Molecular-based Techniques Research Articles

Environmental DNA as a complementary tool for biodiversity monitoring: A multi-technique and multi-trophic approach to investigate cetacean distribution and feeding ecology.

The use of environmental DNA (eDNA) to assess the presence of biological communities has emerged as a promising monitoring tool in the marine conservation landscape. Moreover, advances in Next-Generation Sequencing techniques, such as DNA metabarcoding, enable multi-species detection in mixed samples, allowing the study of complex ecosystems such as oceanic ones. We aimed at using these molecular-based techniques to characterize cetacean communities, as well as potential prey on the northern coast of Mainland Portugal. During four seasonal campaigns (summer 2021 to winter 2022/2023), seawater samples were collected along with visual records of cetacean occurrence. The eDNA isolated from 64 environmental samples was sequenced in an Illumina platform, with universal primers targeting marine vertebrates. Five cetacean species were identified by molecular detection: common dolphin (Delphinus delphis), bottlenose dolphin (Tursiops truncatus), Risso's dolphin (Grampus griseus), harbor porpoise (Phocoena phocoena) and fin whale (Balaenoptera physalus). Overall, except for the latter (not sighted during the campaigns), this cetacean community composition was similar to that obtained through visual monitoring, and the complementary results suggest their presence in the region all year round. In addition, the positive molecular detections of Balaenoptera physalus are of special relevance since there are no records of this species reported on scientific bibliography in the area. The detection of multiple known prey of the identified dolphins indicates an overlap between predator and prey in the study area, which suggests that these animals may use this coastal area for feeding purposes. While this methodological approach remains in a development stage, the present work highlights the benefits of using eDNA to study marine communities, with specific applications for research on cetacean distribution and feeding ecology.

A Recent Advance in the Diagnosis, Treatment, and Vaccine Development for Human Schistosomiasis.

Schistosomiasis, which affects a large number of people worldwide, is among the most overlooked parasitic diseases. The disease is mainly prevalent in sub-Saharan Africa, southeast Asian countries, and South America due to the lack of adequate sanitation. The disease is mainly associated with poor hygiene, sanitation, and contaminated water, so it is also known as a disease of poverty. Three Schistosoma species (S. mansoni, S. japonicum, and S. haematobium) cause significant human infections. Co-infections with Schistosoma and other parasites are widely common. All these parasites may cause intestinal or urogenital schistosomiasis, where the disease may be categorized into the acute, sensitized, and chronic phases. The disease is more prevalent among school children, which may cause anemia and reduce development. Chronic infections frequently cause significant liver, intestinal, and bladder damage. Women exposed to contaminated water while performing normal duties like washing clothes might acquire urogenital schistosomiasis (UGS), which can cause tissue damage and raise the risk of blood-borne disease transmission, including human immunodeficiency virus (HIV) transmission. Praziquantel (PZQ) is the World Health Organization (WHO)-prescribed treatment for individuals who are known to be infected, but it does not prevent further re-infections with larval worms. Vaccine development and new molecular-based diagnosis techniques have promised to be a reliable approach to the diagnosis and prevention of schistosomiasis. The current review emphasizes the recent advancement in the diagnosis of schistosomiasis by molecular techniques and the treatment of schistosomiasis by combined and alternative regimes of drugs. Moreover, this review has also focused on the recent outbreak of schistosomiasis, the development of vaccines, and their clinical trials.

Systematic review and meta-analysis protocol: Diagnostic accuracy assessment for soil-transmitted helminth using microscopy and molecular-based methods

Soil-transmitted helminth (STH) is a part of NTDs that remains to be eliminated because of its significant global impact. Diagnostics are essential in aiding the elimination of STH infection. Therefore, it is imperative to evaluate the progress of diagnostic techniques, especially molecular-based techniques. Nevertheless, there is currently no comprehensive systematic review and meta-analysis (SRMA) evaluating the performance of molecular-based techniques for diagnosing STH. This protocol, registered on PROSPERO (CRD42024563432), intends to assess the diagnostic accuracy of molecular-based techniques compared to microscopic-based techniques for four common STH infections (A. lumbricoides, T. trichiura, hookworms, and S. stercoralis) across global studies. Two authors will independently conduct the screening, study selection, data extraction, and quality assessment of this SRMA. The obtained studies will be assessed using QUADAS-2 and will be analyzed for publication bias and diagnostic accuracy, including sensitivity, specificity, NPV, PPV, and DOR, and SROC. This developed protocol is expected to be versatile enough to be implemented in the range of studies assessing the diagnostic accuracy of the advance compared to the convention methods with adjustments. The findings of this SRMA are expected to provide a valuable contribution towards achieving the goal of eradicating STH infection by 2030.

