An approach to lifting self-isolation for health care workers with prolonged shedding of SARS-CoV-2 RNA.

PURPOSE: According to the European Public Health Authority guidance for ending isolation in the context of COVID-19, a convalescent healthcare worker (HCW) can end their isolation at home and resume work upon clinical improvement and two negative RT-PCR tests from respiratory specimens obtained at 24-h intervals at least 8 days after the onset of symptoms. However, convalescent HCWs may shed SARS-CoV-2 viral RNA for prolonged periods. METHODS: 40 healthy HCWs off work because of ongoing positive RT-PCR results in combined nasopharyngeal (NP) and oropharyngeal (OP) swabs following SARS-CoV-2 infection were invited to participate in this study. These HCWs had been in self-isolation because of a PCR-confirmed SARS-CoV-2 infection. NP and OP swabs as well as a blood sample were collected from each participant. RT-PCR and virus isolation was performed with each swab sample and serum neutralization test as well as two different ELISA tests were performed on all serum samples. RESULTS: No viable virions could be detected in any of 29 nasopharyngeal and 29 oropharyngeal swabs taken from 15 long-time carriers. We found SARSCoV- 2 RNA in 14/29 nasopharyngeal and 10/29 oropharyngeal swabs obtained from screening 15 HCWs with previous COVID-19 up to 55 days after symptom onset. Six (40%) of the 15 initially positive HCWs converted to negative and later reverted to positive again according to their medical records. All but one HCW, a healthy volunteer banned from work, showed the presence of neutralizing antibodies in concomitantly taken blood samples. Late threshold cycle (Ct) values in RT-PCR [mean 37.4; median 37.3; range 30.8-41.7] and the lack of virus growth in cell culture indicate that despite the positive PCR results no infectivity remained. CONCLUSION: We recommend lifting isolation if the RT-PCR Ct-value of a naso- or oropharyngeal swab sample is over 30. Positive results obtained from genes targeted with Ct-values > 30 correspond to non-viable/noninfectious particles that are still detected by RT-PCR. In case of Ct-values lower than 30, a blood sample from the patient should be tested for the presence of neutralizing antibodies. If positive, non-infectiousness can also be assumed.

Prevalence of PCV2 in Austrian and German boars and semen used for artificial insemination.

Since epidemiologically-based science on PCV2 in porcine semen is patchy, we investigated 806 Austrian (A) and German (G) AI boars from five studs, and boars from Austrian farms used for on-farm semen collection, for the presence for IgG/IgM in blood by ELISA (n=754) as well as for PCV2 DNA in semen (n=472) and if positive, also in blood of a few boars by nested PCR and sequencing. A total of 420 boars were tested for both PCV2 in semen and antibodies in blood. Boars were aged between 8 and 82 months at sampling. None of the boars tested positive for IgM but 60.1% did for IgG. PCV2 DNA was detected in 86 (18.2%) semen samples. Minor differences were found between boar populations with respect to the number of antibody positive boars and no differences for DNA in semen. Phylogenetic analysis of 28 sequences revealed a genetic diversity of PCV2 in semen within and between boar populations, with sequences belonging to both PCV2 genotypes 1 and 2. Mean nucleotide sequence identity was 95.7%, with maximum pairwise difference of 8.8%. Boars < or =16 months were tested more frequently positive for IgG (P<0.001) and for PCV2 DNA in semen (P<0.05) than older boars. Of 80 boars tested positive in semen, 34 (42.5%) were antibody negative. A total of 58 semen positive boars with (n=33) and without (n=25) IgG were all tested negative for PCV2 DNA in serum. In conclusion, this study demonstrated the ubiquity of PCV2 in the Austrian and German boar population. Genetically diverse PCV2 can be encountered in boar semen. Shedder boars cannot be detected on the basis of serology. There is an apparent possibility of PCV2 being transmitted through semen.

Studies on the aetiology of non-suppurative encephalitis in pigs.

