Prevalence of Mycobacterium avium subspecies paratuberculosis and hepatitis E in New World camelids in Austria.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis in domestic ruminants and New World Camelids (NWC). Hepatitis E virus (HEV) is an important public health concern worldwide. The virus has been identified in several species, some of them serving as a reservoir for zoonotic HEV strains. Husbandry and breeding of llamas and alpacas have increased in Austria in recent years. Therefore, the aim of the present study was to evaluate the prevalence of MAP and HEV in NWC in Austria. Altogether 445 animals, originating from 78 farms were enrolled in the study. Of the animals sampled, 184 (41.35%) were llamas and 261 (58.65%) were alpacas. 443 blood samples for MAP-ELISA and 399 faecal samples for quantitative PCR (qPCR) and culture for MAP as well as for HEV detection by RT-qPCR have been collected. All of the 399 animals tested for shedding of MAP were negative by faecal solid culture. Using qPCR, 15 (3.8%) of the animals were MAP positive and 384 (96.2%) negative. Out of the 443 serum samples examined for specific antibodies against MAP by ELISA, 6 (1.4%) were positive, 1 (0.2%) was questionable and 436 (98.4%) samples were negative. All faecal samples were tested negative for HEV.

The detection and quantification of Bacillus thuringiensis spores from soil and swabs using quantitative PCR as a model system for routine diagnostics of Bacillus anthracis.

AIMS: To optimize the DNA isolation for the routine detection and quantification of bacillary spores in soil and swabs. The procedure is primarily intended for diagnostics of Bacillus anthracis spores, but due to its high pathogenicity, B. thuringiensis served as its surrogate organism. METHODS AND RESULTS: Various commercial kits for soils and swabs in combination with quantitative PCR were tested with different results. The PowerSoil DNA kit and the Ultra Clean Microbial DNA kit gave the best results for the extraction from soil and swabs, respectively. Extra beating led to considerably higher yields of DNA. The effectiveness of isolation reached 23% for DNA isolation from soil and 13% from swabs. The limit of detection was assessed to be 8·85 × 103 from 250 mg of soil and 2·79 × 103 from a swab inoculated with 100 µl of spore suspension. CONCLUSIONS: The optimized protocol is suitable for direct isolation and quantification of bacillary spores without any previous culturing. SIGNIFICANCE AND IMPACT OF THE STUDY: In contrast to previous studies, the isolation and quantification of spores was performed directly from the sample, without previous culture of spores on plates. Therefore, the method is suitable for such conditions where previous culturing is not possible, such as in military installations under field conditions.

Impaired Albumin Uptake and Processing Promote Albuminuria in OVE26 Diabetic Mice.

The importance of proximal tubules dysfunction to diabetic albuminuria is uncertain. OVE26 mice have the most severe albuminuria of all diabetic mouse models but it is not known if impaired tubule uptake and processing are contributing factors. In the current study fluorescent albumin was used to follow the fate of albumin in OVE26 and normal mice. Compared to normal urine, OVE26 urine contained at least 23 times more intact fluorescent albumin but only 3-fold more 70 kD fluorescent dextran. This indicated that a function other than size selective glomerular sieving contributed to OVE26 albuminuria. Imaging of albumin was similar in normal and diabetic tubules for 3 hrs after injection. However 3 days after injection a subset of OVE26 tubules retained strong albumin fluorescence, which was never observed in normal mice. OVE26 tubules with prolonged retention of injected albumin lost the capacity to take up albumin and there was a significant correlation between tubules unable to eliminate fluorescent albumin and total albuminuria. TUNEL staining revealed a 76-fold increase in cell death in OVE26 tubules that retained fluorescent albumin. These results indicate that failure to process and dispose of internalized albumin leads to impaired albumin uptake, increased albuminuria, and tubule cell apoptosis.

Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens.

RNA viruses are pathogenic agents of many serious infectious diseases affecting humans and animals. The detection of pathogenic RNA viruses is based on modern molecular methods, of which the most widely used methods are the reverse transcription polymerase chain reaction (RT-PCR) and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All steps of RT-PCR and qRT-PCR should be strictly controlled to ensure the validity of obtained results. False-negative results may be caused not only by inhibition of RT or/and PCR steps but also by failure of the nucleic acid extraction step, particularly in the case of viral RNA extraction. The control of nucleic acid extraction generally involves the utilization of a non-pathogenic virus (process control virus) of similar structural properties to those of the target virus. Although in clinical samples the use of such process control virus is only recommended, in other kinds of settings such as food matrices its use is necessary. Currently, several different process control viruses are used for these purposes. Process control viruses can also be constructed artificially using technology for production of MS2 phage-like particles, which have many advantages in comparison with other used controls and are especially suited for controlling the detection and quantification of certain types of RNA viruses. The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Nevertheless, the practical use of MS2 phage-like particles in routine diagnostics is relatively uncommon. The current situation with regard to the use of MS2 phage-like particles as process control viruses in detection of RNA viruses and different methods of their construction, purification and use are summarized and discussed in this review.

Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables.

UNLABELLED: Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes.

