Polymerase Chain Reaction Amplicons Research Articles

Establishment of dual reverse transcriptase-polymerase chain reaction for detection system for Areca palm velarivirus 1.

Areca palm velarivirus 1 (APV1) is one of the main pathogen causing yellow leaf disease, and leading to considerable losses in the Areca palm industry. The detection methods for APV1 are primarily based on phenotype determination and molecular techniques, such as polymerase chain reaction (PCR). However, a single PCR has limitations in accuracy and sensitivity. Therefore, in the present study, we established a dual RT-PCR APV1-detection system with enhanced accuracy and sensitivity using two pairs of specific primers, YLDV2-F/YLDV2-R and YLDV4-F/YLDV4-R. Moreover, two cDNA fragments covering different regions of the viral genome were simultaneously amplified, with PCR amplicon of 311 and 499 bp, respectively. The dual RT-PCR detection system successfully amplified the two target regions of the APV1, demonstrating high specificity and sensitivity and compensating for the limitations of single-primer detection methods. We tested 60 Areca palm samples from different geographical regions, highlighting its advantages in that the dual RT-PCR system efficiently and accurately detected APV1 in samples across diverse areas. The dual RT-PCR APV1 detection system provides a rapid, accurate, and sensitive method for detecting the virus and offers valuable technical support for research in preventing and managing yellow leaf diseases caused by APV1 in Areca palms. Moreover, the findings of this study can serve as a reference for establishing similar plants viral detection systems in the future.

Discovery of Terminal Oxazole‐Bearing Natural Products by a Targeted Metabologenomic Approach

AbstractA targeted metabologenomic method was developed to selectively discover terminal oxazole‐bearing natural products from bacteria. For this, genes encoding oxazole cyclase, a key enzyme in terminal oxazole biosynthesis, were chosen as the genomic signature to screen bacterial strains that may produce oxazole‐bearing compounds. Sixteen strains were identified from the screening of a bacterial DNA library (1,000 strains) using oxazole cyclase gene‐targeting polymerase chain reaction (PCR) primers. The PCR amplicon sequences were subjected to phylogenetic analysis and classified into nine clades. 1H−13C coupled‐HSQC NMR spectra obtained from the culture extracts of the hit strains enabled the unequivocal detection of the target compounds, including five new oxazole compounds, based on the unique 1JCH values and chemical shifts of oxazole: lenzioxazole (1) possessing an unprecedented cyclopentane, permafroxazole (2) bearing a tetraene conjugated with carboxylic acid, tenebriazine (3) incorporating two modified amino acids, and methyl‐oxazolomycins A and B (4 and 5). Tenebriazine displayed inhibitory activity against pathogenic fungi, whereas methyl‐oxazolomycins A and B (4 and 5) selectively showed anti‐proliferative activity against estrogen receptor‐positive breast cancer cells. This metabologenomic method enables the logical and efficient discovery of new microbial natural products with a target structural motif without the need for isotopic labeling.

Discovery of Terminal Oxazole-Bearing Natural Products by a Targeted Metabologenomic Approach.

A targeted metabologenomic method was developed to selectively discover terminal oxazole-bearing natural products from bacteria. For this, genes encoding oxazole cyclase, a key enzyme in terminal oxazole biosynthesis, were chosen as the genomic signature to screen bacterial strains that may produce oxazole-bearing compounds. Sixteen strains were identified from the screening of a bacterial DNA library (1,000 strains) using oxazole cyclase gene-targeting polymerase chain reaction (PCR) primers. The PCR amplicon sequences were subjected to phylogenetic analysis and classified into nine clades. 1H-13C coupled-HSQC NMR spectra obtained from the culture extracts of the hit strains enabled the unequivocal detection of the target compounds, including five new oxazole compounds, based on the unique 1JCH values and chemical shifts of oxazole: lenzioxazole (1) possessing an unprecedented cyclopentane, permafroxazole (2) bearing a tetraene conjugated with carboxylic acid, tenebriazine (3) incorporating two modified amino acids, and methyl-oxazolomycins A and B (4 and 5). Tenebriazine displayed inhibitory activity against pathogenic fungi, whereas methyl-oxazolomycins A and B (4 and 5) selectively showed anti-proliferative activity against estrogen receptor-positive breast cancer cells. This metabologenomic method enables the logical and efficient discovery of new microbial natural products with a target structural motif without the need for isotopic labeling.