Thirty-eight natural cases of aetiologically unclear non-suppurative encephalitis in pigs were studied retrospectively. Brain samples were examined for the presence of porcine circovirus type 2 (PCV-2), porcine respiratory and reproductive syndrome virus (PRRSV), porcine enteroviruses (PEVS), ovine herpesvirus type 2 (OvHV-2), Borna disease virus (BDV) and suid herpesvirus type 1 (SuHV-1) by molecular biological and immunohistochemical methods. Histological examination of the brains revealed variable degrees of lymphohistiocytic encephalitis or meningoencephalitis, characterised predominantly by perivascular mononuclear infiltrates. Two cases could be attributed to PCV-2 infection by in situ hybridisation: viral nucleic acid was found in the mesencephalon, the cerebellum and the medulla oblongata, mainly in the cytoplasm of macrophages, endothelial cells and some glial cells, which were predominantly found in the meninges and around blood vessels. Real-time PCR detected PCV-2 dna in brain samples from seven other pigs. There was no evidence of PRRSV, BDV, SuHV-1, PEVS or OvHV-2 in any of the brain samples examined.

Porcine dermatitis and nephropathy syndrome (PDNS) is associated with a systemic cytokine expression profile indicative of proinflammation and a Th1 bias.

Porcine dermatitis and nephropathy syndrome (PDNS) is broadly discussed as a porcine circovirus type 2 (PCV2)-associated disease, although PCV2, in contrast to postweaning multisystemic wasting syndrome (PMWS), has to date not been proven to be the aetiologic agent. In order to better understand the complex immunopathology of PDNS, the systemic cytokine expression profiles of (i) five pigs suffering from PDNS, (ii) five animals suffering from naturally acquired PMWS and (iii) five controls were investigated at mRNA and protein levels by means of multiplex real-time RT-PCR and flow cytometric intracellular cytokine detection, respectively. IL-1alpha, IL-6 and IFN-gamma mRNA expressions were found to be elevated in PDNS pigs. At the protein level, an increased capacity of peripheral blood mononuclear cells to produce IL-2, IL-4, IL-6, IL-12, TNF-alpha and IFN-gamma was evident. Hematological investigations revealed a hypochromic anemia while basophils and monocytes were relatively and neutrophils absolutely increased in PDNS pigs. PCV2 antibody levels did not differ significantly between PDNS and PMWS affected animals. Taken results together, the cytokine profile of the PDNS affected animals together with hematological data pointed towards a proinflammatory condition supporting a Th1 bias. Cytokine data of PMWS affected animals exhibited only minor non-significant differences when compared to controls, only IL-10 was significantly decreased at the mRNA level.

[Correlation between antibodies against porcine reproductive and respiratory syndrome virus and pathological-anatomical organ findings in slaughter pigs at farm level]. / Zusammenhang zwischen dem Betriebsstatus für das Virus des Porzinen Reproduktiven und Respiratorischen Syndroms und Organbefunden an Schlachtkörpern.

