Targeting 16S rDNA Research Articles

Comparative Molecular Virulence Typing and Antibiotic Resistance of Campylobacter Species at the Human-Animal-Environment Interface.

This study holds significant importance due to its focus on Campylobacter, the leading bacterial cause of gastroenteritis worldwide, responsible for ∼96 million cases each year. By investigating the prevalence of both Campylobacter jejuni and Campylobacter coli in humans, animals, and the environment, this research sheds light on the broader impact of these pathogens, which can harm both human and animal populations. Traditional microbiological methods were implemented followed by optimized multiplex polymerase chain reaction targeting 16S rDNA and virulence gene markers by using specific primers. The findings revealed that a total of 219 Campylobacter isolates were recovered from 528 collected specimens from human, animal, and environmental sources. Campylobacter species showed a prevalence of 41.5%, with C. jejuni accounting for 53% and C. coli for 47%. Antimicrobial resistance rates were high, with tetracycline at 89%, ceftriaxone at 75%, cefotaxime at 70%, erythromycin at 69%, nalidixic acid at 54%, ciprofloxacin at 39%, and gentamicin at 23%. Commonly prevalent virulence-associated genes observed in the Campylobacter were cadF at 93%, flaA at 91%, cdtB at 88%, cheY at 86%, sodB at 78%, and iamA at 32%. The study confirmed multidrug-resistant Campylobacter prevalence at the human-animal-environment interface, harboring virulence-associated genes with potential harm to humans. Data analysis showed a nonsignificant (p ≥ 0.05) correlation between virulence genes and antibiotic susceptibility. To effectively manage Campylobacter infections, a multifaceted strategy incorporating preventative interventions at different levels is required. This strategy should take into account practicability, effectiveness, and sustainability while strengthening surveillance systems and addressing the economics of disease prevention.

Aerosolized Cyanobacterial Harmful Algal Bloom Toxins: Microcystin Congeners Quantified in the Atmosphere.

Cyanobacterial harmful algal blooms (cHABs) have the potential to adversely affect public health through the production of toxins such as microcystins, which consist of numerous molecularly distinct congeners. Microcystins have been observed in the atmosphere after emission from freshwater lakes, but little is known about the health effects of inhaling microcystins and the factors contributing to microcystin aerosolization. This study quantified total microcystin concentrations in water and aerosol samples collected around Grand Lake St. Marys (GLSM), Ohio. Microcystin concentrations in water samples collected on the same day ranged from 13 to 23 μg/L, dominated by the d-Asp3-MC-RR congener. In particulate matter <2.5 μm (PM2.5), microcystin concentrations up to 156 pg/m3 were detected; the microcystins were composed primarily of d-Asp3-MC-RR, with additional congeners (d-Asp3-MC-HtyR and d-Asp3-MC-LR) observed in a sample collected prior to a storm event. The PM size fraction containing the highest aerosolized MC concentration ranged from 0.44 to 2.5 μm. Analysis of total bacteria by qPCR targeting 16S rDNA revealed concentrations up to 9.4 × 104 gc/m3 in aerosol samples (≤3 μm), while a marker specific to cyanobacteria was not detected in any aerosol samples. Concentrations of aerosolized microcystins varied even when concentrations in water were relatively constant, demonstrating the importance of meteorological conditions (wind speed and direction) and aerosol generation mechanism(s) (wave breaking, spillway, and aeration systems) when evaluating inhalation exposure to microcystins and subsequent impacts on human health.

The alteration of oral microbiota before and after training in swimmers.

We aimed to analyze the effect of acute exercise on oral microbiota in regularly trained swimmers. As environmental factors may affect the oral microbiota; we also aimed to analyze the short-duration effect of swimming training on the oral bacteria relative difference in swimmers. Saliva samples of 20 swimmers both before and after the training were used for the oral microbiota metagenesis. The next-generation sequencing method targeting 16S rDNA gene fragments was used for genotyping. The Wilcoxon signed-rank test was used for the statistical evaluation of the taxons. The alfa diversity comparisons were assessed with the One-Way ANOVA, and the Kruskal-Wallis test was used to determine bacterial diversity. Decayed- Missed- Filled total (DMF-T) scores were the indicators of oral hygiene. A comparison of the before and after exercise microbiota of the swimmers gave rise to a statistically significant difference for Firmicutes (p=0.014) and Bacteroidetes (p=0.007) phylum; Clostridia (p=0.006) and Bacilli (p=0.048) classes; Clostridiales (p=0.004), Entomoplasmatales (p=0.009) and Bacillales (p=0.006) for ordo; Lachnospiraceae (p=0.001) family and Stenotrophomonas (p=0.013) genus. Although there were some differences within the other taxa of the bacteria, all were statistically insignificant. Streptococcus, Pseudomonas and Rothia mucilaginosa showed a correlation with the DMF-T values in swimmers. This study was the first in Turkish swimmers to investigate the relative abundance of oral microbiota. We showed that exercise within the pool water changed the oral bacteria's relative abundance. To confirm our results and clarify the effect of pool water on oral bacteria relative abundance, more studies on dietary intake should be carried out.

