OPTIMIZATION OF IN VITRO SELECTIVE MEDIA FOR SELECTING ANTHRACNOSIS-RESISTANT FLAX CELLS

Цель исследований – оптимизация селективных сред для проведения отбора in vitro каллусных клеток льна, устойчивых к культуральному фильтрату штаммов возбудителя антракноза и создание in vitro новых генотипов, устойчивых к болезни. В результате исследований уточнен состав культурального фильтрата штаммов антракноза. Выявлено, что токсичность культуральных фильтратов не зависела от вирулентности используемых штаммов – более токсичными оказались культуральные фильтраты штаммов 784 (сильновирулентного) и 780 (средневирулентного) (загнивание и отмирание первичных корешков на 5 сутки наблюдали у 67 – 88% проросших семян), менее токсичны – штаммы 793 (сильновирулентный) и 788 (слабовирулентный) (на 5 сутки загнивание и отмирание первичных корешков отмечено у 9 – 15% проросших семян). Установлено, что морфогенные очаги формировались активнее у генотипов, морфогенный каллус которых переносили на среду с аналогичной или более высокой концентрацией культурального фильтрата. Показано, что на 14 сутки во втором пассаже с большей частотой формировались морфогенные каллусы, почки и побеги при использовании в первом и втором пассажах селективной среды, содержащей культуральный фильтрат в концентрации 40 мл/л, или в первом пассаже – 40 мл/л, а во втором – 44 мл/л. Выделены генотипы, сохраняющие устойчивость к антракнозу в течение трёх поколений на уровне 50 – 60%: НО-78 х Ленок, HJI-103-2 х Ленок, НЛ-40-1 х Ленок, HЭ-38 х Росинка, НЭ-36 х Ленок, НЭ-17 х Ленок, HЭ-16-2 х Росинка. Research objective – optimization of selective media for in vitro selection of flax callus cells resistant to culture filtrate of anthracnose pathogen strains and in vitro creation of new disease-resistant genotypes. As a result of the research, the composition of the culture filtrate of anthracnose strains was clarified. It was revealed that the toxicity of cultural filtrates did not depend on the virulence of the strains used - cultural filtrates of strains 784 (highly virulent) and 780 (medium virulent) turned out to be more toxic (decay and death of primary roots on day 5 was observed in 67 - 88% of germinated seeds), less toxic - strains 793 (strongly virulent) and 788 (weakly virulent) (on the 5th day, decay and death of primary roots was noted in 9-15% of germinated seeds). It was found that morphogenic foci were formed more actively in genotypes, the morphogenic callus of which was transferred to a medium with a similar or higher concentration of the culture filtrate. It was shown that on the 14th day in the second passage, morphogenic callus, buds and shoots were formed with a greater frequency when using in the first and second passages a selective medium containing a culture filtrate at a concentration of 40 ml/l, or in the first passage - 40 ml/l, and in the second - 44 ml/l. Genotypes were identified that retain resistance to anthracnose for three generations at a level of 50 - 60%: NO-78 x Lenok, HJI-103-2 x Lenok, NL-40-1 x Lenok, NE-38 x Rosinka, NE-36 x Lenok, NE-17 x Lenok, NE-16-2 x Rosinka.

Genomic Characterization of Streptococcus suis Serotype 24 Clonal Complex 221/234 From Human Patients

Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. Although S. suis serotype 2 is prevalent among patient and swine infections, other serotypes are occasionally detected in humans. Of these, serotype 24 clonal complex (CC) 221/234 are recognized as emerging clones of human infection. Genomic exploration of three S. suis serotype 24 CC221/234 strains revealed antimicrobial resistance genes, pathotyping, virulence-associated gene (VAG) profiles, minimum core genome (MCG) typing, and comparison of the genomes. Based on these analyzes, all three serotype 24 strains were MCG7-3 and should be classified in the intermediate/weakly virulent (I/WV) group. All selected serotype 24 strains were susceptible to several antibiotics including β-lactam, fluoroquinolone, and chloramphenicol. Resistance to tetracycline, macrolide, and clindamycin was observed and attributed to the genes tet(O) and erm(B). Genomic comparison revealed the strains S12X, LSS66, LS0L, LS0E, 92–4,172, and IMT40201 that had phylogenetic affinity with serotype 24 CC221/234. Analysis of 80 virulence-associated genes (VAG) showed that all three serotype 24 strains lacked 24 genes consisting of adhesin P, epf, hyl, ihk, irr, mrp, nadR, neuB, NisK/R, ofs, permease (SSU0835), rgg, revS, salK/R, sao, sly, spyM3_0908, srtBCD, srtF, srtG, SSU05_0473, virA, virB4, and virD4. Eleven specific sequences were identified in the 3 serotype 24 genomes that differed from the genomes of the representative strains of epidemic (E; SC84), highly virulent (HV; P1/7), I/WV (89–1,591), and avirulent (T15 and 05HAS68).

