primer pairs for 116- and 247-bp amplicons sharing the C-terminus. For conversion of the copy number of the 18S rRNA gene into the mass concentration of genomic DNA, the accurate copy number of 18S rRNA genes in HEK293 genomic DNA was determined by comparison with copy numbers of three reference genes (EIF5B, DCK, and HBB). Results showed that 88.6–97.9% of HEK293 genomic DNA spiked into rAAV preparations was recovered. The ddPCR-based assay was applied to rAAV preparations to quantitate residual host cell DNA as an impurity. Our findings indicate that the assay can be used for the quantitation and size distribution of residual host cell DNA in rAAV products.

Suitable reference genes are one of the necessary conditions for obtaining reliable results by real-time fluorescence quantitative PCR (qPCR). In this study, the expression of the 10 common candidate reference genes (18s rRNA, rps27, cnbp, rpl7, ube2, hsp-at, gapdh, β-actin, rpl13a, 1-ef1a) at different developmental stages of gonad and embryo of Takifugu bimaculatus were analyzed by qPCR. And the expression stability of these reference genes is analyzed by GeNorm, NormFinder and Bestkeeper softwares. The results showed that the expression stability of 1-ef1a was the highest (p

MDFRC item.We compared the relative values of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) for profiling fungal communities in wastewater treatment plants using both ITS and 18S rRNA gene fragments as phylogenetic markers. A similar number of fungal ribotypes was obtained with both methods for the same treatment plant when the ITS primer set was used, while a greater number of ribotypes was obtained with T-RFLP compared to DGGE with the 18S rRNA primer set. Non-metric multi-dimensional scaling of presence/absence data and analysis of similarity showed that both methods could distinguish between the different plant communities at a statistically significant level (p < 0.05), regardless of which phylogenetic marker was used. The data suggest that both methods can be used preferably together to profile activated sludge fungal communities. A comparison of profiles generated with both these phylogenetic markers based on the number of ribotypes/bands, suggests that the 18S rRNA region is more discriminatory than the ITS region. Detected differences in fungal community compositions between plants probably reflect differences in their influent compositions and operational parameters.

Ribosome RNA (rRNA) accounts for more than 80% of the cell's total RNA, while the physiological functions of rRNA modifications are poorly understood. Mutations of 18S rRNA m6A methyltransferase METTL5 cause intellectual disability, microcephaly, and facial dysmorphisms in patients, however, little is known about the underlying mechanisms. In this study, we identified METTL5 protein complex and revealed that METTL5 mainly interacts with RNA binding proteins and ribosome proteins. Functionally, we found that Mettl5 knockout in mESCs leads to the abnormal craniofacial and nervous development. Moreover, using Mettl5 knockout mouse model, we further demonstrated that Mettl5 knockout mice exhibit intellectual disability, recapitulating the human phenotype. Mechanistically, we found that Mettl5 maintains brain function and intelligence by regulating the myelination process. Our study uncovered the causal correlation between mis-regulated 18S rRNA m6A modification and neural function defects, supporting the important physiological functions of rRNA modifications in human diseases.

The biostimulant BC204 is a commercially available biostimulant based on an extract from Citrus aurantium, comprising a mixture of bioflavonoids, other plant extracts and organic acids. In this study, the potential of BC204 to cause positive effects on plant growth and overall plant health by altering the rhizosphere microbial community of Solanum lycopersicum L. was investigated. Mycorrhizal colonization of plant roots was analysed by trypan blue-staining and light microscopy, followed by polymerase chain reaction (PCR) amplification of a region of the 18S rRNA gene conserved among arbuscular mycorrhizal fungi. No difference in mycorrhizal colonization of roots was observed between control and treated plants. The structural and functional diversity of rhizosphere bacteria between different treatment groups was assessed using 16S rRNA gene restriction fragment length polymorphism (RFLP) analysis and community-level physiological profiling (CLPP), respectively. Nearest sequence matches for three isolates from the rhizosphere of plants treated with BC204 as a soil drench in soil supplemented with Mycoroot™ revealed bacterial genera which play a role in promoting plant growth. Furthermore, the microbial community from this treatment group showed increased richness of substrate utilization on Biolog EcoPlates™. These results indicate some form of interaction between BC204 and Mycoroot™ which causes a shift in the structural and functional diversity of the rhizosphere bacterial community.

