DHA upregulates FADS2 expression in primary cortical astrocytes exposed to vitamin A.

The fads2 gene encoding delta6-desaturase, the rate-limiting enzyme of the LCPUFA biosynthesis is expressed in astrocytes. Dietary fatty acids, which cross the blood-brain barrier, may regulate the transcription of lipogenic enzymes through activation of transcription factors such as peroxisome proliferator-activated receptors (PPARs). The PPARs form the transcription complex with retinoid X receptors (RXRs) that are activated by 9-cis retinoic acid, a metabolite of vitamin A (VA). The study examines whether challenge of astrocytes with VA, prior 24-h treatment with palmitic acid (PA), alpha-linolenic acid (ALA) or docosahexaenoic acid (DHA) has the effect on the FADS2 expression. RT-qPCR showed that in astrocytes not challenged with VA, PA increased fads2 gene expression and DHA decreased it. However, in VA-primed astrocytes, PA doubled the FADS2 mRNA levels, while DHA increased fads2 gene expression, oppositely to non-primed cells. Furthermore, similar changes were seen in VA-primed astrocytes with regard to delta6-desaturase protein levels following PA and DHA treatment. ALA did not have any effect on the FADS2 mRNA and protein levels in either VA-primed or non-primed astrocytes. These findings indicate that in the presence of vitamin A, DHA upregulates fads2 gene expression in astrocytes.

Evaluation of growth conditions and DNA extraction techniques used in the molecular analysis of dermatophytes.

AIMS: Recent molecular methods for diagnosis of superficial mycoses have determined the need for a rapid and easy method of extracting DNA. The aim of study was to determine growth conditions and techniques of DNA extraction for Microsporum canis, Trichophyton mentagrophytes and T. verrucosum. METHODS AND RESULTS: Samples were prepared of each of the DNA extraction methods (phenol-chloroform, CTAB and four different kits) for all of the incubation periods (4, 7 and 10 days) of the cultures on the solid and in the liquid medium. The highest DNA concentrations were obtained using the phenol-chloroform method. The concentration of DNA extracted with the CTAB method accounted for 62·21%, for kits it corresponded from 35·53 to 15·41%. The analysis of the DNA weight yield revealed the highest isolation efficiency of the phenol-chloroform method, 1 mg of mycelium yielded 223·8 µg DNA. Lower DNA yield (by 39·32%) was obtained with the CTAB method; in the case of kits by 68·46-85·32%. In most of the techniques, the DNA yield on the solid medium was higher. CONCLUSION: In summary, the highest DNA yield was noted in the 7-day cultures and extraction with the phenol-chloroform method. Importantly, the type of culture was not relevant for the diagnostic result. SIGNIFICANCE AND IMPACT OF THE STUDY: Most mycoses are caused by fungi that reside in nature. The severity of the infection depends on the pathogenic attributes, socioeconomic factors and local environmental conditions. Recent diagnosis increasingly relies on not only the clinical features. Molecular identifications have determined the need for a rapid and easy method of extracting DNA. Usually two factors have to be considered: maximize the DNA yield and ensure that the extracted DNA is susceptible to enzymatic reactions. These data suggest that phenol-chloroform methods and a 7-day culture period may be useful for validation and constitute the first step of molecular diagnosis of dermatophytes.

Antimicrobial activity of some plant extracts against bacterial pathogens isolated from faeces of red deer (Cervus elaphus).

Antibacterial activity is the most widely studied aspect of plant extracts. Antibiotics extensively produced and consumed in large quantities, have proved to be problematic due to various types of adverse effects. The development of bacterial resistance to currently available antibiotics has necessitated the search for new antibacterial agents. One of the alternative strategies for fighting antibiotic- resistant bacteria is the use of natural antimicrobial substances such as plant extracts. We tested the antimicrobial activity of nine extracts from different plants against pathogenic bacteria isolated from the faeces of red deer (Cervus elaphus). Selected bacteria commonly contaminated the natural environment and constitute a source of infection in other animals and humans. Extracts obtained from the following plants were tested: Hypericum perforatum L., Chamomilla recutita L., Achillea millefolium L., Salvia officinalis L., Thymus vulgaris L., Pinus sylvestris L., Mentha x piperita L., Valeriana officinalis L. and Foeniculum vulgare Mill. The highest degree of antibacterial properties was observed for Mentha x piperita L., narrower spectrum of activity possessed Hypericum perforatum L. Extracts of Achillea millefolium L. had the lowest spectrum of antibacterial activity. Our study confirms that many plant extracts shows in vitro antibacterial activity.

Experimental studies of microbial populations and incidence of zoonotic pathogens in the faeces of red deer (Cervus elaphus).