Severe mycobacterium bovis BCG infection in an infant with partial dominant interferon gamma receptor deficiency-a case report

Mendalian susceptibity to mycobacterial disease (MSMD) is a condition caused by selective susceptibility to weakly virulent bacteria in otherwise healthy patients without additional immunological abnormalities. It is an inherited, genetic disorder with variety of clinical presentation. Diagnosis is mandatory because the illness may get precipitated by BCG and other live vaccines. Estimating interleukin in serum can be considered as a diagnostic test. Immunological analysis is mandatory for confirming the diagnosis. Mutation analysis can be done to confirm the mutation and hence, prevent the disease in the next sibling by testing in utero. This condition can be treated with ATT as the first line treatment. If ineffective, can be given other modalities of treatment described. But relapses are common. Stem cell transplantation is the definitive treatment. We describe an infant diagnosed as partial dominant interferon gamma receptor deficiency (IFNGR1) deficiency, who responded to ATT

Leukocytoclastic vasculitis in patients with IL12B or IL12RB1 deficiency: case report and review of the literature

Background Mendelian susceptibility to mycobacterial disease (MSMD) is an inborn error of immunity, resulting in susceptibility to weakly virulent mycobacteria and other intramacrophagic pathogens. Rheumatologic manifestations and vasculitis are considered rare manifestations in MSMD patients. Case presentation In this study, we reported a 20-year-old female who was presented with recurrent lymphadenitis following bacillus Calmette-Guérin (BCG) vaccination and a history of recurrent disseminated rash diagnosed as leukocytoclastic vasculitis (LCV). A slight reduction in lymphocyte subsets including CD4+, CD19+, and CD 16 + 56 T-cell count, as well as an elevation in immunoglobulins level (IgG, IgA, IgM, IgE), were observed in the patient. Whole exome sequencing revealed a homozygous Indel-frameshift mutation, c.527_528delCT (p. S176Cfs*12), at the exon 5 of the IL12B gene. She experienced symptom resolution after treatment with anti-mycobacterial agents and subcutaneous IFN-γ. We conducted a manual literature search for MSMD patients reported with vasculitis in PubMed, Web of Science, and Scopus databases. A total of 18 MSMD patients were found to be affected by a variety of vasculitis phenotypes mainly including LCV and Henoch-Schönlein purpura (HSP) with often skin involvement. Patients were all involved with vasculitis at the median age of 6.8 (2.6–7.7) years, nearly 6.1 years after the initial presentations. Sixteen patients (88.9%) had IL12RB1 defects and concurrent Salmonella infection was reported in 15 (88.2%) patients. Conclusion The lack of IL-12 and IL-23 signaling/activity/function and salmonella infection may be triggering factors for the development of leukocytoclastic vasculitis. IL12B or IL12RB1 deficiency and salmonellosis should be considered in MSMD patients with vasculitis.

Disseminated Mycobacterium simiae Infection in a Patient with Complete IL-12p40 Deficiency

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare group of genetic disorders characterized by infections with weakly virulent environmental mycobacteria (EM) or Mycobacterium bovis bacillus Calmette-Guérin (BCG). Herein, we described the case of a 4.5-yearold boy with protein-losing enteropathy, lymphoproliferation, and candidiasis, who was found to have disseminated Mycobacterium simiae infection. A homozygous mutation in the IL12B gene, c.527_528delCT (p.S176Cfs*12) was identified, responsible for the complete IL-12p40 deficiency. He was resistant to anti-mycobacterial treatment and finally died due to sepsis-related complications.

