A Simplified Protocol for Reversing Phenotypic Conversion of Ralstonia solanacearum during Experimentation

Background: Ralstonia solanacearum has the problem of losing the virulence in laboratory conditions, during prolonged experimentation. Since pure colonies of R. solanacearum contain cell fractions differing in virulence, it was considered worthwhile to find a way of selecting the cells with lower attenuation. Therefore, a methodology for inducing virulent-type colonies occurrence in Ralstonia solanacearum was developed. Methods: Nutrient gradient was created by swabbing R. solanacearum culture in a slanted KMTTC medium, and Phyllanthus emblica extract was given by well diffusion. Live–dead cell imaging using BacLight, effects of ascorbic acid on cell viability, and production of virulence factors (exopolysaccharides, cellulase, and pectinase) supported this hypothesis. The tagging of R. solanacearum with green fluorescent protein and further confocal scanning laser microscopic visualization confirmed the colonization in vascular bundles of tomato. Results: P. emblica extract suppressed R. solanacearum initially in well diffusion, but further developed virulent-type colonies around the wells. Nutrient deprivation was found to have synergistic effects with P. emblica extract. The converted fluidal (virulent type) colonies could be able to colonize vascular bundles and cause wilting symptoms. Conclusion: This method will be useful in the laboratories working on biocontrol of R. solanacearum for maintaining virulent-type colonies. Moreover, it could form the basis for studies on the stability of phenotypic conversion and cell fractions in R. solanacearum.

BIO-TECHNOLOGY: Newer Biology – An Impression

In this Impression a modern scope of bio-technology is discussed. The word Bio-Technology literally means a combinatorial hybrid of classical Biological - Science with some Chemical Industrial aspects. The micro biomes – the origin of this newer biology ie MICROBIOLOGY , MOLECULAR BIOLOGY ,rDNA science , Virology ( Most important biological entity – Corona Virus –Latest Global Issue !!! ) .The recombination power of Microbial Kinetics including bacterial as well as Viral cloning to more powerful as well as Virulent type – This type of cloning is irreproducible very often as per Statistical Relevance – a Limitation of Genetic Engineering.

Dynamics of Gene Expression in Mice Infected with Different Genotypes of Toxoplasma gondii

Background: Toxoplasma gondii is genetically diverse and different genotypes differ markedly in phenotype. The present study aims to define transcriptional patterns and biological processes that characterize host response to distinct strains of T. gondii. Methods: We conducted a time course study of gene expression microarray in mice during acute infection (days 1 to 7) with the highly virulent type I (GT1 strain), intermediately virulent type II (PTG strain) and non-virulent type III (CTG strain) parasites. Results: Overall, the number of genes affected increased from day 1 to day 5, and decreased on day 7. However, type III and type II infections up-regulated more genes than did type I at the very early phase, whereas type I infection up-regulated more genes at the late phase. Gene ontology (GO) analysis showed that the genes related to inflammatory and immune response were mostly affected and the majority were up-regulated, with type III infection inducing a higher degree of change and affecting more genes than did type I at the early phase. However, this pattern was reversed at the late phase. The change of expression during type II infection was between that of types I and III. Many genes associated with inflammatory and immune responses showed bimodal effects, with the first peak expression mostly at day 3 and then a second peak expression mostly at day 5. Several differentially expressed genes, including INF-γ, iNOS, CXCL10/IP-10, and numerous immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs) were previously experimentally confirmed important host factors in controlling T. gondii infection. Bioinformatic analysis of biological pathways enriched during infection revealed upregulation of pathways relating to cell-mediated immunity and the inflammatory response during all three infection types, though such enrichment was most expansive and pronounced during type I infection, and much less pronounced during type III infection. Conclusions: The findings in our study revealed dynamic differences of gene expression and different pathways of immune response in mice infected with three distinct strains of T. gondii.

Clonal expansion of a virulent Streptococcus suis serotype 9 lineage distinguishable from carriage subpopulations

Streptococcus suis is a porcine pathogen, causing severe invasive infections. S. suis serotype 9 is increasingly causing disease in Dutch and Chinese pig herds, but it is unknown whether all serotype 9 isolates are equally virulent and markers that can identify virulent strains are not available. Therefore, discrimination between virulent isolates and carriage isolates typically not associated with disease, is currently not possible. We collected tonsillar S. suis isolates from 6 herds not previously diagnosed with S. suis infections, and clinical S. suis isolates of previously diseased pigs. We confirmed the virulence of a virulent type strain and one representative clinical isolate, and the lack of virulence of two carriage isolates, in a pig infection model. Phylogenetic analysis of whole genome sequences of 124 isolates resulted in 10 groups, of which two were almost uniquely populated by clinical isolates. The population structure of S. suis serotype 9 appears highly diverse. However, analysis of the capsule loci sequences showed variation in a single region which fully correlated with a virulent genotype. Transmission electron microscopy suggested differences in capsule thickness between carriage and clinical genotypes. In conclusion, we found that that the S. suis serotype 9 population in the Netherlands is diverse. A distinct virulence-associated lineage was identified and could be discriminated based on the capsule locus sequence. Whilst the difference in virulence cannot be directly attributed to the DNA sequence, the correlation of capsule locus sequence with virulence could be used in the development of diagnostic tests to identify potential virulent S. suis serotype 9 in pigs.