A low-cost homogenizing device for in-field and remote DNA and RNA extraction

Environmental monitoring of microorganisms is critical for the protection and enhancement of human and ecosystem health. Even though these molecular methods have overtaken traditional culture-based methods and become more accessible, these techniques still require expensive equipment and dedicated facilities to process samples which in the context of a global pandemic, remote sampling areas or low-income countries can be extremely challenging. Sample preparation and sample homogenisation are critical steps for molecular-based techniques, especially for the extraction of DNA and RNA. This study developed a low-cost, open-source, freely available 3D printed homogenizer for the processing of DNA and RNA extraction. The BoSL Beater 3D is a portable device that allows researcher to perform bead-beating steps commonly required for environmental sample extraction protocols in the field and without access to main’s power. The BoSL Beater 3D was tested on filtered wastewater samples and passive samplers exposed to wastewater over a 24-hour period and showed similar or better performance to the traditional laboratory bead beater for both the extraction of DNA and RNA. The cost of this 3D homogeniser is roughly $18 AUD ($296 AUD with the jigsaw, which is roughly 57 times cheaper than a traditional bead beater) and has the added usability of being portable and easily adaptable to any type of jigsaw. In combination to newly developed field extraction kits as well as portable PCR machines, this 3D homogeniser could provide the tool necessary to enable access to molecular testing in remote setting as well as developing countries, which may not have access to fully equipped laboratories, but also allow for timely reporting. In addition, the BoSL Beater 3D, in combination with field extraction kit, can allow more flexibility to researchers while sampling, shipping, and processing DNA and RNA samples, whilst maintaining quality of these samples.

Detection of multidrug-resistance in Mycobacterium tuberculosis by phenotype- and molecular-based assays

BackgroundThe whole-genome sequencing (WGS) is becoming an increasingly effective tool for rapid and accurate detection of drug resistance in Mycobacterium tuberculosis complex (MTBC). This approach, however, has still been poorly evaluated on strains from Central and Eastern European countries. The purpose of this study was to assess the performance of WGS against conventional drug susceptibility testing (DST) for the detection of multi-drug resistant (MDR) phenotypes among MTBC clinical strains from Poland and Lithuania.MethodsThe study included 208 MTBC strains (130 MDR; 78 drug susceptible), recovered from as many tuberculosis patients in Lithuania and Poland between 2018 and 2021. Resistance to rifampicin (RIF) and isoniazid (INH) was assessed by Critical Concentration (CC) and Minimum Inhibitory Concentration (MIC) DST as well as molecular-based techniques, including line-probe assay (LPA) and WGS. The analysis of WGS results was performed using bioinformatic pipeline- and software-based tools.ResultsThe results obtained with the CC DST were more congruent with those by LPA compared to pipeline-based WGS. Software-based tools showed excellent concordance with pipeline-based analysis in prediction of RIF/INH resistance. The RIF-resistant strains demonstrated a relatively homogenous MIC distribution with the mode at the highest tested MIC value. The most frequent RIF-resistance conferring mutation was rpoB S450L. The mode MIC for INH was two-fold higher among double katG and inhA mutants than among single katG mutants. The overall rate of discordant results between all methods was calculated at 5.3%. Three strains had discordant results by both genotypic methods (LPA and pipeline-based WGS), one strain by LPA only, three strains by MIC DST, two strains by both MIC DST and pipeline-based WGS, and the remaining two strains showed discordant results with all three methods, compared to CC DST.ConclusionsConsidering MIC DST results, current CCs of the first-line anti-TB drugs might be inappropriately high and may need to be revised. Both molecular methods demonstrated 100% specificity, while pipeline-based WGS had slightly lower sensitivity for RIF and INH than LPA, compared to CC DST.

