Molecular Genetic Techniques Research Articles

Metal-Tolerant Bacteria of Wastewater Treatment Plant in a Large City

Biological treatment methods are the most important part of the treatment process for domestic wastewater, the amounts of which are increasing every year due to rapid, unregulated urbanization and the rising number of people living in such areas. At the same time, microorganisms existing in treatment facilities should not only effectively utilize organic pollutants, but also be resistant to a variety of organic and inorganic contaminants. This study’s objective is to isolate and identify—using molecular genetic techniques—strains of bacteria that tolerate concentrations of heavy metals (Ni, Cd, Pb, Zn and Cu) in the 3–5 mM range. They were sourced from water and sludge samples obtained from sewage treatment facilities in a large city. Outcomes of phylogenetic analysis of 16S rRNA gene sequences revealed that tolerant strains of bacteria belonged to the genera Pseudomonas, Serratia and Klebsiella; strains belonging to the genus Pseudomonas dominated. Of ten resistant strains, nine were isolated from sludge and water samples of the secondary sedimentation tank, and the other one from a treatment plant’s digester. Changes in the color of microorganisms’ colonies became evident when cultivated on media enriched with heavy metals. Cultivating nonpathogenic strains of these bacteria and their introduction into communities of other activated sludge microorganisms could have practical application to biological decontamination of wastewater.

Can molecular genetic techniques improve our understanding of the role the microbial biomass plays in soil aggregation?

AbstractSoil structure plays an integral role in regulating the physical, chemical, and biological functioning of soil systems and is essential for maintaining agricultural productivity. Many studies have investigated soil structure formation and stability. However, the underlying mechanisms behind its formation are still often unclear, including how soil microorganisms regulate this process via the aggregation of soil particles. In this review, we seek to summarise current information regarding how microorganisms influence aggregation and explore whether the application of molecular genetic techniques has potential to increase our understanding in this important area. Specifically, we review current information regarding the exact nature and role of microbially produced soil binding agents (extracellular polymeric substances, glomalin, hydrophobins and chaplins) and how different soil microorganisms (bacteria, archaea, fungi, protists) regulate and influence the production of these substances. Molecular genetic techniques have the capacity to provide new information regarding the genetic make‐up of the soil microbial biomass, which could potentially be related to their function in soil and role in the aggregation of soil particles. However, more work is required to identify the key functional genes important for studying aggregation processes. Techniques better able to study the fine scale distribution of microorganisms and microbial products are also required to fully understand microbial interactions and functioning on a scale relevant to aggregation. Future developments in this area may offer an opportunity to improve our understanding of, and potentially manipulate, soil aggregation for the benefit of soil functioning and environments.

Transgenic Rodent Models in Toxicological and Environmental Research: Future Perspectives

The coexistence of humans and animals has existed for centuries. Over the past decade, animal research has played a critical role in drug development and discovery. More and more diverse animals, including transgenic animals, are used in basic research than in applied research. Transgenic animals are generated using molecular genetic techniques to add functional genes, alter gene products, delete genes, insert reporter genes into regulatory sequences, replace or repair genes, and make changes in gene expression. These genetically engineered animals are unique tools for studying a wide range of biomedical issues, allowing the exhibition of specific genetic alterations in various biological systems. Over the past two decades, transgenic animal models have played a critical role in improving our understanding of gene regulation and function in biological systems and human disease. This review article aims to highlight the role of transgenic animals in pharmacological, toxicological, and environmental research. The review accounts for various types of transgenic animals and their appropriateness in multiple types of studies.

