Voluntary and compulsory eradication of bovine viral diarrhoea virus in Lower Austria.

A voluntary BVDV eradication program without vaccination was introduced in Austria in 1997, according to the Swedish model employing herd-level antibody tests. Since this time, identified persistently infected (PI) animals have to be slaughtered. In order to protect non-infected herds, the major routes of introduction of BVDV infection into a herd, such as communal grazing and livestock trade, have to be controlled. In 1998 PI animals were identified in 7.5% of affiliated herds of herd book breeders. During the voluntary program, the majority of the 2455 herd book breeders who actively participated in the BVDV program had cleared up their herds. Hence, in 2005 only nine infected herds (0.36%) remained under clearance. These data show that BVDV control can be achieved at the farm level without a nation-wide BVDV eradication program. A federal law was passed in 2004, obligating all herd owners to follow the BVDV eradication program. Between 2005 and 2007, the number of herds with a certified BVDV-free status increased from 7931 to 9952 (2006) and 11,166 (2007), respectively. Currently (2008), 11,017 of 12,031 existing herds have been certified as BVDV-free herds (91.57%). Nearly all certified BVDV-free dairy herds can now be monitored by testing milk samples because the cows have no antibodies to BVDV. This signifies a marked improvement since 1998, when 46% of the 5024 tested dairy herds had a high level of BVDV antibodies in bulk tank milk. Total eradication of BVDV-infected herds will need a further 1 or 2 years.

Isolated cytochrome c oxidase deficiency as a cause of MELAS.

BACKGROUND: MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) is one of the more common mitochondrial encephalomyopathies. About 80% of MELAS cases are caused by transition 3243A-->G in the mitochondrial tRNA(Leu(UUR)) gene (MT-TL1). Other mutations in MT-TL1, other mitochondrial tRNA genes and mitochondrial-encoded subunits of respiratory complex I account for the remainder of cases. OBJECTIVE: To characterise the molecular basis of a MELAS case without a mutation in any recognised MELAS target gene. RESULTS AND METHODS: Deletion of a single nucleotide (7630delT) within MT-CO2, the gene of subunit II of cytochrome c oxidase (COX), was identified by mitochondrial DNA (mtDNA) sequencing. The deletion-induced frameshift results in a stop codon close to the 5' end of the reading frame. The lack of subunit II (COII) precludes the assembly of COX and leads to the degradation of unassembled subunits, even those not directly affected by the mutation. Despite mitochondrial proliferation and transcriptional upregulation of nuclear and mtDNA-encoded COX genes (including MT-CO2), a severe COX deficiency was found with all investigations of the muscle biopsy (histochemistry, biochemistry, immunoblotting). CONCLUSIONS: The 7630delT mutation in MT-CO2 leads to a lack of COII with subsequent misassembly and degradation of respiratory complex IV despite transcriptional upregulation of its subunits. The causal association of the resulting isolated COX deficiency with MELAS is at odds with current concepts of the biochemical deficits underlying this common mitochondrial disease, and indicates that the genetic and pathobiochemical heterogeneity of MELAS is greater than previously appreciated.

Salmonella diagnosis in pig production: methodological problems in monitoring the prevalence in pigs and pork.

Salmonellosis is an important foodborne infection in industrialized and developing countries. Especially for human Salmonellosis caused by Salmonella enterica serovar Typhimurium, pigs and pork are the major sources of infection. Mitigation and control strategies that result from surveillance programs attempt to reduce or even eradicate Salmonella in pork to lower consumers' risks. The methodology for Salmonella screening in pigs is generally based on antibody detection at slaughter with meat juice as the sample matrix. The instructions to most commercial enzyme-linked immunosorbent assay (ELISA) kits for the detection of Salmonella antibodies state that their product is suitable for antibody detection in meat juice and sera. In the present study, we show that it is essential to recalculate the percent optical density (OD%) data obtained from meat juice by the ELISA (IDEXX HerdCheck swine Salmonella) by the following regression equation: OD%sera = -70.5587 + 128.1490/ {1 + exp[(-18.8969 - OD%meatjuice)/27.6032]}(1.1771), r = 0.87, to compare results with those obtained from sera. By this regression equation, we were able to compare the Salmonella antibody levels (classified as <10, 10 to <20, 20 to <40, and > or =40 OD%) for sows, growers, and slaughter pigs. We identified significantly higher numbers of growers with lower OD% levels than for sows and slaughter pigs. Without a recalculation of the meat juice results, the higher fraction of samples with low OD% values led to an underestimation of the actual seroprevalence.