Evolution of dirofilariasis diagnostic techniques from traditional morphological analysis to molecular-based techniques: a comprehensive review

Dirofilariasis, caused by the nematode Dirofilaria spp., poses significant challenges in diagnosis due to its diverse clinical manifestations and complex life cycle. This comprehensive literature review focuses on the evolution of diagnostic methodologies, spanning from traditional morphological analyses to modern emerging techniques in the context of dirofilariasis diagnosis. The review traces the historical progression of diagnostic modalities, encompassing traditional approaches such as microscopic examination, serological tests (including ELISA and IFA), radiographic imaging, ultrasonography, and necropsy, which laid the foundation for subsequent advancements. The integration of molecular diagnostics marks a significant turning point in dirofilariasis diagnosis with the adoption of polymerase chain reaction (PCR) assays and real-time PCR (qPCR) facilitating enhanced sensitivity and specificity. Furthermore, recent strides in next-generation sequencing (NGS) technologies, including whole–genome sequencing (WGS), targeted sequencing (TS), metagenomic sequencing (MS), and RNA sequencing (transcriptome sequencing), have revolutionized the landscape of dirofilariasis diagnostics. Emerging techniques such as loop-mediated isothermal amplification (LAMP), digital PCR (dPCR), and digital microfluidics are also explored for their potential to augment diagnostic accuracy. The review addresses challenges associated with standardizing molecular protocols, tackling false positives/negatives, and discusses the advantages and limitations of each technique. By providing a comprehensive overview of dirofilariasis diagnostic strategies, from traditional to cutting-edge methods, this review aims to enhance understanding of the disease’s diagnostic landscape. The insights gained have implications for improved disease management and guide future research endeavors toward refining diagnostic protocols and advancing therapeutic interventions.

Rapid susceptibility of Carbapenem resistant Pseudomonas aeruginosa and its resistance gene to non-thermal plasma treatment in a batch reactor

The critically ranked carbapenem-resistant Pseudomonas aeruginosa has been observed to infect immunocompromised patients that consume polluted waters, leading to critical infections and more hospital costs. To save lives and unburden the public health sectors of preventable costs, non-thermal plasma (NTP) technology was investigated as an alternative disinfection step that could be applied in wastewater treatment plants (WWTPs) to inactivate this bacterium and its prominent carbapenem resistance gene (blaNDM-1). Culture and molecular-based techniques were employed to confirm carbapenem resistance in P. aeruginosa (27853). Culture suspensions of carbapenem-resistant ATCC P. aeruginosa (16 h culture) were prepared from confirmed isolates and subjected to plasma treatment at varying time intervals (3 min, 6 min, 9 min, 12 min and 15 min) in triplicates. The plasma treated samples were evaluated for re-growth and the presence of blaNDM-1. The treatment resulted in a 0.68 log reduction after 3 min and the highest log reduction of ≥8 after 12 min, suggesting that plasma disinfection has a great potential to be an efficient tertiary treatment step for WWTPs. Moreover, the gel image showed that band intensity of blaNDM-1 reduced with treatment time, thereby suggesting a probable reduction of amplified genes. Notwithstanding, longer treatment time, a grounded electrode with a larger surface (≥ 40 mm diameter) and/or oxygen-containing feeding gas is warranted to completely inactivate its antibiotic resistance gene (ARG), which might be bound by biofilms as they seem to protect P. aeruginosa from the action of non-thermal plasma (NTP) disinfection.

A review of urinary bladder microbiome in patients with bladder cancer and its implications in bladder pathogenesis.

The recent discovery of the urinary microbiome has led to an emerging field of investigation about the potential role of microorganisms in the pathogenesis of urinary bladder cancer. Few preliminary data have been reported so far implicating urobiome as causative and prognostic factor of bladder tumorigenesis. In the present study, a review of the current evidence is presented about microbiome composition among patients with bladder cancer and healthy individuals as well as possible implications of microbiome on urothelial carcinoma of the bladder. A literature review was conducted using PubMed/MEDLINE, Scopus, and the Cochrane library until December 2023. Search algorithm was constructed using the following terms and their associated Mesh terms and Boolean operators: "urinary microbiome" and "urinary microbiota". Studies written in English language, identifying, and comparing urinary microbiome among bladder cancer patients and healthy control group were included in the review. A total of 2,356 reports were identified. From this total 16 articles complied with the inclusion criteria were selected for analysis. These articles represent a total of about 486 bladder cancer patients. Recent studies revealed the colonization of the urinary tract and the bladder by micro-organisms using both enhanced culture- and molecular-based techniques for microbial characterization. However, several limitations exist in the literature decreasing the reliability of the current reports. As a result, urinary microbiome consist an ambitious era in bladder cancer research with an increasing number of evidence about its potential pathogenetic, prognostic and therapeutic role.