CREB serine 133 is necessary for spatial cognitive flexibility and long-term potentiation

The transcription factor cAMP response element-binding protein (CREB) is widely regarded as orchestrating the genomic response that underpins a range of physiological functions in the central nervous system, including learning and memory. Of the means by which CREB can be regulated, emphasis has been placed on the phosphorylation of a key serine residue, S133, in the CREB protein, which is required for CREB-mediated transcriptional activation in response to a variety of activity-dependent stimuli. Understanding the role of CREB S133 has been complicated by molecular genetic techniques relying on over-expression of either dominant negative or activating transgenes that may distort the physiological role of endogenous CREB. A more elegant recent approach targeting S133 in the endogenous CREB gene has yielded a mouse with constitutive replacement of this residue with alanine (S133A), but has generated results (no behavioural phenotype and no effect on gene transcription) at odds with contemporary views as to the role of CREB S133, and which may reflect compensatory changes associated with the constitutive mutation. To avoid this potential complication, we generated a post-natal and forebrain-specific CREB S133A mutant in which the expression of the mutation was under the control of CaMKIIα promoter. Using male and female mice we show that CREB S133 is necessary for spatial cognitive flexibility, the regulation of basal synaptic transmission, and for the expression of long-term potentiation (LTP) in hippocampal area CA1. These data point to the importance of CREB S133 in neuronal function, synaptic plasticity and cognition in the mammalian brain.

The systematic position of puzzling Sino-Himalayan Lophocoleasikkimensis (Lophocoleaceae, Marchantiophyta) is identified.

Lophocoleasikkimensis, a little-known Sino-Himalayan species, was collected in North Vietnam and its taxonomic position was identified by molecular genetic techniques. The species is characterized by generally narrowly pointed leaves, which are not seen in other representatives of Lophocoleaceae. We found that it belongs to the recently described genus Cryptolophocolea, although it is clearly morphologically dissimilar to other members of the genus. We propose a corresponding nomenclature combination: Cryptolophocoleasikkimensis comb. nov. This species is the only one in its genus with a predominantly Sino-Himalayan distribution; the vast majority of congeners are distributed in the Southern Hemisphere (mostly in Australasia). Reports of this species in Vietnam further confirm the close phytogeographic relationships of the flora of northern Indochina with those of the Sino-Himalayas and suggest that this species is found in other parts of the Hoang Lien Range and the southern Hengduan Range.

MULTIDISCIPLINARY APPROACH IN A PATIENT WITH SD22Q11 DELETION SYNDROME: CASE REPORT

The 22q11.2 deletion syndrome (SD22q11), known as Digeorge syndrome, has a wide phenotypic spectrum with more than 180 clinical signs described in the literature, related to both physical and behavioral aspects, which makes the diagnosis challenging in relation to the condition. The 22q11.2 microdeletion is detected only by molecular genetic techniques such as comparative hybridization of 2 DNA samples. A 13-year-old female patient diagnosed with SD22q11 was referred for specialized dental care for special patients. During anamnesis, heart disease; immunologic, hematologic, neurocognitive, psychiatric disorders; and craniofacial malformation were reported, characteristics consistent with the syndrome in question. Dental procedures, such as extractions and endodontic accesses, were made possible through the medical report of the analysis of systemic conditions, associated with periodic complementary exams. Neurologic management through the administration of carbamazepine and valproic acid was essential to reduce hallucinations while in the dental chair.

Yeast Genomics and Its Applications in Biotechnological Processes: What Is Our Present and Near Future?

Since molecular biology and advanced genetic techniques have become important tools in a variety of fields of interest, including taxonomy, identification, classification, possible production of substances and proteins, applications in pharmacology, medicine, and the food industry, there has been significant progress in studying the yeast genome and its potential applications. Because of this potential, as well as their manageability, safety, ease of cultivation, and reproduction, yeasts are now being extensively researched in order to evaluate a growing number of natural and sustainable applications to provide many benefits to humans. This review will describe what yeasts are, how they are classified, and attempt to provide a rapid overview of the many current and future applications of yeasts. The review will then discuss how yeasts—including those molecularly modified—are used to produce biofuels, proteins such as insulin, vaccines, probiotics, beverage preparations, and food additives and how yeasts could be used in environmental bioremediation and biocontrol for plant infections. This review does not delve into the issues raised during studies and research, but rather presents the positive outcomes that have enabled several industrial, clinical, and agricultural applications in the past and future, including the most recent on cow-free milk.

