Molecular characterization of Aeromonas hydrophila isolates from diseased fishes in district Kasur, Punjab, Pakistan

Pakistan is an agricultural country and fisheries play a very important role in the economic development of the country. Different diseases are prevalent in Pakistani fish but information related to the causative agents is not well-known. Keeping in view the significance of bacterial pathogens as the causative agents of multiple fish diseases, the present study was conducted for identification, characterization and analysis of virulence genes of Aeromonas spp. isolated from diseased fishes. A total of fifty fish samples having multiple clinical indications were collected from different fish farms of district Kasur, Punjab Pakistan. For isolation of Aeromonas spp. samples were enriched and inoculated on Aeromonas isolation medium. Isolates were identified and characterized by different biochemical tests, Analytical Profile Index (API) 20E kit and Polymerase Chain Reaction (PCR) assays. All isolates were screened for three putative virulence genes including aerolysin (aer), haemolysin (hyl) and heat labile cytotonic enterotoxin (alt). Seven isolates of Aeromonas (A.) hydrophila were retrieved and identified based on API 20E. These isolates were further confirmed as A. hydrophila on the basis of PCR assays. Three isolates were detected positive for the presence of virulence genes (alt and hyl). Whereas aerolysin (aer) gene was not present in any of A. hydrophila isolates. The present study confirmed A. hydrophila as the causative agent of epizootic ulcerative syndrome and motile Aeromonas septicemia in fish farms of district Kasur, Punjab Pakistan. Moreover, detection of two virulence genes (alt and hyl) in A. hydrophila isolates is a threat for fish consumers of study area.

SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection

Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples.

Autochthonous Transmission of Angiostrongylus Cantonensis in a Domestic Rabbit (Oryctolagus Cuniculus) in Hawai`i: Detection in Multiple Tissues Using AcanITS1 and AcanR3990 PCR Assays

Background: Hawaii is the hotspot for rat lungworm disease (angiostrongyliasis) caused by the nematode Angiostrongylus cantonensis in the USA. In humans, PCR of the CSF is typically used for diagnosis, however, collection of CSF requires hospitalization. Here, we evaluate the efficacy of two different PCR tests to detect A.cantonensis DNA in multiple tissues including blood from a rabbit presumably infected by eating contaminated lettuce. Methods: Two different PCR assays (AcanR3990, and AcanITS1) were used comparatively to test DNA extracted from slug and rabbit tissues. Assays were conducted using established protocols and were run in triplicate, with negative (dH20) controls included throughout. Results: A juvenile Parmarian martensi (semi-slug) found in local lettuce tested positive for the presence of Angiostrongylus cantonensis DNA. A family and their two domestic rabbits (Oryctolagus cuniculus) consumed this lettuce twice within the five days preceding testing. One rabbit exhibited symptoms consistent with eosinophilic meningitis 3-6 days after being fed the lettuce. Appropriate veterinary treatment was ineffective and the rabbit was subsequently euthanized. This study comparatively applies two different PCR assays to detect A. cantonensis DNA in the peripheral blood, cerebrospinal fluid, brain, heart, and lung tissue of this rabbit, and provides data implicating parasite transmission via contaminated home-grown lettuce. Six of the nine brain DNA samples, as well as the CSF sample, tested positive in replicate or triplicate for A. cantonensis DNA with both PCR assays. The AcanR3990 assay also detected A. cantonensis DNA from the lung, heart septum, all nine samples from the brain, and blood products (plasma, EDTA-treated whole blood, and buffy coat/red blood cells) in replicate or triplicate.