UNLABELLED: Wild animals can serve as hosts, amplifiers or reservoirs for various zoonotic diseases. Most species of deer in highly fragmented agricultural landscapes, search out maximum cover from intrusive human activity. Hence, the likelihood of zoonosis transmission is likely to increase the more humans and wildlife interact. In our study, we conducted a comparative analysis of bacteria isolated from the faeces of red deer (Cervus elaphus) living in their natural environment in south-western Poland and brought in from Hungary and Slovakia under a species reintroduction programme. The faecal bacterial flora from 120 specimens of deer were examined, with particular attention to potentially pathogenic agents. We isolated 458 micro-organisms, of which 13 (2·84%) were identified as EHEC (Enterohaemorrhagic Escherichia coli) strains, and of these one strain, produced the Shiga toxin. No strain was identified as having ESBL (Extended-Spectrum Beta-Lactamase) resistance. Other bacteria that are important in terms of the health of humans and animals included Yersinia enterocolitica (4, 0·67%) and Staphylococcus aureus (4, 0·67%), but without methicillin resistance, and Listeria monocytogenes (8, 1·75%). Of all the micro-organisms 138 (30·13%) were bacteria of the genus Enterococcus, including 12 (2·62%) of the species Enterococcus faecium. The results of the study indicate that red deer may play an important role in the environmental maintenance of zoonotic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: A particularly important factor in the epidemiology of bacterial infections is the introduction of pathogens posing a risk to other animals and humans into the soil, plants and especially water, as contaminants together with faeces. Our study presents screening of potentially pathogenic bacteria in different populations of deer that were displaced under reintroduction programmes. Based on our own research and the literature data, it seems that wild ruminants play an important role in the maintenance of zoonotic pathogens and information about zoonoses from red deer will become increasingly important as deer populations continue to grow, especially in Europe.

The genetic analysis of new Polish strains of European brown hare syndrome virus.

In this paper we describe recently occurring outbreaks of European brown hare syndrome (EBHS) in a captive hare population. The aim of our study was to evaluate the phylogenetic position of detected Polish strains compared to other European strains of EBHSV. Investigations were undertaken in hares from different provinces of Poland. Liver or spleen samples were tested for viral RNA using the RT-nested PCR method and the products were subsequently sequenced. The genetic analysis was based on the fragment of gene encoding viral capsid protein; it revealed a high homology and close relationship between Polish and European EBHSV strains isolated between 2001 and 2011.

Phylogenetic analysis of canine parvovirus CPV-2 strains and its variants isolated in Poland.

Canine parvovirus disease appeared in the world and in Europe during the second half of the 1970s. Over the course of 40 years the original CPV-2 strains mutated and variants 2a, 2b and 2c appeared. Their appearance is connected with specific amino acid changes, mainly in the capsid protein VP2. Strains isolated by the authors were adapted for in vitro cell culture. Phylogenetic analysis revealed differences between strains isolated in Poland in 1982-1985 and in 1995-2009. Strains from the 1980s were shown to belong to variant CPV-2a (11 strains) and variant 2b (2 strains), while no fundamental differences were found among the genetic profiles of the strains from 1995-2009, which were classified as belonging to variant 2c.

Various dietary fats differentially change the gene expression of neuropeptides involved in body weight regulation in rats.

Various high-fat diets are obesogenic but not to the same extent. The aim of the present study was to investigate the effects of saturated fat n-6 and n-3 polyunsaturated fatty acids (PUFAs) on the central neuropeptidergic system in adult rats. Using reverse transcriptase-polymerase chain reaction and in situ hybridisation, we evaluated the net effect of feeding in these fats, comparing the effects of a high- to low-fat diet, and the diversity of the effects of these fats in the same amount within the diet. We also determined plasma lipids, glucose, insulin and leptin concentrations. Six-week feeding with high-saturated fat evoked hyperpahagia and the largest weight gain compared to both high-PUFA diets. Rats fed high-saturated fat were found to have decreased neuropeptide Y (NPY) mRNA expression in the arcuate nucleus (ARC) and the compact zone of the dorsomedial nucleus (DMHc), unchanged pro-opiomelanocortin (POMC), galanin-like peptide (GALP) mRNA expression in the ARC, as well as melanin-concentrating hormone (MCH) and prepro-orexin (preORX) mRNA expression in the lateral hypothalamus, compared to low-saturated fed rats. By contrast, feeding with both high-PUFA diets increased POMC and GALP mRNA expression in the ARC compared to the corresponding low-fat diet and the high-saturated fat diet. Furthermore, feeding with both low-PUFA diets reduced NPY mRNA expression compared to the low-saturated fat diet exclusively in the DMHc. Uniquely, the high n-3 PUFA feeding halved MCH and preORX mRNA expression in the lateral hypothalamus compared to the other high-fat and low n-3 PUFA diets. In rats fed three high-fat diets, plasma insulin and leptin concentrations were significantly increased and the type of fat had no effect on these hormone levels. Rats fed high-saturated fat had both hyperglycaemia and hypertriacylglycerolemia and rats fed high n-3 PUFA only had hyperglycaemia. The present study demonstrates that various forms of dietary fat differentially change the expression of neuropeptide genes involved in energy homeostasis.