Assessment of plant species diversity (Lamiaceae Lindle.) in Uzbekistan based on DNA barcoding

Biological diversity preservation is important concept in modern science and environmental policy of Uzbekistan, improving the status of global biodiversity. Flora of Uzbekistan accounts over 4380 species of vascular plants. 378 taxa representing approximately 9% of currently recognized species diversity of Uzbekistan are considered as a national endemics. The present study aimed at species identification and inventory of wild, rare and endangered plant species based on molecular genetic techniques. For effective species identification and high results reliability, the genotyping of three-four loci of the combinations nuclear and chloroplast DNA-barcodes was performed for all studied species. The genotyping of marker sequences ITS, rbcL, trnL-trnF, matK for Dracocephalum adylovii I.I. Malzev is conducted the first time in Uzbekistan. The reliable similarity in the BLAST database is determined at the genus taxonomic level. 78 consensus nucleotide sequences were obtained for Lamiaceae species. The results of molecular phylogenetic analysis based on marker sequence encoding the matK fragment are presented for representative of three subfamilies: Lamioideae, Nepetoideae, Scutellarioideae recorded in the flora of Uzbekistan. Currently, the solution of biological diversity preservation is global. The possibility of successful application of DNA-barcoding for ecological monitoring is demonstrated.

Prenatal diagnosis and molecular cytogenetic characterization of an inherited microdeletion of chromosome 16p11.2.

Copy number variations (CNVs) in chromosome 16p11.2 (deletions and duplications) are not rare. 16p11.2 microdeletion is among the most commonly known genetic etiologies of autism spectrum disorder, overweightness, and related neurodevelopmental disorders. Here, we report the prenatal diagnosis and genetic counseling of a maternally inherited 16p11.2 microdeletion. In this family, the mother and fetus both have a normal phenotype and the same microdeletion. Following the use of molecular genetic techniques, including array-based methods, the number of reported cases has rapidly increased. The combination of prenatal ultrasound, karyotype analysis, chromosomal microarray analysis (CMA), and genetic counseling is helpful for the prenatal diagnosis of chromosomal microdeletions/microduplications.

Investigation of genetic polymorphism of Russian rape and turnip rape varieties using SSR and SRAP markers.

Rapeseed (Brassica napus L.) and turnip rape (B. rapa L. subsp. campestris (L.)) are important agricultural plants widely used for food, fodder and technical purposes and as green manure. Over the past decades, a large number of perspective varieties that are being currently cultivated in every region of Russia have been developed. To increase the breeding eff iciency and facilitate the seed production, modern molecular-genetic techniques should be introduced as means to estimate species and varietal diversity. The objective of the presented research study was to investigate DNA polymorphism of the rapeseed and turnip rape varieties developed at Federal Williams Research Center of Forage Production and Agroecology and detect informative markers for varietal identif ication and genetic certif ication. To genotype 18 gDNA samples, 42 and 25 combinations of respective SSR and SRAP primers were used. The results obtained demonstrate that SRAP markers were more effective for polymorphism analysis: 36 % of the tested markers revealed genetic polymorphism compared with only 16.7 % of microsatellite loci. Molecular markers to detect differences at interspecif ic and intervarietal levels have also been found. For the investigated set, such microsatellite loci as Na12A02, Ni2C12, Ni02-D08a, Ra02-E01, Ni03H07а and SRAP-marker combinations as F13-R9, Me4- R7, F11-Em2, F10-R7, F9-Em2 and F9-R8 proved to be informative. Application of the two marker techniques made it possible to detect a higher level of DNA polymorphism in plants of different types (spring and winter varieties) if compared against the intervarietal differences within a species or a group. According to Nei’s genetic diversity index, in the cluster of winter rapeseed, VIK 2 and Gorizont varieties had the longest genetic distance, and in the spring cluster, these were Novosel and Veles. A high level of similarity was found between Vikros and Bizon winter rapeseed varieties. The results obtained have a high practical value for varietal specif ication of seed material and genetic certif ication of rapeseed and turnip rape varieties.