Development and Validation of a Real-Time PCR for the Molecular Identification of Thaumatotibia leucotreta (Lepidoptera: Tortricidae: Olethreutinae) Intercepted in Trade

The false codling moth, Thaumatotibia leucotreta (Meyrick), is a polyphagous pest indigenous to most of sub-Saharan Africa. This species is considered to be quarantined throughout much of the world, in part due to its extensive host range and significant damage caused by larval feeding. We developed a specific real-time PCR assay that allows for rapid and reliable identification of T. leucotreta. More than 150 target specimens were sequenced using an Illumina whole-genome shotgun approach to identify the most suitable loci for assay development. A hydrolysis probe that binds to a segment of the internal transcribed spacer 2 (ITS2) region was designed and used with a generic internal control targeting 18S rDNA. The assay was examined for cross-reactivity by testing additional Thaumatotibia species and representatives of related Olethreutine leafroller genera such as Cryptophlebia, Cydia, and Grapholita, which are often encountered in the same geographic region and on the same hosts as T. leucotreta. We compared our newly developed test to previously published TaqMan real-time PCR, SYBR Green real-time PCR, and loop-mediated isothermal amplification (LAMP) tests. Our newly developed real-time PCR assay outperformed all three tests in terms of analytical specificity, with 100% accuracy. These results will help to further improve diagnostic standards for molecular identification of T. leucotreta. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Altered gut microbiota in temporal lobe epilepsy with anxiety disorders.

Patients with epilepsy are particularly vulnerable to the negative effects of anxiety disorders. In particular, temporal lobe epilepsy with anxiety disorders (TLEA) has attracted more attention in epilepsy research. The link between intestinal dysbiosis and TLEA has not been established yet. To gain deeper insight into the link between gut microbiota dysbiosis and factors affecting TLEA, the composition of the gut microbiome, including bacteria and fungi, has been examined. The gut microbiota from 51 temporal lobe epilepsy patients has been subjected to sequencing targeting 16S rDNA (Illumina MiSeq) and from 45 temporal lobe epilepsy patients targeting the ITS-1 region (through pyrosequencing). A differential analysis has been conducted on the gut microbiota from the phylum to the genus level. TLEA patients' gut bacteria and fungal microbiota exhibited distinct characteristics and diversity as evidenced by high-throughput sequencing (HTS). TLEA patients showed higher abundances of Escherichia-Shigella (genus), Enterobacterales (order), Enterobacteriaceae (family), Proteobacteria (phylum), Gammaproteobacteria (class), and lower abundances of Clostridia (class), Firmicutes, Lachnospiraceae (family), Lachnospirales (order), and Ruminococcus (genus). Among fungi, Saccharomycetales fam. incertae sedis (family), Saccharomycetales (order), Saccharomycetes (class), and Ascomycota (phylum) were significantly more abundant in TLEA patients than in patients with temporal lobe epilepsy but without anxiety. Adoption and perception of seizure control significantly affected TLEA bacterial community structure, while yearly hospitalization frequency affected fungal community structures in TLEA patients. Here, our study validated the gut microbiota dysbiosis of TLEA. Moreover, the pioneering study of bacterial and fungal microbiota profiles will help in understanding the course of TLEA and drive us toward preventing TLEA gut microbiota dysbiosis.

