Real-time Screening of Foods Using Repetitive Element PCR Reveals a DNA Marker Characteristic for Enterotoxigenic Bacillus Species

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables, and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic, including not just food-associated gastrointestinal toxicoinfections, but human endophthalmitis as well. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Repetitive element PCR was used to compare the banding patterns of each sample against B. cereus ATCC 14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe and were capable of causing gastroenteritis in consumers.

Improved diagnosis of viable parenchymal neurocysticercosis by combining antibody banding patterns on enzyme-linked immunoelectrotransfer blot (EITB) with antigen ELISA assay

The diagnosis of NCC depends on neuroimaging and serological confirmation. While antibody detection by enzyme-linked immunoelectrotransfer blot (EITB) fails to predict viable NCC, EITB banding patterns provide information about the host’s infection course. Adding antigen ELISA results on EITB banding patterns may improve their ability to predict or rule out of viable NCC. We assessed whether combining EITB banding patterns with Ag-ELISA improves discrimination of viable infection in imaging-confirmed parenchymal NCC. EITB banding patterns were grouped into classes using latent class analysis. True-positive and false-negative Ag-ELISA results in each class were compared using Fisher’s exact test. Four classes were identified: 1 (EITB-negative or positive to GP50 alone [GP50 antigen family]), 2 (positive to GP42-39 and GP24 [T24/42 family], with or without GP50), 3 and 4 (positive to GP50, GP42-39 and GP24, and reacting to bands in the 8-kDa family). Most cases in classes 3 and 4 had viable NCC (82% and 88%) compared to classes 2 and 1 (53% and 5%). Adding positive Ag-ELISA results to class 2 predicted all viable NCC cases (22/22 [100%]), whereas 11/40 patients (27.5%) Ag-ELISA negative had viable NCC ( P < 0.001). Only 1/4 patients (25%) Ag-ELISA positive in class 1 had viable NCC, whereas 1/36 patients (2.8%) Ag-ELISA negative had viable NCC ( P = 0.192). In classes 3 and 4, adding Ag-ELISA was not contributory. Combining Ag-ELISA with EITB banding patterns improves discrimination of viable from non-viable NCC, particularly for class-2 responses. Together, these complement neuroimaging more appropriately for the diagnosis of viable NCC.

Characterization of Xanthomonas citri pv. glycines Population Genetics and Virulence in a National Survey of Bacterial Pustule Disease in Korea

Xanthomonas citri pv. glycines (Xcg) is a major pathogen of soybean (Glycine max) in South Korea, despite the availability of soybean varieties with some resistance. We conducted a nationwide survey of the incidence and severity of bacterial pustule caused by Xcg. The percentage of infected fields was 7% to 17% between 2015 and 2017. We characterized the diversity of a nationwide collection of 106 Xcg isolates based on avrBs3 banding patterns. The isolates fell into 11 groups, each represented by a type strain; only two of these were similar to isolates collected from 1999 to 2002. The diversity of Xcg strains increased and the dominant strains changed between 1999 and 2017, with three new type strains comprising 44% of the isolates examined in 2012 to 2017. Pathogenicity tests did not show evidence for a shift in the races or aggressiveness of Xcg strains. Korean soybean cultivars, including the widely-grown Daewon cultivar, were susceptible to the 11 new type strains. The cultivar CNS, which carries the rxp resistance gene, was susceptible to most type strains, including two representing 83% of the Korean Xcg strains. In contrast, Williams 82, which also carries rxp, showed resistance to at least five type strains. Collectively, these results suggest that Williams 82 has resistance loci in addition to rxp. The widespread distribution of Xcg, the high virulence of the current endemic strains, and the low resistance of most Korean soybean cultivars collectively favor widespread disease in Korea in years that are favorable to pustule development.