Ceratium furcoides (Levander) Langhans in reservoirs at the Ebro watershed, Spain and Sao Paulo state, Brazil

This study compares the morphological characteristics of the dinoflagellate Ceratium furcoides from three Ebro basin reser¬voirs (Spain) and from six reservoirs at São Paulo state (Brazil) with the aim to identify the distinct morphotypes of Ceratium furcoides in Spain and Brazil. We studied the physical and chemical variables to determine the trophic state of each reservoir. Unlike Ceratium hirundinella, Ceratium furcoides is not a frequent species within the plankton community at Ebro Basin reservoirs, however, this species has been responsible of one monospecific bloom at El Val reservoir. Nevertheless, both Ce-ratium species are considered invasive in South America. Ceratium furcoides has displaced C. hirundinella at São Paulo state reservoirs, reaching high densities, to the extent of forming monospecific blooms. Morphometric analysis was performed by optic and electronic microscopy (DIC and SEM respectively). The analysis shows significant differences in: i) total cell length, ii) horns length and iii) presence or absence of a fourth horn. While the microscopic study allows differentiating morphotypes according to the reservoir typology, a deeper study using molecular genetic techniques on the different populations of C. fur¬coides would allow to know better the relationship between the dinoflagellate dynamics and the reservoirs trophic states.

Prevalence of bacterial infections and the use of multiplex PCR assay for rapid detection of pathogens in cultured fish in Ghana.

The modern and rapid avenue for detecting pathogens provided by molecular genetic techniques including polymerase chain reaction (PCR) was explored in the present study to identify prevalent disease pathogens, from six aquaculture farms and in two commonly cultured fish in Ghana. The specific detection was carried out directly on clinical samples of naturally infected fish (O. niloticus and C. gariepinus) based on syber-mix reaction protocol in traditional PCR. Molecular diagnostic techniques allowed the detection of the six most common and important bacterial pathogens in aquaculture farms in Ghana. Also, three of the pathogens (Streptococcus agalactiae, Streptococcus iniae, and Staphylococcus aureus) were simultaneously isolated in a multiplex reaction. The results indicated 90-100% sensitivity and specificity for each of the six bacterial pathogens tested. Streptococcosis and motile aeromonad septicemia were found to be highly prevalent in most aquaculture farms in Ghana with severity in infections traced to the 85.7% and 14.9% co-infections with all six target pathogens in catfish and tilapia respectively. The prevalence rate of infections significantly correlated with variations in salinity, conductivity, and dissolved oxygen concentrations in the thermal stressed condition of the culture water. Multiplex techniques employed in this study represent one of the first to be used by a fish health laboratory in Ghana for rapid detection of pathogens in diseased fish and could be a useful alternative to the culture-based method for routine diagnosis of fish diseases in Ghana.

