Random Amplification Research Articles

First report from Bangladesh on genetic diversity of multidrug-resistant Pasteurella multocida type B:2 in fowl cholera.

Background and Aim:Fowl cholera (FC) caused by Pasteurella multocida is a highly contagious bacterial disease of global importance for poultry production. The severity and incidence of FC caused by P. multocida may vary considerably depending on several factors associated with the host (including species and age of infected birds), the environment, and the bacterial strain. This study aimed to investigate the genetic diversity of multidrug-resistant P. multocida strains isolated from FC outbreaks in laying hens from commercial farms of Bangladesh.Materials and Methods:We collected 57 samples of suspected FC, including 36 live and 21 dead laying hens. P. multocida isolates were characterized by biochemical and molecular-biological methods.Results:Twenty-two strains of P. multocida were isolated from these samples through phenotypic and genotypic characterization. The strains were grouped into two distinct random amplification of polymorphic DNA (RAPD) biotypes harboring a range of pathogenic genes; exbB, ompH, ptfA, nanB, sodC, and hgbA. In this study, 90.90% and 81.82% P. multocida strains were multidrug-resistant and biofilm formers, respectively. Whole-genome sequencing of the two representative RAPD phylotypes confirmed as P. multocida type B: L2:ST122, harboring a number of virulence factors-associated genes (VFGs), and antimicrobial resistance (AMR) genes (ARGs). In addition, pan-genome analysis revealed 90 unique genes in the genomes of P. multocida predicted to be associated with versatile metabolic functions, pathogenicity, virulence, and AMR.Conclusion:This is first-ever report on the association of P. multocida genotype B: L2:ST122 and related VFGs and ARGs in the pathogenesis of FC in laying hens. This study also provides a genetic context for future researches on the evolutionary diversity of P. multocida strains and their host adaptation.

DNA Damage in Human Amniotic Cells: Antigenotoxic Potential of Curcumin and α-Lipoic Acid.

Oxidative imbalances in the gestational phase are responsible for certain complications during pregnancy and for foetal and neonatal genetic disorders. In this work, using human amniocytes, we aimed to evaluate the protection provided to foetal DNA by two concentrations of antioxidant molecules, α-lipoic acid (LA) and curcumin (Cur), against hydrogen peroxide (H2O2)-induced damage. Genotoxicity tests, performed by the random amplification of polymorphic DNA (RAPD-PCR) technique and TUNEL tests, showed that the lowest concentration of LA-protected cells and DNA from H2O2 insults. However, a greater ability to protect the amniocytes’ DNA against H2O2 was observed following co-treatment with the highest concentration of Cur with H2O2. In fact, a genomic template stability (GTS%) similar to that of the negative control and a statistically significant reduction in the DNA fragmentation index (DFI) were revealed. Moreover, following a combined treatment with both antioxidants and H2O2, no statistical difference from controls was observed, in terms of both induced mutations and DNA breaks. Furthermore, no effect on morphology or cell viability was observed. The results demonstrate the ability of LA and Cur to protect the genetic material of amniocytes against genotoxic insults, suggesting their beneficial effects in pathologies related to oxidative stress.

Strain Typing of Trichosporon asahii Clinical Isolates by Random Amplification of Polymorphic DNA (RAPD) Analysis.

Objective The aim of this study was to identify and isolate Trichosporon asahii ( T. asahii ) from clinical samples and to assess the genetic relatedness of the most frequently isolated strains of T. asahii using random amplification of polymorphic DNA (RAPD) primers GAC-1 and M13. Methods All the clinical samples that grew Trichosporon species, identified and confirmed by polymerase chain reaction (PCR) using Trichosporon genus-specific primers, were considered for the study. Confirmation of the species T. asahii was carried out by T. asahii- specific PCR. Fingerprinting of the most frequently isolated T. asahii isolates was carried out by RAPD using random primers GAC-1 and M13. Results Among the 72 clinical isolates of Trichosporon sp. confirmed by Trichosporon -specific PCR, 65 were found to be T. asahii as identified by T. asahii- specific PCR. Fingerprinting of the 65 isolates confirmed as T. asahii using GAC-1 RAPD primer yielded 11 different patterns, whereas that of M13 primer produced only 5 patterns. The pattern I was found to be the most predominant type (29.2%) followed by pattern III (16.9%) by GAC-1 primer. Conclusions This study being the first of its kind in India on strain typing of T. asahii isolates by adopting RAPD analysis throws light on genetic diversity among the T. asahii isolates from clinical samples. Fingerprinting by RAPD primer GAC-1 identified more heterogeneity among the T. asahii isolates than M13.