Highly divergent karyotypes and barcoding of the East African genus Gonatoxia Karsch (Orthoptera: Phaneropterinae)

East Africa is a hotspot of biodiversity of many orthopteran taxa, including bushcrickets. Gonatoxia Karsch, 1889 species are fully alate Phaneropterinae, which are perfectly adapted to the foliage of forests. We examined five species using combined cytogenetic and molecular data to determine the inter- and intraspecific genetic diversity. The variation in the diploid number of chromosomes in males ranged from 2n = 28 + X0 and 26 + X0 to 2n = 6 + X0. Fluorescence in situ hybridization showed from one to many 18S rDNA loci as well as interstitial sequences, especially in G. helleri. 18S rDNA loci coincided with active NOR and C-banding patterns. The isolation of populations of the species explains differences in the number of chromosomes (G. maculata), chromosomal polymorphism and chromosomal heterozygosity (G. helleri). Our molecular phylogeny based on the COI locus supported the monophyly of the genus Gonatoxia and separateness of the five examined species in accordance with their morphological features and chromosome numbers as well as the species’ distribution.

Effects of radiation dose and nitrogen purge on collagen scaffold properties

Sterilization of medical devices is commonly performed using radiation methods. However, collagen materials can be damaged when using standard radiation doses (25 kGy). Small increases of radiation dose can allow for increases in the acceptable initial bioburden load of aseptically manufactured devices while maintaining required sterility assurance levels, which is often critical in early stage translational settings. In this study, we hypothesized that small increases in radiation dose from 15 to 20 kGy would result in significant changes to several key characteristics of collagen scaffolds. Scaffolds were manufactured by lyophilizing the pepsin digest of dense bovine connective tissue in cylindrical molds and were irradiated at either 0, 15, 17.5, or 20 kGy with an additional group packaged in nitrogen and irradiated at 17.5 kGy. Groups were evaluated for changes to the soluble collagen and glycosaminoglycan mass fractions, protein banding patterns in electrophoresis, a collagen fragmentation assay, and resistance to enzymatic degradation. All parameters were statistically analyzed using one-way analysis of variance with Tukey’s correction for multiple comparisons. The soluble collagen mass fraction was significantly decreased in the 20 kGy group; however, there was no significant effect of radiation dose or a nitrogen-rich environment on the other measured parameters, including protein banding patterns, fragmented collagen content, and resistance to enzymatic degradation. Statement of Clinical Significance: Collagen scaffolds have proven useful in clinical applications but can be damaged by standard radiation doses. Low-dose sterilization may be a viable alternative that minimally impacts key properties of these scaffolds.

Species-specific PCR assay for the detection of Babesia odocoilei

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

Screening of Mango Landraces for Polyembryony and Confirmation of Seedling Origin using Microsatellite Markers

Background: The introduction of polyembryonic rootstocks in the area of propagation is of great importance since they produce one zygotic and several nucellar plantlets. Proper identification of sexual embryo from each hybrid seed is necessary in order to preserve only the nucellar seedlings, which would help to maintain the rootstock’s genetic characteristics as well as to overcome the major constraints in the area of fruit breeding especially in hybridization programme by eliminating the nucellar ones to advanced generations. Contrasting reports exists regarding the vigour of zygotic seedlings of polyembryonic mango genotypes. It is necessary to identify/ distinguish the zygotic seedling from the nucellar population at an early stage, for which, microsatellite analysis could be a reliable tool. Methods: The experiment was laid out in completely randomized design (CRD) with 20 treatments replicated thrice. The twenty local mango landraces from Thiruvananthapuram (Kerala) were screened for polyembryony and were geo-referenced. Germination studies were conducted. Microsatellite analysis of all the plantlets from two varieties which exhibited the highest polyembryony were done using SSR primers and their banding patterns were compared with those of their respective mother plants. Result: Out of twenty mango varieties screened, seventeen were polyembryonic. Kappa Manga recorded the highest germination, germination index and seedling vigour index-I. Kotookonam Varikka recorded the highest polyembryony and followed by Kochu Kilichundan. Microsatellite analysis revealed that all the seedlings obtained from the respective stones of Kotookonam Varikka and Kochu Kilichundan had identical SSR profile to the mother plant, which indicated nucellar origin of seedlings having similar genetic composition to the mother plant.

