Changes in the Level of DNA Methylation in Candida albicans under the Influence of Physical and Chemical Factors.

The effects of physical factors such as radiation (electromagnetic, microwave, infrared, laser, UVC, and X-ray) and high temperature, as well as chemical factors (controlled atmosphere) on the level of global DNA cytosine methylation in C. albicans ATCC 10231 cells were investigated. Prolonged exposure to each type of radiation significantly increased the DNA methylation level. In addition, the global methylation level in C. albicans cells increased with the incubation temperature. An increase in the percentage of methylated DNA was also noted in C. albicans cells cultured in an atmosphere with reduced O2. In contrast, in an atmosphere containing more than 3% CO2 and in anaerobic conditions, the DNA methylation level decreased relative to the control. This study showed that prolonged exposure to various types of radiation and high temperature as well as reduced O2 in the atmosphere caused a significant increase in the global DNA methylation level. This is most likely a response protecting DNA against damage, which at the same time can lead to epigenetic disorders, and in consequence can adversely affect the functioning of the organism.

Genetic differences in variants of the AMD virus at the site of a disease outbreak.

Aleutian Mink Disease Virus (AMDV) results in mink breeding losses due to frequent abortion, low fecundity and high juvenile mortality. Due to the high persistence of pathogen in the environment and lack of causative treatment there is a need for research on alternative methods to eliminate the pathways of the spread of the virus and extinguish current outbreaks. The aim of the study was to investigate molecular variation of AMDV on a farm where mass deaths of mink took place. The material for the research was obtained from a mink farm located in Latvia. Mass deaths had occurred on the farm among symptoms typical of Aleutian disease. Spleen samples were collected from the dead animals during post-mortem examination. Sequencing and bioinformatic analysis made it possible to distinguish the variants occurring in the groups. The presence of the genetic material of the virus was confirmed by PCR and qPCR in each of the spleen samples. The isolates were divided into two main groups: the dominant group A, with more than 83% of all isolates, and group B. Comparison of the variants with the nonpathogenic strain AMDV-G revealed that isolates from group A were more than 95% similar to that strain, whereas the similarity of group B isolates was just over 86%. The average viral load in both groups was 108 copies; no differences in viral load were noted between groups. Testing based on serological analysis produces fairly effective screening results, but these methods do not enable complete elimination of the virus from a population. Only their use in combination with modern testing techniques as tools for identification of vectors and the directions of the spread of the AMD virus can make it possible to block the routes of its spread and to extinguish its current outbreaks.

Genotypes and Variants of BKPyV in Organ Donors after Brain Death.

Kidney transplantation from a donor with latent BKPyV might be the cause of serious complications, such as BK virus-associated nephropathy. The aim of the study was to determine the prevalence of BKPyV infection in donors after brain death (DBDs), to analyse the molecular variation of BKPyV and to compare clinical and inflammation parameters of DBDs infected with various genotypes of BKPyV. BKPyV was investigated in blood and urine samples of 103 DBDs using PCR followed by sequencing and bioinformatic analysis, and the viral load was assessed by qPCR. Clinical parameters, including cellular markers of inflammation were assessed. The results confirm high prevalence of BKPyV (48%),and genotype IV (49%) over genotype I (43%) and the co-infection with genotypes I and IV in 8.2%. Viral load ranged from 102 to 107 copies/mL, with an average of 1.92 × 106 copies/mL. No specific markers for BKPyV infection were detected among the parameters tested. Infection with genotype I may be associated with the adverse impact on thekidney function, while infection with genotype IV was associated with the anemia Not only the viral load but also the genotype of BKPyV may have an impact on the course of infection.

