Programmes to control or eradicate Salmonella in animal production in Denmark.

The number of diagnosed cases of salmonella infections in humans has been increasing during the latest 10 years, for the last 5 years mainly because of an increase in infections with Salmonella Enteritidis. As far as Danish produced animal products is concerned, it is assumed that the most important sources of human salmonella infections are, in order of priority: eggs, poultry meat and pork. In Denmark there are at the moment public and voluntary salmonella pre-harvest reduction programmes in the production of pigs, broilers and eggs. The programme in the pig production is a control programme, that means that the aim is to maintain a generally low level of salmonella in pig herds. At the same time the goal of a low level of salmonella contamination of pork is also pursued through general and specific hygiene measures in the slaughterhouses. The programmes in the poultry production are limited to broilers and hens eggs. They are, at least in theory, eradication programmes where the aim is total freedom from salmonella. According to the rules of Council Directive 92/117/EEC, flocks of hens producing eggs for hatching must be free from Salmonella Enteritidis and Typhimurium, whereas according to the Danish national requirements (Veterinary Service Orders to come into effect shortly), these two salmonella serotypes must be eradicated from flocks of hens producing eggs for sale to consumers and all salmonella serotypes must be eradicated from flocks of hens producing eggs for hatching.

Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements.

Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086. Several characteristics suggest that this element is a transposon, which we call Tn5090. Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins. The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein. The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly. Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism. The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.

Equine herpesvirus type 1: detection of viral DNA sequences in aborted fetuses with the polymerase chain reaction.

Primers and probes were selected from the gene encoding glycoprotein 13 (gp 13) of equine herpesvirus 1 (EHV-1). The polymerase chain reaction (PCR) was run on infected and noninfected cultured cells and on 63 specimens from 29 aborted equine fetuses. The results were evaluated by electrophoresis and dot-blot hybridization using an oligonucleotide probe labeled with biotin. In the infected samples electrophoresis showed a PCR product of about 280 base pairs. The dot-blot hybridization confirmed that this product contained EHV-1 DNA sequences. PCR took 4 h and hybridization another 14 h; the results were thus achieved within 24 h and were highly specific for EHV-1. Close concordance was found between the results of PCR and virus isolation.

The effect of a control program for enzootic bovine leukosis. Changes in herd prevalence in Denmark, 1969-1978.

As part of the Danish control program for enzootic bovine leukosis, three hematologic screenings of all cattle herds in Denmark were performed in the period 1969-1978. Herds with multiple cases of persistent lymphocytosis and/or leukotic tumors were classified as "leukosis herds." During nationwide blood testing, 369 leukosis herds were discovered, and an additional 77 herds were found due to follow-up of tumor cases or special testing in connection with movements of cattle in 1969-1978. In the present study, the prevalence of leukosis herds in the three screening rounds is related to geographic area and herd size. By means of a statistical log-linear model, the authors show that prevalence increased proportionally with herd size. The observed number of leukosis herds per 10,000 herds tested declined during the screening program from 105 to 38 in east Denmark and from 12 to 4 in west Denmark.

Detection of pseudorabies virus DNA sequences by the polymerase chain reaction.

Genomic sequences of pseudorabies virus, a porcine herpesvirus, were amplified by the polymerase chain reaction from cells of infected cultures, nasal cells and organs from acutely diseased as well as from organs of latently infected pigs.

Bacteriophage P4 DNA replication. Nucleotide sequence of the P4 replication gene and the cis replication region.

A 3100 base piece of DNA from the 11,500 base genome of bacteriophage P4 was analyzed for its nucleotide sequence. This segment of DNA contains two open reading frames of 106 and 777 amino acid residues; the latter of which is the coding sequence for the Mr 84,841 alpha protein, which is necessary for P4 DNA replication and is thought to act as a P4-specific DNA primase. A region of about 300 base-pairs localized just beyond the alpha gene and about 4500 bases from the origin of replication (ori), was defined as the locus for P4's cis replication region (crr). This region is required for replication both in vivo and in vitro, and consists of two directly repeated sequences of 120 base-pairs that match one another at 98 positions. These directly repeated sequences are separated by 60 base-pairs, which are not necessary for replication. Each repeat in crr contains three copies of the octamer TGTTCACC that is found six times in ori. Either of the 120 base-pair repeat sequences in crr is sufficient for replication, and the entire crr can function in an inverted orientation. crr is also active at a distance of 1800 bases from the P4 origin of replication.

Massive overproduction of dihydrofolate reductase in bacteria as a response to the use of trimethoprim.