Exploring the Correlation between Interleukin-17A Promoter Polymorphism at its -197 G/A and Rheumatoid Arthritis: Impact on Disease Severity and Activity

T helper 17 (Th17) cells have been reported to be the most powerful factor in autoimmune disorder pathogenesis, which points to the Th17 master cytokine, interleukin (IL)-17A, as the crucial mediator. We aimed to determine the impact of IL-17A polymorphism in the -197 G/A promoter region on level of IL-17 and intensity of rheumatoid arthritis (RA) disease symptoms. This case-control study was conducted at the Department of Clinical Rheumatology of Aswan university Hospital and included 35 people suffering RA and 30 volunteer controls, matched for age and sex. Rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies, erythrocyte sedimentation rate (ESR), serum IL-17, and C-reactive protein (CRP) were measured in the RA patient group. Restriction fragment length polymorphism (RFLP) was conducted by polymerase chain reaction (PCR) amplicon obtained by IL-17A -197 G /A primers. Of the 35 RA patients, RF was positive in 33 (94.29%) and anti-CCP antibodies in 25 (71.43%), CRP in 31 (88.57%). Of the 35 RA patients, 5 (14.29%) patients carried the G/G genotype, 18 (51.43%) G/A and 12 (34.29%) A/A. IL-17 serum level was significantly greater in the more active RA (DAS28 >5.1) group than the less active (DAS28 ≤5.1) group. Of the RA patients carrying wild type G/G genotype, 60% had more active disease (DAS 28> 5.1), as compared to those with lower activity (DAS 28 ≤5.1), 40% carried the wild type G/G genotype. In conclusion, the study findings imply that IL-17A gene polymorphism is connected to RA clinical severity rather than with RA susceptibility.

Natural occurrence of tomato chlorosis virus on tomatillo (Physalis philadelphica) in the United States.

Tomatillo (Physalis philadelphica L.) is an annual plant native to Mexico and Guatemala, and cultivated in other tropical and subtropical regions. In October 2023, tomatillo plants with interveinal yellowing of leaves, marginal chlorosis, leaf thickening, and leaf rolling symptoms (Figure 1) were observed at Colquitt and Tift County, Georgia, US. The disease incidence ranged from 80-100 % which reduced fruit quality and marketability. Twenty tomatillo leaves exhibiting severe symptoms were collected, and, sub-sampled of the leaves were pooled into microcentrifuge tubes. Further, MagMAX 96 viral RNA isolation kit (Thermo Fisher Scientific, US), was used for the extraction of (n=4) total nucleic acid (TNA) (Kavalappara et al. 2021). Symptomatic leaves were tested for the presence of insect-transmitted viruses such as begomovirus (tomato yellow leaf curl virus, TYLCV), potyvirus (turnip mosaic virus, TuMV), crinivirus (tomato infectious chlorosis virus, TICV; tomato chlorosis virus, ToCV), and tospovirus (orthotospovirus tomatomaculae, TSWV). Polymerase chain reaction (PCR) was performed for detecting TYLCV, using gene-specific primers (Kumar et al., 2023). However, for ToCV, TuMV and TICV detection, cDNA was prepared using 100 ng of TNA as a template, followed by the PCR ( Liu et al., 2012). Moreover, the detection of TSWV was conducted using immuno-strips (Adgia, US) following the manufacturer's instructions. ToCV was detected from all the tested samples, while TuMV, TICV, TYLCV and TSWV were not detected in any symptomatic tissues. In addition, RT-PCR was performed using gene-specific primers targeting the RNA-dependent RNA polymerase (RdRP) gene and the heat-shock protein 70 (Hsp70) gene of ToCV. The PCR amplicon of 439 bp encoding Hsp70 and 643 bp corresponding to RdRP was gel-purified and Sanger sequenced (Azenta Life Sciences, US). BLASTn analysis shows RdRP gene from ToCV-tomatillo (OR905600) has 100 % identity with ToCV of RNA1 segment (RdRP, GenBank accession no. AY903447, Florida, US), while Hsp70 gene (OR900219) has 100 % identity with ToCV of RNA2 segment (Hsp70, GenBank accession no. LC778246, Cairo, Egypt). In addition, the symptomatic tomatillo leaves were studied for transmission assay using tomato, employing non-viruliferous whiteflies (Bemisia tabaci) with 48 h of acquisition access period. Further, two weeks post-infection, the presence of ToCV was detected from the test plants while other whitefly-transmitted viruses remins undetected. In 2023, ToCV is widespread in tomato-growing counties, infecting commercially grown tomato cultivars with intermediate resistance against TYLCV-IL (Israel strain). However, tomatillo plants infected with TuMV in California (Liu et al., 2012), TSWV in Georgia, (Díaz-Pérez and Pappu 2000) and TYLCV in Mexico (Gámez-Jiménez et al. 2009) were reported. This study suggests that tomatillo could be a permissive host for ToCV while restrictive to other prevalent viruses in the region. A recent investigation speculates a potential synergistic interaction between ToCV and TYLCV-IL, exacerbating the breakdown of host resistance in tomato (Fiallo-Olivé et al. 2019, Kumar et al. 2023). To the best of our knowledge, this is the first report for the natural incidence of ToCV on tomatillo within the US. The findings will contribute to developing more effective management strategies against emerging viral threats.