OBJECTIVE: The porcine reproductive and respiratory syndrome (PRRS) worldwide causes important economic losses in pig production. Its causative agent, the PRRS virus (PRRSV), is one of the most frequently detected infectious agents in relation to respiratory diseases in pigs in Austria. We investigated the correlation between the PRRSV status of pig farms, determined by detection of PRRSV antibodies in the serum of slaughter pigs, and the prevalence of pathological-anatomical lung lesions in slaughter pigs of the respective farms. MATERIAL AND METHODS: Between December 1, 2011 and April 16, 2012, a total of 1056 serum samples of slaughter pigs from 66 pig farms were collected at an Austrian abattoir. The presence of PRRSV antibodies was tested by enzyme-linked immunosorbent assay in each sample and the PRRSV status of the respective farms was determined. No PRRSV vaccination was performed on any of the farms. In addition, the pathological-anatomical lung lesions of all slaughter pigs of the 66 farms that were slaughtered between September 1, 2011 and December 11, 2012 were recorded by authorized veterinarians at the abattoir. The prevalence of lung lesions and pleuritis in PRRSV-positive and unsuspected farms was compared and statistically interpreted. RESULTS: Slaughter pigs of PRRSV positive farms had a significantly higher prevalence of severe lung lesions and pleuritis visceralis and parietalis than slaughter pigs of PRRSV unsuspected farms. Pigs of combined farms (nursery and fattening unit at the same location) displayed a tendency for more moderate and severe lung lesions than pigs of exclusive fattening farms. CONCLUSIONS AND CLINICAL RELEVANCE: In the present study, the PRRSV status of pig farms displayed a significant influence on the prevalence of lung lesions in the slaughter pigs. Findings untypical for PRRS, including pleuritis, were also found significantly more often on those farms. This leads to the conclusion that other primary and/or secondary infections are involved, which can be exacerbated by the immunosuppressive effect of the PRRSV. There was a tendency for combined farms to be more severely affected than fattening farms.

Comparison of different commercial serological tests for the detection of Toxoplasma gondii antibodies in serum of naturally exposed pigs.

Toxoplasma gondii is the aetiological agent of the zoonotic disease toxoplasmosis and transmitted among other ways by chemically and physically untreated, that is, raw pork to humans. The detection of Toxoplasma gondii is impossible by currently practiced meat inspection, but serological tests can be used to detect Toxoplasma gondii antibodies in pig herds and can consequently be helpful to identify potentially contaminated pork. Therefore, appropriate serological tests are required. In this study, serum samples of 1368 naturally exposed slaughter pigs from 73 Austrian farms were collected. Serum samples of at least 16 slaughter pigs per farm were tested. The prevalence of Toxoplasma gondii antibodies in serum was measured by a commercial available modified agglutination test (MAT) and compared to three different commercial available enzyme-linked immunosorbent assays (ELISA). The MAT detected 6.5%, ELISA I 6.7%, ELISA II 4.8% and ELISA III 4.3% of the pigs as Toxoplasma gondii antibody positive. The agreement, according to the kappa coefficient (κ), was substantial between the MAT and ELISA I (κ = 0.62), II (κ = 0.64) and III (κ = 0.67). A better agreement was determined between ELISA I and II (κ = 0.715), ELISA I and III (κ = 0.747) and ELISA II and III (κ = 0.865). At least one pig per farm was detected Toxoplasma gondii antibody positive in 17 (23.3%) farms by the MAT, 26 (35.6%) farms by ELISA I, 16 (21.9%) farms by ELISA II and 11 (15.1%) farms by ELISA III. Pig farms with a high number of Toxoplasma gondii antibody-positive pigs or high antibody titres were identified by all of the four used serological tests. Concerning the occurrence of Toxoplasma gondii antibodies in Austrian pig farms, a monitoring and surveillance programme would be reasonable to find high-risk farms.

Activation of mouse oocytes requires multiple sperm factors but not sperm PLCgamma1.

A sperm cytosolic factor is responsible for oocyte activation at fertilization in mammals. The molecular identity of this factor is not yet known, although a sperm phospholipase Cgamma (PLCgamma) is a potential candidate. In this study, cation-exchange chromatography with a Heparin column was used for the fractionation of porcine sperm cytosolic extracts. Oocyte activation potential of the resulting fractions was tested and active fractions were subjected to Western blot analysis using antibodies specific to PLCgamma1. PLCgamma1 was detected in fractions other than those supporting oocyte activation (Ca(2+)-release and pronuclear formation). The active Heparin fraction was then purified on a Mono Q anion-exchange column. One of the resulting fractions still contained Ca(2+)-releasing activity, but pronuclear formation did not occur. We conclude that sperm PLCgamma1 is not involved in oocyte activation and that Ca(2+)-release and pronuclear formation requires multiple factors from sperm cytosol.