A new species of the nereidid annelid, genus Nicon Kinberg, 1866, from KIOST Seamount, Northwest Pacific deep water is described. Nicon is a genus characterized by lacking paragnaths or papillae on the pharynx and composed of nine species worldwide, distributed from shallow water to deep sea. Nicon ablepsia sp. nov. here described is characterized by the lack of eyes on the prostomium, prolonged tentacular cirri reaching to chaetiger 6, notochaetae homogomph spinigers, neurochaetae homogomph spinigers and heterogomph falcigers. Phylogenetic relationships of Nicon remain undetermined based on molecular data. In this study, we constructed molecular Maximum-Likelihood phylogenetic tree from 29 nereidid species based on four marker genes: mitochondrial 16S rRNA gene and cytochrome c oxidase subunit I (COI) gene; nuclear 18S rRNA gene and 28S rRNA gene. Our analysis suggest the Nicon is clustered within Nereidinae, and nereidinae is not recovered as monophyletic. A key to species of Nicon is provided.

Nematodes of the family Philometridae Baylis & Daubney, 1926 (Dracunculoidea Stiles, 1907) are generally poorly known, and there are many taxonomic issues within the family. Philometrids are parasites of fish and are found in various locations throughout the host, including within the subcutaneous tissues and musculature, the abdominal cavity and gonads; vast sexual dimorphism often means the males are not collected, leading to many species being described solely on female characteristics. Although there have been a number of studies utilising molecular data, the vast majority of species are yet to be sequenced. This study undertook genetic sequencing of 15 recently described species of philometrids across 4 genera, many of which were from specimens collected from waters off Australia. All of the sequences obtained were closely related with representatives of the family Philometridae. Species were found to be distributed in the phylogenetic trees within 4 clades based on a combination of site of infection within the host and host habitat. Family of host and geographical location was not as important for position within the trees. Clade A contained philometrids collected from the abdominal cavities and head tissues of South American freshwater fish. Clade B contained philometrids primarily from the abdominal cavities of freshwater European cyprinids. Clade C contained philometrids primarily from the ovaries of marine fish. Clade D contained philometrids from the body tissues of marine and freshwater fish. The potential co-evolutionary patterns between philometrids and their fish hosts are highlighted as an area of future research. This research also highlighted the importance of correct identification of any sequenced specimen.

The English grain aphid, Sitobion avenae (Fabricius), exhibits classic and dramatic phenotypic plasticity in wing development. Both genetic and environmental inputs contribute to the wing polyphenism in aphids, an extreme form of phenotypic plasticity in which a single genotype produces discrete winged and wingless morphs. Validated reference genes are needed to accurately normalize temporal and spatial variation in gene expression estimates by RT-qPCR. In this research, the stability of 11 candidate reference genes selected from S. avenae transcriptomes was evaluated under an array of abiotic and biotic conditions relevant to wing development. RefFinder, a comprehensive software integrating rankings from delta Ct, BestKeeper, NormFinder, and geNorm, offered a series of reference genes for every experimental condition. Overall, helicase (HEL) and ubiquitin ribosomal protein S27A fusion protein (RpS27) are suited for most of the conditions examined in this study, although exceptions do exist. Specifically, NADH dehydrogenase (Ap-NADH) and 28S ribosomal RNA (28S) are recommended for insecticide and antibiotic treatments, while ribosomal RNA L14 (RPL14) and 18S ribosomal RNA (18S) are selected for density treatment, respectively. This study provides a suite of reference genes to investigate the wing polyphenism in S. avenae, and is important for application of RT-qPCR in future experiments of novel tactics to control aphids.

Didelphis marsupialis is a primary reservoir of Trypanosoma cruzi, etiologic agent of American Trypanosomiasis-AT or Chagas Disease-CD, in America. Some findings of Trypanosoma rangeli have been recorded in this mammal, in sympatry with T. cruzi. In Los Montes de María, Bolívar, Colombian Caribbean, triatomine insects and potential parasite host has been registered, but little is known about the relationship between these parasites and D. marsupialis. We investigated the natural trypanosomatids infection rate in D. marsupialis, applying a parasitological and molecular diagnosis. Twenty D. marsupialis was investigated between 2018 and 2019 using 21 Tomahawk® traps placed on the sylvatic/domestic corridors. Blood was drawn by cardiopuncture after sedation. An aliquot of blood samples was cultured in Novy, Nicolle, McNeal/Roswell Park Memorial Institute medium at 24 °C/60 days for the detection of motile trypomastigotes. Parasite DNA was obtained by salting out methods from positive blood cultures. Trypanosomatids diagnosis was done by Polymerase Chain Reaction-sequencing of V7V8 region of 18S ribosomal RNA (18S-rRNA) gene. Amplicons were sequenced, and consensus sequences were aligned with reference sequences from GenBank. Four isolates corresponded to T. rangeli (20%) and one to T. cruzi (5%). The natural infection of D. marsupialis by T. rangeli and T. cruzi constitutes the first record of these parasites in didelphids in Los Montes de María and the first record of T. rangeli in this marsupial, in the Colombian Caribbean.