A Comprehensive Review of Innovative Paradigms in Microbial Detection and Antimicrobial Resistance: Beyond Traditional Cultural Methods.

Microbial detection and antimicrobial resistance (AMR) surveillance are critical components of public health efforts to combat infectious diseases and preserve the efficacy of antimicrobial agents. While foundational in microbial identification, traditional cultural methods are often laborious, time-consuming, and limited in their ability to detect AMR markers. In response to these challenges, innovative paradigms have emerged, leveraging advances in molecular biology, genomics, proteomics, nanotechnology, and bioinformatics. This comprehensive review provides an overview of innovative approaches beyond traditional cultural methods for microbial detection and AMR surveillance. Molecular-based techniques such as polymerase chain reaction (PCR) and next-generation sequencing (NGS) offer enhanced sensitivity and specificity, enabling the rapid identification of microbial pathogens and AMR determinants. Mass spectrometry-based methods provide rapid and accurate detection of microbial biomarkers, including matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and biosensor technologies. Nanotechnology approaches, such as nanoparticle-based assays and nanopore sequencing, offer novel platforms for sensitive and label-free detection of pathogens and AMR markers. Embracing these innovative paradigms holds immense promise for improving disease diagnosis, antibiotic stewardship, and AMR containment efforts. However, challenges such as cost, standardization, and integration with existing healthcare systems must be addressed to realize the full potential of these technologies. By fostering interdisciplinary collaboration and innovation, we can strengthen our ability to detect, monitor, and combat AMR, safeguarding public health for generations.

Pathogenic bacteria prevalence in cultured Nile tilapia in Southwest Mexico: A real-time PCR analysis.

The present study investigates molecular-based PCR techniques to estimate the prevalence of fish pathogens in southwest Mexico where recurrent mortality in the tilapia cultures has been observed. Sample of internal organs and lesions of Nile tilapia were taken and analysed in 2018, 2019, 2020 and 2022 to detect bacterial pathogens using PCR. No samples were taken in 2021 due to the COVID-19 pandemic. The real-time PCR conditions were optimized to allow a qualitative reliable detection of the bacteria from fixed fish tissue. A total of 599 pond- and cage-cultured tilapia from the southwestern Mexican Pacific (Guerrero, Oaxaca and Chiapas states) were analysed. In this tropical region, during 2018 and 2019 water temperatures of the tilapia cultures were generally with the optimal range to grow Nile tilapia, although extreme values were recorded on some farms. Most of the tilapia sampled were apparently healthy. No Francisella sp. was detected in any sample, and Staphylococcus sp. was the most prevalent (from 0% to 64%) bacteria from the three states over time. Low prevalence of Aeromonas sp. was found, from 0% to 4.3%, although the fish pathogen Aeromonas dhakensis was not detected. Sterptococcus iniae was only detected in Chiapas in 2019 at a low prevalence (1.4%), while the major tilapia pathogen S. agalactiae was detected at a high prevalence (from 0% to 59%) in the three Mexican states. This is the first detection of these pathogenic bacteria in rural farms using real-time PCR and constitutes a great risk for tilapia aquaculture in Mexico, as well as a potential dispersion of these pathogens to other aquaculture areas.

Microbial Detection and Quantification of Low-Biomass Water Samples Using an International Space Station Smart Sample Concentrator.

