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.

Effect of caponization on performance and quality characteristics of long bones in Polbar chickens.

The effect of caponization on the mechanical properties of Polbar chicken bones was the subject of investigation. The biomechanical strength of bones and the risk of their deformation or fracture were determined on the basis of their geometric, structural, material, and densitometric properties. Eight-week-old cockerels of Polbar breed were surgically castrated and then fattened until the 24th week of life. It was observed that caponization did not influence the weight and length of either of the long bones. It did however significantly reduce their ash content and the Ca and P contents in their femur, and P content in their tibia. Dual energy x-ray absorptiometry densitometry revealed that bone mineral content of the mid-diaphyseal part of both bones were reduced. Similarly, the bone tissue density of bones decreased. Caponization influenced the bone's geometric structure increasing the internal and external diameters of the bones. The bone cross-sectional area was greater in capons, and, consequently, the cross-sectional moment of inertia and the radius of gyration significantly increased. However, the relative wall thickness and cortical index were not altered. The three-point bending tests revealed the negative effect of caponization on the mechanical endurance of bones. Yield strength, Young modulus, and yield stress, characterizing the strength of the bone's material, decreased in capons. That suggests a higher risk of permanent deformation as capon bones become less elastic. Concluding, caponization negatively influenced the quality characteristics of Polbar chicken long bones.

Impact of lymphocyte culture media on the number of metaphases and chromosome band resolution.

A special type of differential staining of chromosomes is replication banding. This staining technique reveals the band pattern characteristic of each homologous pair of chromosomes, which is a reflection of heterogeneous euchromatin structure. Banding enables identification of homologous chromosomes and detection of chromosomal aberrations, both structural and numerical. Slide preparation requires knowledge of many techniques, and the procedure is often different for each laboratory. The aim of the study was to determine the effect of selected media for lymphocyte cultures on the number of metaphases and the band resolution of chromosomes. The study was carried out using cell cultures from whole peripheral blood. The slides were stained by the GTG method. After their removal from the water bath they were immersed in trypsin solution, then rinsed in PBS solution and stained in Giemsa solution. After staining they were rinsed again and left to dry. The study confirmed the effect of selected commercially available cell media on the number of metaphases and band resolution of chromosomes, which have not previously been described. In all of the tests performed, the cell culture, fixation, slide preparation (automatic method), staining, and number of reagents were identical.

Activity and Safety of Inhaled Itraconazole Nanosuspension in a Model Pulmonary Aspergillus fumigatus Infection in Inoculated Young Quails.

Pulmonary aspergillosis is frequently reported in parrots, falcons, and other birds held in captivity. Inhalation is the main route of infection for Aspergillus fumigatus, resulting in both acute and chronic disease conditions. Itraconazole (ITRA) is an antifungal commonly used in birds, but its administration requires repeated oral dosing, and the safety margin is narrow. To investigate the efficacy of inhaled ITRA, six groups of ten young quails (Coturnix japonica) were inoculated intratracheally with 5 × 10(6) spores (3 groups) or 5 × 10(7) spores (3 groups). Animals were exposed to nebulized ITRA nanosuspension as 10 % suspension or 4 % suspension, once daily for 30 min, starting 2 h after inoculation for 6 days. Control groups were exposed to nebulized saline for the same period of time. Survival and clinical scores were evaluated, and animals were subjected to gross pathology. In control animals, aspergillosis resulted in systemic disease without pulmonary or air sac granulomas. Animals died from multiple organ failure. Inhalation of 10 % ITRA nanosuspension blocked lethality and prevented disease-related symptoms in the quails exposed to the low dose of spores, while the disease course in quails inoculated with the high-spore dose was retarded. Inhalation of 4 % ITRA nanosuspension was less effective. Both inhalations were well tolerated, and gross pathology did not reveal signs of local toxicity. The data indicate that inhaled administration of 10 % ITRA nanosuspension is capable of alleviating an acute A. fumigatus infection in quails. A lower ITRA concentration may be only active in chronic pulmonary aspergillosis.

Genome organization and DNA methylation patterns of B chromosomes in the red fox and Chinese raccoon dogs.

The molecular structure of B chromosomes (Bs) is relatively well studied. Previous research demonstrates that Bs of various species usually contain two types of repetitive DNA sequences, satellite DNA and ribosomal DNA, but Bs also contain genes encoding histone proteins and many others. However, many questions remain regarding the origin and function of these chromosomes. Here, we focused on the comparative cytogenetic characteristics of the red fox and Chinese raccoon dog B chromosomes with particular attention to the distribution of repetitive DNA sequences and their methylation status. We confirmed that the small Bs of the red fox show a typical fluorescent telomeric distal signal, whereas medium-sized Bs of the Chinese raccoon dog were characterized by clusters of telomeric sequences along their length. We also found different DNA methylation patterns for the B chromosomes of both species. Therefore, we concluded that DNA methylation may maintain the transcriptional inactivation of DNA sequences localized to B chromosomes and may prevent genetic unbalancing and several negative phenotypic effects.