Control of BVDV-infection on common grassland--the key for successful BVDV-eradication in Lower Austria.

A bovine viral diarrhea/mucosal disease (BVD/MD) control and eradication program was introduced in Lower Austria in 1996, according to the Swedish model. At present 9800 out of 17,000 herds are part of this program. An important risk factor for BVDV-transmission under local conditions is communal grazing. Approximately 3-4% of livestock share common pastures, in which susceptible pregnant cattle may be mixed with unrecognised persistently infected (PI) animals. Rules and regulations were defined to allow only herds free from BVDV-infection on to common grassland. At the moment, 5067 herds are certified free from BVDV. The percentage of BVDV-free herds in regions with intensive pasture utilisation is higher (57.3%) than in the other regions (43.0%) of Lower Austria. With a reliable system for identification of PI-animals and a high certainty of prevention of PI-animals on common grassland, the main transmission of BVDV infection can be stopped, even if the animals are derived from infected herds and transiently infected animals cannot be excluded.

Mitochondrial genotype and risk for Alzheimer's disease: cross-sectional data from the Vienna-Transdanube-Aging "VITA" study.

The Vienna Transdanube Aging (VITA) study searches for early markers of Alzheimer's disease (AD) by examining the mental status in a community-based cohort of 606, 75-years old volunteers that are then related to various clinical and genetic analyses. To determine whether mutations in mtDNA are involved in expression of AD, the mtDNA of 79 "control" participants is screened for alterations by sequencing of "hot-spot-regions". This study on mtDNA mutations has eliminated the influence of aging on the occurrence of mtDNA alterations by sequencing samples from persons at the age of exactly 75 years. Thus, our cohort reveals a snap-shot of mitochondrial sequences of elderly persons. So far, a high percentage (56%) of persons with known or unknown mutations in the fragments analyzed were found. These data will be compared in due time to a cohort of participants with proven late-onset AD.

Estrogen receptor-alpha and estrogen receptor-beta are present in the human growth plate in childhood and adolescence, in identical distribution.

OBJECTIVE: To localize estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) within the growth plate and adjacent bony tissue of children in the prepubertal and pubertal age period. METHODS: Tissue was taken during orthopedic surgery (epiphysiodesis) for correction of congenital or traumatic leg length difference in 2 prepubertal females and 2 adolescent males. Immunohistochemistry was performed on paraffin-embedded or cryostat sections by using commercially available rabbit polyclonal antibodies for ER-alpha and ER-beta. RESULTS: Both ER-alpha and ER-beta were detected within the growth plate in all sections investigated. Immunostaining was restricted to hypertrophic chondrocytes. In the bony tissue adjacent to the growth plate, osteoblasts stained positive for both ER-alpha and ER-beta, whereas osteocytes and osteoclasts were negative. Staining with ER-alpha was mainly nuclear but some cells also showed cytoplasmic signals, while ER-beta staining was predominantly cytoplasmic, only few nuclei stained positive. There was no difference in the local distribution of both ERs between tissue from prepubertal and pubertal patients. CONCLUSION: Our findings indicate that the hypertrophic chondrocyte is the main target cell for estrogen action within the growth plate. The presence of ER in prepubertal children suggests that estrogens play a role in skeletal maturation under physiological conditions also in this age-group.

Expression and dimerization of the rat activin subunits betaC and betaE: evidence for the ormation of novel activin dimers.

Activins are cytokines of the transforming growth factor beta family, which plays a central role in the determination of cell fate and the regulation of tissue balance. Family members are composed of two subunits and this dimerization is critical for liganding their cognate receptors and execution of proper functions. In the current study we focused on the localization of activin betaA, betaB, betaC and betaE subunits in the adult rat and analyzed the composition of putative activin beta dimers. By dissecting tissue distribution of various activins, we found that the liver, in particular the hepatocytes, is the major source for activin betaC and betaE transcripts, since other tissues almost failed to express these isoforms. In sharp contrast, the emergence of activin betaA and betaB appeared ubiquitous. Using a highly selective proteome approach, we were able to identify homo- as well as heterodimers of individual activin subunits, indicating a high redundancy of ligand composition. Certainly, this broad potential to homo- and heterodimerize has to be considered in future studies on activin function.