Objectives Three species of protozoal Hepatozoon species ( H felis, H canis and H silvestris) are known to infect cats in Europe. The objective of this study was to determine the prevalence of Hepatozoon species in samples from cats living in Germany that were submitted to a veterinary laboratory. Methods The study included cats tested for Hepatozoon species by PCR between 2007 and 2020 by the Laboklin laboratory. Travel history and haematological results were documented for cats with positive test results. From 2018 onwards, a partial 18S rRNA Hepatozoon gene fragment was sequenced from cats with positive PCR results. Results Sixty-four of 931 cats (7%) tested positive for Hepatozoon species. Sex and age did not have a statistically significant impact. Sequencing was carried out for 16 samples and revealed H felis in all cases. All cats with positive test results and a relevant travel history had been imported from the Mediterranean or south-eastern Europe. There were no autochthonous infections with Hepatozoon species. Leukocytosis, haemoconcentration and anaemia were the most common haematological abnormalities. Conclusions and relevance Although infections with Hepatozoon species in cats are usually subclinical, it may be useful to screen cats imported from the Mediterranean and south-eastern Europe for these pathogens to prevent local transmission cycles. There was no evidence of autochthonous infections in Germany; however, further investigations regarding a possible transmission of Hepatozoon species from infected cats to blood-feeding arthropods in Germany may be of interest. To avoid potential spread of the pathogens, ectoparasite prophylaxis is advisable.

Cryptosporidium spp. are significant zoonotic parasites in humans and animals worldwide. This study aimed to investigate the prevalence of Cryptosporidium infection among raccoon (Procyon lotor) in north of Iran. The fecal samples (n = 30) were collected from raccoons. After DNA extraction, all samples were examined by nested PCR amplification of the 18S ribosomal RNA (rRNA) gene. From 30 raccoon samples, 4 (13.3%) were positive, and the isolates were identified as Cryptosporidium skunk genotype based on sequence analysis. The large distribution of raccoons in northern provinces of Iran and their potency for carrying some human-infecting parasites like Cryptosporidium spp. propose this mammalian as a source for zoonotic parasites.

How does the evolution of bioinformatics tools impact the biological interpretation of high-throughput sequencing datasets? For eukaryotic metabarcoding studies, in particular, researchers often rely on tools originally developed for the analysis of 16S ribosomal RNA (rRNA) datasets. Such tools do not adequately account for the complexity of eukaryotic genomes, the ubiquity of intragenomic variation in eukaryotic metabarcoding loci, or the differential evolutionary rates observed across eukaryotic genes and taxa. Recently, metabarcoding workflows have shifted away from the use of Operational Taxonomic Units (OTUs) towards delimitation of Amplicon Sequence Variants (ASVs). We assessed how the choice of bioinformatics algorithm impacts the downstream biological conclusions that are drawn from eukaryotic 18S rRNA metabarcoding studies. We focused on four workflows including UCLUST and VSearch algorithms for OTU clustering, and DADA2 and Deblur algorithms for ASV delimitation. We used two 18S rRNA datasets to further evaluate whether dataset complexity had a major impact on the statistical trends and ecological metrics: a “high complexity” (HC) environmental dataset generated from community DNA in Arctic marine sediments, and a “low complexity” (LC) dataset representing individually-barcoded nematodes. Our results indicate that ASV algorithms produce more biologically realistic metabarcoding outputs, with DADA2 being the most consistent and accurate pipeline regardless of dataset complexity. In contrast, OTU clustering algorithms inflate the metabarcoding-derived estimates of biodiversity, consistently returning a high proportion of “rare” Molecular Operational Taxonomic Units (MOTUs) that appear to represent computational artifacts and sequencing errors. However, species-specific MOTUs with high relative abundance are often recovered regardless of the bioinformatics approach. We also found high concordance across pipelines for downstream ecological analysis based on beta-diversity and alpha-diversity comparisons that utilize taxonomic assignment information. Analyses of LC datasets and rare MOTUs are especially sensitive to the choice of algorithms and better software tools may be needed to address these scenarios.