The prevalence of virulence determinants in methicillin-resistant Staphylococcus aureus isolated from different infections in hospitalized patients in Poland.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for hard-to-treat infections. The presence of 19 virulence genes in 120 MRSA isolates obtained from hospitalized patients and genetic relationships of these isolates were investigated. The eno (100%) and ebps (93.3%) genes encoding laminin- and elastin binding proteins, respectively, were ubiquitous. Other adhesion genes: fib (77.5%), fnbB (41.6%), bbp (40.8%), cna (30.8%) encoding proteins binding fibrinogen, fibronectin, bone sialoprotein and collagen, respectively, and map/eap (62.5%), encoding Eap, were also frequent. The etB and etD genes, encoding exfoliative toxins, were present in 15.6% and 12.5% isolates, respectively. The splA, splE and sspA, encoding serine protease were detected in 100%, 70.8% and 94.2% isolates, respectively. The tst gene, encoding toxic shock syndrome toxin-1 was found in 75% isolates. The cna, map/eap and tst genes were the most common in wound isolates and much less common in blood isolates. We identified 45 different spa types, t003 (21.7%) and t008 (18.8%) being the most common. The t003 was the most frequent among isolates from the respiratory tract (35.5%), while t008 in blood isolates (40%). Identification of virulence factors of MRSA is important for evaluation of pathogen transmission rate and disease development.

Molecular Epidemiology and Variation of the BK Polyomavirus in the Population of Central and Eastern Europe Based on the Example of Poland.

The BK polyomavirus (BKPyV) is a widespread pathogen in humans. Polymorphism of the region encoding the VP1 protein of BKPyV provides the basis for classifying the virus into types and subtypes, whose frequency varies depending on geographic location. The aim of our study was to determine the frequency of BKPyV in the Polish population and to assess its variation by analysing polymorphism in the typing region. The study was conducted on 168 healthy, Polish volunteers, whose blood (plasma) and urine were sampled. The virus was detected using PCR, products, sequenced and subjected to bioinformatic analysis. In addition, viral load was assessed by qPCR. The presence of the genetic material of the BK virus was noted in 61/168 urine samples but in none of the plasma sample. Sequencing and phylogenetic analysis confirmed that the BKPyV isolates were of types I and IV, dominant in Europe (63.93% and 36.07%, respectively). All isolates from genotype I belonged to subtype Ib-2, showing polymorphism at position 1809 with a frequency of 61.54% (G1809A) and 38.46% (G1809C). To the best of our knowledge, this is the first study of this magnitude on the genetic variation of BKPyV among healthy volunteers in Poland.

BK Polyomavirus-Biology, Genomic Variation and Diagnosis.

The BK polyomavirus (BKPyV), a representative of the family Polyomaviridae, is widespread in the human population. While the virus does not cause significant clinical symptoms in immunocompetent individuals, it is activated in cases of immune deficiency, both pharmacological and pathological. Infection with the BKPyV is of particular importance in recipients of kidney transplants or HSC transplantation, in which it can lead to the loss of the transplanted kidney or to haemorrhagic cystitis, respectively. Four main genotypes of the virus are distinguished on the basis of molecular differentiation. The most common genotype worldwide is genotype I, with a frequency of about 80%, followed by genotype IV (about 15%), while genotypes II and III are isolated only sporadically. The distribution of the molecular variants of the virus is associated with the region of origin. BKPyV subtype Ia is most common in Africa, Ib-1 in Southeast Asia, and Ib-2 in Europe, while Ic is the most common variant in Northeast Asia. The development of molecular methods has enabled significant improvement not only in BKPyV diagnostics, but in monitoring the effectiveness of treatment as well. Amplification of viral DNA from urine by PCR (Polymerase Chain Reaction) and qPCR Quantitative Polymerase Chain Reaction) is a non-invasive method that can be used to confirm the presence of the genetic material of the virus and to determine the viral load. Sequencing techniques together with bioinformatics tools and databases can be used to determine variants of the virus, analyse their circulation in populations, identify relationships between them, and investigate the directions of evolution of the virus.

The Effects of Prenatal Supplementation with ß-Hydroxy-ß-Methylbutyrate and/or Alpha-Ketoglutaric Acid on the Development and Maturation of Mink Intestines Are Dependent on the Number of Pregnancies and the Sex of the Offspring.