Microscopy, culture, and quantitative real-time PCR examination confirm internalization of mycobacteria in plants.

The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10(8) to 10(10) cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10(4) cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.

Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods.

Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16 h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12 weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

Short communication: Examination of milk filters by real-time PCR as a herd-level indicator of the presence of Mycobacterium avium subspecies paratuberculosis in dairy herds.

The aim of this study was to assess the suitability of real-time quantitative PCR (qPCR) for the detection of Mycobacterium avium ssp. paratuberculosis (MAP) in milk filters as a herd level indicator of paratuberculosis infection. Seventy-nine samples from textile or metal milk filters from 15 herds with defined MAP prevalence (infection status = noninfected, 0-5%, 5-10%, or >10% of animals with clinically confirmed paratuberculosis) were analyzed. The MAP DNA was isolated by a modified commercially available protocol for feces, and detection and quantification of the pathogen was performed by the IS900 qPCR. Mycobacterium avium ssp. paratuberculosis DNA was detected in 63 (79.7%) samples. Determination of MAP infection established by fecal and tissue culture was correctly confirmed by the analysis of milk filters on 11 of 12 infected farms; MAP was not detected in filters from 3 farms where paratuberculosis was never diagnosed. Statistical analysis of the data supports the evidence that milk filters can be used as a template for the direct detection of MAP on the herd level. The probability of successful MAP detection in milk filters in a herd with MAP-infected cows is at least 94.3%. Absolute numbers of MAP detected on the milk filter can be used for a rough estimation of paratuberculosis prevalence >10% in the herd. Analysis of milk filters for the presence of MAP can be a useful tool for the detection of paratuberculosis on the herd level before any individual control strategies.

Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) is known to be a very slow-growing organism. The fact that cells typically need several weeks to form visible colonies severely compromises the suitability of plate counting for assessment of viable cell counts. This problem might be overcome by the application of fast molecular methods containing a viability component. We have evaluated a promising technology combining sample treatment with propidium monoazide (PMA) prior to DNA extraction for selective detection of cells with intact cell membranes with detection of sequence element F57 by quantitative PCR (F57 qPCR). Element F57 is unique for MAP and is not known to exist in any other bacterial species. Conditions of PMA treatment were optimised for MAP isolate 7082 using live and heat-killed cells and comparing different DNA extraction procedures. The subsequent successful application of the optimised protocol to four other MAP isolates of different origins suggested that the optimised protocol might be broadly applicable to different MAP strains. Furthermore, different equations were compared to use the data resulting from this technology to optimally predict the percentage of live MAP cells in mixtures containing both live and dead cells. The presented protocol holds promise to be used routinely for detecting MAP with intact cell membranes in research applications.

Distribution of Mycobacterium avium subsp. avium and M. a. hominissuis in artificially infected pigs studied by culture and IS901 and IS1245 quantitative real time PCR.

Mycobacterium avium subsp. avium (MAA) and M. a. hominissuis (MAH) belong to the Mycobacterium avium complex (MAC) and are frequently associated with diseases in animals and humans. The aim of this study was to develop a system for rapid and accurate real time quantitative PCR (qPCR) identification and quantification of MAA and MAH. This study included 22 per os infected pigs, of which 10 were infected with MAA, 10 with MAH and 2 were present as a negative control group. From each animal, 21 different tissue samples as well as blood were tested by microscopy, culture and triplex qPCR. The developed triplex qPCR reaction was based on the simultaneous detection of specific insertion sequences, IS901 and IS1245, and also contained an internal amplification control. In both groups of experimentally infected animals, the newly developed triplex qPCR assay proved to be more specific and sensitive in comparison with the other methods used. Contrary to culture examination, triplex qPCR confirmed the infection in all animals infected with MAA, and in eight animals infected with MAH. In conclusion, we developed a quick and sufficiently sensitive triplex qPCR for MAA and MAH detection in tissue and blood samples. From the food safety point of view the presence of MAH in muscles should be considered as a possible threat to human health.

Detection and genetic characterisation of Hepatitis E virus in Czech pig production herds.

The objective of the present study was to carry out a small surveillance programme in Czech pig production herds using the nested reverse transcriptase-polymerase chain reaction (nRT-PCR) technique to trace Hepatitis E virus (HEV) in different biological samples and to characterise the detected swine HEV isolates by phylogenetic analysis. A total of 32 piglets from 11 herds clinically suspected of postweaning multisystemic wasting syndrome (PMWS) were examined. Bile, liver tissue and serum samples were collected from each animal. Due to the high genetic variability of HEV, three sets of primers targeting each of the open reading frames (ORFs) of its genome were used. HEV RNA was most frequently detected in the bile samples (40.0%), followed by liver tissue (16.1%) and serum (3.2%). Seven (63.6%) of the 11 monitored farms were found to have at least one HEV RNA positive piglet. Specific 242 bp sequences within the ORF1 coding non-structural proteins were sequenced and phylogenetically analysed. Phylogenetic analysis using the neighbor-joining and maximum parsimony method confirmed that all detected Czech swine HEV isolates belonged to genotype III. Comparison of the Czech swine HEV isolates with corresponding sequences of swHEV available in GenBank failed to find any 100% homologous HEV isolate.