Improved antigen and nucleic acid detection in a bovine virus diarrhoea eradication program.

A bovine viral diarrhoea/mucosal disease (BVD/MD) control and eradication program was introduced in Lower Austria in 1996, according to the Swedish model. An important risk factor for BVD transmission under local conditions is communal grazing where susceptible pregnant cattle from several herds may be mixed with unrecognised persistently infected (PI) animals. A reliable system for identification of PI animals is therefore essential for BVD eradication and steps were taken to improve a commercially available antigen-capture ELISA (Ag-ELISA) by modifying the method for leukocyte preparation and adjusting the negative cut-off value. A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) employing panpestivirus 324/326 primers targeting the 5'-untranslated region of the virus genome was also simplified and used on pooled blood samples to facilitate larger sample throughputs. RT-PCR positive pools were analysed individually to identify infected animals. Seven hundred eighty-six samples were tested by Ag-ELISA according to the instruction manual and 5324 samples with the modified method. All 6110 samples were retested by RT-PCR. The percentage of RT-PCR positive results with doubtful and negative Ag-ELISA samples significantly diminished using the modified method (from 4.71 to 0.82%). Selected BVD viruses were genetically typed by PCR product sequencing; special attention being paid to RT-PCR amplicons from samples which were negative or doubtful by ELISA. However, no correlation was found between the phylogenetic grouping of the viruses and the Ag-ELISA results.

Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups.

Seventy-eight bovine viral diarrhoea viruses (BVDV) recently collected in Austria, France, Hungary, Italy, Slovakia, Spain and UK were genetically typed in the 5'-untranslated (5'UTR) and autoprotease (Npro) regions of the pestivirus genome. Seventy-six of the isolates were BVDV-1 and two French isolates were of the BVDV-2 genotype. Phylogenetic analysis of the 5'UTR (245 nt), including additional BVDV-1 sequences from USA, Canada, Germany, New Zealand, Mozambique and Sweden, taken from GenBank and from our previous works, indicated that these viruses were clustered not only into the two generally accepted groups (BVDV-1a-"NADL like" and BVDV-1b-"Osloss like"), but altogether into 11 phylogenetic groups. Similar clustering was observed with Npro region sequences (385 nt) and the highest bootstrap values (over 95%) were obtained by phylogeny combining 5'UTR and Npro sequences. Some associations between the genetic grouping and the origin of the isolates were apparent, probably reflecting historical trade contacts. Considering the variability of isolates it is recommended that diagnostic PCR primers should be re-examined to ensure coverage of all BVDV-1 groups. The genogroups were less clearly differentiated by monoclonal antibody typing, suggesting significant antigenic similarities within the BVDV-1 genotype.

Expression of galanin in human placenta.

The neuropeptide galanin was originally implicated in the regulation of feeding behaviour. Today, galanin is implicated in several physiological functions including reproduction and feeding. Many hypothalamic neurohormones of the hypothalamo--pituitary axis (HPA) are also expressed in the placenta where the specialized topological compartments of the HPA are missing and where paracrine and autocrine regulatory mechanisms consequently prevail. Since galanin influences gonadotrophin-releasing hormone secretion in the HPA, we argued that a similar regulatory role for galanin might exist in human placenta. Since the presence of galanin in human placenta had not been previously reported, we analysed galanin expression in the human placenta by immunohistochemistry and quantitative polymerase chain reaction (PCR) throughout gestation. We found that the peptide hormone localizes to the syncytio- and cytotrophoblast layers; its RNA could be detected. By quantitative PCR we observed that throughout gestation, there is a loss of galanin mRNA which parallels the fall in signal intensity from immunohistochemical detection of the galanin oligopeptide. Furthermore, we detected secretion of galanin from isolated trophoblastic cells. We conclude that galanin may be an important and novel regulator of placental function.

Expression of muscarinic receptor types in the primate ovary and evidence for nonneuronal acetylcholine synthesis.