Abstract 246: Method to analyze mutational and phenotypic profiles from single cell for clonal evolution

Abstract Single cell multiomics assays targeting RNA and Protein from the same cell provide a high-resolution view of the heterogeneity of the sample. However both analytes target the phenotype of the cells and unambiguous inference that a cellular phenotype is caused by a genotype can only be achieved by their measurement from the same single cell. To address this gap, we have developed the Tapestri multi-omics workflow to analyze the DNA and Protein information from the same cell. After pre-processing the reads, cell calling is performed using DNA reads. Variant calling and filtering is carried out using DNA reads to identify the high quality genetic variants within each cell and the variant-cell matrix is then analyzed to identify clones. The Protein reads for the valid cells are log normalized. A systemic artefact resulting in random uniform amplification of antibodies is corrected for by learning the scaling factor from the read count distribution. This reduces the number of false positives. Furthermore read depth dependence of expression is corrected by identifying the correlation of the mean expression and background counts with the total reads in the cell. The mean signal and background for each cell is learned using a 2 component gaussian mixture model. Then, z-score normalization is applied to prevent high expressors from skewing further analysis. The scaled values are dimensionally reduced using PCA. A graph structure is created using KNN with weights calculated using Jaccard similarity following which community detection is employed to identify the cell types. We test this method on a model system with two donor PBMC, 2 cell lines mixture titrated at a 47:47:5:1 ratio with a 312 amplicon DNA panel and 45 plex antibody panel. We filter cells using isotype controls from the antibody panel and mixed cell signatures using the DNA panel. We can identify 4 clones using the genetic variants. We can identify multiple cell types including the major populations such as T-cells, B-cells, Monocytes and NK-cells using the phenotypic expression in the PBMC donors. Moreover, we are able to correlate the proportions of each PBMC cell type for the identified clones with that of the individual donors. Due to the corrections applied during the normalization of protein counts we are also able to accurately identify various expected expression profiles in PBMCs such as CD45RA-CD45RO and CD8-CD4 mutual exclusion, and the trimodal expression of CD4. Citation Format: Saurabh Parikh, Manimozhi Manivannan, Jacqueline Marin, Kate Thompson, Daniel Mendoza, Benjamin Schroeder, Saurabh Gulati, Shu Wang, Aik Ooi. Method to analyze mutational and phenotypic profiles from single cell for clonal evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 246.

Genetic Resources and Diversity among Sheep Breeds of Asia and Europe

Sheep has been domesticated southwestern Asia for about 8000-9000 years ago and represented one of earliest livestock species. Ancestor relationship can be investigated through mtDNA data. There are many genetic markers to determine the relationship between and among the sheep breeds. Restriction fragment length polymorphisms are basic technique and have less variability and many restrictions as compared to Random amplification of polymorphic DNA. Due to specific amplification of Amplified fragment length polymorphisms it is more suitable than Restriction fragment length polymorphisms and Random amplification of polymorphic DNA. Microsatellites are widely used technique for the determination of genetic diversity. This technique provides information about the classification and characterization of sheep breeds. However this technique cannot provides information on breed functional traits. On the basis of mtDNA analysis haplotypes groups differ in sequences by any extent of Ovis species. Haplotype A and B are two of most important haplotype groups. Haplotype A carries Asiatic mouflon (Ovis orientalis) while Haplotype B carries European mouflon (Ovis musimon). Actually Haplotype A and B are both found in Asia while Haplotype B only dominates in Europe so sheep descent from one or more Asiatic mouflon. Haplotype C also found in Turkey, Portugal, Caucasus and China while Haplotype D present in Karachai sheep from Caucasian. Haplotype E which is very rare and found only in Turkey.