Misidentification of Plasmodium Species by Cross-Reacting Primers and Cerebral Malaria Caused by Plasmodium vivax

Abstract Introduction The clinical presentation of a case as cerebral malaria with molecular identification confirming it as Plasmodium vivax underlines the importance of using molecular tools to identify the species and type of malaria. The possibility of the relationship between the complication observed during clinical diagnosis and the multifactorial molecular changes could likely be the reason for terming it cerebral malaria. Methods We report four cases analyzed using the quantitative buffy coat technique followed by classical Giemsa stained thick-film microscopy, and nested polymerase chain reaction for the genus-specific region of Plasmodium targeting 18S rDNA followed by species-specific identification with a different set of primers and products confirmation with sequencing. Results Primers targeting P. knowlesi generated the expected product size of 153 base pairs that, upon sequencing, matched with the P. vivax sequence reflecting the relatedness of the species. Likewise, primers targeting P. ovale generated a 456 product whose sequence matched the P. vivax sequence. Conclusion Infection with P. vivax can potentially cause cerebral malaria, and P. vivax can cause severe malaria complications alone or mixed with other species and can show cerebral malaria signs, which are typically associated with P. falciparum infections. The sequence relatedness reflects the genome similarity between P. knowlesi and P. ovale with P. vivax. The need to reconfirm with an additional set of newly reported primers is mandatory.

Diagnosis, Clinical and Molecular Delineation of Human Plasmodium Species from Mangaluru, Southwest India

Malaria is a global threat and a never-ending battle without appropriate identification and differentiation of the parasite species. This work compared the diagnostic methods including the thick film microscopy technique, quantitative buffy coat, and polymerase chain reaction. The inaccuracy of species determination by microscopy and the consequent treatment regime underlines the necessity to upgrade routine diagnostic methods with molecular techniques. In the study, 436 samples were collected; venous blood was processed for the quantitative buffy coat technique followed by classical Giemsa staining of thin and thick smears and nested Polymerase Chain Reaction (nPCR) for the genus-specific region of Plasmodium targeting 18S rDNA followed by species-specific identification. Of 436 samples screened for malaria, results in PCR showed 78.7% (100/127) to be P. vivax, 4.8% (6/127) as P. falciparum and 16.5% (21/127) to be mixed infection (P. vivax + P. falciparum). The prevalence of malaria was 0.29, and there was good concordance between the methods for detecting Plasmodium (Kappa:0.77). In our investigation, nested PCR and TFM exhibited a sensitivity of 97.7% and a specificity of 100% for malaria detection compared to QBC. Clinical parameters- thrombocytopenia and anemia, were compared in this study. A positive association was observed between thrombocytopenia and malaria (p&lt;0.05), but the association between anemia and malaria infection remains unclear. Primer cross-reactions were also observed in the primer sequence of P. ovale and P. knowlesi, but sequencing confirmed it as P. vivax and the study of phylogeny paved a new way in analyzing the relatedness of the sequences.

A Case of Bovine Eosinophilic Myositis (BEM) Associated with Co-Infection by Sarcocystis hominis and Toxoplasma gondii.

Bovine eosinophilic myositis (BEM) is a specific inflammatory myopathy, often associated with Sarcocystis spp., with multifocal gray-green lesions leading to carcass condemnation with considerable economic losses. Here is described a peculiar case of BEM that occurred in an adult (16 month) cattle, born in France, bred, and slaughtered in Italy at the end of 2021. On inspection, muscles showed the typical multifocal gray-green lesions that were sampled for, cytological, histological, and molecular investigations, while meat juice was subjected to IFAT for Toxoplasma IgG. Genomic DNA was extracted from lesions, portions of healthy muscle and from meat juice pellet and analyzed by PCR targeting 18S rDNA, COI mtDNA and B1 genes, and sequenced. The cytology showed inflammatory cells mostly referable to eosinophils; at histology, protozoan cysts and severe granulomatous myositis were observed. A BEM lesion and meat juice pellet subjected to PCR showed, concurrently, sequences referable both to S. hominis and T. gondii. Meat juice IFAT resulted negative for T. gondii IgG. Our findings highlight the first detection of T. gondii DNA in association with S. hominis in a BEM case, suggesting a multiple parasite infection associated with this pathology, although the actual role of T. gondii infection in the pathophysiology of the diseases should be clarified.