Selective visual detection of multiplex PCR amplicon using magnetic microbeads

Nucleic acid amplification tests (NATs), such as genetic tests using polymerase chain reaction (PCR), are sensitive methods for detecting pathogens and food contamination. The rapid, easy, and inexpensive detection of amplicons, DNA, or RNA is key to realizing on-site NATs. We have previously developed a novel amplicon detection method using magnetic microbeads based on the hydrophobicity of DNA in deionized water. In this study, we aimed to expand the method for the detection of multiplex DNA amplicons. Tagged primers and probes for selective attachment were used to detect amplicons from two strawberry pathogens. The amplicon-labeled magnetic microbeads were placed in the round-bottom well of a hydrophilic glass substrate. The attachment of amplicons to the magnetic microbeads changed their surface from hydrophilic to hydrophobic. The magnetized microbeads concentrated at the bottom when the substrate was placed on a permanent magnet, and the concentrated microbeads were easily recognizable by the naked eye. Microbeads without amplicons were adsorbed over a broad area of the bottom of the glass well owing to their hydrophilicity. The appropriate tag probe was attached to specific amplicons for detection, and each amplicon from multiplex PCR was selectively detected within approximately 15 min. Notably, this method requires no electric power and contributes to the realization of on-site NAT detection. This study presents a simple and rapid method for the selective detection of multiplex PCR amplicons using DNA–DNA hybridization.

Efficient detection of single nucleotide variants in targeted genomic loci.

Single nucleotide variants (SNVs), including single nucleotide polymorphisms, are often associated with morphological and/or physiological abnormalities in various organisms. Targeted genomic DNA can be amplified and directly sequenced to detect these mutations, but this method is relatively time consuming and expensive. We recently established the heteroduplex mobility assay to detect genetic mutations as an easy, low-cost method in genome editing, but detecting such small genetic differences remains difficult. Here, we developed a new, simple method to detect single nucleotide changes in the zebrafish genome by polymerase chain reaction (PCR) and electrophoresis. We first designed a specific single stranded DNA with four tandem guanine nucleotides inserted beside the mutation site, called guanine-inserted primer (GIP). When reannealing, hybridized complexes of GIP and PCR amplicons with or without 1-bp-mutated alleles form different bulge structures, presumably leading to different mobilities on a polyacrylamide gel. This GIP-interacting mobility assay is easy to use; therefore, it could contribute to the detection of SNVs in any organism.

Identification and Functional Assessment of Eight CYP3A4 Allelic Variants *39-*46 Detected in the Chinese Han Population.