Systemic cytokine profile in feeder pigs suffering from natural postweaning multisystemic wasting syndrome (PMWS) as determined by semiquantitative RT-PCR and flow cytometric intracellular cytokine detection.

Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease in pigs caused by porcine circovirus type 2 (PCV2). Development of this disease is presumably associated with an impairment of the immune system. We, therefore, investigated the systemic expression of relevant cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, TNF-alpha, IFN-gamma) and IL-2Ralpha at mRNA (semiquantitative RT-PCR) and at protein level (flow cytometric intracellular cytokine detection after short-time stimulation of peripheral blood mononuclear cells) in 10 feeder pigs aged 14 weeks suffering from natural PMWS and in 10 clinically healthy pen-mates. Hematological examination revealed a significant (p < 0.001) relative lymphopenia in the diseased animals when compared to reference pigs. IL-1alpha and IL-10 mRNA levels were notably increased in the affected pigs, whereas IL-2 and IL-2Ralpha (CD25) mRNA levels tended to be down-regulated. IL-8, TNF-alpha and IFN-gamma mRNA expressions appeared to be slightly increased. Intracellular cytokine levels as measured by flow cytometry revealed an increase of IL-1beta, IL-2, and IL-6, whereas IL-12 and TNF-alpha expressions were not affected. IFN-gamma was slightly decreased in the diseased animals. In conclusion, despite the assumption, that the cellular immune response to PMWS as a virus-induced disease should be characterized by either a Th1 driven cytokine profile or a cytokine profile indicative of T cell immunosuppression, our results did not support that hypothesis. Nevertheless, data from intracellular cytokine detection suggest an even increased percentage of the remaining lymphocytes capable to produce IL-2 upon in vitro stimulation, which is in contrast to the slightly diminished IL-2 mRNA levels reflecting the in vivo situation at least at the mRNA level.

Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes.

Cumulus cell-enclosed bovine oocytes in germinal vesicle (GV) and in metaphase II (MII) stages were cryopreserved. Different concentrations (1 M; 1.5 M) of various cryoprotectants (glycerol, PROH, DMSO) were tested. After thawing, the oocytes were exposed to various carbohydrates (sucrose, lactose, trehalose) at a concentration of 0.1 M and 0.25 M for cryoprotectant removal. Developmental capacity of the frozen-thawed oocytes was studied by in vitro maturation, fertilization and culture. We found no difference in subsequent development using glycerol or PROH for GV and MII oocytes. The DMSO treatment led to significantly better cleavage and development up to 4-cell stage in MII oocytes. Development beyond the 8-cell stage was obtained only when unmatured oocytes were frozen. No difference in the efficiency of the 3 cryoprotectants was detected in MII oocytes. However, in GV oocytes, glycerol and PROH yielded significantly better cleavage and 4-cell rate compared to DMSO (P<0.001). Influence of the concentration of a cryoprotectant on development was not observed in GV or MII oocytes. Among the 3 cryoprotectants, DMSO was less suitable, at both concentrations, than PROH and glycerol for the development of 6- to 8-cell stage embryos in the GV group. In the MII group, 1.5 M DMSO was as efficient as PROH and as glycerol at a 1.5-M concentration, and it was more efficient than 1 M glycerol. The use of carbohydrates during rehydration did not render a beneficial effect at either of the 2 concentrations, and when no carbohydrates were used in the MII group the oocytes cleaved better than GV oocytes.

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice.

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.

Corona radiata density as a non-invasive marker of bovine cumulus-corona-oocyte complexes selected for in vitro embryo production.