Evaluation of the toxicity of substituted benzthioanilides by using in vitro tests.

The cytotoxicity of 12 benzthioanilides substituted in the N-aromatic ring, and of two commercial preparations (imaverol and thiuram) for comparison, was studied with clone 81 cat cells, by determining the highest tolerated dose, and by using the neutral red uptake assay and the kenacid blue assay for total protein. The concentrations that induced 20%, 50% and 80% (IC20, IC50 and IC80) inhibition relative to controls were calculated from dose-response curves. For some compounds, rat LD50 values were also determined. All the benzthioanilide preparations showed in vitro toxicities lower than those of the fungicides imaverol and thiuram. It was confirmed that the cytotoxicities of the compounds depend on the type of substituent. The least toxic compound contained a CONHCH(2)CO(2)H substituent in the para position of the N-aromatic ring, and the most toxic compounds contained chloro and fluoro, or three chloro substituents in the anilide moiety. All the benzthioanilides tested showed fungistatic activity for dermatophytes; two of the compounds (compound 5 and compound 12) also inhibited the development of yeasts at concentrations lower than those which caused toxicity in vitro. The LD50 values and the cytotoxic concentrations in vitro were linearly related.

Increase of gonadotropin-releasing hormone concentration in pituitary portal blood after substance P administration in male rats.

Substance P (SP) affects gonadotropin release from the anterior pituitary gland. In the present study we tested whether SP exerts this effect through GnRH release into pituitary portal blood in intact male rats (INT), orchidectomized rats with s.c. chronically implanted empty Silastic capsule (ORCX), testosterone capsule (ORCX + T), and 17beta-estradiol capsule (ORCX + E2). The pituitary glands were exposed by the transpharyngeal approach under urethane-chloralose anesthesia. Then, the stalk portal vessels were cut and three 30-min portal blood samples were collected. Each first sample of blood was treated as a control before 0.2 ml injection of normal saline, 5 microg, or 25 microg of SP in 0.2 ml of normal saline into the internal carotid artery. GnRH concentration in the purified portal plasma were measured by RIA. Injection of SP into the internal carotid artery caused a significant increase in GnRH concentration in pituitary portal plasma only in INT rats. The higher dose of SP markedly increased GnRH concentration in the 1st blood sample (p < 0.001) and in the 2nd blood sample GnRH concentration was lower but still significant higher than prior SP injection (p < 0.05). The lower dose of SP increased GnRH concentration later, only in the 2nd portal blood sample after intracarotid SP injection (p < 0.001). Injection of normal saline had no effect on GnRH concentration in pituitary portal blood in INT rats. In ORCX, ORCX testosterone- and estrogen-implanted rats portal plasma GnRH concentrations were not changed significantly after injection of both doses of SP. These results indicate that SP stimulates GnRH release into pituitary portal blood and the influence of SP on GnRH neurons depends on the levels of circulating gonadal steroid hormones.

Gonadotropin-releasing hormone (GnRH) content in the median eminence after superior cervical ganglionectomy in ovariectomized and estrogen-treated rats.

The aim of the present study was to examine if superior cervical ganglionectomy (SCGx) modifies the gonadotropin-releasing hormone (GnRH) content in the median eminence in female rats. Intact, ovariectomized (OVX) and ovariectomized, 17 beta-estradiol implanted (OVX + E2) female rats were subjected to SCGx or sham operation (sham-SCGx). After 12 or 48 hours they were decapitated and the GnRH content in the median eminence was determined by radioimmunoassay. SCGx performed 12 hours earlier decreased GnRH content in the median eminence in cycling female rats (p < 0.05) as compared with sham-operated control. No changes in GnRH content were observed 48 hours after SCGx. Ovariectomy decreased GnRH content in the median eminence (p < 0.05) and SCGx caused a further decrease in GnRH content (p < 0.05). Implantation of 17 beta-estradiol suppressed the dramatic decrease in GnRH content in the median eminence observed after SCGx in OVX rats without estrogen treatment. After SCGx the GnRH content in OVX + E2 rats was significantly higher than in OVX rats (p < 0.05). Our present results demonstrate that SCGx has some transient influence on the GnRH content in the median eminence. It may be assumed that noradrenaline released from degenerating sympathetic neurons located at the superior cervical ganglia (SCG) affects the GnRH-ergic terminals in the median eminence. The data support the hypothesis that SCG have a modulatory role in the mechanisms controlling the function of the hypothalamic-hypophyseal axis.