The presence of muscarinic receptors (MR) in the ovary of different species has been recognized, but the identity of these receptors as well as ovarian sources of their natural ligand, acetylcholine (ACh), have not been determined. Because luteinized human granulosa cells (GC) in culture express functional MR, we have determined whether the group of the related MR subtypes, M1R, M3R, and M5R, are present in vivo in human and rhesus monkey ovaries. To this end, ribonucleic acids (RNAs) of different human and monkey ovaries as well as RNAs from human GC and monkey oocytes were reverse transcribed and subjected to PCR amplification, followed by sequencing of the amplified complementary DNAs. Results obtained showed that M1R, M3R, and M5R messenger RNAs are present in adult human and monkey ovaries; oocytes express exclusively the M3R subtype, whereas GC express M1R and M5R. To determine the ovarian source(s) of the natural ligand of these ACh receptors, we attempted to localize the enzyme responsible for its synthesis with the help of a monoclonal antibody recognizing choline acetyltransferase for immunohistochemistry. In neither human nor monkey sections did we detect immunoreactive choline acetyltransferase-positive fibers or nerve cells, but, surprisingly, GC of antral follicles showed prominent staining. To determine whether GC can produce ACh, human cultured GC derived from preovulatory follicles were analyzed using a high pressure liquid chromatography technique. The results showed that these cells contained ACh in concentrations ranging from 4.2-11.5 pmol/10(6) cells. Samples of a rat granulosa cell line likewise contained ACh. Thus, the ovary contains multiple MR, and GC of antral follicles are able to synthesize ACh, the ligand of MR. We propose that ACh may serve as an as yet unrecognized factor involved in the complex regulation of ovarian function in the primate, e.g. regulation of cell proliferation or progesterone production.

Biology of transforming growth factor beta in hepatocarcinogenesis.

TGF-beta is an important factor in the regulation of liver growth. It is an inhibitor of hepatocyte DNA synthesis and may induce active cell death, e.g., to remove excessive tissue mass. Studies using transgenic mice suggest that expression in the resting liver has to be well balanced; either under- or overexpression appear to cause an increased turnover of hepatocytes and to predispose to hepatocarcinogenesis. TGF-beta overexpression is frequently observed in human hepatocellular carcinomas, probably as a late event in tumor development. In men and mice, TGF-beta overexpression appears to be associated with loss of TGF-beta responsiveness often by disruption of TGF-beta signaling. However, mechanisms as mutations in TGF-beta receptor II or Smad2 and 4 genes, frequently observed in other human cancers, have only rarely been observed in hepatocellular carcinomas. Further studies may clarify the mechanisms by which hepatocellular tumors escape TGF-beta growth control, as well as analyze possible roles of TGF-beta overexpression in immunosuppression and angiogenesis.

Storage of bovine viral diarrhoea virus samples on filter paper and detection of viral RNA by a RT-PCR method.

Of four solid carriers tested, Whatman paper No 1 was the best for storing blood and serum samples for the diagnosis of bovine viral diarrhoea (BVD) by means of viral RNA detection. The filter papers were impregnated with 10 microl of blood or serum, followed by air drying. Samples collected in this way from persistently infected animals had lost infectivity within a few days, but viral RNA could still be detected by RT-PCR for up to 6 months. When investigated by RT-PCR, 12 blood and 10 serum samples selected at random from animals persistently infected with BVD virus showed the same results whether samples had been spotted onto filters or examined directly from the liquid state. The filters spotted with blood or serum are convenient for storage and transport of samples to a diagnostic laboratory without the need for cooling. Sequencing of amplified RNA can be used subsequently for genetic typing.

Endogenous regulation of the GnRH receptor by GnRH in the human placenta.

Gonadotrophin releasing hormone (GnRH) plays an important regulatory role in the function and growth of human placenta, but its placental expression sites and co-localization with GnRH receptor (GnRH-R) are not well known. GnRH and GnRH-R expression has been found in both placenta and cultured trophoblasts; however, cultured trophoblastic cells very quickly lose GnRH-R message and subsequently receptor protein. Speculating that endogenously released GnRH induced this down-regulation, we determined GnRH-R in cultures of trophoblastic cells in the presence of a neutralizing anti-GnRH antibody. Cells incubated with this antibody showed a strong signal for the GnRH-R, while those cultured without did not (as analysed by immunofluorescence, reverse transcription-polymerase chain reaction, protein 'dot blot' and Western blotting). Furthermore, addition of the GnRH agonist buserelin led to a reduction of the receptor protein. We have therefore shown that GnRH released from trophoblastic cells down-regulates GnRH-R in these trophoblastic cells. Having previously shown that trophoblast layers were synchronously positive for GnRH and GnRH-R, these new findings support the hypothesis of an ultrashort feedback regulation of trophoblasts by GnRH involving autocrine regulation of GnRH-R.