Ecotoxicological effects of titanium dioxide nanoparticles and Galaxolide, separately and as binary mixtures, in radish (Raphanus sativus)

Nano-TiO2 and Galaxolide (HHCB) are continually released into the environment because they are common ingredients of personal care products. In this study, the effects of nano-TiO2 and HHCB, individually and as binary mixtures, on Raphanus sativus were investigated. Growth indices (germination rate, root length, and shoot elongation), random amplification of polymorphic DNA profiles of DNA damage in the seedling roots, and expression of genes related to DNA damage, repair, and the cell cycle were assessed. Radish germination was not affected by nano-TiO2 (5–200 mg L−1) but was inhibited by HHCB (≥50 mg L−1). Nano-TiO2 and HHCB both caused severe DNA damage, including DNA mismatch damage, DNA double-strand breaks, and chromosomal damage. The binary mixtures indicated antagonistic effects occurred, and 200 mg L−1 nano-TiO2 decreased the genetic toxicity of HHCB. Of the genes that were examined, MRE11 and WRKY40 were the most sensitive to nano-TiO2 and HHCB, indicating that these genes could be used as sensitive biomarkers for exposure of R. sativus to nano-TiO2 and HHCB. The results improve our understanding of the risks posed by nano-TiO2 and HHCB to R. sativus in particular and possibly to other plants.

Molecular Systematic Investigations of Three Fin Fish Cichlid Species of Oreochromis niloticus (Nile Tilapia), Genetically Improved Farmed Tilapia (GIFT) and Astronotus ocellatus (Oscar Cichlid)

Fisheries in India contribute significantly to the total GDP of agriculture and earn significant foreign exchange. Aquaculture is playing an important role in India and is bestowed with a huge biodiversity of aquatic organisms. India ranks second in total fish production in the world. Nile tilapia has become the world’s second most popular farmed fish, after carps. Oscars is a popular aquarium fish around the world. In the present study, efforts were made to analyze the extent of divergence or similarity among three cichlid finfishes (Nile Tilapia, GIFT Tilapia, and Oscar Cichlid) using molecular biology techniques such as Random Amplification of Polymorphic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP). The Phylogenetic tree was constructed using PhyElph software to study the evolutionary relationship between the three cichlid finfish species. The Phylogenetic or evolutionary relationship was established for the three fishes, Nile tilapia, GIFT, and Oscar cichlid, with the Phylogenetic tree. It was found that Nile tilapia and GIFT share a recent common ancestor, while Oscar cichlid does not share any evolutionary relationship with Nile tilapia and GIFT.

Using of RAPD-PCR markers to detect the genetic relationship of number of Bellevalia Lapeyr and Ornithogalum L. species developing in central and northern of Iraq

This study evaluates eleven wild plant species belong to two genera of Asparagaceae family, naming of Bellevalia lapeyr and Ornithogalum L. and discuss the similarities and differences among all species by using eighteen random primers for the Random amplification polymorphic DNA (RAPD) markers were used to study the genetic relationship and genetic distance among these species. The study aimed to determination genetic relationships among all the eleven species by using RAPD markers and the method of extraction DNA from plants was CTAB with liquid nitrogen. The result show 16 primers success in giving 293 bands, of which 263 were polymorphic bands, 25 unique bands and 5 were absent. B.chrisii type also succeeded in showing two unique bands at OPG-08 primer without registering any other bands. The lowest genetic distance was 0.306 between B. longipes and B.parva, while the highest genetic distance was 0.943 between B.chrisii and O.pyrenaicum. It is concluded from our study based on the results of the RAPD test that the studied species were divided into two main groups, each group was divided into subgroups, the first group included three groups and the second main group included two main groups. The study found that the O.pyrenaicum type is distinguished by being far from the rest of the species.

Molecular Characterization and Allelopathic Potential of Radish Species on Wheat and Weed Species

This study was conducted at Salahaddin University/ College of Agricultural Engineering Science, to inspect the allelopathic effects of radish (Raphanus sativus L.) on two wheat species and two endemic weed species. The study included two experiments, first one was bioassay experiment to estimate the aqueous extracts effect of three radish species (red, black and white) at five concentration levels (0, 10, 20, 30, and 40%) on germination and seedling growth of bread wheat (Triticum aesitivum Var. Adana), hard wheat (Triticum durum Def. Var. Smito) and two endemic weed species wild oat (Avena fatua) and wild barely (Hordeum spontaneum). Results indicated that aqueous extracts of black radish possess more allelopathic potential compare to red and white radish, also, aqueous extracts significantly affected on all studied plant species at dissimilar ratio, while most effective concentration was (40%). Whereas the germination of all plant species totally inhibited at this concentration. The second experiment aimed to indicate the genetic relatedness (genetic diversity) among radish species by using Random amplification of polymorphic DNA (RAPD-PCR) technique. The results indicate that the genetic distance among the three studied radish species ranged from 17.33 to 37.6. The lowest genetic distance was recorded between black and red that was 17.33 and the highest genetic distance recorded between black, red with white was 37.6 and white radish with each of black and red radish were 65.067. the results suggested that radish species have different allelopathic potential and genetic variance exist among them.