Parkinson’s disease (PD) is one of the most common neurodegenerative disease, and is so far not considered curable. PD patients suffer from several motor and non-motor symptoms, including gastrointestinal dysfunctions and alterations of the enteric nervous system. Constipation and additional intestinal affections can precede the classical motor symptoms by several years. Recently, we reported effects of PD and related medications on the faecal bacterial community of 34 German PD patients and 25 age-matched controls. Here, we used the same collective and analysed the V6 and V7 hypervariable region of PCR-amplified, eukaryotic 18S rRNA genes using an Illumina MiSeq platform. In all, 53% (18) of the PD samples and 72% (18) of the control samples yielded sufficient amplicons for downstream community analyses. The PD samples showed a significantly lower alpha and a different beta eukaryotic diversity than the controls. Most strikingly, we observed a significantly higher relative abundance of sequence affiliated with the Geotrichum genus in the PD samples (39.7%), when compared to the control samples (0.05%). In addition, we observed lower relative abundances of sequences affiliated with Aspergillus/Penicillium, Charophyta/Linum, unidentified Opisthokonta and three genera of minor abundant zooflagellates in the PD samples. Our data add knowledge to the small body of data about the eukaryotic microbiota of PD patients and suggest a potential association of certain gut eukaryotes and PD.

BackgroundThe microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences.ResultsTo overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected.ConclusionCCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences.

This study was conducted to investigate Babesia parasites infecting sheep in eight districts of Sulaimani governorate/north Iraq from April to October 2017. Forty flocks of small ruminants were selected to collect blood samples randomly from 450 sheep. The samples were examined for babesiosis by microscopic examination and PCR. Primers based on the 18S rRNA were used for Babesia diagnosis, followed by sequencing of the amplicons for confirmation of the PCR product identities. Seventy-four samples (16.44%) showed the presence of Babesia piroplasms microscopically, while 116 (25.78%) samples were positive using PCR. Results showed that B. ovis was reported in 15.78% (n = 71), and B. motasi in 10.0% (n = 45) of the samples. Also, BLAST analysis of the obtained partial sequences of the 18S rRNA gene from current study isolates revealed the existence of both B. ovis and B. motasi, with a high homology degree of nucleotide identity with other nucleotide sequences of Babesia spp. in GenBank database. Distribution of babesiosis, according to the sampling time, revealed that high-frequency rates occur during July and August. Based on the result data, babesiosis was mainly caused by B. ovis and B. motasi.

Plasmodium vivax, the leading cause of malaria in the vast majority of countries outside of Africa and the second most dominant cause of malaria globally, is fast becoming widespread and domiciled in Nigeria and other parts of sub-Saharan Africa. The objective of this cross-sectional survey conducted in Ishielu, a suburb of Abakaliki, southeastern Nigeria was to identify circulating Plasmodium species (with focus on P. falciparum and P. vivax species) in the population. A total of 120 venous blood samples (2-5 ml) of individuals in the community was examined microscopically; while thirty randomlyselected blood samples were used for polymerase-chain reaction (PCR) assays. Genomic DNA was isolated from the blood samples and species-specific nested PCRs targeting the 18S ribosomal ribonucleic acid (rRNA) gene were applied in detecting the presence of the target parasite species. Microscopy showed that 90% (108/120) of the blood samples were positive for parasitaemia and P. falciparum was the only Plasmodium species identified. Of the 30 selected blood samples assayed by PCR, the amplicon of 18S rRNA gene of the malaria parasite species was amplifiable in 80 % (24/30), combined vivax and falciparum positive cases were identified in three blood specimens and from microscopically negative slides. Mixed infections constituted 25% (6/24) while single infections with vivax and falciparum amounted to 42% (10/24) and 33% (8/24) respectively. These call for large-scale surveys of the target parasite species at community level by molecular PCR method for significant reduction of morbidity due to malaria.

Trehala manna is the edible and trehalose-rich cocoons of a few Larinus species (Coleoptera: Curculionidae: Lixinae: Lixini), and manufactured by the feeding activity of larvae on the Echinops plants. Knowledge on the morphological and molecular properties of immature stages of Larinus species, especially trehala-constructing ones, is still very limited. Herein, the mature larva and pupa of Larinus hedenborgi Boheman, 1845, are morphologically described for the first time, illustrated and compared with known immature stages of other Larinus species. Morphological identification keys prepared for all known immature stages of Larinus species. The DNA sequences of three mitochondrial (COI, Cytb, and 16S RNA) and four nuclear (enolase, histon 4, 18S rRNA, and 28S rRNA) markers were generated for L. hedenborgi and compared with the available sequences in the GenBank. Comparative morphology revealed the affinity of L. hedenborgi with the L. vulpes, L. inaequalicollis, and L. capsulatus based on five larval and two pupal characters. Molecular analysis based on 1859 bp of mtDNA and 1618 bp of nDNA sequences indicated a close association between the L. hedenborgi and other Lixinae namely Larinus species. Although both morphological and molecular descriptions supported the taxonomic status of L. hedenborgi within weevils, sufficient molecular data must be available to allow further comparisons. This pioneering study can be expanded to a large-scale zoogeographic study using morpho-molecular characters, simultaneously with population-level sampling of all trehala-constructing Larinus species.