Prenatal and postnatal supplementation with ß-hydroxy-ß-methylbutyrate (HMB) and alpha-ketoglutaric acid (AKG) affects the development and maturation of offspring. Both substances have the potential to stimulate cell metabolism via different routes. However, parity affects development and may alter the effects of dietary supplementation. This study aimed to evaluate the effect of gestational supplementation with HMB and/or AKG to primiparous and multiparous minks on the structure and maturation of the offspring's small intestine. Primiparous and multiparous American minks (Neovison vison), of the standard dark brown type, were supplemented daily with HMB (0.02 g/kg b.w.) and/or AKG (0.4 g/kg b.w.) during gestation (n = 7 for each treatment). Supplementation stopped when the minks gave birth. Intestine samples were collected from 8-month-old male and female offspring during autopsy and histology and histomorphometry analysis was conducted (LAEC approval no 64/2015). Gestational supplementation had a long-term effect, improving the structure of the offspring's intestine toward facilitating absorption and passage of intestinal contents. AKG supplementation affected intestinal absorption (enterocytes, villi and absorptive surface), and HMB affected intestinal peristalsis and secretion (crypts and Goblet cells). These effects were strongly dependent on parity and offspring gender. Present findings have important nutritional implications and should be considered in feeding practices and supplementation plans in animal reproduction.

Detection of mink astrovirus in Poland and further phylogenetic comparison with other European and Canadian astroviruses.

Mink astrovirus infection remains a poorly understood disease entity, and the aetiological agent itself causes disease with a heterogeneous course, including gastrointestinal and neurological symptoms. This paper presents cases of astrovirus infection in mink from continental Europe. RNA was isolated from the brains and intestines of animals showing symptoms typical of shaking mink syndrome (n = 6). RT-PCR was used to amplify astrovirus genetic material, and the reaction products were separated on a 1% agarose gel. The specificity of the reaction was confirmed by sequencing fragment coding RdRP protein (length of sequencing product 170 bp) from all samples. The presence of astrovirus RNA was detected in each of the samples tested. Sequencing and bioinformatic analysis indicated the presence of the same variant of the virus in all samples. Comparison of the variant with the sequences available in bioinformatics databases confirmed that the Polish isolates form a separate clade, closely related to Danish isolates. The dissimilarity of the Polish variant to those isolated in other countries ranged from 2.4% (in relation to Danish isolates) to 7.1% (in relation to Canadian isolates). Phylogenetic relationships between variants appear to be associated with the geographic distances between them. To our knowledge, this work describes the first results on the molecular epidemiology of MAstV in continental Europe. The detection of MAstV in Central Europe indicates the need for further research to broaden our understanding of the molecular epidemiology of MAstV in Europe.

Effectiveness of various methods of DNA isolation from bones and teeth of animals exposed to high temperature.

In the event of fires, natural disasters, and other events associated with high temperature, bones and teeth are the only source of genetic material for identifying human or animal carcasses. To obtain reliable final results of identification tests, the use of appropriate nucleic acid extraction methods is crucial. Therefore, the main objective of this research was to evaluate the effectiveness of selected methods of DNA isolation from animal burnt bones and teeth. In addition, the effect of the duration of high temperature on the stability of nuclear and mitochondrial DNA in these tissues was determined, as well as the possibility of using the genetic material obtained for species identification of remains of unknown origin. Bones and teeth collected during necropsy of dogs were burnt in a laboratory oven at 400 °C (752 °F; 673.15 K) for 5, 10, 15, 30, 45 and 60 min. DNA was isolated according to four different protocols, using three commercial kits, i.e. the PrepFiler® Forensic DNA Extraction Kit from Applied Biosystems, the QIAamp® DNA Investigator Kit from QIAGEN, and the DNA Mini Kit from Syngen, as well as a classic organic method. The effectiveness of these methods was compared by assessing the amount of isolated DNA using Real-Time PCR and its purity using a NanoDrop™ spectrophotometer. Each isolate was also subjected to PCR with primers designed to amplify fragments of dog mitochondrial DNA. The effectiveness of species identification was assessed for the method showing the best DNA recovery and for the organic method, considered the gold standard for analysis of difficult material. The QIAamp® DNA Investigator Kit showed the highest efficiency of DNA isolation from bones and teeth burnt for 15 min (the longest burning time for which DNA could still be recovered from bones and teeth). The results of the experiment clearly indicate that DNA stability in hard tissues depends on how long they burn. In the case of exposure to 400 °C, reliable genetic testing, including species identification, is possible when the burning time does not exceed 15 min. Among the hard tissues examined, bones proved more suitable than teeth for identification purposes. It was also concluded that identification of bone remains with extreme heat damage should be based on mitochondrial DNA analysis.

Breeding parameters on a mink farm infected with Aleutian mink disease virus following the use of methisoprinol.

