mecA positive Staphylococcus spp. in bovine mastitis, milkers, milking environment, and the circulation of different MRSA clones at dairy cows farms in the Northeast region of Brazil

ABSTRACT: This study detected the presence and distribution of mecA in Staphylococcus spp. in the dairy production environment at farm level in Brazil. We analyzed 335 samples of mastitis cow milk, 15 samples of nostrils and hand swabs from milkers, 14 teat cup swabs, and 9 milking buckets swabs. Initially, the samples were subjected to microbiological analysis to detect Staphylococcus spp. and then S. aureus and mecA positive isolates were identified by PCR. All S. aureus isolates carrying the mecA genes were subjected to DNA macro-restriction analysis by Pulsed-Field Gel Electrophoresis (PFGE). The mecA gene was detected in 6/335 (1.78%) of mastitis cow milk, 5/15 (33.3%), and 5/15 (33.3%) of nostrils and hand swab, and 4/14 (28.5%) of the teat cup isolates. MRSA genotyping was performed by PFGE, a total of seven pulsotypes were grouped in two clusters. This study identified the occurrence and spread of MRSA at dairy environment of farms, and also the existence of distinct genetic profiles between isolates.

Detection of Nontuberculous Mycobacterial Species from Tissue Samples of Cattle and Buffaloes by PCR and PRA (PCR-RFLP)

Background: Nontuberculous mycobacteria are opportunistic pathogens and some of them may cause disease in humans and animals causing pulmonary infections, mastitis, lesions in respiratory tract and lymph nodes of cattle, due to which they are being recognized worldwide and also interfere with the diagnosis of bovine tuberculosis. Methods: The present study was conducted for detection of nontuberculous mycobacterial species (NTM) in tissue samples (with and without tubercle lesions) in cattle and buffaloes from postmortem hall GADVASU, Ludhiana. Polymerase Chain Reaction and PCR-RFLP which involved hsp65 gene amplification (439 bp) and restriction analysis of amplified product was performed on 30 tissue samples for detection of nontuberculous mycobacterial species. Result: Three out of 30 samples showed hsp65 gene amplification and 2 were identified as M. kansasii using restriction analysis technique and one could not be identified as the RFLP patterns was different from other known PCR-RFLP profiles. NTM such as M. kansasi may cause infection in animals and PRA (PCR-Restriction Fragment Length Polymorphism Analysis) technique was found to be a rapid tool for identification and differentiation of NTM upto species level.

A simple method for detection of mutations in amino acid 452 of the Spike protein of SARS-CoV-2 using restriction enzyme analysis

Variants of Concern or Interest of SARS-CoV-2 (VOC or VOI), the coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 452 of the Spike protein are particularly important and associated with some of these variants: L452R, present in Delta VOC, and L452Q, present in Lambda VOI. These mutations have been associated with both increased infectivity and evasion of protective immune response. A search on GISAID to detect the number of sequences harboring the L452R mutation and the frequency of Delta VOC among them, showed that since August 2021, most of these sequences belong to the Delta VOC. Restriction enzyme analysis is proposed as a rapid method to detect L452R. A small amplicon from the Spike gene was digested with MspI. A 100% concordance was observed between digestion and sequencing results. The mutation L452Q can also be detected by restriction analysis, allowing the identification of putative Lambda VOIs. The proposed methodology, which allows screening of a great number of samples, could provide a faster information on the prevalence of Delta VOC cases.

Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

Biological potential of Ascophyllum nodosum extract on rhizobial diversity in nodules of mothbean Vigna aconitifolia Jacq. via Amplified Ribosomal DNA Restriction Analysis

Aim: The aim of this study was to use Ascophyllum nodosum for potentially increasing the growth and rhizobial diversity in nodulating rhizobia in Vigna aconitifolia. Methodology: Different concentrations of Ascophyllum nodosum extracts (0.01%, 0.02%, 0.05%, 0.10% and 0.50%) were applied via foliar spray and on roots of Vigna aconitifolia. Growth characteristics and Amplified Ribosomal DNA Restriction Analysis were conducted to detect the morphological and molecular changes in rhizobial diversity. The restriction profiles thus obtained were used to study the rhizobial communities via Cluster analysis and Dendrogram using NTSYS-PC program and UPGMA constructed. Results: Roots treated with 0.05% Ascophyllum nodosum extract showed best growth of plants. This concentration not only proved best for the aggregation of nodules but also for obtaining enormous rhizobial diversity. Interpretation: Ascophyllum nodosum is a modern, cheap, non-toxic natural biofertilizer and Amplified Ribosomal DNA Restriction Analysis represents a favorable alternative to culture dependent method for assessing rhizobial diversity in nodulating bacteria.

Association of insulin-like growth factor 1 (IGF1) gene polymorphism with the reproductive performance of three dual-purpose chicken breeds

The study was designed to investigate the association of Insulin-like growth factor 1 (IGF1) gene polymorphism with the reproductive performance of FUNAAB-Alpha, Sasso, and Kuroiler dual-purpose chicken breeds. To achieve this, a total of 250 healthy hens were selected at 12 weeks of age and were intensively managed in cages for 52 weeks. Blood sample was taken from each chicken at the 34th week and genomic DNA was extracted using Qiagentm DNA extraction kit, PCR was used to amplify the DNA fragments, and the PCR products were electrophoresed. Amplicons obtained were digested with restriction enzyme hinf1, and were further electrophoresed on 1.5% agarose gel. Data obtained were analyzed using the General linear model of SAS (2002) version 9.0 to determine the effect of IGF1 gene polymorphism and the distribution of alleles within the breeds. Results show polymorphism of the IGF1 gene and the restriction analysis indicated two alleles; A 58% and C 42% with the identification of genotypes AA, AC, and CC, and genotypic frequency of 22%, 43% and 35% respectively. Significant associations were observed between the polymorphism of the IGF1 gene, age of the bird at first lay, and weight of the hen at first lay. Chickens with haplotype CC came earlier into lay compared to those with the other two haplotypes (AA and AC). Therefore, the study suggests that haplotype CC could be used as a genetic marker to select for an improved laying performance in chickens.

Epigenetic DNA Modifications Upregulate SPRY2 in Human Colorectal Cancers

Conventional wisdom is that Sprouty2 (SPRY2), a suppressor of Receptor Tyrosine Kinase (RTK) signaling, functions as a tumor suppressor and is downregulated in many solid tumors. We reported, for the first time, that increased expression of SPRY2 augments cancer phenotype and Epithelial-Mesenchymal-Transition (EMT) in colorectal cancer (CRC). In this report, we assessed epigenetic DNA modifications that regulate SPRY2 expression in CRC. A total of 4 loci within SPRY2 were evaluated for 5mC using Combined Bisulfite Restriction Analysis (COBRA). Previously sequenced 5hmC nano-hmC seal data within SPRY2 promoter and gene body were evaluated in CRC. Combined bioinformatics analyses of SPRY2 CRC transcripts by RNA-seq/microarray and 450K methyl-array data archived in The Cancer Genome Atlas (TCGA) and GEO database were performed. SPRY2 protein in CRC tumors and cells was measured by Western blotting. Increased SPRY2 mRNA was observed across several CRC datasets and increased protein expression was observed among CRC patient samples. For the first time, SPRY2 hypomethylation was identified in adenocarcinomas in the promoter and gene body. We also revealed, for the first time, increases of 5hmC deposition in the promoter region of SPRY2 in CRC. SPRY2 promoter hypomethylation and increased 5hmC may play an influential role in upregulating SPRY2 in CRC.