GENETIC DIVERSITY OF Digitaria horizontalis Willd. IN BUILT ENVIRONMENTS IN BENIN CITY, NIGERIA

The influence of humans on biodiversity at the genetic, species, community, and ecosystem levels is enormous. This is even more impactful on plant populations, which, being immobile, are at the mercy of their immediate environment. Digitaria horizontalis is a very common grass species in Benin City. Its ruderal nature accounts mostly for its widespread. The current study investigated the existence of possible genetic diversity of the grass in built environment in Benin City. Plant samples were randomly collected from 21 built environment locations within 6 Local Government Council Areas that make up Benin City. Test plants were allowed to acclimatize in experimental bowls and then allowed to grow for 4 weeks after which morphological characteristics were determined. Genetic diversity was determined by Random Amplification of Polymorphic DNA. Significant differences in plant height (33.0 – 59.0 cm) occurred when plants were compared based on location of collection. Changes in patterns of the association with weeds within the vicinity of the test plant was observed. The most prevalent plant species associated with the test plant was Pennisetum purpureum with a 12.18% prevalence. RAPD analysis using primer OPA 04 revealed a locus (about 600bp) that was absent in one or more of the locations with minimum disturbance. This 600pb fragment was however observed in most samples from highly built up and trampled environments. Primer OPA 03 showed monomorphic bands in 2 test samples with a band size of 400bp. It is therefore reported that there is an existence of the genetic diversity in a landscape of grass species (Digitaria horizontalis) found in different built environment in Benin City, Edo State, Nigeria.

Discrimination of Vitis vinifera varieties using DNA fingerprinting and NMR coupled with chemometrics and their impact on the efficacy of fluoxetine and indomethacin in vivo

The grapes, Vitis vinifera, belong to family Vitaceae. Some fruit juices substantially decrease the oral absorption of certain drugs undergoing intestinal uptake transport. Green seedless “Thompson” type (GT) and reddish black seeded “Magic black” type (BM) of V. vinifera L. were discriminated using deoxyribonucleic acid (DNA) fingerprinting and nuclear magnetic resonance (NMR) coupled with chemometrics. Their effects on the bioavailability of fluoxetine and indomethacin were assessed in vivo. Practical applications Random amplification of polymorphic DNA (RAPD) analysis of GT and BM revealed that primers OPA-02 and OPB-10 indicate a taxonomical relationship between them as they produced high similarity coefficients estimated by 80 and 66.67%, respectively. OPC-03, OPB-13, and OPC-07 primers help in their discrimination based on their minimal levels of similarity coefficients estimated by zero percentages accompanied by the high levels of polymorphism. Principal component analysis score plot obtained from processing of proton nuclear magnetic resonance (1H NMR) showed very good discrimination between GT and BM in PC1 that accounts for most of the variance. Oral administration of 100 mg/kg of GT and BM separately produce no significant change in immobility period of rats dropped individually into a glass cylinder. Concomitant administration of either 100 mg/kg of GT or BM with 20 mg/kg of fluoxetine significantly decreased the efficacy of antidepressant reference drug (fluoxetine) by decreasing time before immobility showing a percentage of depression inhibition estimated by 17.2 and 29.1%, respectively, that obviously decreased when compared to fluoxetine alone. Regarding anti-inflammatory activity, both GT and BM exhibited a notable activity by inhibiting the increase in paw edema and inflammation induced by carrageen in rats where BM is more potent approaching indomethacin. Administration of GT or BM with indomethacin reduces its efficacy revealing 27.5 and 26.1% in edema percentage after 1 hr, respectively, that decreased minimally to be 19.1% and 15.6% after 4 hr, respectively, instead of 7% in case of indomethacin alone. Thus, it can be concluded that DNA and NMR fingerprinting could successfully differentiate between Vitis varieties. Grape juice decreased the efficacy of fluoxetine and indomethacin. Further studies should be conducted to declare the effect of Vitis varieties on the bioavailability of additional drugs that treat chronic diseases as diabetes and hypertension.