Physiological variation of irradiated red radish plants and their phylogenic relationship using SCoT and CDDP markers

Greenhouse experiment is carried out to explore the outcome of γ-radiation on physiological and genetic variation in red radish (Raphanus sativus) for two generations. Gamma rays from 60Co were used to penetrate red radish seeds with different dose levels (0.0, 10, 20, 40 and 80 Gy). Plants generated from irradiated seeds and from self-pollination of these plants, called M1 and M2 generations, respectively. Some morphological and physiological traits were then determined, and the genetic diversity of both generations was studied using Start Codon Targeted (SCoT) and Conserved DNA-Derived Polymorphism (CDDP) molecular markers. All studied morphological traits (number of leaves/plants, leave height, root diameter, and root weight) were steadily improved by raising irradiation dose rate, reaching a cumulative raise at the irradiation doe level 40 Gy and decreased at dose level 80 Gy. Photosynthetic pigments of red radish plants released a notable increase by increasing gamma rays dose level for chlorophyll (a), chlorophyll (b) and carotenoids for 40 Gy dose rate. Proline content was elevated proportionally to the irradiation dose level, with the greatest increase seen at dose level of 80 Gy. Moreover, phytochemical screening was detected for the both two generations. Fourteen SCoT primers generated a total number of banding patterns of 194 with average 13.86 and the primer SCoT-33 released the highest number banding patterns (21). The percentage mean of polymorphism for all the SCoT primers was 74.66% and was 66.49 and 63.74% for M1 and M2 respectively. Furthermore, fifteen CDDP primers generated a total number of banding patterns of 186 and the primer CDDP-5 relieved the highest number of banding patterns (20). The percentage mean of polymorphism for all the CDDP primers was 73.41% and was 64.38 and 65.91% for M1 and M2 respectively. It could be concluded that gamma irradiation exhibited an appropriate variation in red radish M1 and M2 which was detected by SCoT and CDDP molecular markers.

Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris

India holds a significant rank in production and consumption of the age old protein rich crop Lentil with only one cultivated species and a large number of phenotypically similar cultivars. The need for a reliable and cost effective method of genetic characterization to unravel differences within the Lentil cultivars was felt. The present paper adopted EMA based chromosome preparation followed by staining with two contrasting fluorochromes dyes CMA and DAPI that bind directly to GC and AT rich heterochromatic segments on chromosomes. Analysis of fluorochrome banding pattern furnished a comparative account of genetic diversity within the cultivars that could not be achieved by traditional karyotyping. The marker pair of nucleolar chromosomes (4th and 3rd, majorly) occupied a pivotal position to intensify differences between cultivars in terms of banding patterns around secondary constrictions, suggestive of yet unknown variation in heterochromatin composition. Our study has strengthened genetic background and relationships of Lentil cultivars. We observed certain types of unusual fluorochrome bands that put forward the exclusivity of Indian germplasm and have questioned the mainstream heterochromatin elements of plant chromosomes captured by CMA-DAPI stains. The comprehensive fluorescent karyotypes of 30 L. culinaris cultivars prepared for the first time, serve as an archetype for the benefit of future breeding programmes in any Indian crop.

Effects of Condensed Tannins Supplementation on Animal Performance, Phylogenetic Microbial Changes, and In Vitro Methane Emissions in Steers Grazing Winter Wheat

Eighteen growing rumen-cannulated steers, with initial body weight (BW) of 167.4 ± 7.10 kg, were randomly allocated to one of three treatments that included a control (0% CT) and two CT treatment levels (0.05% and 0.07% condensed tannins (CT)/kg BW) with two replicates each. Both in vivo and in vitro experiments were conducted. In Exp. 1, final BW and average daily gain were greater (p < 0.05) for the 0.07% CT treatment compared to either 0.05% CT or control groups. Rumen bacterial populations in steers fed winter wheat in the absence of CT represented large proportions of the moderate-guanines and cytosines (GC) containing bacterial clusters with similarity coefficient (SC) ranging from 64% to 92% In the presence of CT on day 0, day 20, and day 60, however, the SC was 60% or greater (90% SC) with multiple bacterial band clusters as shown by the denaturing gel gradient electrophoresis banding patterns. In Exp. 2, in vitro total gas, potential gas, and CH4 productions decreased (p < 0.01) as CT supplementation increased in steers grazing wheat forage. These results suggested that the administration of CT improved BW gain and induced bacterial community changes in the rumen of steers grazing wheat forage.