First identification of multidrug-resistant Acinetobacter bereziniae isolates harboring blaNDM-1 from hospitals in South China

This study is a first report on the identification of multidrug-resistant (MDR) Acinetobacter bereziniae among non-baumannii acinetobacters that had previously escaped automated laboratory detection, and characterize their clinical courses of infection at two tertiary-care hospitals in Shenzhen city, China (2015–2017). Herein, definitive identification by PCR was performed with universal and species-specific primers targeting 16S rDNA and rpoB genes, respectively, followed by Sanger sequencing and blast analysis. Antimicrobial susceptibility of A. bereziniae isolates was assessed accordingly. Three of the five identified A. bereziniae isolates exhibited carbapenem-resistance and were subjected to a multiplex PCR assay to detect drug-resistance genes. Sequences of the rpoB amplicon were aligned with curated sequences from global databases for phylogenetic analysis on evolutionary relations. Five clinical isolates of A. bereziniae were thereby re-identified, whose infections were primarily nosocomial. Automated identification and susceptibility testing systems (Phoenix-100 and VITEK 2) proved insufficient for discriminating A. bereziniae from other acinetobacters such as Acinetobacter baumannii and Acinetobacter guillouiae. Among these isolates, three exhibited carbapenem-resistant phenotypes indistinguishable from that of carbapenem-resistant A. baumannii. The carbapenem-resistant A. bereziniae isolates were subsequently confirmed to carry a blaNDM-1 (New Delhi metallo-β-lactamase-1) gene downstream of ISAba125. Phylogenetic analysis revealed that A. bereziniae isolates evolved slowly but independently in local habitats. A. bereziniae isolates are difficult to distinguish by traditional automated detection systems. PCR-based identification via amplification and sequencing of selected house-keeping genes provides sufficient resolution for discriminating the isolates.

Glomerales Dominate Arbuscular Mycorrhizal Fungal Communities Associated with Spontaneous Plants in Phosphate-Rich Soils of Former Rock Phosphate Mining Sites.

Arbuscular mycorrhizal fungi (AMF) are key drivers of soil functioning. They interact with multiple soil parameters, notably, phosphorus (P). In this work, AMF communities of native plants grown spontaneously on former mining sites either enriched (P sites) or not enriched with P (nP sites) by mining cuttings of rock phosphate (RP) were studied. No significant differences were observed in the root mycorrhizal rates of the plants when comparing P and nP sites. The assessment of AMF diversity and community structure using Illumina MiSeq metabarcoding and targeting 18S rDNA in roots and rhizospheric soils showed a total of 318 Amplicon Sequence Variants (ASVs) of Glomeromycota phylum. No significant difference in the diversity was found between P and nP sites. Glomeraceae species were largely dominant, formed a fungal core of 26 ASVs, and were persistent and abundant in all sites. In the P soils, eight ASVs were identified by indicator species analysis. A trend towards an increase in Diversisporaceae and Claroideoglomeraceae and a reduction in Paraglomeraceae and Glomeraceae were noticed. These results provide new insights into AMF ecology in former RP mining sites; they document that P concentration is a driver of AMF community structures in soils enriched in RP long term but also suggest an influence of land disturbance, ecosystem self-restoration, and AMF life history strategies as drivers of AMF community profiles.

Microbial community development during syngas methanation in a trickle bed reactor with various nutrient sources

Microbial community development within an anaerobic trickle bed reactor (TBR) during methanation of syngas (56% H2, 30% CO, 14% CO2) was investigated using three different nutrient media: defined nutrient medium (241 days), diluted digestate from a thermophilic co-digestion plant operating with food waste (200 days) and reject water from dewatered digested sewage sludge at a wastewater treatment plant (220 days). Different TBR operating periods showed slightly different performance that was not clearly linked to the nutrient medium, as all proved suitable for the methanation process. During operation, maximum syngas load was 5.33 L per L packed bed volume (pbv) & day and methane (CH4) production was 1.26 L CH4/Lpbv/d. Microbial community analysis with Illumina Miseq targeting 16S rDNA revealed high relative abundance (20–40%) of several potential syngas and acetate consumers within the genera Sporomusa, Spirochaetaceae, Rikenellaceae and Acetobacterium during the process. These were the dominant taxa except in a period with high flow rate of digestate from the food waste plant. The dominant methanogen in all periods was a member of the genus Methanobacterium, while Methanosarcina was also observed in the carrier community. As in reactor effluent, the dominant bacterial genus in the carrier was Sporomusa. These results show that syngas methanation in TBR can proceed well with different nutrient sources, including undefined medium of different origins. Moreover, the dominant syngas community remained the same over time even when non-sterilised digestates were used as nutrient medium.Key points•Independent of nutrient source, syngas methanation above 1 L/Lpbv/D was achieved.•Methanobacterium and Sporomusa were dominant genera throughout the process.•Acetate conversion proceeded via both methanogenesis and syntrophic acetate oxidation.Graphical abstract