Deep viral blood metagenomics reveals extensive anellovirus diversity in healthy humans

Human blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. By far, anelloviruses constitute the viral family that is more frequently found in human blood, although amplification biases and contaminations pose a major challenge in this field. To investigate this further, we subjected pooled plasma samples from 120 healthy donors in Spain to high-speed centrifugation, RNA and DNA extraction, random amplification, and massive parallel sequencing. Our results confirm the extensive presence of anelloviruses in such samples, which represented nearly 97% of the total viral sequence reads obtained. We assembled 114 different viral genomes belonging to this family, revealing remarkable diversity. Phylogenetic analysis of ORF1 suggested 28 potentially novel anellovirus species, 24 of which were validated by Sanger sequencing to discard artifacts. These findings underscore the importance of implementing more efficient purification procedures that enrich the viral fraction as an essential step in virome studies and question the suggested pathological role of anelloviruses.

Sequence-independent single-primer-amplification (SISPA) as a screening technique for detecting unexpected RNA viral adventitious agents in cell cultures

The sequence-independent, single-primer amplification (SISPA) enables the random amplification of nucleic acids, allowing the detection and genome sequencing of different viral agents. This feature of SISPA method provides evidence for application of it in monitoring the presence of adventitious RNA viruses in cell cultures. We evaluated SISPA method for the detection of a challenge RNA virus representing adventitious agent in cell cultures. Besides, by optimizing the SISPA method in our laboratory, we found false-positive results on negative control lanes in electrophoresis gels. To investigate the sources of contamination, false-positive results of SISPA were cloned into Escherichia coli cells, sequenced, and phylogenetically analyzed. This data revealed that the SISPA method can be used as an adjunct method to confirm the absence of unexpected adventitious RNA viruses in cell cultures. The phylogenetic analysis of SISPA contaminant sequences showed that the false-positive results were caused by nucleic acid amplification of commercial cDNA synthesis kit reagents, probably tracing back to expression plasmids and host ribosomal sequences, used for the production of enzymes. Therefore, laboratories using random amplification methods must be constantly aware of the potentials of such contaminations, yielding false-positive results and background noise in the final NGS reads.

On protocols for increasing the uniformity of random bits generated with noisy quantum computers

Generating random numbers is important for many real-world applications, including cryptography, statistical sampling and Monte Carlo simulations. Quantum systems subject to a measurement produce random results via Born’s rule, and thus it is natural to study the possibility of using such systems in order to generate high-quality random numbers. However, current quantum devices are subject to errors and noise, which can make the output bits deviate from the uniform distribution. In this work, we propose and analyse two protocols that can be used to increase the uniformity of the bits obtained when running a circuit with a Hadamard gate and a measurement in a noisy quantum computer. These protocols may be used prior to other standard processes, such as randomness amplification. We conduct experiments on both a quantum simulator and a real quantum computer, obtaining results that suggest that these protocols are useful to improve the probability of the generated bits passing statistical tests for uniformity.

Polymorphism of Sakini Chicken Population From Different Locations/Altitudes of Nepal Using Randomly Amplified Polymorphic Dna Markers

A study was conducted to evaluate genetic polymorphism in three Sakini chicken populations using random amplification of polymorphic DNA (RAPD) with seven highly polymorphic primers. All populations showed polymorphism with these primers that generates 59 different bands with an average of 8.4 bands per primer with 78.6% polymorphism nature. Primer OPA-16 produced the highest number of polymorphism bands 47 % and the lowest number of bands was produced by the OPA-05 primer 24 %. Differences for genetic distance (D) among populations were significant (P<0.05). A consensus dendogram was therefore developed to show the phylogenetic relationship among the populations. The cluster pattern is well supported by the principle component analysis that also separates all three populations of Sakini chicken into six major groups. The results provide evidence of the applicability of RAPD to determining genetic relatedness within and among different poultry populations and in developing reproducible markers useful in evaluating individual variation in poultry.
 SAARC J. Agri., 18(2): 115-124 (2020)