Cytochrome P450 3A4 (CYP3A4), a key enzyme, is pivotal in metabolizing approximately half of the drugs used clinically. The genetic polymorphism of the CYP3A4 gene significantly influences individual variations in drug metabolism, potentially leading to severe adverse drug reactions (ADRs). In this study, we conducted a genetic analysis on CYP3A4 gene in 1163 Chinese Han individuals to identify the genetic variations that might affect their drug metabolism capabilities. For this purpose, a multiplex polymerase chain reaction (PCR) amplicon sequencing technique was developed, enabling us to perform the genotyping of CYP3A4 gene efficiently and economically on a large scale. As a result, a total of 14 CYP3A4 allelic variants were identified, comprising six previously reported alleles and eight new nonsynonymous variants that were nominated as new allelic variants *39-*46 by the PharmVar Association. Further, functional assessments of these novel CYP3A4 variants were undertaken by coexpressing them with cytochromes P450 oxidoreductase (CYPOR) in Saccharomyces cerevisiae microsomes. Immunoblot analysis indicated that with the exception of CYP3A4.40 and CYP3A4.45, the protein expression levels of most new variants were similar to that of the wild-type CYP3A4.1 in yeast cells. To evaluate their catalytic activities, midazolam was used as a probe drug. The results showed that variant CYP3A4.45 had almost no catalytic activity, whereas the other variants exhibited significantly reduced drug metabolism abilities. This suggests that the majority of the CYP3A4 variants identified in the Chinese population possess markedly altered capacities for drug metabolism. SIGNIFICANCE STATEMENT: In this study, we established a multiplex polymerase chain reaction (PCR) amplicon sequencing method and detected the maximum number of new CYP3A4 variants in a single ethnic population. Additionally, we performed the functional characterizations of these eight novel CYP3A4 allele variants in vitro. This study not only contributes to the understanding of CYP3A4 genetic polymorphism in the Chinese Han population but also holds substantial reference value for their potential clinical applications in personalized medicine.

Genotyping of Echinococcus granulosus Human Isolates Obtained from the Puncture, Aspiration, Injection, and Re-Aspiration (PAIR) Process in Türkiye.

Echinococcus granulosus is spread by the excretion of cystic organs into the environment. The dog is infected via eating the cystic organ. It then contaminates the environment with eggs of E. granulosus, which are infective to humans and animals. We aimed to determine the E. granulosus genotypes that cause infection in humans in the Van region, Türkiye. Sixty patients between 18 and 100 years of age, who underwent the puncture, aspiration, injection, re-aspiration (PAIR) procedure in the Department of Radiodiognastics of Van Yüzüncü Yıl University, Van, Türkiye were included in the study. PAIR fluids were examined microscopically and DNA was isolated from the fluids. After DNA isolation, polymerase chain reaction (PCR) was performed using primers that amplify the E. granulosus NADH dehydrogenase subunit 1 (NAD1) gene region. After sequence analysis of the PCR amplicons, Basic Local Alignment Search Tool (BLAST) was performed. In the microscopic examination, protoscolex or hook was detected in 42 (70%) of the samples. DNA was successfully extracted from all of the cyst fluids containing protoscolex and hook, and the NAD1 gene region was PCR-amplified. After using BLAST, all of the samples were determined to be an E. granulosus sensu stricto G1 genotype. Sequence comparison revealed that four (9.5%) isolate sequences showed single nucleotide polymorphism (SNP). Sequences of isolates with SNP submitted to the GenBank with accession numbers OR565864 to OR565867. E. granulosus s.s. G1 genotype, known as sheep strain, is common in human hydatid disease in the Van region of Türkiye.

Tracking the contamination sources of microbial population and characterizing Listeria monocytogenes in a chicken slaughterhouse by using culture-dependent and -independent methods