The pressing need to safeguard the health of astronauts aboard the International Space Station (ISS) necessitates constant and rigorous microbial monitoring. Recognizing the shortcomings of traditional culture-based methods, NASA is deliberating the incorporation of molecular-based techniques. The challenge, however, lies in developing and validating effective methods for concentrating samples to facilitate this transition. This study is dedicated to investigating the potential of an ISS Smart Sample Concentrator (iSSC) as an innovative concentration method. First, the iSSC system and its components were tested and optimized for microgravity, including various testing environments: a drop tower, parabolic flight, and the ISS itself. Upon confirming the system's compatibility with microgravity, we further evaluated its proficiency and reliability in concentrating large volumes (i.e., 1 L) of water samples inoculated with different microbes. The samples carried 102 to 105 colony-forming units (CFUs) of Sphingomonas paucimobilis, Ralstonia pickettii, or Cupriavidus basilensis per liter, aligning with NASA's acceptable limit of 5 × 104 CFU/L. The average retrieved volume post-concentration was ≈450 µL, yielding samples that were ≈2200 times more concentrated for subsequent quantitative PCR (qPCR) and CFU analysis. The average microbial percent recovery, as assessed with CFU counts, demonstrated consistency for C. basilensis and R. pickettii at around 50% and 45%, respectively. For S. paucimobilis, the efficiency oscillated between 40% and 80%. Interestingly, when we examined microbial recovery using qPCR, the results showed more variability across all tested species. The significance of these findings lies not merely in the successful validation of the iSSC but also in the system's proven consistency, as evidenced by its alignment with previous validation-phase results. In conclusion, conducted research underscored the potential of the iSSC in monitoring microbial contamination in potable water aboard the ISS, heralding a paradigm shift from culture-based to molecular-based monitoring methods.

Targeted sequencing analysis pipeline for species identification of human pathogenic fungi using long-read nanopore sequencing

Among molecular-based techniques for fungal identification, Sanger sequencing of the primary universal fungal DNA barcode, the internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), is commonly used in clinical routine laboratories due to its simplicity, universality, efficacy, and affordability for fungal species identification. However, Sanger sequencing fails to identify mixed ITS sequences in the case of mixed infections. To overcome this limitation, different high-throughput sequencing technologies have been explored. The nanopore-based technology is now one of the most promising long-read sequencing technologies on the market as it has the potential to sequence the full-length ITS region in a single read. In this study, we established a workflow for species identification using the sequences of the entire ITS region generated by nanopore sequencing of both pure yeast isolates and mocked mixed species reads generated with different scenarios. The species used in this study included Candida albicans (n = 2), Candida tropicalis (n = 1), Nakaseomyces glabratus (formerly Candida glabrata) (n = 1), Trichosporon asahii (n = 2), Pichia kudriavzevii (formerly Candida krusei) (n = 1), and Cryptococcus neoformans (n = 1). Comparing various methods to generate the consensus sequence for fungal species identification, the results from this study indicate that read clustering using a modified version of the NanoCLUST pipeline is more sensitive than Canu or VSEARCH, as it classified species accurately with a lower abundance cluster of reads (3% abundance compared to 10% with VSEARCH). The modified NanoCLUST also reduced the number of classified clusters compared to VSEARCH, making the subsequent BLAST+ analysis faster. Subsampling of the datasets, which reduces the size of the datasets by approximately tenfold, did not significantly affect the identification results in terms of the identified species name, percent identity, query coverage, percentage of reads in the classified cluster, and the number of clusters. The ability of the method to distinguish mixed species within sub-populations of large datasets has the potential to aid computer analysis by reducing the required processing power. The herein presented new sequence analysis pipeline will facilitate better interpretation of fungal sequence data for species identification.

Scope of Onsite, Portable Prevention Diagnostic Strategies for Alternaria Infections in Medicinal Plants.