Diversity and proteolytic activity of Pseudomonas species isolated from raw cow milk samples across China

Pseudomonas spp. are common microorganisms from cold-storage raw milk, and protease secreted by Pseudomonas spp. can cause the deterioration of stored milk. However, analyses of Pseudomonas spp. diversity and proteolytic activity in raw milk from different regions of China have not been extensively examined. With this aim, the diversity and proteolytic activity of Pseudomonas isolated from 25 raw cow milk samples from Inner Mongolia, Heilongjiang, Gansu, Henan, Anhui, Jiangsu, Chongqing and Hunan of China in different seasons were evaluated by PCR targeting 16S rDNA and rpoD, as well as TNBS method, respectively.A total of 116 Pseudomonas isolates from 25 raw cow milk samples were identified at the species level, including P. fluorescens, P. veronii, P. psychrophila, P. lundensis, P. lactis, P. azotoformans, P. granadensis, P. lurida, P. rhizosphaerae, P. rhodesiae and P. extremorientalis. P. fluorescens accounted for 75.8% of the total. Of all 116 Pseudomonas isolates, 68.9% of them displayed proteolytic activity at 4 °C, 81.9% at 10 °C and 85.3% at 25 °C, respectively. The aprX gene encoded a secreted and heat-resistant metalloprotease that was present in 60.3% of the Pseudomonas isolates tested. The proteases showed residual activity ranged from 73 ± 4% to 84 ± 7% residual activity after the heat treatment at 72 °C for 15 s and 62 ± 3% to 74 ± 2% after the heat treatment at 132 °C for 4 s. This is the first report to compare Pseudomonas spp. diversity and proteolytic activity at species levels in raw milk from different regions of China. The results of this study provide valuable data about the diversity and spoilage potential of Pseudomonas species in raw milk and the thermal resistance of the proteases. Therefore, these findings provide a reference for the importance to prevent Pseudomonas spp. contamination of raw cow milk to ensure the quality and safety of milk and dairy products.

Ornithodoros (Pavlovskyella) ticks associated with a Rickettsia sp. in Pakistan

BackgroundSoft ticks (Ixodida: Argasidae) are medically important ectoparasites that mainly feed on birds and mammals, which play a key role in their geographic distribution and dispersion. Despite their importance, studies on soft ticks are scarce for many regions and countries of the world, including Pakistan.MethodsIn this study, 2330 soft ticks—179 larvae (7.7%), 850 nymphs (36.4%), 711 males (30.5%) and 590 females (25.3%)—were collected from animal shelters in 18 locations within five districts of Khyber Pakhtunkhwa, Pakistan. A subset of the collected ticks was processed for DNA extraction and polymerase chain reaction (PCR) for the amplification of tick 12S ribosomal DNA (rDNA), 16S rDNA and cytochrome c oxidase subunit I (cox1), and rickettsial 16S rDNA gene fragments. The obtained sequences were used for the construction of a phylogenetic tree.ResultsAll the specimens were morphologically identified as Ornithodoros, and were morphologically similar to Ornithodoros tholozani. The genus was confirmed by sequencing partial 12S rDNA, 16S rDNA and cox1 gene fragments. Additionally, a Rickettsia sp. was detected in some of the collected ticks by PCR targeting 16S rDNA. The morphological relatedness of the tick specimens with O. tholozani was confirmed by phylogenetic analysis, in which the Ornithodoros sp. clustered with Ornithodoros tholozani and Ornithodoros verrucosus, both of which belong to the subgenus Pavlovskyella and have been previously reported from Israel, Ukraine and Iran. The phylogenetic tree also indicated that the Ornithodoros sp. from Pakistan corresponds to an undetermined species. Furthermore, the associated Rickettsia sp. grouped with the limoniae group of Rickettsia species previously reported from Argas japonicus ticks from China.ConclusionsThis is the first molecular study of an Ornithodoros species from Pakistan. Further studies are essential to confirm its identity and possible pathogenicity with regard to its associated microorganisms in the studied region.Graphical abstract