PURPOSE The molecular profile of specimens of Mediorhynchus gallinarum (Bhalero, 1937) collected from chickens, Gallus gallus L. in Indonesia was analysed. The aim of this study was to assess the phylogenetic position of species of Mediorhynchus within the order Giganthorhynchida. METHODS We used one mitochondrial gene (cytochrome oxidase 1) and one nuclear gene (18S ribosomal RNA) to infer phylogenetic relationships of class Archiacanthocephala. RESULTS The COI and 18S rDNA genes sequences showed that M. gallinarum had low genetic variation and that this species is sister to Mediorhynchus africanus Amin, Evans, Heckmann, El-Naggar, 2013. The phylogenetic relationships of the Class Archiacanthocephala showed that it is not resolved but, however, were mostly congruent using both genes. A review of host-parasite life cycles and geographic distributions of Archiacanthocephala indicates that mainly small mammals and birds are definitive hosts, while termites, cockroaches, and millipedes are intermediate hosts. CONCLUSIONS While the intermediate hosts have wide geographic distributions, the narrow distribution of the definitive hosts limit the access of archiacanthocephalans to a wider range of prospective hosts. Additional analyses, to increase taxonomic and character sampling will improve the development of a robust phylogeny and provide more stable classification. The results presented here contribute to better understanding of the ecological and evolutionary relationships that allow the host-parasite co-existence within the class Archiacanthocephala.

Diverse communities of symbiotic microbes inhabit the digestive systems of vertebrates and play a crucial role in animal health, and host diet plays a major role in shaping the composition and diversity of these communities. Here, we characterized diet and gut microbiome of fire salamander populations from three Belgian forests. We carried out DNA metabarcoding on fecal samples, targeting eukaryotic 18S rRNA of potential dietary prey items, and bacterial 16S rRNA of the concomitant gut microbiome. Our results demonstrated an abundance of soft-bodied prey in the diet of fire salamanders, and a significant difference in the diet composition between males and females. This sex-dependent effect on diet was also reflected in the gut microbiome diversity, which is higher in males than female animals. Proximity to human activities was associated with increased intestinal pathogen loads. Collectively, the data supports a relationship between diet, environment and intestinal microbiome in fire salamanders, with potential health implications.

Toxoplasma gondii is a parasite of significant public health importance. We attempted to detect T. gondii contamination and assess advantages and disadvantages of contamination indicators through surveilling soil, wildlife, cats (Felis catus), and cows (Bos taurus) on a farm in Tennessee, U.S. in 2016 and 2017. Twenty-two soil samples were collected from the farm and subjected to oocyst flotation, DNA extraction, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting 18S ribosomal RNA (18S rRNA) gene to detect and identify T. gondii. Three samples (13.6%) were positive for the parasite; however, T. gondii DNA was not consistently detected from repeated tests. Blood samples were collected from small mammals, cats, and mesopredators live-trapped on the farm, and serum from 30 of the farm's cows were obtained. Serological testing by the modified agglutination test (MAT; cutoff 1:50) found 2.5% (1/40) of small mammals, 52.9% (9/17) of raccoons (Procyon lotor), and 50% (1/2) of domestic cats were seropositive for T. gondii antibodies. No antibodies were found in 16 opossums (Didelphis virginiana), two skunks (Mephitis mephitis), and 30 cows. Small mammal tissue samples were subjected to PCR-RFLP detection. Four out of 29 (13.7%) tissue samples were positive for T. gondii; however, T. gondii DNA was not consistently detected during repeated PCR-RFLP testing. Our results indicate the ability to detect T. gondii varies greatly by contamination indicator. We found detection of soil oocysts to be challenging, and results suggest limited utility of the method performed. The ability to detect T. gondii in animals was highly variable among species. Our research emphasizes the importance of a holistic approach when surveilling for T. gondii to compensate for shortcomings of each contamination indicator. Future research should be conducted to further investigate the most effective T. gondii surveillance methods and species with increased sample sizes at other agricultural facilities., Graphical abstract Image 1, Highlights • We investigated T. gondii contamination at a farm and the efficacy of T. gondii detection indicators through examining soil, small mammals, mesopredators, domestic cats, and agricultural animals • Soil oocyst detection was low suggesting limited utility of the method performed in the study region • Small rodents and agricultural animals had low detection of T. gondii • Mesopredators (i.e., raccoons and domestic cats) had the greatest affinity for T. gondii detection out of all tested animals and fomites • Multiple indicators of potential contamination should be examined when trying to detect T. gondii