Medicinal plants are constantly challenged by different biotic inconveniences, which not only cause yield and economic losses but also affect the quality of products derived from them. Among them, Alternaria pathogens are one of the harmful fungal pathogens in medicinal plants across the globe. Therefore, a fast and accurate detection method in the early stage is needed to avoid significant economic losses. Although traditional methods are available to detect Alternaria, they are more time-consuming and costly and need good expertise. Nevertheless, numerous biochemical- and molecular-based techniques are available for the detection of plant diseases, but their efficacy is constrained by differences in their accuracy, specificity, sensitivity, dependability, and speed in addition to being unsuitable for direct on-field studies. Considering the effect of Alternaria on medicinal plants, the development of novel and early detection measures is required to detect causal Alternaria species accurately, sensitively, and rapidly that can be further applied in fields to speed up the advancement process in detection strategies. In this regard, nanotechnology can be employed to develop portable biosensors suitable for early and correct pathogenic disease detection on the field. It also provides an efficient future scope to convert innovative nanoparticle-derived fabricated biomolecules and biosensor approaches in the diagnostics of disease-causing pathogens in important medicinal plants. In this review, we summarize the traditional methods, including immunological and molecular methods, utilized in plant-disease diagnostics. We also brief advanced automobile and efficient sensing technologies for diagnostics. Here we are proposing an idea with a focus on the development of electrochemical and/or colorimetric properties-based nano-biosensors that could be useful in the early detection of Alternaria and other plant pathogens in important medicinal plants. In addition, we discuss challenges faced during the fabrication of biosensors and new capabilities of the technology that provide information regarding disease management strategies.

Determination of Genetic variation among Beauveria bassiana and Metarhizium anisopliae Isolates from Palestine and other Geographic regions using ITS and β-tubulin Sequences, RFLP and ISSR Data

In this work we report the genetic variation of ten isolates of entomopathogenic fungi, four of which were isolated from Palestine while the remaining six were from other geographic regions. The variation was determined using molecular-based techniques including; sequence analysis of β-tubulin (BT) gene, the Internal Transcribed Spacers (ITS) 1 & 2 of the rDNA, Restriction Fragment Length Polymorphism (RFLP), and Inter-Simple Sequence Repeat (ISSR) analysis. The sequencing data of ITS regions confirmed the taxonomic identity of six isolates as Metarhizium anisopliae while the identity of the other four as Beauveria bassiana. These data also revealed that the two Palestinian Beauveria b. isolates are probably new strains since theyshowed 99% homology with the best match sequences reported in the gene bank. The restriction digestion of the ITS2 sequences produced a fingerprint for the PAL-M01 isolate. The ITS and BT sequences were able to distinguish the B-Bot genotype from the other Beauveria b. isolates. In contrast, the ITS could discriminate the three Metarhizium isolates: Ma-7, PAL-M01, and PPRC. Moreover, the ISSR analysis detected high levels of polymorphism among the Beauveria b. (80%) and the Metarhizium a. (90%) isolates. The fact that the PPRC and B-Bot isolates areobviously genetically different from their Metarhizium a. and Beauveria b. counterparts could be attributed to their distant geographic origins. The molecular data obtained in this work can be utilized for specific identification of each ofthese isolates which, would facilitate the local use of these fungi as biocontrol agents. To the best of our knowledge, this is the first study, in Palestine, attempting to discriminate between EPF isolates using advanced molecular techniques.

Liver Fluke Species Identification Isolated From Humans and Animals Using PCR-RFLP and DNA Sequencing

Fasciola species are a member of flatworms belonging to the trematodes (flukes), commonly known as liver fluke, they are extremely pathogenic parasites that affect the liver of humans and animals, nowadays, most laboratories and research facilities use molecular-based techniques for identifying and describing Fasciola species. The molecular diagnostic markers such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and PCR-RFLP methods are accurate and more specific than the immunological and microscopical methods. The identification of the species of liver flukes will give a new clue for the treatment and control of fascioliasis. The aim, of this study, is to find the molecular characterization of Fasciola spp. isolated from humans and animals in Sulaimani city. The flukes were isolated from humans using endoscopic techniques and from slaughtered livestock at the new slaughterhouse of Sulaimani, 48 liver flukes were collected from different hosts; human (n = 3), cattle (n = 20), sheep (n = 20), and goats (n = 5) from October 2021 to April 2022. The uinversal primers ribosomal Deoxy Ribo Nuclic Acid (rDNA) were used, then the PCR products were subjected to restriction fragment polymorphism (RFLP) assay and The PCR Product was digested with restriction enzymes DraII, also the DNA sequencing was used for the PCR product of the primer Cytochrome Oxidase subuint 1 (COX1). The results of the PCR-RFLP of the 28s rDNA show the genetic polymorphisms among the flukes and two patterns of RFLP were observed F. hepatica, and F. gigantica, also the sequence analysis of the partial gene of the COX1 showed the isolated flukes belonged to F. hepatica and F. gigantica with some genetic variation, and the result of the sequences was deposited in the Gene Bank under the following Accession numbers; F. gigantica (OP718780 and OP718781) and F. hepatica (OP718782, OP718783, and OP718784). The present study concludes thatF. hepatica and F. gigantica are both responsible for human and animal Fasciolasis in Kurdistan-Iraq, Therefore, RFLP techniques and DNA sequencing are a reliable, and differential method for species and genotyping identification of liver fluke.