A Francisella tularensis L,D-carboxypeptidase plays important roles in cell morphology, envelope integrity, and virulence

SummaryFrancisella tularensis is a Gram-negative, intracellular bacterium that causes the zoonotic disease tularemia. Intracellular pathogens, including F. tularensis, have evolved mechanisms to survive in the harsh environment of macrophages and neutrophils, where they are exposed to cell membrane-damaging molecules. The bacterial cell wall, primarily composed of peptidoglycan (PG), maintains cell morphology, structure, and membrane integrity. Intracellular Gram-negative bacteria protect themselves from macrophage and neutrophil killing by recycling and repairing damaged PG – a process that involves over 50 different PG synthesis and recycling enzymes. Here, we identified a PG recycling enzyme, L,D-carboxypeptidase A (LdcA), of F. tularensis that is responsible for converting PG tetrapeptide stems to tripeptide stems. Unlike E. coli LdcA and most other orthologs, F. tularensis LdcA does not localize to the cytoplasm and also exhibits L,D-endopeptidase activity, converting PG pentapeptide stems to tripeptide stems. Loss of F. tularensis LdcA led to altered cell morphology and membrane integrity, as well as attenuation in a mouse pulmonary infection model and in primary and immortalized macrophages. Finally, an F. tularensis ldcA mutant protected mice against virulent Type A F. tularensis SchuS4 pulmonary challenge.

Western-Type Helicobacter pylori CagA are the Most Frequent Type in Mongolian Patients

Helicobacter pylori infection possessing East-Asian-type CagA is associated with carcinogenesis. Mongolia has the highest mortality rate from gastric cancer. Therefore, we evaluated the CagA status in the Mongolian population. High risk and gastric cancer patients were determined using endoscopy and histological examination. H. pylori strains were isolated from different locations in Mongolia. The CagA subtypes (East-Asian-type or Western-type, based on sequencing of Glu-Pro-Ile-Tyr-Ala (EPIYA) segments) and vacA genotypes (s and m regions) were determined using PCR-based sequencing and PCR, respectively. In total, 368 patients were examined (341 gastritis, 10 peptic ulcer, and 17 gastric cancer). Sixty-two (16.8%) strains were cagA-negative and 306 (83.1%) were cagA-positive (293 Western-type, 12 East-Asian-type, and one hybrid type). All cagA-negative strains were isolated from gastritis patients. In the gastritis group, 78.6% (268/341) had Western-type CagA, 2.9% (10/341) had East-Asian-type, and 18.2% (61/341) were cagA-negative. However, all H. pylori from gastric cancer patients possessed Western-type CagA. Histological analyses showed that East-Asian-type CagA was the most virulent strains, followed by Western-type and cagA-negative strains. This finding agreed with the current consensus. CagA-positive strains were the most virulent type. However, the fact that different CagA types can explain the high incidence of gastric cancer might be inapplicable in Mongolia.

Pathogenic Characterization of European Genotype Porcine Reproductive and Respiratory Syndrome Virus Recently Isolated in Mainland China

Background:Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen in pig that causes tremendous economic loss in the global swine industry. PRRSV is divided into the European and North American genotypes, with virulence ranging from apathogenic-moderately virulent to highly pathogenic. The emergence of new highly virulent type 1 strains and coexistence of the two genotypes complicate the differential diagnosis, disease prevention, and control of PRRSV. Although the emergence of a novel type 1 PRRSV strain in mainland China was first confirmed in 2011, there is no information available concerning the pathogenesis of this strain.Objectives:We sought to determine the pathogenesis of a newly emerged Chinese type 1 PRRSV strain HLJB1.Methods:Pigs were infected with HLJB1 and characterized using serological and histopathological tests.Results:HLJB1 infection induced transient chemosis, reddened conjunctiva, skin cyanosis, mild transient pyrexia, dyspnea, and tachypnea between 7 and 13 days post-infection. Gross pneumonic lesions were characterized by multifocal, tan-mottled areas. Lymph nodes and spleen were enlarged. Characteristic microscopic lesions consisted of pulmonary consolidation and alveolar septal thickening with red blood cell infiltration, depletion of splenic lymphocytes, and hyperplasia and activation of macrophage. No pigs infected with HLJB1 died during the experiment.Conclusion:Our findings indicate that Chinese type I PRRSV strain HLJB1 caused classic PRRSV-specific lesions. As it caused lower viremia in pigs compared with other classic type 1 isolates, HLJB1 is less virulent than other type I strains.