Coinfections with the tick-borne pathogens Theileria luwenshuni and Anaplasma phagocytophilum can cause significant economic losses in sheep and goat farming. The difficulty in detecting these two pathogens by microscopic examination warrants the development of a rapid detection test to discriminate them. In this study, a duplex polymerase chain reaction (PCR) assay was developed to simultaneously detect T. luwenshuni and A. phagocytophilum. Alignment of the sequences from related pathogens allowed us to design a primer pair targeting the 18S ribosomal RNA gene in T. luwenshuni and generate a target product of 962 bp, whereas a previously reported species-specific primer (SSAP2f/SSAP2r) for A. phagocytophilum was used in the same reaction to generate a product of 641 bp. Genomic DNA from T. luwenshuni and A. phagocytophilum was 10-fold serially diluted for testing PCR sensitivity. Under the optimal PCR conditions we established, the lower limit of detection of the assay was 29.13 fg/μL for T. luwenshuni and 1.53 fg/μL for A. phagocytophilum, and PCR primers used in this study were confirmed to be 100% species-specific using other hemoparasites previously identified by other methods. No significant difference was found between conventional and duplex PCR protocols used to detect the two species. Our study provides an effective, sensitive, specific, and accurate tool for the diagnosis and epidemiological surveillance of mixed infections of the two pathogens in sheep and goats.

Selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function research. Our study selected suitable reference genes for analyzing the gene expression of Agrilus zanthoxylumi Hou (Coleoptera: Buprestidae). Six candidate genes were detected by real-time quantitative polymerase chain reaction: the histone gene, the β-tubulin gene, the actin gene, 18S ribosomal RNA, and 28S-1 and 28S-2 ribosomal RNA. The expression of the candidate reference genes in different tissue samples (head, thorax, abdomen, legs, and wings) of A. zanthoxylumi was evaluated and analyzed by using GeNorm, NormFinder, and BestKeeper software programs. Gene expression stability results show that the expression of the 28S-2 gene is the most stable of the six candidate genes in all tissues of female A. zanthoxylumi, followed by the 28S-1 gene. The actin gene has the most stable expression of the six genes in male tissues, followed by the 28S-2 gene. The screening results of reference genes with the most stable expression in different sexes and tissues obtained in this study can be used for the subsequent quantitative expression research of related genes and provide theoretical basis and reference materials for the research of related gene expression levels of A. zanthoxylumi.

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

The current study was carried out to evaluate and identify the genetic variation between Local Black chicken lines with two commercial Layer (Isa Brown) and Broiler (Rose) breeds using RAPD markers and sequencing approach of 18s rRNA gene. From the result of the RAPD marker, all primers used were produced 152 scorable bands ranging from 2 to 9 with the size ranging from 320 to 2990 bp with percentage polymorphic loci 64.86% among chicken populations. The highest amplified fragments by primer OPC-11 and lowest by OPAA-03 were 24 and 8, respectively. The mean of the observed number of alleles (na), effective number of alleles (ne), gene diversity (h), Shannon's information index (I) for all loci found to be 1.6486, 1.5189, 0.2883 and 0.4129, respectively. The existence of a high level of polymorphisms and targeted (74) loci throughout all chicken populations/primers indicate sufficient genetic distance and more genetic variability among chicken populations using RAPD-PCR techniques. Result of blasted sequences of 18srRNA gene of local chicken has GenBank accession number MT808178 and MT808179 by BLAST tool against Gallus gallus, it showed the highest identity 95.74% and 94.88% for data of first and second part, respectively. The overall dendrograms clustered showed that the local chicken was closer to the commercial layer than to the broiler chicken lines. Therefore, it suggests that improving the local Black chicken line according to the layer breeding program to collect genetically invaluable genetic resources

Microorganisms exhibit seasonal succession governed by physicochemical factors and interspecies interactions, yet drivers of this process in different environments remain to be determined. We used high-throughput sequencing of 16S rRNA and 18S rRNA genes to study seasonal dynamics of bacterial and microeukaryotic communities at pelagic site of Lake Baikal from spring (under-ice, mixing) to autumn (direct stratification). The microbial community was subdivided into distinctive coherent clusters of operational taxonomic units (OTUs). Individual OTUs were consistently replaced during different seasonal events. The coherent clusters change their contribution to the microbial community depending on season. Changes of temperature, concentrations of silicon, and nitrates are the key factors affected the structure of microbial communities. Functional prediction revealed that some bacterial or eukaryotic taxa that switched with seasons had similar functional properties, which demonstrate their functional redundancy. We have also detected specific functional properties in different coherent clusters of bacteria or microeukaryotes, which can indicate their ability to adapt to seasonal changes of environment. Our results revealed a relationship between seasonal succession, coherency, and functional features of freshwater bacteria and microeukaryotes.