Listeria monocytogenes is the etiologic agent of listeriosis, a foodborne disease that poses a threat to public health globally. Chicken meat exhibits heightened susceptibility to L. monocytogenes contamination during butchery. The persistence of this pathogen in the slaughterhouse environment enables recurring contamination of meat products. This study aimed at identifying the sources and transmission routes of L. monocytogenes contamination within an abattoir where it was consistently detected for three consecutive years (2019–2021). Furthermore, the environmental factors aiding contamination along chicken processing lines were determined by surveying the microbiome within the facility. Samples collected in 2019 to 2021 were subjected to culture-dependent analysis to assess the prevalence, serotypes, and multi-locus sequence typing (MLST) of L. monocytogenes. Additionally, the specimens collected in 2021 underwent culture-independent analysis via real-time quantitative polymerase chain reaction (qPCR) and 16S rRNA gene amplicon sequencing to identify the contamination sources and characterize the entire microbial community within the slaughterhouse. L. monocytogenes was isolated only from the clean zone, where the final slaughtering stage occurs. Most strains isolated from the final carcasses showed the same genetic cluster as the isolate in the chilling water and were assigned to MLST profile ST3. Culture-independent qPCR confirmed L. monocytogenes contamination in all samples, excluding post-scalding carcasses, prewashed post-evisceration carcasses, and the bleeding areas. Consequently, qPCR enabled more comprehensive identification of L. monocytogenes contamination points than culture-dependent approaches. Moreover, 16S rRNA gene amplicon sequencing demonstrated that psychro-tolerant and spoilage-related bacteria with L. monocytogenes-like attributes exhibited enhanced viability in the clean zone and immersion-chilling water. Metagenomics-based source tracking analysis further revealed that the shackles and chilling waters represent predominant sources of cross-contamination between different slaughterhouse zones, whereas the grading and packaging workstations and chilling water in the clean zone were deemed crucial sources affecting final carcass contamination. Collectively, these findings demonstrate through culture-dependent and -independent methods that L. monocytogenes spreads along the slaughter line, contaminating the slaughterhouse. Moreover, by investigating changes in microbial community and bacterial flow along the slaughter line within the facility, the sources influencing carcass contamination can be effectively traced.

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes

BackgroundTheileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype.MethodsA pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically.ResultsEach genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays.ConclusionsThe genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.Graphical abstract

Molecular occurrence and genetic diversity of Ehrlichia canis in naturally infected dogs from Thailand

Canine monocytic ehrlichiosis is cause by Ehrlichia canis resulting in hematologic disorders and severe clinical signs. The aim of this study was to scrutinize the molecular detection and genetic diversity of E. canis based on the trp36 gene in dogs from Thailand’s northern and central regions. A total of 120 dogs blood samples were amplified for trp36 gene of E. canis using the polymerase chain reaction (PCR). Forty-seven out of 120 dog blood samples (39.16%, 47/120) were positive for E. canis the trp36 DNA with 790 bp of PCR amplicon size. The factor significantly associated with E. canis infection is animal housing status (p < 0.05). Sequence and phylogenetic analysis showed that E. canis trp36 gene of Thailand isolates was clustered into 1st clade with similarity ranging from 95.65 to 100% together with the US genogroup. The 14 haplotypes of the trp36 gene shown in TCS network exhibited that haplotype #1–4 was found in Thailand. The entropy analysis of the trp36 gene illustrated 751 polymorphic sites and 271 entropy peaks of nucleic and amino acid sequences, respectively. Hence, these findings are crucial for better understanding the epidemiology of Ehrlichia infection and could be helpful for implementing control measures in Thailand.

Uncovering the prevalence and drivers of antibiotic resistance genes in soils across different land-use types

The emergence and spread of antibiotic resistance genes (ARGs) in soil due to animal excreta and organic waste is a major threat to human health and ecosystems, and global efforts are required to tackle the issue. However, there is limited knowledge of the variation in ARG prevalence and diversity resulting from different land-use patterns and underlying driving factors in soils. This study aimed to comprehensively characterize the profile of ARGs and mobile genetic elements and their drivers in soil samples collected from 11 provinces across China, representing three different land-use types, using high-throughput quantitative polymerase chain reaction and 16S rRNA amplicon sequencing. Our results showed that agricultural soil had the highest abundance and diversity of ARGs, followed by tea plantation and forest land. A total of 124 unique ARGs were detected in all samples, with shared subtypes among different land-use patterns indicating a common origin or high transmission frequency. Moreover, significant differences in ARG distribution were observed among different geographical regions, with the greatest enrichment of ARGs found in southern China. Biotic and abiotic factors, including soil properties, climatic factors, and bacterial diversity, were identified as the primary drivers associated with ARG abundance, explaining 71.8% of total ARG variation. The findings of our study demonstrate that different land-use patterns are associated with variations in ARG abundance in soil, with agricultural practices posing the greatest risk to human health and ecosystems regarding ARGs. Our identification of biotic and abiotic drivers of ARG abundance provides valuable insights into strategies for mitigating the spread of these genes. This study emphasizes the need for coordinated and integrated approaches to address the global antimicrobial resistance crisis.

Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures.

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N-N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H-15N heteronuclear multiple bond correlation (HMBC) along with 1H-15N heteronuclear single quantum coherence (HSQC) and 1H-15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade-structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.

Molecular characterization and detection of multidrug-resistant gene in bacterial strains in a health care centre located in Iwo, Osun State, Nigeria

The importance of bacterial isolates from hospital wastewater as a reservoir of antibiotic resistance and a potential source of resistance genes to clinical pathogens is underestimated. This study is aimed to isolate and molecularly characterize bacteria from healthcare wastewater in addition to evaluating the distribution of multiple drug-resistant bacteria in the study area. A study was conducted in a Healthcare Centre, in Iwo, Osun State Nigeria. The wastewater was taken from four sections of the wastewater discharge point. Collection of samples was aseptically carried out, transported, and, analyzed immediately following standard procedures. Identification was carried out using biochemical tests and 16S rRNA sequencing. Antibiotic susceptibility was carried out on Mueller Hinton agar following the Kirby-Bauer disk diffusion method. Detection of resistant genes was done using Polymerase Chain Reaction (PCR). A total of 86 bacterial isolates were obtained from wastewater discharge locations. Twenty-five isolates were picked for molecular identification and six different bacterial genera belonging to Bacillus (68%) the most occurring, followed by Cronobacter, Acinetobacter, Enterobacter, with 8%, Klebsiella, and Enterococcus (4%) were identified. Out of 30 randomly picked PCR amplicons, the result revealed 19 isolates (63%) and 10 isolates (33%) were positive for blaTEM and blaSHV respectively, while 9 isolates (30%) harbored both the two genes concurrently. The contamination of wastewater by antibiotics may lead to the rise of antibiotic resistance in the environment, so there is a need to monitor and treat the hospital wastewater that is discharged into the environment.

The first molecular isolation of Halicephalobus gingivalis from horses in Iran.

Parasitic infections pose significant threats to humans' and animals' well-being worldwide. Among these parasites, Halicephalobus spp., a genus of nematodes, has gained attention due to its ability to cause severe infections in various animal species, including horses. This study aimed to determine the prevalence of Halicephalobus spp., specifically focusing on Halicephalobus gingivalis in horses. In July 2022, a cross-sectional study was conducted in northern Iran to determine the prevalence of Halicephalobus spp. Using standard coprological techniques, 141 fecal samples from randomly selected horses were analyzed for GI helminth eggs. The Halicephalobus spp. eggs present in faeces were identified by molecular methods. Polymerase Chain Reaction (PCR) was used to amplify the partial 5' variable region (~ 390 base pairs) of 18S DNA using SSUA_F and SSU22_R primers. Furthermore, the PCR products obtained were sequenced, and phylogenetic analysis was performed using available sequences from GenBank. Microscopic examination of 141 fresh faecal samples revealed 5 fecal samples were infected with small ellipsoidal nematode eggs ranging between 40 and 50 × 50-60μm. This study's PCR amplicons showed ~ 390bp bands on 2.0% agarose gel. A partial sequence of 18S DNA (363bp) was obtained herein (GenBank accession no. OQ843456). Overall, using molecular tools represents a significant step forward in diagnosing and managing the Halicephalobus gingivalis infections in horses.

Molecular detection and pyrG sequence analysis of Avibacterium paragallinarum using clinical samples of infraorbital exudates from layer chickens with infectious coryza symptoms in Indonesia.