Aleutian mink disease virus is one of the greatest threats to modern mink farming. The disease reduces fecundity and causes high mortality among kits. The aim of this study was to evaluate the effectiveness of methisoprinol in counteracting the effects of Aleutian disease, both by inhibiting replication of the virus and by mitigating the harmful effects of the disease on the fecundity and weight of infected animals. The study included 300 individuals with confirmed infection, divided according to antibody titres into three experimental groups, which received a 20% methisoprinol solution, and three control groups, which did not receive the immunostimulant. In the mink from the experimental groups, the number of copies of the genetic material of the virus in the spleens and lymph nodes was one order of magnitude lower than in the case of the control groups. Mink receiving the supplement also showed higher fecundity (on average 5.83 in the experimental groups and 4.83 in the control groups), and the weight of their offspring before slaughter was over 200 g higher. Given the lack of effective methods for immunoprophylaxis and treatment, methisoprinol supplementation can be an effective means of counteracting the effects of AMDV on persistently infected farms.

Aleutian Mink Disease Virus in the breeding environment in Poland and its place in the global epidemiology of AMDV.

Aleutian mink disease (AMD) leads to an increase in mortality of animals and causes losses in mink farming. The study investigated the presence of AMDV in tissue and environmental samples from farmed mink in Poland, and selected samples were genetically characterized. Blood, spleens and swabs from the breeding environment were collected on 27 farms in seven voivodeships in Poland (n = 250). DNA was isolated, amplified by PCR and subsequently subjected to sequencing to reveal information on the molecular epidemiology of the samples. A qPCR method was used to determine the viral load in test samples. The presence of AMDV was confirmed in tissues and the farm environment on 26 of the 27 farms. The average viral load in spleens was 108 copies. The virus was also present in the blood (average - 105 copies) and the farm environment (average - 103 copies). Isolates from the West Pomeranian Voivodeship showed high similarity within the voivodeship (over 99%). Variants from the Lublin and Podlaskie Voivodeships differed 5% from any of the AMDV isolates present in the NCBI database. Isolates from the Greater Poland, Pomeranian, Podkarpackie and Lesser Poland Voivodeships formed heterogeneous clades, showing over 97% similarity to variants previously isolated in Poland, the Netherlands and Lithuania. A high degree of genetic variation was identified among the majority of the samples, which indicates that AMDV has been introduced to Poland multiple times. However, the results within one area showed high identity between isolates, suggesting that one common ancestor was the source of these outbreaks.

Molecular markers used in forensic genetics.

Forensic genetics is a field that has become subject to increasing interest in recent years. Both the technology and the markers used for forensic purposes have changed since the 1980s. The minisatellite sequences used in the famous Pitchfork case introduced genetics to the forensic sciences. Minisatellite sequences have now been replaced by more sensitive microsatellite markers, which have become the basis for the creation of genetic profile databases. Modern molecular methods also exploit single nucleotide polymorphisms, which are often the only way to identify degraded DNA samples. The same type of variation is taken into consideration in attempting to establish the ethnicity of a perpetrator and to determine phenotypic traits such as the eye or hair colour of the individual who is the source of the genetic material. This paper contains a review of the techniques and molecular markers used in human and animal forensic genetics, and also presents the potential trends in forensic genetics such as phenotyping.

A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection.

The Aleutian mink disease virus (AMDV) is one of the most serious threats to modern mink breeding. The disease can have various courses, from progressive to subclinical infections. The objective of the study was to provide a comparative molecular characterization of isolates of AMDV from farms with a clinical and subclinical course of the disease. The qPCR analysis showed a difference of two orders of magnitude between the number of copies of the viral DNA on the farm with the clinical course of the disease (105) and the farm with the subclinical course (103). The sequencing results confirm a high level of homogeneity within each farm and variation between them. The phylogenetic analysis indicates that the variants belonging to different farms are closely related and occupy different branches of the same clade. The in silico analysis of the effect of differences in the sequence encoding the VP2 protein between the farms revealed no effect of the polymorphism on its functionality. The close phylogenetic relationship between the isolates from the two farms, the synonymous nature of most of the polymorphisms and the potentially minor effect on the functionality of the protein indicate that the differences in the clinical picture may be due not only to polymorphisms in the nucleotide and amino acid sequences, but also to the stage of infection on the farm and the degree of stabilization of the pathogen-host relationship.