Genetic Diversity of Porcine Reproductive and Respiratory Syndrome Virus and Evaluation of Three One-Step Real-Time RT-PCR Assays in Korea

Background Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV. Methods In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Results A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated. Conclusion The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

NATIONAL SCALE REAL-TIME SURVEILLANCE OF SARS-COV-2 VARIANTS DYNAMICS BY WASTEWATER MONITORING IN ISRAEL

In this report, we describe a national-scale monitoring of the SARS-COV-2 (SC-2) variant dynamics in Israel, using multiple-time sampling of twelve wastewater treatment plants. We used a combination of inclusive and selective quantitative PCR assays that specifically identify variants A19 or B.1.1.7 and tested each sample for the presence and relative viral RNA load of each variant. We show that between December-2020 and March-2021, a complete shift in the SC-2 variant circulation was observed, where the B.1.1.7 replaced the A19 in all examined test points. We further show that the normalized viral load (NVL) values and the average new cases per week reached a peak in January 2021, and then decreased gradually in almost all test points, in parallel with the progression of the national vaccination campaign, during February-March 2021. This study demonstrates the importance of monitoring SC-2 variant dynamics on a national scale through wastewater sampling. It also provides a proof-of-concept methodology for continuous surveillance by using a combination of inclusive and selective PCR tests, which is far more amendable for high throughput monitoring compared with sequencing. This approach may be useful for real-time dynamics surveillance of current and future variants, such as the Omicron (BA.1) variant.

Surveillance and correlation of SARS-CoV-2 viral RNA, antigen, virus isolation, and self-reported symptoms in a longitudinal study with daily sampling

The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of FDA emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, i.e., the presence of replicable virus. As the number of tests conducted increased, persistent SARS-CoV-2 RNA positivity by RT-PCR in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR (qRT-PCR) assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike (S), nucleocapsid (N), membrane (M), envelope (E), and ORF8. The absolute copy number of each RNA target was determined in longitudinal specimens from a household transmission study. Calculated viral RNA levels over the 14-day follow up period were compared with antigen testing and self-reported symptoms to characterize the clinical and molecular dynamics of infection and infer predictive values of these qRT-PCR assays relative to culture isolation. When detection of sgS RNA was added to the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel, we found a qRT-PCR positive result was 98% predictive of a positive culture (negative predictive value was 94%). Our findings suggest sgRNA presence correlates with active infection, may help identify individuals shedding culturable virus, and that similar multiplex assays can be adapted to current and future variants.

Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™

Probiotic health benefits are now well-recognized to be strain specific. Probiotic strain characterization and identification is thus important in clinical research and in the probiotic industry. This is becoming especially important with reports of probiotic products failing to meet the declared strain content, potentially compromising their efficacy. Availability of reliable identification methods is essential for strain authentication during discovery, evaluation and commercialization of a probiotic strain. This study aims to develop identification methods for strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07 (Bi-07™) based on real-time PCR, targeting single nucleotide polymorphisms (SNPs). The SNPs were targeted by PCR assays with locked nucleic acid (LNA) probes, which is a novel application in probiotic identification. The assays were then validated following the guidelines for validating qualitative real-time PCR assays. Each assay was evaluated for specificity against 22 non-target strains including closely related Bifidobacterium animalis subsp. lactis strains and were found to achieve 100% true positive and 0% false positive rates. To determine reaction sensitivity and efficiency, three standard curves were established for each strain. Reaction efficiency values were 86, 91, and 90% (R square values > 0.99), and 87, 84, and 86% (R square values > 0.98) for B. animalis subsp. lactis DSM 15954 and Bi-07 assays, respectively. The limit of detection (LOD) was 5.0 picograms and 0.5 picograms of DNA for DSM 15954 and Bi-07 assays, respectively. Each assay was evaluated for accuracy using five samples tested at three different DNA concentrations and both assays proved to be highly repeatable and reproducible. Standard deviation of Cq values between two replicates was always below 1.38 and below 1.68 for DSM 15954 and Bi-07 assays, respectively. The assays proved to be applicable to mono-strain and multi-strain samples as well as for samples in various matrices of foods or dietary supplement ingredients. Overall, the methods demonstrated high specificity, sensitivity, efficiency and precision and broad applicability to sample, matrix and machine types. These methods facilitate strain level identification of the highly monophyletic strains B. animalis subsp. lactis DSM 15954 and Bi-07 to ensure probiotic efficacy and provide a strategy to identify other closely related probiotics organisms.