The density of the corona radiata as a marker for the quality of cumulus-corona-oocyte complexes (CCOC's) for in vitro embryo production was tested. The CCOC's in which the corona radiata appeared as a dark rim surrounding the zona pellucida (Group 1) and CCOC's in which the corona had the same density as the rest of the cumulus investment (Group 2) were assessed with respect to nuclear ultrastructure, corona-cumulus expansion and capacity for sustaining embryonic development in vitro. An intermediate Group 3 with characteristics between Groups 1 and 2 was also assessed for in vitro development capacity. The CCOC's in Group 1 were typically meiotically unactivated and presented a nonundulating nuclear envelope. More than half of the CCOC's in Group 2 showed some degree of meiotic activation, and those that were nonactivated displayed "holes" in the nuclear envelope or dilatations of the perinuclear cisterna. The CCOC's in Group 2 were characterized by partial corona-cumulus expansion already at collection. The CCOC's from Groups 1 and 3 sustained embryonic development in vitro at a significantly higher rate than CCOC's from Group 2. It is concluded that CCOC's in which the corona radiata has the same density as the rest of the cumulus investment are less competent candidates for in vitro embryo production.

Granulosa-cumulus-corona expansion and aromatase localization in preovulatory follicles in superovulated heifers.

Granulosa-cumulus and cumulus-corona expansion as well as aromatase localization within ovarian follicles were monitored during the preovulatory period in superovulated cattle that were blood sampled every 2'nd h for LH analyses. Granulosa-cumulus as well as cumulus-corona expansion were studied by means of transmission electron microscopy and computerized image analysis. Localization of aromatase, an enzyme involved in estrogen synthesis, was determined immunocytochemically using anti-human placental aromatase cytochrome P-450 antisera. Nuclear oocyte maturation was determined by aceto:orcein staining. Significant cell dissociation within the granulosa-cumulus stalk occurred before the breakdown of the germinal vesicle, i.e. the oocyte nucleus, during the period up to 5-7 h after the LH peak, i.e. the highest LH concentration during the surge. Significant increase in intercellular spacing between the cumulus-corona cells occurred at 13-15 and 19-21 h after the LH peak. Before the LH peak all layers of granulosa cells were immunocytochemically stained for aromatase. At 5-7 h after the LH peak, however, only the granulosa cell layers located near the basal lamina were stained, and at all later intervals staining was absent. The granulosa cells of primary and secondary follicles, the interstitial gland cells, the theca interna cells and the oocytes in all follicles were immunocytochemically unstained.

[Selection for disease resistance in domestic ruminants and swine by indicator traits as well as marker and causal genes--a review. Part 1: Short introduction to immunogenetics of MHC and non-MHC genes and special immunogenetics of cattle and swine]. / Selektion beim domestizierten Wiederkäuer und Schwein auf Krankheits- und Seuchenresistenz mit Hilfe von Indikatormerkmalen, Marker- und Kausalgenen--ein Uberblick. 1. Mitteilung: Kurze Einführung in die Immungenetik der MHC- und Nicht-MHC-Gene und spezielle Immungenetik bei Rind und Schwein.

The introduction deals with immunogenetic investigations on the field of life-stock. The main chapter is outlined as a tabular overview of current opportunities of the application of indicator traits as well as marker and causal genes in breeding for disease resistance in cattle, sheep, goats and swine. In the discussion of the second part, emphasis was layed on diseases of small ruminants in central and western Europe. Indicator traits are discussed with respect of their advantages and disadvantages. The rigorous selection of specific traits is connected with an increase of the number of homozygotes. In contrary, pathogens do undergo mutations, thus escaping the host's immune system. Out of this point of view it is advisable, to set on selection very cautiously. The role of technologies of modern immunogenetics is pointed out in respect of constructing disease resistant animals.

[Selection for disease and epidemic resistance in domestic ruminants and swine by indicator traits, marker and causal genes--a review. Part 2: Special immunogenetics of sheep and goats with particular regard for endoparasitoses, scrapie, foot rot and maedi-visna virus infection]. / Selektion beim domestizierten Wiederkäuer und Schwein auf Krankheits- und Seuchenresistenz mit Hilfe von Indikatormerkmalen, Marker- und Kausalgenen--ein Uberblick. 2. Mitteilung: Spezielle Immungenetik bei Schaf und Ziege unter besonderer Berücksichtigung der Endoparasitosen, Scrapie, Moderhinke und der Maedi-Visna-Virusinfektion.