35 Characterization of the promoter region of ZNFO, an oocyte-specific gene in cattle

Maternal factors are essential aspects of oocyte competence, which orchestrate early embryonic development. ZNFO is a Krüppel-associated box domain (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocyte. Previous studies have demonstrated that ZNFO is essential for early embryonic development. However, the mechanisms regulating ZNFO transcription remain elusive. The objective of present study is to elucidate regulatory mechanisms of ZNFO transcription invitro, and specifically to identify putative core promoter and transcription factor(s) regulating ZNFO expression. 5′ Random amplification of cDNA ends (RACE) was performed using RNA isolated from 100 germinal vesicle (GV) stage oocytes to identify the transcription start site (TSS) of ZNFO. To elucidate the molecular mechanisms of ZNFO transcription, a 1.7-kb fragment of the 5′ regulatory region was obtained and cloned into pGL4.14 promoterless vector. The luciferase reporter assay was performed to confirm the promoter activity of the regulatory region. To further identify the core promoter region of the putative ZNFO promoter, a series of 5′ deletions in the ZNFO promoter followed by luciferase reporter assay was performed. The luciferase results indicated that the core promoter region of ZNFO was located within a region 57 to 31bp upstream of the transcription start site. Bioinformatics analysis indicated that a putative USF1/USF2 binding site (GGTCTCGTGACC) is located within the core promoter region. USF1 is a basic helix–loop–helix leucine zipper transcription factor, which regulates the expression of various maternal genes, which are essential for oocyte maturation and early embryonic development in cattle. To confirm that USF1 regulated ZNFO expression, bovine USF1 open reading frame (ORF) was cloned into pcDNA3.1A-HA vector, generating a USF1 overexpression construct. Overexpression of USF1 by transfecting USF1 plasmid enhanced ZNFO promoter activity within HEK293 cells, confirming that ZNFO expression is regulated by USF1. From these results, we conclude that USF1 activates the ZNFO promoter by binding to its target site, GGTCTCGTGACC.

Characteristic of Pseudomonas syringae pv. atrofaciens Isolated from Weeds of Wheat Field

The aim of this study was the identification of the causative agent of the basal glume rot of wheat Pseudomonas syringae pv. atrofaciens from the affected weeds in wheat crops, and determination of its virulent properties. Isolation of P. syringae pv. atrofaciens from weeds of wheat crops was carried out by classical microbiological methods. To identify isolated bacteria, their morphological, cultural, biochemical, and serological properties as well as fatty acids and Random Amplification of Polymorphic DNA (RAPD)-PCR (Polymerase chain reaction) profiles with the OPA-13 primer were studied. Pathogenic properties were investigated by artificial inoculation of wheat plants and weed plants, from which bacteria were isolated. For the first time, bacteria that are virulent both for weeds and wheat were isolated from weeds growing in wheat crops. It was shown that the fatty acids profiles of the bacteria isolated from the weeds contained typical for P. syringae pv. atrofaciens fatty acids, in particular, hydroxy acids: 3-hydroxydecanoic, 2-hydroxydodecanoic, and 3-hydroxydodecanoic. RAPD-PCR profiles of the newly isolated strains were identical to those of the collection strains P. syringae pv. atrofaciens UCM B-1011 and P. syringae pv. atrofaciens UCM B-1014 and contained a dominant fragment of 700 bp. The isolated strains, according to their phenotypic and genotypic properties, were identified as P. syringae pv. atrofaciens. It was established that the causative agent of basal glume rot of wheat P. syringae pv. atrofaciens is polyphagous and capable of infecting a wide range of plants. The main control measure for cereals diseases caused by P. syringae pv. Atrofaciens—crop rotations with nonhost species, should be revised, and alternative control methods must be proposed.

GENETIC DIVERSITY OF SABAH RICE CULTIVARS USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS

Rice is the most important staple crop in Malaysia and is cultivated all over the country, including the state of Sabah. The uniqueness of rice cultivation in Sabah lies in the type of rice itself, deriving mainly from local or non-commercial cultivars but with distinctive characteristics including long grains, aromatic properties, and drought tolerance. However, despite having these important agricultural traits, information on the genetic diversity of Sabah rice remains limited. Hence, the purpose of this study was to determine the genetic polymorphisms of Sabah rice using random amplification of polymorphic DNA (RAPD) markers. A total of 101 alleles were profiled, from which 94% were identified as polymorphic. Phylogenetic analysis grouped the rice samples into three clusters, with two clusters classifying the ability of rice to grow under different planting conditions, suitable for growth irrigate and upland condition. The first cluster was dominated by cultivars that could survive in wet (irrigated) areas, while the other featured those that were found in dry (upland) areas. Furthermore, two alleles, OPA-05-B2 and OPA-01-B11, were found to be unique to cultivars within the upland cluster and were thus proposed to be involved in dry environmental adaptation. The results of the present study provide an insight into the genetic relationships and diversity of Sabah rice.