Infectious coryza (IC) or snot, is caused by Avibacterium paragallinarum and leads to upper respiratory disease in poultry. Various diagnostic methods are available, including isolation and identification through bacterial culture and biochemical tests. However, the isolation and subsequent identification of A. paragallinarum are challenging because the bacteria are fastidious and require specific growth factors. This study aimed to detect A. paragallinarum in clinical samples taken from the exudate of the infraorbital sinus of layer hens showing clinical signs of IC. Samples were collected from 10 layer hens with IC symptoms. Following DNA extraction, HPG-2 polymerase chain reaction (PCR) assays were performed. The PCR amplicons underwent electrophoresis to determine those of the correct target size (511 bp), and these were sequenced. The resultant sequences were analyzed using the National Center for Biotechnology Information (NCBI) basic local alignment search tool. MEGA X was used for bioinformatics analysis. The presence of A. paragallinarum was confirmed by HPG-2 PCR in 4/10 samples. Bioinformatics analysis showed that the amino acid sequence of the samples and the A. paragallinarum reference sequences in the NCBI database were grouped within the same cluster. Furthermore, the nucleotide sequences showed 98.64%-100% of similarity with the reference sequences. The phylogenetic reconstruction of partial pyrG sequences from 55 A. paragallinarum strains/isolates deposited in GenBank confirmed that the four HPG-2 PCR-positive samples fell within the A. paragallinarum cluster, separate from the Pasteurella multocida, Avibacterium spp., and Rodentibacter pneumotropicus clusters. Avibacterium paragallinarum infection was molecularly confirmed in 4/10 (40%) samples by HPG-2 PCR amplicon detection. Clustering of the pyrG partial gene sequences revealed that the positive samples fell within the A. paragallinarum cluster.

First Report of Pectobacterium brasiliense Causing Bacterial Blackleg and Soft Rot of Potato in Pennsylvania.

Potato (Solanum tuberosum) plants showing blackleg and soft rot symptoms were collected at a commercial vegetable farm near Newmanstown, PA in August 2021 (Fig. S1). The incidence of potato blackleg in the unirrigated field was about 5 to 8%, but approximately 30% in the irrigated field. The diseased stems were cut into 5 cm and surface disinfected. The stem segments were placed into a 50-mL tube containing 15 mL of sterile water for 15 min for bacterial release. The bacterial suspension was streaked on crystal violet polypectate (CVP) (Hélias et al. 2012) plates and incubated at 28°C for 48 h. Three single colonies produced pits on CVP were picked and purified. Genomic DNA of all three isolates were extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). Polymerase chain reaction (PCR) was performed using all three extracted DNAs as a template with the primer pairs gapA 7F/938R (Cigna et al. 2017), recA F/R (Waleron et al. 2001), dnaA F/R (Schneider et al. 2011) and dnaX F/R (Sławiak et al. 2009) targeting the gapA, recA, dnaA and dnaX genes, respectively. Isolate 21PA01 was further studied as a representative isolate. PCR amplicons derived from both forward and reverse primers were sequenced and analyzed using the BLAST algorithm against the NCBI database (https://www.ncbi.nlm.nih.gov). The regions of gapA (GenBank accession No. ON989738), recA (ON989739), dnaA (OP121183), and dnaX (OP121184) had 99.86%, 100%, 98.88%, and 100% identities with Pectobacterium brasiliense strains S1.16.01.3M (MN167062.1), BL-2 (MW721598.1), IPO:4132 (CP059956.1), and BL-2 (MW721603.1), respectively. A phylogenetic maximum-likelihood tree of the concatenated genes with the length of 2551 bp was constructed to visualize the relationship among different species of Dickeya and Pectobacterium. As a result, 21PA01 was in a single monophyletic cluster with other Pectobacterium brasiliense reference strains (Fig. S2 C). To confirm the pathogen, Koch's postulates were performed. Seed pieces of potato 'Lamoka' were planted in potting mix in one-gallon plastic pots in a greenhouse. Three weeks after emergence, the stems of three plants were each injected with 10 µL of bacteria suspension of either 21PA01 at 107 CFU/mL, P. parmentieri ME175 in tryptic soy broth (TSB) at 107 CFU/mL or TSB at 2 cm above the soil line. Seven days after inoculation, stems inoculated with 21PA01 and ME175 showed black and rotten symptoms, whereas the TSB-injected control plants remained symptomless. In addition, 'Lamoka' tubers were inoculated by placing 10 µL 21PA01 and ME175 suspensions at 107 CFU/mL, and TSB in a 1-cm-deep hole poked in a tuber separately and then sealed with petroleum gel, followed by incubation in a moist chamber at 22 °C for 4 d. The 21PA01 and ME175 inoculated tubers showed soft rot symptoms, but the TSB treatment had no symptoms. Bacterial colonies were isolated from the infected stems and confirmed by the DNA sequences as described above. PCR result was negative on control plant samples. Both stem and tuber inoculation trials were repeated two times, and the results were consistent. Thus, 21PA01 was identified as Pectobacterium brasiliense. To our knowledge, this is the first report of P. brasiliense infecting potatoes in Pennsylvania, USA, although it has been reported somewhere else (van der Merwe et al. 2010, Zhao et al. 2018). This could be a new species in Northeastern US.