The occurrence of tick‐borne infectious diseases, including zoonotic babesiosis, has become a serious concern in recent years. In this study, we detected Babesia spp. using polymerase chain reaction (PCR) amplification of the 18S rRNA of the parasites isolated from ixodid ticks collected from small mammals in the Republic of Korea (ROK). Sequence analysis of the PCR amplicon revealed the presence of B. duncani, B. venatorum, B. capreoli/divergens, and, the most prevalent, B. microti in the ticks. The molecular phylogenetic analysis showed that the four species‐specific18S rRNA sequences clustered in four distinct clades. This is the first study to provide molecular evidence for the presence of zoonotic Babesia spp. other than B. microti in ticks in the ROK., Babesiosis is an emerging malaria‐like disease caused by tick‐borne parasites. This study is the first to identify zoonotic Babesia spp. besides the predominant Babesia microti in ticks parasitizing on small mammals inhabiting different regions in the Republic of Korea. This indicates the need for nationwide surveillance of wildlife to prevent the occurrence of various zoonotic Babesia parasites.

The present study was aimed at screening suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in tiger puffer (Takifugu rubripes), an important aquaculture species in Asia and also a good model species for lipid research. Specifically, this reference gene screening was targeted at standardization of gene expression in different tissues (liver, muscle, brain, intestine, heart, eye, skin, and spleen) or under different nutritional conditions (starvation and different dietary lipid levels). Eight candidate reference genes (ribosomal protein L19 and L13 (RPL19 and RPL13), elongation factor-1 alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase1 (HPRT1), beta-2-Microglobulin (B2M), 18S ribosomal RNA (18SrRNA), and beta actin (ACTB)) were evaluated with four algorithms (geNorm, NormFinder, BestKeeper, and comparative ΔCt method). The results showed that different algorithms generated inconsistent results. Based on these findings, RPL19, EF1α, 18SrRNA, and RPL13 were relatively stable in different tissues of tiger puffer. During starvation conditions, ACTB/RPL19 was the best reference gene combination. Under different dietary lipid levels, ACTB/RPL13 was the most suitable reference gene combination. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in tiger puffer.

In South America, apicomplexan parasites of the genus Hepatozoon have been sporadically detected in mammals. Previous studies in wild canids from Brazil and Argentina demonstrated infections by species genetically related to Hepatozoon americanum. The aim of the present work was to detect the presence of Hepatozoon in road-killed foxes encountered in Uruguayan highways. Blood samples from 45 crab-eating (Cerdocyon thous) and 32 grey pampean (Lycalopex gymnocercus) foxes were analyzed by PCR for Hepatozoon 18S rRNA gene. Eight foxes (10.4%) were found to be infected with an H. americanum-like protozoan, an Hepatozoon closely related to H. americanum. Bayesian and maximum-likelihood phylogenetic analyses revealed that the sequences obtained in this study cluster with H. americanum from the United States, and with an H. americanum-like species from dog and foxes from Brazil and Argentina. In the Unites States, H. americanum causes severe disease in dogs. In addition to this, an increasing habitat overlap between dogs and foxes makes the presence of H. americanum-like protozoan in foxes acquires veterinary relevance. This work represents the first report of L. gymnocercus infected with an H. americanum-like protozoan, and of wild canids infected with Hepatozoon in Uruguay.

During samplings aimed at isolating microalgal strains, a coccoid greyish-green alga was collected along the North Adriatic coasts (Mediterranean Sea, Italy) and grown in culture. The microalgal st...

Background Canine babesiosis is a common and clinically significant tick‐borne disease caused by obligate haematozoan parasites of the genus Babesia. Purpose To report Babesia canis canis genotype A infection in a dog. Methods A 2‐year‐old female Shih Tzu dog was submitted with the history of anorexia and depression for one week and no prior surgery. Fever, anorexia, depression and vomiting as well as mucosal pallor were noticed on physical examination. Microscopic examination of the Giemsa‐stained blood smear disclosed large form of Babesia, and single to four pear‐shaped merozoites within erythrocytes (RBCs). The specific primers were used for detecting Babesia canis. Results The result of PCR was confirmed by 18S rRNA gene sequence analyzing and has been registered in GenBank under following accession numbers for Babesia canis canis (MW199108). The sequences were compared to those in GenBank, and alignments showed that the B. canis canis isolate belonged to genotype A. Conclusions This is the first description of B. canis canis genotype A in dog from Iran., A dog infected by B. c. canis genotype A. This is the first description of B. canis canis genotype A in dog from Iran.