Gonadotropin - releasing hormone (GnRH) content in the medial basal hypothalamus after substance P injection into the 3rd cerebral ventricle in female rats.

The effect of substance P (SP) on gonadotropin releasing hormone (GnRH) content in the medial basal hypothalamus (MBH) was studied. To evaluate this effect, 5 microg of SP in saline or saline alone were injected into the 3rd cerebral ventricle in conscious ovariectomized (OVX) and ovariectomized with subcutaneously implanted 17beta-estradiol capsules (OVX+E[_2]) rats. Two hours later the animals were decapitated and GnRH was estimated by radioimmunoassay in the tissue extracts from MBH. SP injected i.c.v. had no effect on the GnRH content in MBH in OVX rats. However, SP significantly decreased GnRH content in OVX+E[_2] rats. These results provide evidence that SP participates in the control of GnRH neurons within MBH. It is suggested that SP may stimulate GnRH release from GnRH neuron terminals and that estrogen may be involved in this process.

Sensitivity and specificity of a modified agar gel precipitation test and its application to the diagnosis of enzootic bovine leukosis.

Sensitivity and specificity of a modified agar gel immuno-diffusion (AGID) test for the diagnosis of enzootic bovine leukosis was compared in 609 sera using the standard method, the modified test, and ELISA. The modifications concerned the composition of agar gel (1.8% Bacto-agar Difco), addition of polyethylene glycol (PEG 6000, 4%) and enlarging the diameters of the wells from 6 to 10 mm for sera. The change in well diameters and the addition of PEG 6000 increased the sensitivity of the test by about 60-100%, favourably influenced the proper interpretation of the results, especially with weak positive sera. ELISA proved to be more sensitive either than the modified (by 4.1%) or standard AGID (by 5.25%) tests. The modified AGID test may find wide application in laboratory practice, especially when a more sensitive technique is not available.

Studies on the role of humoral and cell-mediated immunity in pigs after vaccination with Aujeszky's disease virus.

Humoral and cellular immunity in pigs vaccinated twice with Aujeszky's disease virus (ADV) was studied by seroneutralizing test and direct leucocyte migration inhibition technique. Significant migration inhibition of leucocytes (LMI) was found on the fifth day, whereas specific antibodies began to appear at that time only in very low titers. Anamnestic reaction due to the second injection of ADV did not bring about a significant increase of migration inhibition of leucocytes, instead the level of antibodies elevated markedly.

Immunological experimental arthritis in pigs. I. Propagation dynamics of the attenuated strain of Aujeszky's disease virus in the pig organism and the humoral immunity formation.

Studies on the propagation dynamics of Aujeszky's disease virus and the formation of general and local joint immunity were to provide information explaining the mechanism of experimental rheumatoid arthritis in animals. It was shown that: Aujeszky's disease virus administered intramuscularly caused viremy on 3-4th day after its application and disappeared from the blood circulation on 5-6th day, introduced into the pig ankle joint, the virus was eliminated from the articular fluid two hours after injection, but it appeared again 3-7 days later probably due to the propagation in the cells of the synovial membrane, and that first parenteral administration of the virus induced the formation of neutralizing antibodies in low titer (5-10 units); application of the virus for the second time brought about a considerable increase of antibodies titer within 40-80 units.

Immunological experimental arthritis in pigs. IV. Reaction of the synovial membrane of the non-immunized and immunized pigs after intraarticular administration of the virus of Aujeszky's disease.

7 days after intramuscular administration of the attenuated virus of Aujeszky's disease, strain TK 300 L, it was found: in non-immunized animals--immunological arthritis with fibrinoid necrosis and abundant infiltrations from lymphoid cells; in animals of low antibodies titer--only few infiltrations from lymphoid and plasmic cells round the sub-synovial vessels; in animals of high antibodies titer--vast areas of fibrinoid necrosis, abundant infiltrations from lymphoid cells and presence of gigantic cells.

Immunological experimental arthritis in pigs. V. Reaction of the synovial membrane in the pigs non-immunized and immunized with the virus of Aujeszky's disease after the intraarticular administration of the immunological complexes.

Immunized animals were given intraarticular complexes formed in vitro from the autologous serum and virus AD. 7 days after complex administration with excess of the virus, weak inflammatory reactions of the immunological type were noted. After neutral complex administration virulent immunological inflammation of the synovial membrane took place. The histological picture resembled rheumatoid arthritis in the man and the changes obtained after the virus administration to the joints with a high level of antibodies. Administration of complexes and homological serum alone to the joints of the nonimmunized animals caused the occurrence of superficial focuses of fibrinoid necrosis, but there was a lack of cellular reactions.