Molecular evidence and phylogenetic delineation of spotted fever group Rickettsia species in Amblyomma ticks from cattle in Gauteng and Limpopo Provinces, South Africa

Objective: To determine the prevalence of tick-borne pathogens with a particular focus on Rickettsia spp. in ticks collected from cattle in Gauteng and Limpopo Provinces, South Africa. Methods: A total of 200 ticks were collected from cattle within the Madala livestock, Pretoria, Gauteng Province and in Mankweng Township, Polokwane, Limpopo Province in 2019. The ticks were morphologically identified and processed individually for a total genomic DNA extraction. Specific primers targetting ompA, ompB, and the 17KDa genes were used for a molecular screening and delineation of Rickettsia from the extracted genetic materials using polymerase chain reaction (PCR) technique. PCR amplicons of positive samples were sequenced bidirectionally using the Sanger sequencing method. Sequences generated were processed and analysed using appropriate bioinformatics software. Results: The ticks were morphologically identified as Amblyomma spp. PCR profiling of the genomic DNA samples revealed the presence of the Rickettsia pathogen in 42 (21%) of the ticks collected from both Provinces. Out of the genes profiled, 14 (7%) were positive for 17KDa, 42 (21%) for ompA and 32 (16%) were positive for ompB genes respectively. The nucleotide blast of the sequenced genomes showed high similarity, as high as 100% with other reference Rickettsia (R.) africae in the GenBank. The phylogenetic analysis of the sequences further validated them as R. africae with their characteristic clustering pattern with related reference sequences. Conclusions: There is an abundance of R. africae in Amblyomma ticks collected from cattle in the study areas. This has serious public health implications as individuals who accidentally get infested with the ticks could acquire R. africae. Hence, adequate precautions in terms of sensitization of farmers about the risk and mass mobilization drive to control the vectors in the areas are highly recommended to safeguard public health.

Comparison of Kato-Katz, PCR and coproantigen for the diagnosis of Taenia solium taeniasis.

Four methods were compared for the diagnosis of human taeniasis caused by Taenia solium. Fecal samples from persons living in a T. solium endemic region of Madagascar were examined for taeniid eggs by the Kato–Katz method. Subsequently, samples positive (n = 16) and negative (n = 200) for T. solium eggs were examined by (i) amplification of the fragment of small subunit of the mitochondrial ribosomal RNA (rrnS) gene using conventional polymerase chain reaction (PCR) and (ii) a nested PCR of a fragment of the T. solium Tso31 gene. Additionally, 12 egg-positive and all egg-negative samples were tested for coproantigen detection. A further 9 egg-positive fecal samples were examined using both PCRs. Of the 12 egg-positive samples tested by PCRs and coproantigen methods, 9 (75%) were positive by rrnS PCR, 3 (25%) using Tso31-nested PCR and 9 (75%) by coproantigen testing. None of the 200 egg-negative fecal samples was positive in either rrnS or Tso31-nested PCR. Twenty of the 25 egg-positive samples (80%) were positive in rrnS PCR, and DNA sequencing of PCR amplicons was obtained from 18 samples, all confirmed to be T. solium. Twelve of the 25 egg-positive samples (48%) were positive in the Tso31-nested PCR, all of which were also positive by rrnS PCR. It is suggested that species-specific diagnosis of T. solium taeniasis may be achieved by either coprological examination to detect eggs or coproantigen testing, followed by rrnS PCR and DNA sequencing to confirm the tapeworm species in egg-positive or coproantigen-positive samples.