Development of an RNA anti-parasite based on CRISPR-Cas9 against Chilodonella piscicola

Chilodonella piscicola is a gill and body surface parasite of freshwater fish, affecting the growth of juvenile and adult freshwater fishes. To develop an effective method to manage C.piscicola in aquaculture, we used CRISPR/Cas9 technology to design and validate small guide RNA targeting DNA barcoding ITS-1 and 18S rDNA sequences of C.piscicola to develop a species-specific pesticide. In this study, effective sgRNA targets were found in ITS-1 and 18S rDNA sequences of C.piscicola, respectively. In the soaking experiment, we found that the mixing of Cas9 mRNA and sgRNAs could significantly reduce the survival rate of C.piscicola. The Real-time quantitative PCR results showed that both Cas9 works with the single sgRNA or the mixture of the two sgRNA could destroy the parasite DNA at a specific region. DNA sequencing also verified the effectiveness of single sgRNA or two sgRNAs mixed with cas9 induced mutation on the target fragment of the parasite DNA in the experimental group. The results of this study once again verified the effectiveness of genetic barcoding in the application of anti-parasitic and provided a new idea for anti-parasitic methods.

First molecular detection of Hemolivia and Hepatozoon parasites in reptile-associated ticks on Iriomote Island, Japan.

Hepatozoon and Hemolivia are members of the haemogregarines and are reported in reptiles and reptile-associated ticks. However, no studies have reported on Hepatozoon and Hemolivia in Japanese reptile-associated ticks. This study aimed to molecularly identify and to characterize Hepatozoon and Hemolivia in Japanese reptile-associated ticks, Amblyomma geoemydae (Cantor, 1847) and Amblyomma nitidum (Hirst & Hirst, 1910). A total of 41 and 75 DNA samples from A. geoemydae and A. nitidum ticks, respectively, were used for screening of Hepatozoon and Hemolivia with polymerase chain reaction targeting 18S rDNA. As a result, Hemolivia and Hepatozoon were detected in two A. geoemydae and one A. nitidum, respectively. The sequences of Hemolivia spp. showed a 99.5% (1,050/1,055bp) identity with Hemolivia parvula (KR069083), and the Hemolivia spp. were located in the same clade as H. parvula in the phylogenetic tree. The sequences of Hepatozoon sp. showed a 98.4% (1,521/1,545bp) identity with Hepatozoon colubri (MN723844), and the Hepatozoon sp. was distinct from validated Hepatozoon species in the tree. Our findings highlight the first molecular record of Hemolivia in Japan and present the first detection of Hepatozoon in A. nitidum. Further investigations on these tick-borne protozoa are required to understand their life cycle and pathogenicity.

Evaluation of Some Potential Protocols to Extract DNA from Paddy Soil

Isolation of DNA from environmental samples is a crucial step in microbial community analyses through molecular methods. The present study was conducted to evaluate a DNA extraction protocol from paddy soil with a comparison on quality, quantity and integrity of the isolated DNA and to determine the suitability of extracted DNA for downstream applications in microbial community analyses. Three protocols (i.e. PEG/NaCl, Mannitol/C TAB and Sodium Phosphate Buffer) used for the extraction of DNA from different types of soil were attempted on paddy soil. The quality and quantity of the extracted genomic DNA was quantified spectrophotometrically and integrity was checked by gel electrophoresis. The efficiency of DNA extraction by the three protocols was compared with a commercia l soil DNA extraction kit (Norgen’s Soil DNA Isolation Plus Kit). Further, quality of the extracted DNA for PCR amplification was assessed using universal primer pairs for bacteria and fungi. DNA extracted using PEG/NaCl method resulted in the highest DNA concentration, while the highest purity was recorded by the DNA extracted by Mannitol/CTAB method (A 260/A280 = 1.61 and A260/A230 = 1.15). Expected PCR products targeting 16s rDNA and ITS regions were obtained from the DNA samples extracted by Mannitol/CTAB method. Therefore, Mannitol/CTAB method used in the present study is suitable to extract high-quality DNA from paddy soil for molecular microbial studies.

Capture by hybridization for full-length barcode-based eukaryotic and prokaryotic biodiversity inventories of deep sea ecosystems.