Influence of intestinal dysbacteriosis on the course of pregnancy

BACKGROUND: Due to the active development of molecular genetic diagnostic techniques, much attention is now being paid to the study of intestinal microflora. A number of studies have demonstrated the major influence of the intestinal microbiota on the course of pregnancy. However, the number of publications devoted to this problem is limited.
 AIM: The aim of the investigation is to assess the qualitative and quantitative composition of the intestinal microflora in pregnant women.
 MATERIALS AND METHODS: To achieve this goal, 200 pregnant women in the age group 18 to 43 years and with a gestational age of 6 to 22 weeks were examined. A quantitative and qualitative analysis of the microbiocenosis of the vagina and rectum was performed using the real-time polymerase chain reaction method.
 RESULTS: The results of the study demonstrated the presence of intestinal dysbacteriosis in 100% of pregnant women. Grade I dysbacteriosis was diagnosed in 64.5% of cases, grade II in 26.5% and grade III in 9%. In patients with moderate to severe dysbacteriosis, opportunistic pathogens Enterobacter spp., Clostridium difficile, Campylobacter spp. and Streptococcus spp. in concentrations exceeding formally permissible values. High quantitative indicators of clinically significant opportunistic pathogens in the vaginal biotope were detected amid the decrease in normal flora and a high concentration of opportunistic pathogens in the intestinal biocenosis. Grade II and III intestinal dysbiosis should be considered as a risk factor for the formation of a complicated course of the gestational period in the early stages.
 CONCLUSIONS: Thus, pregnancy is a predisposing factor to the formation of dysbiotic changes in the intestine. In turn, rational management of intestinal dysbacteriosis can contribute to the correct course of the gestational period in the early stages.

Методика пренатальной молекулярно-генетической диагностикиврожденной гиперплазии коры надпочечников, обусловленной дефицитом 21-гидроксилазы

Цель: представление метода пренатальной молекулярно-генетической диагностики (ПД) врожденной гиперплазии коры надпочечников (ВГКН) с использованием анализа частых патогенных вариантов, косвенной диагностики и анализа пола. Методы. Молекулярно-генетические исследования выполнены на образцах ДНК, выделенных из образцов цельной крови и ворсин хориона. Стратегия исследования основана на методах из литературных источников, собственных модификациях и разработках. Исследование включало анализ частых патогенных вариантов гена CYP21A2 (P30L, IVS2AS A/C-G, -13, 8 bp del, I172N, V281L, Q318X, R356W, P453S), перестройки между геном и псевдогеном (делеция 30 т.п.н.), косвенных STR-маркеров (TNFa, D6S273, D6S2781, LH1, D6S1666, D6S2443) и пола плода (SRY и AMELX/AMELY). Базовым методом для всех этапов служила полимеразная цепная реакция (ПЦР). При анализе точковых мутаций гена CYP21A2 использовали подход двухраундовой ПЦР и последующего рестрикционного анализа. Для детекции применяли методы гель-электрофореза и фрагментного анализа на генетическом анализаторе. Результаты. В нашем исследовании детально описано пренатальное молекулярно-генетическое исследование ВГКН с особенностями всех этапов тестирования. В семье, планирующей ПД и имеющей ребенка с сольтеряющей формой ВГКН, проводилось предварительное молекулярно-генетическое обследование. У больного ребенка выявлен компаундный генотип с патогенными вариантами IVS2AS A/C-G, -13 материнского происхождения и комплексным мутантным аллелем P30L + 8 bp del + IVS2AS A/C-G, -13 отцовского происхождения. В образце плода нами выявлен гетерозиготный генотип с патогенным материнским вариантом во втором интроне. Исследованные полиморфные STR-локусы, фланкирующие ген CYP21A2, подтвердили нормальный отцовский и патогенный материнский гаплотипы у плода. Дополнительно данные по STR свидетельствовали об отсутствии контаминации материала плода материнской ДНК. У матери неинформативными (гомозиготными) были два микросателлитных маркера, у отца информативны были все шесть. Заключение. Предложенная нами система молекулярно-генетического тестирования, включающая анализ патогенных вариантов в сочетании с косвенной диагностикой по STR-маркерам и анализом пола позволяет обеспечить надежный вариант ПД ВГКН. Aim: to present a technique for prenatal molecular genetic diagnosis (PD) of congenital adrenal hyperplasia (CAH), including test of common pathogenic CYP21A2 variants, linkage microsatellite markers and sex markers analysis. Methods. DNA samples were isolated from whole blood samples and chorionic villi sample. The research based on methods from literature and own developments. The assay included the analysis of common mutations in the CYP21A2 gene (P30L, IVS2AS A/C-G,-13, 8 bp del, I172N, V281L, Q318X, R356W, P453S), gene-pseudogene rearrangement (deletion of 30 kb), STR markers (TNFa, D6S273, D6S2781, LH1, D6S1666, D6S2443) and sex markers (SRY and AMELX/AMELY). PCR was baseline method for all steps. We performed a nested PCR approach and subsequent restriction analysis for point mutations of the CYP21A2 gene. Methods of gel electrophoresis and fragment analysis on a genetic analyzer were used for detection. Results. Our study describes the detailed approach of prenatal molecular genetic investigation of CAH. Preliminary molecular genetic examination of a family with a salt-wasting form of CAH child was carried out. A compound genotype with pathogenic maternal variants IVS2AS A/C-G, -13 and a complex mutant paternal allele P30L + 8 bp del + IVS2AS A/C-G, -13 was revealed in the affected child. In the fetal sample we identified a heterozygous genotype with a pathogenic maternal allele of the 2nd intron. The polymorphic microsatellite markers flanking the CYP21A2 gene confirmed the normal paternal and pathogenic maternal haplotypes in the fetus. Additionally STR data indicated that there was no contamination of maternal DNA fetal material. The mother was uninformative (homozygous) with two microsatellite markers, the father was informative with all six. Conclusions. We devised an improved stepwise molecular diagnostic strategy includes the direct analysis of pathogenic variants in combination with indirect diagnostics by STR markers and sex analysis for reliable PD of CAH.

