Fate of salmonellas and competing flora in meat sample enrichments in buffered peptone water and in Muller-Kauffmann's tetrathionate medium

Studies have been carried out in which growth patterns of a Salmonella sp. and competing micro-organisms, especially other Enterobacteriaceae, were followed during pre-enrichment in buffered peptone water (BPw) and subsequent selective enrichment in tetrathionate broth (TBB). Pre-enrichment cultures were inoculated with minced meat and three reference samples containing nalidixic acid-resistant salmonellas. Irrespective of their initial numbers in BPw, Enterobacteriaceae increased to 10(8)/ml or more. During incubation in TBB at 43 degrees C, numbers of lactose-positive Enterobacteriaceae decreased in most enrichments which resulted in a positive salmonella isolation, but remained constant in the majority of those that did not. Levels of lactose-negative Enterobacteriaceae did not decrease in most salmonella-positive tests, but did so in half of the salmonella-negative ones. In the salmonella-positive tests the numbers of salmonellas had increased to 10(3)-10(7)/ml in BPw and after transfer to TBB slowly reached 10(4)/ml or more. In all cases the numbers of salmonellas exceeded those of the competing flora on brilliant green agar (BGA). In the salmonella-negative tests the numbers of salmonellas had increased less in BPw and decreased in most of the TBB enrichments. In none of these negative tests did the numbers of salmonellas exceed those of the competing flora on BGA. Escherichia coli dominated in most of the salmonella-negative tests. The results suggest more influence of lactose-positive than lactose-negative Enterobacteriaceae on the detection of salmonellas. The effect of competing microorganisms seems to depend not only upon their initial numbers, but also upon the types that can interact with salmonellas during selective enrichment.     

Are antibodies responsible for a decreased superovulatory response in goats which have been treated repeatedly with porcine follicle-stimulating hormone?

Repeated administration of xenogenic gonadotropins in human or animal species may be responsible for antibody production and refractoriness. An experiment was conducted in which goats were treated with porcine FSH (p-FSH) at 6-week intervals for a period of 7 months. A sensitive radioimmunoassay (RIA) was used to detect antibodies to p-FSH in plasma samples taken at short-term intervals during a 7-month period. Antibodies appeared after the first injection, and levels increased following booster injections. A high correlation rate existed between antibody level and superovulatory response. Refractoriness in goats was associated with a high level of antibodies.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

Collaborative study on the isolation of salmonella from artificially contaminated milk powder

The suitability of artificially contaminated milk powder as a substrate for salmonella reference samples and its stability under different storage conditions were studied. The need for a reconstitution step in the standard isolation method for salmonellas from milk powders was also investigated. When milk powder was examined in this way with a reconstitution step, differences in laboratory methods and/or storage times had no significant effect on the results after storage at 4 degrees C. With powder stored at room temperature there was a systematic decrease in the number of samples positive as the storage time increased. It is concluded therefore that milk powder contaminated with salmonellas should be stored at 4 degrees C. Examination of such milk powder with a reconstitution step yielded better results than without it and this step is therefore necessary for improving the reproducibility of the method. No significant differences were encountered between the standard isolation method and that used in the authors' laboratories. The results of this study indicate that milk powder is suitable as basic material for reference samples and that a reconstitution step should be included in the standard salmonella isolation procedure.     

Lysosomal hydrolases in calf thyroid

1. Two acid phosphatases (β-glycerophosphatase and phenylphosphatase), acid β-glucuronidase and cathepsin were demonstrated in the 0·25m-sucrose homogenates from whole calf thyroid tissue and from isolated calf thyroid cells. 2. The main kinetic characters of these enzymes were studied. 3. All these acid hydrolases are partially sedimentable and display a latency that is unmasked by treatment with Triton X-100 and on dilution in hypo-osmotic media. It is concluded that these acid hydrolases belong to the lysosomes.     

Transgenic Analysis of a PotentialHoxd-11Limb Regulatory Element Present in Tetrapods and Fish

Genes of theHoxDcomplex related to theDrosophila Abd-Bgene are involved in the morphogenesis of vertebrate paired appendages.Hoxd-11,for instance, is necessary in combination with otherHoxgenes for the proper development of different parts of the tetrapod limbs. Sequence comparisons between the mouse, chicken, and zebrafishHoxd-11loci have revealed the conservation of several blocks of DNA sequence which may be of importance for the regulation ofHoxd-11expression. We have used transgenic mice to show that one of these conserved elements specifically drives expression in a proximal-posterior part of developing forelimbs. Production of mice transgenic for a full fishHoxd-11construct as well as for mouse–fishHoxd-11chimeric constructs shows that the fish counterpart of this sequence is able to elicit expression in mouse forelimbs as well, though in a slightly different domain. However, this fish element requires the presence of the mouse promoter and does not work in its own context. These results are discussed in light of both the control ofHoxdgene expression during limb development and the use of a comparative interspecies approach to understand the regulation of genes involved in vertebrate development.     

Pregnancies, calves and calf viability after transfer of in vitro produced bovine embryos

Pregnancy, parturition and calf survival following the transfer of embryos produced in vitro were monitored. A total of 44 blastocysts was transferred in pairs to 1 uterine horn ipsilateral to the corpus luteum (CL) of 22 synchronized heifers. At Day 42 of development 14 recipients (64%) were pregnant; the calving rate was also 64%. The twinning rate was 9/14 at Day 42 and 7/14 at birth, for an overall fetal mortality rate of 9%. The average gestation length was 281 and 275 d for single and twin pregnancies, respectively. Blood samples from recipients were collected for determination of bovine pregnancy associated glycoprotein (bPAG) from 2 wk after transfer and throughout the pregnancy. During the first trimester of pregnancy, the bPAG concentration was significantly higher in twin than in single bearing heifers, and the perinatal increase in bPAG was correlated positively with the total weight of the fetus(es). The percentage of male calves was 43%. The birth weight of twin individuals was 25 +/- 1 kg, which was 78% of the birthweight of the singletons (32 +/- 2 kg). One singleton calf was oversized, weighing 58 kg (80% more than the median weight of the other singletons). Stillbirths occurred in 21% of the twins, butin none of the singletons. Calf mortality during the first 14 d was higher for twins (4/11) than for singletons (1/7) due to infections and cerebellar hypoplasia. Karyotyping the calves detected no cytogenetically recognizable abnormalities. All calves were negative for BVD virus and IBR antibodies. The results of this study showed that although the incidence of fetal loss was low, there was an unacceptable high perinatal mortality of the calves. Thus it is likely that the blood supply through the placenta of animals pregnant with twins was impaired or it is possible that these fetuses and calves had increased stress susceptibility caused by the in vitro conditions. Furthermore, the birth of 1 oversized calf, 2 calves with cerebellar hypoplasia and 5 calves succumbing to infections seems to indicate that a proportion of in vitro produced calves may suffer from factors inherent in the in vitro production system.     

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.     

Species-Specificity of the BamA Component of the Bacterial Outer Membrane Protein-Assembly Machinery

The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP) in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA.