Results of the German investigation on the EU project "Salmonella in Pork (Salinpork)"--1. Investigations in the farms

Reports of human salmonellosis caused by the consumption of pork and the introduction of control and surveillance programmes in different countries of the European Union were the reason for carrying out an international study under the title "Salmonella in Pork (Salinpork)": Six different EU-countries were involved in this study, which was supported by the Commission of the European Community and which was carried out over a period of April 1996 to April 1999. The aim of the investigation presented was to determine the prevalence of Salmonella in fattening, breeding and farrow-to-feeder herds as well as the determination of risk factors for the introduction of Salmonella into the farm (Part 1). In addition, sources of contamination of pork should be detected by taking samples of the product and the environment in the slaughterhouse (Part 2). In Germany, the investigation into Salmonella infections of 60 fattening, 20 breeding and 20 farrow-to-feeder herds were carried out in Schleswig-Holstein. The investigation included bacteriological examinations of feed and faecal samples for sero- and phagetyping and serological examinations by using the Danish Mix-ELISA. From 2,947 serological investigated fattening pigs were 7.3% (n = 213) positive, from 797 breeding sows 9.2% (n = 73) were serological positive and 4.5% (n = 18) of the investigated sows (N = 399) in farrow-to-feeder herds were serological positive. Altogether, 28.3% of the fattening, 50.0% of the breeding and 15.0% of the farrow-to-feeder herds were serological positive. A questionnaire was used to capture data about management, hygiene measures, feeding systems and the occurrence of diseases in the herd. After statistical analysis the common risk factor of fattening herds and sow herds was the use of pelleted feed. But in a control study with 17 different fattening herds the result could not be proven. Other factors which can influence the occurrence of Salmonella infections were discussed.     

Results of the German investigation in the EU Project "Salmonella in Pork (Salinpork)"--2. Investigations in a slaughterhouse

In 1997 bacteriological examinations for the distribution of Salmonella in slaughterhouses were carried out in Germany within the framework of an international study "Salmonella in Pork (Salinpork)". During 6 days, 1,200 swab and water samples from slaughtered pigs and the environment were taken. 4.4% of the samples (n = 53) were Salmonella positive. S. typhimurium was isolated mainly (69.8%; n = 37), and 6 phagetypes were differentiated. In addition, S. derby and S. panama could be demonstrated. The resistance pattern of the different isolated S. typhimurium-phagetypes are presented. The phagetype DT 104 was multiresistant to ampicillin, spectinomycin, streptomycin, sulphonamide and tetracycline. In comparison with the serological prevalence of 7.3% of the fattening pigs in the farms (Part 1), only 1.0% of the samples taken from the surface of the carcass were Salmonella-positive. Swabs taken from the liver were in 2.7% positive and samples from the tongue gave in 5.3% of the cases Salmonella-positive results. In the examination of the environment Salmonella was demonstrated mainly from the water outlets, whereas Salmonella could not be isolated from water of the scalding tank. There was only one case (0.7%) in which Salmonella could be isolated from the hands of the personnel, and also only one swab of the polishing machine was positive (1.1%). But 6.7% samples of the saw were Salmonella-positive. A comparison of repeated, at intervals taken samples showed that the number of Salmonella-positive samples was higher in the last examination round of the particular slaughter days. The reason is suspected in the increasing number of slaughtered pigs and supplying farms, which may increase the probability of bringing in Salmonella.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

Spontaneous production of nitric oxide (NO), prostaglandin (PGE2) and neutral metalloproteinases (NMPs) in media of explant cultures of equine synovial membrane and articular cartilage from normal and osteoarthritic joints

Nitric oxide (NO), prostaglandin E2 (PGE2), and the activity of neutral metalloproteinases (NMPs) were measured in conditioned media of equine synovial membrane and articular cartilage explant cultures from horses with normal joints (n = 7) and from horses affected with moderate (n = 7) or severe osteoarthritis (n = 14) as judged by macroscopic appearance. Normal articular cartilage appeared glossy and bluish-white, was of normal thickness and showed no evidence of discolouration, fibrillation or other cartilage discontinuity. Slight discolouration and fibrillation or minor clefts of the cartilage were considered as moderate OA, whereas erosions of articular cartilage down to the subchondral bone were considered as cases of severe OA. Explant cultures of equine synovial membrane and articular cartilage released the local mediators, NO and PGE2, as well as detectable levels of NMP activity into culture media. Concentrations of NO were higher in articular cartilage explants compared to synovial membrane explants, whereas concentrations of PGE2 were higher in synovial membrane explants. The NMPs with collagenolytic activities were similar in both explant cultures, whereas gelatinolytic activities were higher in synovial membrane explant cultures and caseinolytic activities were generally higher in articular cartilage explant cultures. Furthermore it was shown that concentrations or enzyme activities increased according to the severity of disease of the joints. Concentrations for NO, collagenolytic and gelatinolytic NMPs were relatively stable, whereas PGE2 and caseinolytic NMP concentrations increased over time in culture.     

THE PHYSIOLOGIC RESPONSE OF DOMESTIC ANIMALS TO IONIZING RADIATION: A REVIEW

With respect to clinical signs of the radiation syndromes, some remarkable species variations exist. For example the marked delayed reaction of the acute hematologic response in cows. An unusually high sensitivity of the central nervous system is found in burros, which is probably caused by acute vascular and/or metabolic changes in the brain. The species-specific number of intestinal crypt and hemopoietic stem cells may explain the early survival differences among species after high doses of irradiation. Mortality due to acute radiation syndromes is lowest in chickens. Regarding late effects, various neoplasms are typical in dogs, and cattle more commonly develop cataracts.     

Charcoal affects early development and hormonal concentrations of somatic embryos of hybrid larch

Embryogenic tissue of hybrid larch (Larix x marschlinsii Coaz) was multiplied on Medium M (modified MSG medium supplemented with the plant growth regulators (PGRs) 2,4-dichlorophenoxyacetic acid (2,4-D; 9 microM) and N-6-benzyladenine (2.25 microM)). After 1 week, cultures were transferred to either MSG lacking PGRs (Medium C-) or MSG lacking PGRs but supplemented with 1% activated charcoal (Medium C+). Embryos were sampled after 1 week on Medium M, C- or C+. Embryos were analyzed by ELISA for abscisic acid (ABA), abscisic acid-glucose ester, 2,4-D, indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (iP) and isopentenyladenosine (iPA). Transfer of embryos to Medium C+ reduced the embryo concentrations of 2,4-D and iPA, but resulted in elevated concentrations of IAA, IAAsp, ABA, Z, ZR and iP. Charcoal reduced 2,4-D concentrations of embryos by an order of magnitude greater than PGR-free medium alone. Charcoal affected embryo concentrations of five of the eight PGRs quantified. Use of either C+ or C- medium as part of the maturation protocols also affected germination and plantlet establishment of the embryos. A 1-week treatment on Medium C+ positively influenced plantlet establishment and generally reduced variability during both germination and plantlet establishment.     

High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.