Longitudinal study on the effects of intramammary infection with non-aureus staphylococci on udder health and milk production in dairy heifers

We conducted a longitudinal study to evaluate the effect of non-aureus staphylococci (NAS) causing subclinical intramammary infections (IMI) on quarter milk somatic cell count (qSCC) and quarter milk yield (qMY). In total, 324 quarters of 82 Holstein Friesian heifers were followed from calving to 130 d in milk (DIM) and were sampled 10 times each at 14-d intervals. The IMI status of each quarter was determined based on bacterial culture results at the current and previous or next sampling day, or both. The qSCC was determined on each sampling day and the average qMY on sampling day was available through stored daily milk weight data in the management program of the automatic milking system. A transient IMI (tIMI) was defined as a case where a specific pathogen was isolated from a quarter on only one sampling day and not on the previous or next sampling day. When the same bacterial strain, as defined by random amplification of polymorphic DNA-PCR, was isolated from the same quarter on multiple sampling days, it was defined as a persistent IMI (pIMI) status on those sampling days; a pIMI episode was defined as the combination of multiple consecutive pIMI statuses with the same bacterial strain on different sampling days. During this study, 142 subclinical IMI with NAS occurred in 116 different quarters from 64 animals, yielding in total 304 NAS isolates belonging to 17 different species. The prevalence of NAS was highest in the first 4 DIM. Overall, the predominant species was Staphylococcus chromogenes (52% of the isolates), followed by S. epidermidis (9.2%), S. xylosus (8.2%), and S. equorum (5.9%). Staphylococcus chromogenes was the only species for which an effect on qSCC and qMY could be analyzed separately; the other NAS species were considered as a group because of their low prevalence. Eighteen out of 40 IMI (45%) caused by S. chromogenes persisted over at least 2 sampling days, whereas only 10 of 102 (9.8%) IMI caused by other NAS species persisted for at least 2 sampling days. The average duration of pIMI episodes was 110.4 d for S. chromogenes and 70 d for the other NAS species. Remarkably, 17 of the 18 pIMI episodes with S. chromogenes started within the first 18 DIM. The qSCC was highest in quarters having a pIMI with a major pathogen, followed by quarters having a pIMI with S. chromogenes, and a pIMI with other NAS. Transient IMI with other NAS or with a major pathogen caused a small but significantly higher qSCC, whereas the qSCC in quarters having a tIMI with S. chromogenes was not statistically different compared with noninfected quarters. No significant differences in qMY were observed between quarters having a pIMI or tIMI with S. chromogenes or with the other NAS species compared with noninfected quarters, despite the higher qSCC. Quarters having a pIMI with major pathogens showed significantly lower daily milk production. Surprisingly, quarters that cured from an IMI with S. chromogenes had a significantly lower qMY than noninfected quarters.

Isolation and Antimicrobial Sensitivity of Mycoplasma synoviae and Mycoplasma gallisepticum from Vaccinated Hens in Mexico.

Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) strains were isolated at 39.5 °C to rule out temperature-sensitive strains (ts+) and identified using random amplification of polymorphic DNA. Then, their minimum inhibitory concentrations (MIC100) were calculated in isolated strains from broiler breeders and laying hens vaccinated with ts+ MS-H and ts+ MG TS-11 vaccines in Mexico. We sampled 631 lots of hens. A total of 28 of the 123 MS isolates and 12 of the 23 MG isolates were analyzed using random amplification of polymorphic DNA, of which 24 and 3 matched the DNA banding patterns of the MS-H and MG-F strains, respectively. The isolated MS and MG strains were sensitive to tiamulin and tylosin and showed intermediate sensitivity or resistance to lincomycin, florfenicol, erythromycin, enrofloxacin, and curcumin. Although both the MS and MG strains were sensitive to the same antibiotics (MIC100 lower than 1 mg mL−1), the MG strains were 5 to 10 times more sensitive than the MS strains. MS is the most frequently isolated mycoplasma in Mexican poultry production. The MS vaccine used (ts+ MS-H) could reverse its thermosensitivity and therefore could regain its virulence. MS was less sensitive to tiamulin and tylosin compared to MG.