The introduction of the first part deals with immunogenetic investigations on the field of life-stock. The main chapter is outlined as a tabular overview of current opportunities of the application of indicator traits as well as marker and causal genes in breeding for disease resistance in cattle, sheep, goats and swine. In the discussion of the second part, emphasis was laied on diseases of small ruminants in central and western Europe with special respect of endoparasitoses, scrapie, foot-rot and maedi-visna virus infection. Indicator traits are discussed with respect of their advantages and disadvantages. The rigorous selection on specific traits is connected with an increase of the number of homozygotes. In contrary, pathogens do undergo mutations, thus escaping the host's immune system. Out of this point of view it is advisable, to set on selection very cautiously. The role of technologies of modern immunogenetics is pointed out in respect of constructing disease resistant animals.

[Serological detection of emerging viral infections in wild boars from different hunting regions of Southern Germany]. / Serologische Erfassung von viralen Infektionskrankheiten in der Wildschweinpopulation verschiedener Jagdgebiete Süddeutschlands.

OBJECTIVE: Wild boars represent a possible virus reservoir for notifiable diseases of farm animals, including Aujeszky's disease (AD) and classical swine fever (CSF). Monitoring of the epidemiological situation in the wild boar population is especially relevant in countries that are officially free from these diseases. Apart from OIE-notifiable diseases, other viral agents that are widely distributed and play a significant role in farm animals, such as the porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type2 (PCV-2), and swine influenza virus (SIV), are sporadically detected in wild boars. Thus, the wild boar population is a potential source for maintenance of these infections in farm animals. The aim of this study was therefore to test for antibodies to the indicated emerging viral infections in wild boars in several hunting regions of Southern Germany. MATERIAL AND METHODS: Blood serum of 94 shot wild boars from 19 hunting regions in Bavaria, Rhineland-Palatinate and Baden-Württemberg were collected. Antibodies to AD virus (ADV), CSF virus (CSFV), PRRSV, SIV (H1N1) (all by IDEXX ELISA) and PCV-2 (IgM and IgG by Ingenasa ELISA) in blood serum were determined. RESULTS: Antibodies to ADV were detected in four animals (4.2%), to PRRSV in one animal (1.2%), to SIV (H1N1) in two animals (2.1%) and to PCV-2 (IgG) in 15 animals (16.0%) of which three animals also had antibodies to PCV-2 (IgM) (3.2%). CSFV antibodies were not detected in the examined wild boars. CONCLUSIONS AND CLINICAL RELEVANCE: Compared to other studies in several European and American states, the seroprevalence to the tested emerging diseases was low in this study. Nevertheless, the wild boar population may still be a virus reservoir and therefore a source of infection for domestic pigs. This is especially important in the case of notifiable diseases, like AD and CSF. Therefore, a continuous monitoring of those diseases in the wild boar population would be advisable.

Minipigs and potbellied pigs as pets in the veterinary practice--a retrospective study.

Minipigs have become popular pets in recent years. Therefore, an increasing number of veterinarians are being challenged by specific problems of these animals. This retrospective study gives an overview on the diagnoses and therapeutic interventions of the patients submitted to the clinic for swine at the University of Veterinary Medicine Vienna during the last 6 years (n=48). Most frequently, colic symptoms of the gastro-intestinal tract (n=12) and orthopaedic locomotion disorders (n=10), mainly due to accidents or long claws, could be observed, followed by urogenital tract and skin disorders (n=4 each). Therapeutic interventions are discussed with regard to medical aspects as well as statutory provisions.

Fibroepithelial hamartoma in a domestic pig.