Bot Gummosis of Lemon (Citrus × limon) Caused by Neofusicoccum parvum

Neofusicoccum parvum, in the family Botryosphaeriaceae, was identified as the causal agent of bot gummosis of lemon (Citrus × limon) trees, in the two major lemon-producing regions in Italy. Gummy cankers on trunk and scaffold branches of mature trees were the most typical disease symptoms. Neofusicoccum parvum was the sole fungus constantly and consistently isolated from the canker bark of symptomatic lemon trees. It was identified on the basis of morphological characters and the phylogenetic analysis of three loci, i.e., the internal transcribed spacer of nuclear ribosomal DNA (ITS) as well as the translation elongation factor 1-alpha (TEF1) and β-tubulin (TUB2) genes. The pathogenicity of N. parvum was demonstrated by wound inoculating two lemon cultivars, ‘Femminello 2kr’ and ‘Monachello’, as well as citrange (C. sinensis × Poncirus trifoliata) ‘Carrizo’ rootstock. In artificial inoculations, the fungus was very aggressive on lemons and weakly virulent on citrange, consistently with symptoms observed in the field as a consequence of natural infections. This is the first report of N. parvum, both in a wide and in a strict taxonomic sense, as a pathogen of lemon in Italy.

USAGE OF CULTURAL FILTRATES IN IN VITRO SELECTION FOR ANTHRACNOSE RESISTANCE

The research was carried out on the basis of the laboratory of selection technologies of the Federal State Budgetary Scientific Institution “Federal Scientific Center of Fiber Crops” (Tver region) in 2018–2020. The aim of the research is in vitro development of new flax genotypes resistant to anthracnose, one of the most harmful fungal diseases. As a result of the research, the composition of the cultural filtrate of the anthracnose causative agent was clarified. It was revealed that toxicity of the cultural filtrates did not depend on the virulence of the strains used in the present studies, the cultural filtrates of strains 784 (highly virulent) and 780 (medium virulent) turned out to be more toxic (decay and death of radicle was observed on the 5th day in 67 - 88% of germinated seeds), less toxic are strains 793 (highly virulent) and 788 (weakly virulent) (decay and death of radicle was observed on the 5th day in 9-15% of germinated seeds). It was found that morphogenic foci were formed more actively in genotypes the morphogenic callus of which was transferred to a medium with a higher concentration of the cultural filtrate; it was shown that in the second passage, when transferring morphogenic calli from a selective medium, which contains 40 ml / L of cultural filtrate on a selective medium also containing 40 ml / L of cultural filtrate, as well as on a selective medium containing 44 ml / L of cultural filtrate, the number of formed morphogenic calli and green buds on the 14th day is significantly higher than in case of transferring on a selective medium containing 36 ml / L of cultural filtrate. Viable regenerant plants were obtained and genotypes were isolated, which retained resistance to anthracnose for three generations at a level of 50 - 60%: NO-78 x Lenok, NL-103-2 x Lenok, NL-40-1 x Lenok, NE-38 x Rosinka, NE-36 x Lenok, NE-17 x Lenok, NE-16-2 x Rosinka.

DEPENDENCE OF PHYTOTOXICITY OF CULTURAL FILTRATES OF THE FLAX ANTHRACNOSE PATHOGEN COLLETOTRICHUM LINI MANNS ET BOLLEY STRAINS ON THE AMINO ACID COMPOSITION