Among several observations of greatly increased levels of chromosomal dihydrofolate reductase as a cause of resistance to high concentrations of the antifolate drug trimethoprim, in clinically isolated bacteria, one is described here of a strain of Escherichia coli overproducing dihydrofolate reductase several hundredfold. The chromosomally located resistance gene of this strain was isolated, inserted into a plasmid vector, and analyzed for its nucleotide sequence. The structural gene for the overproduced dihydrofolate reductase was found to be identical to that of E. coli K12, with nine exceptions, of which seven resulted in synonymous codon usage. Two transversions resulted in a substitution of Gly or Trp at amino acid position 30, and of Gln for Glu at position 154. Six of the nine base changes resulted in codons more frequently used. The Gly substitution which leads to a less commonly used codon, was thought to relate to the observed threefold increase in Ki for trimethoprim. Furthermore, a C----T transition was found in the -35 region of the promoter, increasing its homology with the E. coli consensus promoter sequence. In the ribosome-binding area of the resistant strain, finally, seven base changes were observed, two of which resulted in a five-base sequence of complementarity with the 3'-end of ribosomal 16S RNA. The distance between the -10 site of the promoter and the start codon for translation was finally increased one base pair by the insertion of an A at position +9 in the resistant strain. These genetic changes towards more efficient transcriptional and translational start sequences and towards increased mRNA expressivity are interpreted to reflect an evolutionary adaptation to the presence of antifolates.

Regulatory changes in the formation of chromosomal dihydrofolate reductase causing resistance to trimethoprim.

High resistance to trimethoprim mediated by the several hundredfold overproduction of the drug target enzyme, dihyrofolate reductase, in a clinically isolated Escherichia coli strain, 1810, was cloned onto several vector plasmids and seemed to be comprised of a single dihydrofolate reductase gene, which by DNA-DNA hybridization and restriction enzyme digestion mapping was very similar to the corresponding gene of E. coli K-12. Determination of mRNA formation in the originally isolated resistant strain and strains with cloned trimethoprim resistance determinant demonstrated an about 15-fold increase in production of dihydrofolate reductase mRNA compared with that in E. coli K-12. This was explained by the occurrence of a promoter up mutation in the resistant isolate accompanied by changes in the restriction enzyme digestion pattern found by comparison with the corresponding pattern from E. coli K-12.

An outbreak of duck virus enteritis among ducks and geese in Denmark.

Duck virus enteritis occurred in the spring of 1982 among domesticated mallards, Pekin ducks and geese producing eggs for the same hatchery. Wild mallards may have introduced the infection to the domestic birds. High mortality occurred in one flock of Pekin ducks and in young geese. Mallards were also affected, but less severely. Gross and microscopic lesions were in general typical for DVE. Virus was demonstrated by electron microscopy of Bursa fabricii from experimentally infected ducklings. Neutralizing antibodies were found in serum from ducks, surviving an acute outbreak in the flock. Vaccination was performed and hygienic precautions taken, and transmission from infected flocks to progeny was negligible.

Testing blood plasma from cattle in leukosos-free herds for antibodies against bovine leukemia virus (BLV).

As a prelude to the introduction of serological methods in the Danish leukosis eradication programme, an examination for antibody to Bovine Leukemia Virus (BLV) was carried out in 215 randomly selected leukosis-free herds in three areas where 3 routine haematological screening tests had bee made over a period of 6 years. A gel diffusion test was applied to plasma samples. Of 3319 animals screened, none were found to have detectable levels of antibody to BLV in their plasma. This would seem to indicate that false positive reactions are not a serious problem in connection with this test, and also that herds classified as leukosis-free after a series of haematological herd tests carried out within a period of some years, are in fact free from infection with Bovine Leukemia Virus.

The control of bovine leukosis in denmark, Epidemiologic and diagnostic aspects.

The Danish programme to control bovine enzootic leukosis was initiated in 1959 and intensified in 1969. The programme has resulted in a sharp decline in total and relative numbers of leukotic tumor cases in adult animals. However, total eradication of bovine leukosis has not yet been achieved, and the statistics from recent years seem to indicate that the present programme based on haematological and histological examinations will control but not eradicate the disease. An immunodiffusion test based on an internal protein (p 24) antigen has been used experimentally. This test is a valuable tool in herd diagnosis, but it is not sensitive enough for accurate single animal diagnosis.

Studies on age specific leukocyte and lymphocyte counts in normal Friesian, Red Danish and Jersey cattle in Denmark.

To determine reference values for leukocyte and lymphocyte counts of the most common cattle breeds in Denmark, especially regarding the evaluation procedure in the leukosis control work, blood samples from normal cattle of all ages within the Friesian, the Red Danish and the Jersey breeds were collected, transported and counted as in the routine leukosis control programme. Evaluation of breed- and age specific counts showed numerically small, but statistically significant differences in mean values of leukocytes and lymphocytes between on the one hand Jersey cattle and on the other hand Red Danish and Friesian cattle. However, it was found that the key values used in the leukosis control work are generally in good agreement with the calculated 99.87 percentiles of all age- and breed specific lymphocyte count distributions, which were approximated with logarithmic normal distribution curves.