Background and Aim: The Sunda pangolin (Manis javanica) is on the International Union for Conservation of Nature Red List of Threatened Species (critically endangered) due to high levels of illegal trafficking for its products. Thailand is one of the habitats of this species, and it has become the main hub for its illegal trafficking. Rehabilitating these captive pangolins and reintroducing them back to the wild are challenging due to the limited knowledge on their diet, management, and diseases. Hemoparasites, including Babesia spp., can cause important protozoal infections in both domestic and wild animals, resulting in the failure of rehabilitation and conservation programs. However, Babesia spp. has not been reported in pangolins. The aim of the study was to determine the prevalence of Babesia spp. in the Sunda pangolin of Thailand. Materials and Methods: A total of 128 confiscated Sunda pangolins from across different regions in Thailand were investigated. These pangolins had been admitted to a regional Wildlife Quarantine Center for rehabilitation before release in the forest. Routine physical examinations were conducted on the animals. We collected blood samples from each pangolin for hematological analysis and to detect Babesia spp. using polymerase chain reaction (PCR) targeting the partial 18s rRNA gene. Results: Babesia-specific PCR detected 53 animals (41.4%) that were positive for Babesia spp. Blood smears were obtained from the positive samples and investigated under a light microscope to observe for trophozoites of Babesia spp. Examination of 40 PCR-positive and -negative samples found no significant differences between the hematological parameters of Babesia-positive and Babesia-negative samples. Eight PCR-positive samples were randomly selected and their DNA was sequenced. Seven and one of sequences match uncharacterized Babesia spp. with 100% and 99.2% similarity, respectively. Phylogenetic analysis demonstrated that our samples form a unique monophyletic clade along with other Babesia spp. detected in the wild. This clade is clearly separated from other Babesia spp. from small carnivores, ruminants, and rats. Conclusion: Our results provide evidence of infection of Sunda pangolins in Thailand by Babesia spp. These pangolins originated from different regions and had not lived together before blood collection. Thus, we suggest that the uncharacterized Babesia spp. found in this study constitute a new group of pangolin-specific Babesia spp. The prevalence of the uncharacterized Babesia spp. was not correlated to pangolin health. Further studies are required to characterize the genomes and phenotypes, including the morphology and pathogenicity of these protozoa. Such information will be helpful for the conservation and health management of the Sunda pangolin.

One major difficulty in identifying the gelatinous bodied bullet-shaped Siphonophore, Diphyids, is that their shape is deformed following ethanol fixation. Ethanol often is preferred over other fixatives, since samples fixed in ethanol can be used for molecular studies that can supplement morphological findings. To overcome this problem, we obtained protein mass spectra of ten species of Diphyidae found in the waters of the Kuroshio Current (Northwest Pacific and South Coast of South Korea) to test whether MALDI-ToF MS could be used as a methodology for species identification. In addition, a number of morphological characteristics that can be used with ethanol-treated samples was summarized. Concatenated phylogenetic analysis was also performed to determine the phylogenetic relationship by obtaining partial sequences of four genes (mtCOI, 16S rRNA, 18S rRNA, and ITS regions). Based on our integrative analysis, MALDI-ToF MS was evaluated as a potentially fast, inexpensive, and accurate tool for species identification along with conventional morphological and DNA barcoding for Diphyidae.

Objective Blastocystis is a zoonotic protozoan that infects a wide range of animals, including humans and rodents. This study aimed to determine the frequency and subtype distribution of Blastocystis in laboratory rats at a laboratory animal facility in Turkey. Methods This study included 54 male Sprague-Dawley rats from Aydin Adnan Menderes University Laboratory Animal Center. Among these rats, 30 were fed with high-fat diet (obese group) and the remaining 24 received standard chow (non-obese group). Blastocystis positivity was determined with amplification of small subunit 18S rRNA gene following their nucleic acid extraction from faecal samples. Subtypes were detected by submitting the partial 18S rRNA gene sequences to the database (pubmlst. Results Blastocystis infection was detected in 33 (61.1%) of 54 laboratory rats. The frequency of Blastocystis was significantly different between obese and non-obese rats (p