The aim of this work is to determine the amino acid composition of the cultural filtrates of the flax anthracnose fungus Colletotrichum lini Manns et Bolley strains to adjust the concentration of the selective agent in the nutrient medium when creating new flax genotypes resistant to anthracnose in vitro. It was found that the cultural filtrates of strains 527 and 608 contain such amino acids as alanine, glycine, asparagine, cysteine, threonine, aspartic acid, glutamic acid, as well as arginine in the highly virulent strain 527. The traces of tyrosine and lysine in the weakly virulent strain 608 were also found. On the day of cultivation, the supply of nutrients in the cultivation medium was apparently depleted, and the fungus began to use the products of its vital activity for life support. In the culture filtrate of the highly virulent strain 527, the concentration of all certain amino acids was significantly higher than in the culture filtrate of the weakly virulent strain 608. It was shown that the 23-day culture filtrate of the highly virulent strain 527 had the highest toxicity which is lower than in all genotypes taken in the study. The toxicity of the culture filtrate depends on the virulence of the anthracnose pathogen strain. The culture filtrate of a highly virulent strain is more toxic than a weakly virulent one. The presence of cysteine in the culture filtrates of the strains increases the possibility of inhibiting the growth and development of flax cells in in vitro culture. When using the culture filtrate of anthracnose pathogen strains containing asparagine, glutamine, serine, glycine, aspartic and glutamic acids, it is possible to induce the growth and development of flax cells in vitro. As the fungal mycelium grew in the culture filtrates, the concentrations of alanine, asparagine, glycine, aspartic and glutamic acids decreased. Due to the high concentration of cysteine and tyrosine, the culture filtrates of strains 419 and 639 were toxic during the entire study period (up to 42 days).

Forms of bacterial hemorrhagic septicemia course in fisheries of the North Caucasus regions

Summary. Bacterial hemorrhagic septicemia of fish is widespread in the Russian Federation, including in the regions of the North Caucasus. Clinically, the disease is manifested by the presence of separate serous-hemorrhagic sites on the skin, usually in the head and abdomen, inflammation of the fins, ulcerations (abscesses) on the skin, ascites and occasionally protrusion of the anus. The etiological structure of bacterial hemorrhagic septicemia of fish includes a wide range of pathogens belonging to both the Aeromonadaceae family and bacteria of other families: Pseudomonadaceae, Enterobacteriaceae, Streptococcaceae, Bacillaceae, Staphylococccaceae, Moraxellaceae, Flavobacteriaceae, Microavocacteriaceae, Microavocactaeceae. The main causative agents of bacterial hemorrhagic septicemia of fish are opportunistic bacteria of the genus Aeromonas. This disease can occur in three forms: acute, subacute and chronic. The form of the course of bacterial hemorrhagic septicemia of fish depends on the temperature of the water, the species of the fish, the contamination of the reservoir, the amount of oxygen, the density of fish and other factors that reduce the resistance of the fish organism. The incubation period of bacterial hemorrhagic septicemia of fish on average lasts from three to thirty days. Bacterial hemorrhagic septicemia of fish most often occurs in the form of endogenous autoinfection, which is caused by weakly virulent pathogens located in the body of fish - representatives of the normal intestinal microflora. At the acute form of the disease, the death of fish reaches 90-100%, at subacute - up to 75%. The climatic conditions of the regions of the North Caucasus - high year-round water temperature, sharp fluctuations in the temperature of water and air in the winter-spring and autumn-winter periods contribute to the spread of bacterial hemorrhagic septicemia of fish in natural waters.

Patient iPSC-Derived Macrophages to Study Inborn Errors of the IFN-γ Responsive Pathway

Interferon γ (IFN-γ) was shown to be a macrophage activating factor already in 1984. Consistently, inborn errors of IFN-γ immunity underlie Mendelian Susceptibility to Mycobacterial Disease (MSMD). MSMD is characterized by genetic predisposition to disease caused by weakly virulent mycobacterial species. Paradoxically, macrophages from patients with MSMD were little tested. Here, we report a disease modeling platform for studying IFN-γ related pathologies using macrophages derived from patient specific induced pluripotent stem cells (iPSCs). We used iPSCs from patients with autosomal recessive complete- and partial IFN-γR2 deficiency, partial IFN-γR1 deficiency and complete STAT1 deficiency. Macrophages from all patient iPSCs showed normal morphology and IFN-γ-independent functionality like phagocytic uptake of bioparticles and internalization of cytokines. For the IFN-γ-dependent functionalities, we observed that the deficiencies played out at various stages of the IFN-γ pathway, with the complete IFN-γR2 and complete STAT1 deficient cells showing the most severe phenotypes, in terms of upregulation of surface markers and induction of downstream targets. Although iPSC-derived macrophages with partial IFN-γR1 and IFN-γR2 deficiency still showed residual induction of downstream targets, they did not reduce the mycobacterial growth when challenged with Bacillus Calmette–Guérin. Taken together, we report a disease modeling platform to study the role of macrophages in patients with inborn errors of IFN-γ immunity.