Biodiversity inventory of marine systems remains limited due to unbalanced access to the three ocean dimensions. The use of environmental DNA (eDNA) for metabarcoding allows fast and effective biodiversity inventory and is forecast as a future biodiversity research and biomonitoring tool. However, in poorly understood ecosystems, eDNA results remain difficult to interpret due to large gaps in reference databases and PCR bias limiting the detection of some major phyla. Here, we aimed to circumvent these limitations by avoiding PCR and recollecting larger DNA fragments to improve assignment of detected taxa through phylogenetic reconstruction. We applied capture by hybridization (CBH) to enrich DNA from deep-sea sediment samples and compared the results with those obtained through an up-to-date metabarcoding PCR-based approach (MTB). Originally developed for bacterial communities and targeting 16S rDNA, the CBH approach was applied to 18S rDNA to improve the detection of species forming benthic communities of eukaryotes, with a particular focus on metazoans. The results confirmed the possibility of extending CBH to metazoans with two major advantages: (i) CBH revealed a broader spectrum of prokaryotic, eukaryotic, and particularly metazoan diversity, and (ii) CBH allowed much more robust phylogenetic reconstructions of full-length barcodes with up to 1900 base pairs. This is particularly important for taxa whose assignment is hampered by gaps in reference databases. This study provides a database and probes to apply 18S CBH to diverse marine systems, confirming this promising new tool to improve biodiversity assessments in data-poor ecosystems such as those in the deep sea.

Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis

BackgroundCattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis.MethodsTo reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification.ResultsSarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses.ConclusionsOur study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis.Graphical

Spiroplasma Infection among Ixodid Ticks Exhibits Species Dependence and Suggests a Vertical Pattern of Transmission.

Members of the genus Spiroplasma are Gram-positive bacteria without cell walls. Some Spiroplasma species can cause disease in arthropods such as bees, whereas others provide their host with resistance to pathogens. Ticks also harbour Spiroplasma, but their role has not been elucidated yet. Here, the infection status and genetic diversity of Spiroplasma in ticks were investigated using samples collected from different geographic regions in Japan. A total of 712 ticks were tested for Spiroplasma infection by PCR targeting 16S rDNA, and Spiroplasma species were genetically characterized based on 16S rDNA, ITS, dnaA, and rpoB gene sequences. A total of 109 samples originating from eight tick species were positive for Spiroplasma infection, with infection rates ranging from 0% to 84% depending on the species. A linear mixed model indicated that tick species was the primary factor associated with Spiroplasma infection. Moreover, certain Spiroplasma alleles that are highly adapted to specific tick species may explain the high infection rates in Ixodes ovatus and Haemaphysalis kitaokai. A comparison of the alleles obtained suggests that horizontal transmission between tick species may not be a frequent event. These findings provide clues to understand the transmission cycle of Spiroplasma species in wild tick populations and their roles in host ticks.

Genetic and Morphological Diversity of Indigenous Bradyrhizobium Nodulating Soybean in Organic and Conventional Family Farming Systems

Organic farming systems are gaining popularity as agronomically and environmentally sound soil management strategies with potential to enhance soil microbial diversity and fertility, environmental quality and sustainable crop production. This work aimed at understanding the effect of organic and conventional farming on the diversity of soybean nodulating bradyrhizobia species. Field trapping of indigenous soybean Bradyrhizobium was done by planting promiscuous soybeans varieties SB16 and SC squire as well as non-promiscuous Gazelle in three organic and three conventional farms in Tharaka-Nithi County of Kenya. After 45 days of growth, 108 nodule isolates were obtained from the soybean nodules and placed into 13 groups based on their morphological characteristics. Genetic diversity was done by polymerase chain reaction (PCR) targeting 16S rDNA gene using universal primers P5-R and P3-F and sequencing was carried out using the same primer. High morphological and genetic diversity of the nodule isolates was observed in organic farms as opposed to conventional farms. There was little or no genetic differentiation between the nodule isolates from the different farms with the highest molecular variation (91.12%) being partitioned within populations as opposed to among populations (8.88%). All the isolates were identified as bradyrhizobia with close evolutionary ties with Bradyrhizobium japonicum and Bradyrhizobium yuanminense. Organic farming systems favor the proliferation of bradyrhizobia species and therefore a suitable environmentally friendly alternative for enhancing soybean production.