Editorial

Editorial

Thomas Michael Jessell. 2 August 1951—28 April 2019

Tom Jessell was a British neuroscientist who made pioneering discoveries in developmental neurobiology. Beginning with a neuropharmacology training in London and Cambridge, Tom moved to Harvard and finally Columbia University. There, Tom embraced the dramatic late twentieth-century developments in molecular biology to unravel the genetic basis of spinal cord neuronal diversity and developmental mechanisms of circuit formation. Combining encyclopaedic knowledge of the classical neurobiology literature with virtuoso molecular genetic, anatomical and electrophysiological techniques, exploited by his team of gifted students, post-docs and collaborators, Tom made seminal contributions to broad areas of neuroscience. Notable contributions included the discovery of extracellular factors that induce distinct neuronal cell types and guide their connectivity, as well as the identification of the transcriptional repertoires that led to neuronal complexity in the developing spinal cord. Important insights into circuitry and behaviour followed these defining studies. At the same time, Tom was heavily involved in neuroscience education and the development of new neuroscience research institutes. For all his Herculean energy for research, he still found great pleasure in fine art, cricket and jazz, and his life was greatly enriched by his partner Jane Dodd, also a Cambridge-trained neuroscientist, and their three daughters. Tom's wit, energy and incisive intelligence will be sorely missed. His legacy is not only a paradigm-shifting body of papers, but also his educational and institutional achievements and the remarkable group of scientists, once his trainees, who, in their turn, continue to advance neuroscience research around the world.

Verification of a cryptic t(Y;15) translocation in a male with an apparent 45,X karyotype