This case report is the first description of a fibroepithelial hamartoma in a pig. The dysplasia, which covered half of the face of the newborn piglet, did not increase in relative size until the animal was euthanatized at 6 months of age. Histologic examination revealed a moderate orthokeratotic hyperkeratosis with variable degrees of epithelial proliferation. The main body of the dysplasia consisted of collagenous fibers. In addition, some proliferating small blood vessels as well as focally gathered dilated apocrine glands were evident. Given morphologic and clinical features, the diagnosis of a hamartoma seemed to be justified.

Immunophenotypic characterization of peripheral blast cells in a leukemic miniature pig.

The health status of a 4-year-old female, dd-haplotype miniature pig deteriorated rapidly, so the animal finally had to be euthanized because of poor clinical condition. Necropsy revealed a massive leukocytic infiltration in the parenchymatous organs of the abdominal cavity. On hematologic cell counting, severe leukocytosis (69.3 x 10(9) cells/liter) and high-grade basophilia (6.9 x 10(9) cells/liter) were evident. Cytologic examination, as well as analysis of expression of leukocyte differentiation antigens by means of flow cytometry, classified blasts, which accounted for about 22% of leukocytes, as biphenotypic cells co-expressing the myeloid marker SWC3 (CD172a) and the lymphoid markers CD5 and CD25. Hematologic features resembled those seen in humans with chronic myeloid leukemia at blast phase.

Analysis of theFusarium toxins Deoxynivalenol and Zearalenone in Austrian feeds of the crop years 2002 and 2003.

164 feed samples were collected in Lower Austria, Upper Austria, Styria and Carynthia in the crop years 2002 and 2003 and were analysed by means of the RIDASCREEN(®) ELISA-assay for theFusarium toxins Deoxynivalenol (DON) and Zearalenone (ZON). The collected samples consisted of swine feeds, cereals and silages. DON concentrations were found up to the range of mg/kg (ppm), in contrast ZON results remained significantly lower.

Characterization of the cytokine pattern of porcine bone marrow-derived cells treated with 1alpha,25(OH)D.

The biologically active form of vitamine D(3) [1alpha,25(OH)(2)D(3)] has recently been described not only to influence bone metabolism but also to exert immunomodulating activities, which may have an impact on bone formation/resorption as well. In this study, we analysed the effects of 1alpha,25(OH)(2)D(3) on the cytokine pattern of porcine bone marrow-derived cells from piglets aged 1-3 weeks. After culture for 1 week, the number of osteoclasts was determined, with tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells being considered osteoclasts. Cultured bone marrow cell-derived mRNA was subjected to semiquantitative RT-PCR specific for a panel of porcine cytokines (IL-1alpha, IL-6, IL-8, IL-10, and TNF-alpha). In addition, an immunofluorescence analysis using anti-porcine mAbs specific for IL-1beta, IL-2, IL-4, IL-6, IL-12, TNF-alpha, and IFN-gamma was performed. In order to prove the existence of a porcine homologue of the receptor activator of NF-kappaB ligand (RANKL) bone marrow cell- as well as porcine white blood cell-derived mRNA was investigated by RT-PCR using primer pairs specific for murine RANKL. Cell culture supernatant was analysed for soluble RANKL by means of an ELISA designed for quantification of human RANKL. By means of RT-PCR, expression of IL-1alpha, IL-6, IL-8, IL-10 and TNF-alpha mRNA could be found in cells cultured with and without 1alpha,25(OH)(2)D(3). Immunofluorescence analysis revealed that IL-1, IL-6, and TNF-alpha were produced by both stromal cells and osteoclasts. Besides its known osteoclastogenic effects, 1alpha,25(OH)(2)D(3) tended to downregulate the respective cytokines, but significantly upregulated RANKL expression. The homology between the porcine RANKL-specific sequence and the corresponding human RANKL sequence was 79%. The data found support the idea that porcine bone marrow cell cultures may provide a suitable alternative to murine systems in human osteological research.