Newcastle disease in pigeon review article

Newcastle disease (ND) is a viral disease of poultry and other bird species like pigeon (Columbi livia), it characterizes as devastating and contagious and it has been distributed worldwide. ND in pigeons is caused by pigeon paramyxovirus type 1(PPMV-1) which is an antigenic and host variant of the avian paramyxovirus serotype 1 (APMV-1), the disease in pigeons is termed paramyxovirosis and cause nervous signs with high mortality usually in the bird with kidneys infected with viscerotropic strains, and neural signs appeared individually. PPMV-1 was initially detected by hemagglutination activity (HA) and confirmed with hemagglutination inhibition assay (HI), and molecular-based techniques. Serological diagnosis of paramyxovirosis is necessary because of the similarity of the PPMV-1 infection with sodium chloride poisoning, pigeon herpes virus infection (PHV), and ornidazole overdose that cause same neurological signs. PPMV-1 infection can be controlled effectively by vaccination by specific vaccines associated with good biosecurity aspect

Detecting Embryo Developmental Potential by Single Blastomere RNA-Seq.

Recent advances in preimplantation embryo diagnostics enable a wide range of applications using single cell biopsy and molecular-based selection techniques without compromising embryo production. This study was conducted to develop a single cell embryo biopsy technique and gene expression analysis method with a very low input volume to ensure normal embryo development and to see if there are differences in gene expression profiles between day-5 biopsied bovine embryos that developed into blastocysts and embryos arrested at morula stage. Out of the 65 biopsied morulae, 32 developed to blastocysts (49.2%). Out of the 13,580 successfully annotated genes, 1204 showed a difference in mRNA expression level. Out of these, 155 genes were expressed in embryos developing to blastocysts. The pathway enrichment analysis revealed significant enrichment in "organelle biogenesis and maintenance", "mRNA splicing" and "mitochondrial translation" pathways. These findings suggest principal differences in gene expression patterns and functional networks of embryos able to reach the blastocyst stage compared to embryos arrested in development. Our preliminary data suggest that single blastomere biopsy and selected gene expression profiles at morula stage could offer additional possibilities for early preimplantation embryo selection before transfer.

Development and Maturation of Microbiota in Cow Rumen, Plant-Fibers Degradation and Influences on the Immune System and Cow Health

Rumen is part of the forestomach of ruminants and plays a key role in the conversion of feed into metabolites that are absorbed and used by the host. The rumen is also the place of formation of proteins of microbial origin, which represent a source of energy for the host animal. From a functional point of view, ruminants are monogastric at birth as they have an undeveloped forestomach system. Microbial communities in the rumen first show colonization by bacteria, followed by that of methanogenic Archaea and then anaerobic fungi and protozoa. In newborn calves, molecular-based techniques evidenced initial rumen colonisation by facultative anaerobic bacteria, as the phyla Proteobacteria and Firmicutes, with genera Enterococcus and Streptococcus and the species Escherichia coli, followed by Archaea within a few hours after birth. These early colonizers utilize the oxygen available in the rumen, thus creating an anaerobic environment conducive to the growth of rigorous anaerobic communities, including Bifidobacterium and Bacteroides. The strict anaerobic bacterial community, including cellulolytic and proteolytic bacteria, establishes and dominates the rumen microbiome within the first two weeks of life. The entire microbial community allows ruminants to use ligno-cellulosic materials and non-protein nitrogen to produce high-quality food. Importantly, these close anaerobic bacterial communities in the rumen of newborns play an essential role in the development of the mucosal immune system. A healthy rumen leads to healthy ruminants with optimal performance. It is worth highlighting the importance of the microbiome in maintaining the health of cattle and its potential in alleviating disease. This mini-review described the development of the cow microbiome in the rumen, the degradation abilities and influence of the feed on the rumen microbiota, and the microbiota effects on the cow’s immune system and health.