BackgroundA rare disease is that an individual with a non-chimeric karyotype of 45,X develops into a male. We explored the genetic aetiology of an infertile male with an apparent 45,X karyotype, which was subsequently verified as cryptic translocation between chromosomes Y and 15.MethodsDNA was extracted from the patient's peripheral blood. A range of genetic testing was performed, including conventional chromosomal karyotyping, short tandem repeat (STR) analysis for azoospermia factor (AZF) region, fluorescence in situ hybridization (FISH) with specific probes groups of DXZ1/DYZ3, DYZ3/D15Z1/PML and SRY/D15Z1/PML, and chromosomal microarray analysis (CMA) for genomic copy number variations (CNVs).ResultsThe patient was found to have an apparent 45,X karyotype. STR analysis showed that he possessed a short arm of the Y chromosome, including the SRY gene; however, he was missing the long arm of the Y chromosome, including AZFa + b + c and Yqter. A FISH assay of DXZ1 and DYZ3 probes showed a green signal of the X centromere and a red of the Y centromeric signal on a D-group-sized chromosome. By FISH assaying with D15Z1 and DYZ3 probes, chromosomes 15 and Y centromeric signals appeared closely on a single chromosome, as the PML control probe ascertained. A further FISH assay with D15Z1 and SRY probes revealed a signal of the SRY gene at the end of one arm of chromosome 15. The result of the CMA indicated a deletion with an approximate size of 45.31 Mb spanning from Yq11 to Yter.ConclusionOur study enriched the karyotype-phenotype correlation of Y and 15 chromosomes translocation. It strengthened the critical roles of molecular genetic techniques in identifying the chromosomal breakpoints and regions involved. Genetic aetiology can guide early intervention in childhood and assisted reproduction in adulthood.

Axonemal Symmetry Break, a New Ultrastructural Diagnostic Tool for Primary Ciliary Dyskinesia?

Diagnosis testing for primary ciliary dyskinesia (PCD) requires a combination of investigations that includes study of ciliary beat pattern by high-speed video-microscopy, genetic testing and assessment of the ciliary ultrastructure by transmission electron microscopy (TEM). Historically, TEM was considered to be the “gold standard” for the diagnosis of PCD. However, with the advances in molecular genetic techniques, an increasing number of PCD variants show normal ultrastructure and cannot be diagnosed by TEM. During ultrastructural assessment of ciliary biopsies of patients with suspicion of PCD, we observed an axonemal defect not previously described that affects peripheral doublets tilting. To further characterize this defect of unknown significance, we studied the ciliary axonemes by TEM from both PCD-confirmed patients and patients with other sino-pulmonary diseases. We detected peripheral doublets tilting in all the PCD patients, without any significant difference in the distribution of ciliary beat pattern or mutated gene. This defect was also present in those patients with normal ultrastructure PCD subtypes. We believe that the performance of axonemal asymmetry analysis would be helpful to enhance diagnosis of PCD.

The use of internal transcribe spacer (ITS) as a molecular diagnostic tool for understanding variation among population of Metarhizium spp.

Direct analysis of DNA polymorphisms now is a general approach to identify and compare fungi at intraspecific, species, genus, or higher level. In this research using molecular genetic techniques with RAPD-PCR technique with ITS1 and ITS2 as promers were used. The samples are Metrahizium from Indonesia (M4 and M5) and Philippines (M6) using RAPD-PCR amplification, DNA sequencing and Analysis of ITS DNA sequences and ITS DNA sequence homologous alignment by search similarities used Blast Biological Software and multi-aligned with CLUSTALW Biological Software. It confirmed three isolates are: M. anisopliae and by multiple sequence alignment revealed transmission translation and transversion and deletion among them. The phylogram showed biogeographic area and their virulence.

Prevalence and molecular characterization of Cryptosporidium sp. in pigs in Northwestern Russia

Cryptosporidiosis is a widespread parasitic disease of many species of domestic and wild animals, as well as humans, which is a significant problem in the field of veterinary medicine and medicine. Farm animals, in particular piglets, are most often susceptible to this disease, however, the species composition of representatives of the genus Cryptosporidium in this animal species in the Russian Federation has remained unknown to this day. For the first time in the Russian Federation, in the conditions of the North-West, on the example of the Vologda region in pig farms with industrial technology of maintenance, as well as in private farms using the latest molecular genetic techniques, namely, using high-performance sequencing of amplicon libraries of fragments of the 18S rRNA gene obtained as a result of nested (nested) PCR, we have established the parasitism of C. scrofarum in all age groups of the examined animals. The infection rate of animals kept in pig farms was 34% (51/150), in farms – 32.4% (81/250). The most susceptible to infection are animals that are fattening at the age of 13-24 weeks.