Cytogenetic mapping of immunity-related genes in the domestic horse.

Chromosomal locations of 19 horse immunity-related loci (CASP1, CD14, EIF5A, FCER1A, IFNG, IL12A, IL12B, IL12RB2, IL1A, IL23A, IL4, IL6, MMP7, MS4A2, MYD88, NOS2A, PTGS2, TFRC and TLR2) were determined by fluorescence in situ hybridization. For IFNG and PTGS2, this study is a confirmation of their previously reported position. In addition, microsatellite (HMBr1) was localized in the same region as IFNG. All genes were assigned to regions of conserved synteny and the data obtained in this study enhance the comparative human-horse map. Cytogenetic localization of IL6 to ECA4q14-q21.1 suggested a new breakage point that changes the order of loci compared with HSA7. The map assignments of these loci serve as anchors for other loci and will aid in the search for candidate genes associated with traits in the horse.

Effect of drugs affecting synthesis or degradation of connexin on onset or completion of ethylene glycol induced inhibition of intercellular gap junctional communication.

The role of a connexin synthesis and degradation in the onset or completion of the ethylene glycol-induced inhibition of the gap junctional intercellular communication (GJIC) in V79-4 Chinese hamster cell line was studied as a model of an interaction between the cells and a potential tumor promoter. GJIC was assessed on two levels: on the cytophysiological level - the dye coupling method, and on the immunocytochemical level - the immunolabeling of connexin43. The specific activator of connexin synthesis - Dibutyryl cAMP made the onset of the EG-induced inhibition of GJIC slower, but its effect was only temporary. On the other hand it also speeded up the re-establishment of standard values of GJIC after the removal of EG. Although the non-specific inhibitor of protein degradation via proteasomes - leupeptin increased the amount of connexin plaques on cell membranes, its effect on GJIC remained insignificant. The non-specific inhibitors of transcription - actinomycin D and translation - cycloheximide significantly inhibited the re-establishment of the standard values of GJIC after the removal of EG. The results indicate that although the storage of connexins in Golghi complex probably plays the principal role in the control of the gap junctional communication, the extensive changes in GJIC activity depend on the de novo synthesis of connexin per se.

Catalase transfection decreases hydrogen peroxide toxicity in a pancreatic beta cell line.

BetaTC6-F7 cells like normal Beta cells were found to be highly sensitive to hydrogen peroxide and to possess very low levels of catalase. Therefore we tested whether overexpression of catalase could enhance resistance to hydrogen peroxide. Enzyme activity was increased forty fold by transient transfection of a catalase transgene. To assess protection from hydrogen peroxide a cotransfection method using a human growth hormone reporter gene was developed. Human growth hormone secretion was shown to be a suitable marker for insulin secretion since both hormones demonstrated virtually identical glucose dose response curves. Catalase transfection was found to provide significant protection against hydrogen peroxide indicating that low catalase may contribute to the sensitivity of cells to hydrogen peroxide.

Kinetic evaluation of platelet monoamine oxidase following relaxation in chronic anxiety.

The effect of relaxation training, utilizing EMG biofeedback, on platelet monoamine oxidase (MAO) activity was examined in patients with a history of chronic anxiety. Anxiety scores and MAO activity were significantly lowered after 4 weeks of therapy. Kinetic studies, using phenylethylamine as substrate, indicated a significant increase of the Km constant while the Vmax showed no significant or consistent variation. It is thought that this phenomenon represents an adaptive response by the individual to maintain a homeostatic level of the biogenic amines.

Effects of neuroleptics on platelet monoamine oxidase activity.

Platelet MAO activity in schizophrenics was significantly decreased, by about 15%, after 3 weeks of treatment with haloperidol. Treatment with thioridazine or butaperazine also tended to decrease platelet MAO activity. The neuroleptic-induced decrease began to appear within a few days of treatment and did not show tolerance over 1-2 months of treatment with haloperidol. Platelet MAO activity of schizophrenic patients measured during drug-free base-line was not significantly different from that of normal controls, but MAO activity of schizophrenics was significantly lower than normals after 3 weeks of treatment with neuroleptics. The extent of decrease in platelet MAO activity correlated negatively with base-line prolactin and its increase after 24 hr. With PEA as substrate, the decrease in activity correlated positively with steady state plasma haloperidol.

Catecholamines and monoamine oxidase activity in anxiety.

Levels of platelet monamine oxidase activity, state anxiety and trait anxiety were quantified twice in 20 drug-free subjects with generalized anxiety and an equal number of healthy drug-free controls at 4-week intervals. Fifteen subjects in each groups also had plasma epinephrine and norepinephrine measured. The index group received relaxation training during the interval between the two evaluation sessions. Post-relaxation training values for monoamine oxidase, epinephrine, norepinephrine and the anxiety levels were found to be significantly lower than the pre-treatment values for the index group. No significant changes were seen in the control group values. For the index group, catecholamine levels and monoamine oxidase activity were seen to correlate significant before and after relaxation training.