Internal transcribed spacer 2 (ITS2) based molecular identification of malaria vectors from Bangsring Banyuwangi-Indonesia

Since the malaria outbreak in 2011, the breeding place of Anopheles in Bangsring Village on Banyuwangi District has been monitored by District Public Health Office as part of a vector surveillance program. Morphological identification is still a standard tool to observe Anopheles occurrence and diversity, but the presence of cryptic species made it unreliable. In this study, a molecular approach called DNA barcoding technique was used to assist the morphology-based techniques to identify Anopheles species found in Bangsring. The internal transcribed spacer 2 (ITS2) sequence was used as molecular marker. Based on the morphological features, we were able to identify Anopheles (An.) vagus, An. subpictus, An. sundaicus and An. aconitus. ITS2 sequences from the four identified species were then analyzed simultaneously with eighteen reference sequences from NCBI which had a high similarity of 98-100%. The NJ phylogenetic tree formed three major clades, where the two clades as monophyletic clades were An. vagus and An. aconitus. Another clade was formed as polyphyletic clade containing An. subpictus and An. sundaicus. Although An. subpictus and An. sundaicus were placed in the same clade, seven nucleotide differences were observed in their ITS2 sequence. The intra-specific variation of those two species was 0.08 and 0.49%, respectively, while the interspecific variation was 1.39%. Interspecific variation which was higher than the mean intra-specific variation might indicate that An. sundaicus and An. subpictus were a distantly species. However, the value of interspecific variation lower than 3% might also indicate that those species were classified as a complex species. All ITS2 sequences from morphologically identified species had similar results with molecular-based techniques. This result showed that molecular identification using the ITS2 sequence was reliable in supporting morphological identification among closely related anopheline mosquitoes and gave further information about their evolutionary divergence.

The Simpson Grading: Is It Still Valid?

Simple SummarySince the initial publication of the Simpson Grade scale, the management paradigm for intracranial meningiomas has significantly evolved. The generalized application of the Simpson Grade in modern neurosurgery management of meningiomas is controversial. We conducted a review of the literature to determine the prognostic significance of the Simpson Grade and find it to be an antiquated grading scale with limited utility in the modern era. The Simpson Grade was introduced in the era of limited resources, outdated techniques, and rudimentary surgical and imaging technologies. With the advent of modern techniques including pre- and post-operative imaging, microsurgical and endoscopic techniques, advanced histopathology and molecular analysis and adjuvant radiotherapy, the utility of the Simpson Grade scale for prognostication of recurrence after meningioma resection has become less useful. While the extent of resection remains an important factor in reducing recurrence, a subjective naked-eye criteria to Grade extent of resection cannot be generalized to all meningiomas regardless of their location or biology. Achieving the highest Simpson Grade resection should not always be the goal of surgery. It is prudent to take advantage of all the tools in the neurosurgeons’ armamentarium to aim for maximal safe resection of meningiomas. The primary goal of this study was to review the literature highlighting the Simpson Grade and its association with recurrence in modern meningioma practice. A PubMed search was conducted using terms “Simpson”, “Grade”, “meningioma”, “recurrence”, “gross total resection”, “extent of resection” “human”. A separate search using the terms “intraoperative imaging”, “intraoperative MRI” and “meningioma” were conducted. All studies reporting prognostic value of Simpson Grades were retrospective in nature. Simpson Grade I, II and III can be defined as gross total resection and were associated with lower recurrence compared to Simpson Grade IV or subtotal resection. The volume of residual tumor, a factor not considered in the Simpson Grade, is also a useful predictor of recurrence. Subtotal resection followed by stereotactic radiosurgery has similar recurrence-free survival as gross total resection. In current modern meningioma surgery, the Simpson Grade is no longer relevant and should be replaced with a grading scale that relies on post-operative MRI imaging that assess GTR versus STR and then divides STR into > or <4–5 cm3, in combination with modern molecular-based techniques for recurrence risk stratification.