CPEO associated with a single nucleotide deletion in the mitochondrial tRNA(Tyr) gene.

In the muscle biopsy of a female patient with chronic progressive external ophthalmoplegia (CPEO), myopathy, and exercise intolerance, the heteroplasmic deletion of a single nucleotide (DeltaT5885) in the mitochondrial tRNA tyrosine gene (tRNA(Tyr)) was found. The mutation was associated with the mitochondrial phenotype of individual muscle fibers, suggesting a causal association of DeltaT5885 with the mitochondrial disease phenotype. The microdeletion was absent from the patient's and her relatives' blood, indicating a spontaneous somatic origin.

Reproductive outcome of 32 patients with primary or secondary infertility and uterine pathology.

We did a retrospective study on 102 patients who had a laparoscopy and a hysteroscopy during investigations for primary or secondary infertility. 32 of the 102 patients had uterine pathology. Seven of them had septate uteri, eight had uterine synechiae, another six had uterine fibroids, four had a bicornuated uterus, while the remaining had either a combination of all or other uterine anomalies. After surgical treatment of these conditions ten women conceived and five pregnancies including one twin pregnancy resulted in term deliveries.

Analysis of ultrashort feedback regulation in human placenta: synthesis and secretion of GnRH by human trophoblastic cells.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) presumably controls placental growth and functions by autocrine/paracrine mechanisms, and is therefore an important part of the neuroendocrine network in human placenta. AIM: Our earlier work had indicated that GnRH was expressed in human placenta; in extension to these findings, we wanted to analyse synthesis and release of GnRH by trophoblastic cells. GnRH-associated peptide, co-linearly synthesised with GnRH, was used as indicator of actual peptide synthesis. METHOD: First, we immunised rabbits with lipopeptides containing partial sequences of GnRH-associated peptide (GAP) and developed antibodies for immunohistochemical staining. Second, we set up a competitive enzyme immunoassay to measure GnRH: Non-biotinylated GnRH, GnRH analogues or trophoblastic cell culture supernatants were used to inhibit binding of biotinylated des-pGlu1-GnRH to a monoclonal anti-GnRH antibody. RESULTS: a) Placental sections stained positive for GAP in the layers of trophoblastic cells. b) GnRH could be detect by a competitive EIA in supernatants of placental cultures in concentrations between 200 and 5 nM. CONCLUSIONS: GnRH is synthesised and released by trophoblastic cells.

Inhibition of ovulation by a novel progestogen (drospirenone) alone or in combination with ethinylestradiol.

OBJECTIVE: To investigate ovulation inhibition with drospirenone, a novel progestogen that has a profile similar to natural progesterone, when given alone or in combination with ethinylestradiol. METHOD: Hormonal parameters (LH, FSH, 17beta-estradiol and progesterone) and peripheral parameters (cervical score, spinnbarkeit and crystallization), as well as follicle size assessed by ultrasonography, were measured in two groups of healthy women. Forty-eight women aged 19-35 years were randomly assigned to receive 0.5 mg, 1.0 mg, 2.0 mg or 3.0 mg of drospirenone over a single treatment cycle, and 52 women aged 20-35 years were randomized to receive either 2 mg drospirenone/30 microg ethinylestradiol or 3 mg drospirenone/30 microg ethinylestradiol over three treatment cycles. Baseline measurements were taken during a control pretreatment cycle. RESULTS: Adequate ovarian suppression with drospirenone alone was evident at dose levels of 2 and 3 mg, and at 3 mg all subjects had anovulatory cycles. Although both combined preparations (2 mg and 3 mg drospirenone/30 microg ethinylestradiol) inhibited the hypothalamic-pituitary-ovarian axis, follicular maturation leading to escape ovulation was observed in three subjects in the 2 mg drospirenone/30 microg ethinylestradiol group. Only one of these ovulations was considered to be definitely the result of treatment failure. All cycles in the 3 mg drospirenone/30 microg ethinylestradiol group were anovulatory. No statistically significant difference was found between treatment groups. CONCLUSION: The combination of 3 mg drospirenone/30 microg ethinylestradiol (Yasmin, Schering AG) reliably inhibits ovulation, with a low frequency of follicular maturation, and provides a reasonable safety margin.