Comparative molecular analysis of strains of the Aleutian Disease Virus isolated from farmed and wild mink.

INTRODUCTION AND OBJECTIVE: Aleutian Disease is a significant biological factor causing substantial losses in mink farming. The virus inducing the disease also infects wild populations which may constitute an asymptomatic reservoir. To compare genetic variants of the AMD virus occurring in wild and farmed mink populations, an analysis was performed on a fragment of the VP2 protein sequence of the virus infecting both populations, taken from different living environments. MATERIAL AND METHODS: Genetic material was isolated from 11 farmed animals in which anti-AMDV antibodies had been detected and from 20 wild animals. The DNA obtained was amplified using primers specific for the fragment encoding the VP2 protein. The product obtained was sequenced and bioinformatic analysis was performed. RESULTS: Viral material was detected in 11 farmed and 7 free-living animals. Similarity of sequences averaged 99% within groups and 94% between groups. The sequencing results made it possible to identify characteristic changes for each group. In the isolates from the wild animals, the following changes were observed in the epitope region with respect to the reference sequence: C3704T, G3710A, T3722C, T3746C and A3749G. In the isolates from the farmed animals a G3779A transition was noted. Phylogenetic analysis showed that the variants infecting the two groups occupy separate branches of the phylogenetic tree. CONCLUSIONS: The variants of the virus infecting the two groups may have a common origin, but at present they constitute two separate groups, with characteristic differences making it possible to recognize their genotype.

Analysis of genomic instability in primary spermatocytes of interspecific hybrids of the red fox (Vulpes vulpes) and the Arctic fox (Alopex lagopus).

The aim of this study was to analyse meiotic cells of male interspecific hybrids of the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus). To this end we determined stages of meiotic cells as well as carried out FISH analyses with probes specific to heterosomes and a TUNEL assay on synaptonemal complex preparations. The meiotic cell analysis revealed only the presence of stages of the first meiotic division from leptotene to pachytene. Moreover, we observed an increased level of early dissociation of the X-Y bivalent as well as a high percentage of apoptotic cells. These results indicate the disruption of meiotic division in male hybrids manifested through meiotic arrest of the cells. Faulty pairing of the heterosomes can be considered as one of the causes leading to the initiation of the apoptotic process.

Comparative cytogenetic analysis of sex chromosomes in several Canidae species using zoo-FISH.

Sex chromosome differentiation began early during mammalian evolution. The karyotype of almost all placental mammals living today includes a pair of heterosomes: XX in females and XY in males. The genomes of different species may contain homologous synteny blocks indicating that they share a common ancestry. One of the tools used for their identification is the Zoo-FISH technique. The aim of the study was to determine whether sex chromosomes of some members of the Canidae family (the domestic dog, the red fox, the arctic fox, an interspecific hybrid: arctic fox x red fox and the Chinese raccoon dog) are evolutionarily conservative. Comparative cytogenetic analysis by Zoo-FISH using painting probes specific to domestic dog heterosomes was performed. The results show the presence of homologous synteny covering the entire structures of the X and the Y chromosomes. This suggests that sex chromosomes are conserved in the Canidae family. The data obtained through Zoo-FISH karyotype analysis append information obtained using other comparative genomics methods, giving a more complete depiction of genome evolution.

Evaluation of genetic biodiversity in farm-bred and wild raccoon dogs in Poland.

The aim of the study was to analyze the intra- and inter-group diversity in farm-raised and wild raccoon dogs with the use of molecular markers. Genetic differences between the particular raccoon dog groups were observed, accompanied by a relatively high intra-group genetic variation. It was noted that the wild raccoon dogs were characterized by the highest genetic diversity, compared to the three study groups of farm-bred raccoon dogs. Wild raccoon dogs and farm-bred raccoon dogs constitute separate phylogenetic groups. The results obtained suggest that farm breeding may lead to differentiation into a different phylogenetic lineage than that of the wild raccoon dogs. In each case, the genetic distance between the animals bred on the individual farms was lower than the distances between the farm-raised and wild animals. Since the Polish farm breeding is based entirely on phenotype ranking, the genotype of "native" animals is still closely related to that of wild animals.