Estimation of global content of 5-methylcytosine in DNA during allantoic and pulmonary respiration in the chicken embryo.

DNA methylation is an epigenetic modification that plays an important role in the proper development and functioning of an organism. The DNA methylation level is species-, tissue- and organelle-specific, and the methylation pattern is determined during embryogenesis. A correlation between methylation and age is also observed. Epigenetic phenomena are an enormously interesting research subject, not only from the perspective of pure science, but also due to their possible applications in medicine. The aim of this study was to determine the global DNA methylation level in relation to the developmental stage of the embryo. The global level of 5-methylcytosine in the DNA during pulmonary respiration was found to be higher than during allantoic respiration. The analysis shows a clear dependence between the stage of individual development and the global DNA level of 5-methylcytosine. In the future, methylation level may be a determinant of age and perhaps even a tool for predicting life expectancy. Abnormalities in the methylation process result in premature ageing at the cellular and individual level.

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.

Polymorphism of cytogenetic markers in wild and farm red fox (Vulpes vulpes) populations.

Analysis of the origin of domestic animals is of wide interest and has many practical applications in areas such as agriculture and evolutionary biology. Identification of an ancestor and comparison with the domesticated form allows for an analysis of genetic, physiological, morphological and behavioral effects of domestication. Because fox breeding has been an ongoing process for over a century, differences are expected between farm and wild populations at the chromosomal level. The aim of this work was to analyse polymorphisms at the chromosomal level in foxes raised on farms and those living in the wild. Blood samples and lung tissue served as the experimental material and were obtained after slaughter of 35 foxes, including 28 breeding animals and 7 wild animals. The classical cytogenetic method was used including AgNOR technique, as well as molecular methods such as fluorescence in situ hybridization (FISH), and primed in situ labeling (PRINS). Analysis of the number of B chromosomes showed the presence of polymorphisms in foxes from both studied populations, but there was no correlation between the number of B chromosomes and the origin and gender of particular animals. An analysis ofactive nucleolar organizers showed the presence of a large number of polymorphisms and a tendency towards reduction of the number of NORs in the captive-raised population.

A model for treating avian aspergillosis: serum and lung tissue kinetics for Japanese quail (Coturnix japonica) following single and multiple aerosol exposures of a nanoparticulate itraconazole suspension.

Aspergillosis is frequently reported in parrots, falcons and other birds held in captivity. Inhalation is the main route of infection for Aspergillus fumigatus, resulting in both acute and chronic disease conditions. Itraconazole (ITRA) is an antifungal commonly used in birds, but administration requires repeated oral dosing and the safety margin is narrow. We describe lung tissue and serum pharmacokinetics of a nanoparticulate ITRA suspension administered to Japanese quail by aerosol exposure. Aerosolized ITRA (1 and 10% suspension) administered over 30 min did not induce adverse clinical reactions in quail upon single or 5-day repeated doses. High lung concentrations, well above the inhibitory levels for A. fumigatus, of 4.14 ± 0.19 µg/g and 27.5 ± 4.58 µg/g (mean ± SEM, n = 3), were achieved following single-dose inhalation of 1% and 10% suspension, respectively. Upon multiple dose administration of 10% suspension, mean lung concentrations reached 104.9 ± 10.1 µg/g. Drug clearance from the lungs was slow with terminal half-lives of 19.7 h and 35.8 h following inhalation of 1% and 10% suspension, respectively. Data suggest that lung clearance is solubility driven. Lung concentrations of hydroxy-itraconazole reached 1-2% of the ITRA lung tissue concentration indicating metabolism in lung tissue. Steady, but low, serum concentrations of ITRA could be measured after multiple dose administration, reaching less than 0.1% of the lung tissue concentration. This formulation may represent a novel, easy to administer treatment modality for fungal lung infection, preventing high systemic exposure. It may also be useful as metaphylaxis to prevent the outbreak of aspergillosis in colonized animals.

Analysis of age-related global DNA methylation in chicken.

DNA methylation is an epigenetic modification that plays an important role in the normal development and function of organisms. The level of DNA methylation is species-, tissue-, and organelle-specific, and the methylation pattern is determined during embryogenesis. DNA methylation has also been correlated with age. The aim of this study was to determine the global DNA methylation levels and their correlation with age in the chicken, using a Polish autosexing chicken breed, Polbar. A quantitative technique based on an immunoenzymatic assay was used for global DNA methylation analysis. The results show increased global DNA methylation levels with older Polbar embryos. Global DNA methylation levels decrease with the age of hens in the postembryonic stage. This study expands the current knowledge of the Polbar epigenome and the general knowledge of the function of epigenetic mechanisms in birds.

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.