Surveillance of antimicrobial resistance in bacteria isolated from food animals to antimicrobial growth promoters and related therapeutic agents in Denmark.

This study was conducted to describe the occurrence of acquired resistance to antimicrobials used for growth promotion among bacteria isolated from swine, cattle and poultry in Denmark. Resistance to structurally related therapeutic agents was also examined. Three categories of bacteria were tested: 1) indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium), 2) zoonotic bacteria (Campylobacter, Salmonella, Yersinia enterocolitica), and 3) animal pathogens (E. coli, Staphylococcus aureus, coagulase-negative staphylococci (CNS), Staphylococcus hyicus, Actinobacillus pleuropneumoniae). All antimicrobials used as growth promoters in Denmark and some structurally related therapeutic agents (in brackets) were included: Avilamycin, avoparcin (vancomycin), bacitracin, carbadox, flavomycin, monensin, olaquindox, salinomycin, spiramycin (erythromycin, lincomycin), tylosin (erythromycin, lincomycin), and virginiamycin (pristinamycin). Bacterial species intrinsically resistant to an antimicrobial were not tested towards that antimicrobial. Breakpoints for growth promoters were established by population distribution of the bacteria tested. A total of 2,372 bacterial isolates collected during October 1995 to September 1996 were included in the study. Acquired resistance to all currently used growth promoting antimicrobials was found. A frequent occurrence of resistance were observed to avilamycin, avoparcin, bacitracin, flavomycin, spiramycin, tylosin and virginiamycin, whereas resistance to carbadox, monensin, olaquindox and salinomycin was less frequent. The occurrence of resistance varied by animal origin and bacterial species. The highest levels of resistance was observed among enterococci, whereas less resistance was observed among zoonotic bacteria and bacteria pathogenic to animals. The association between the occurrence of resistance and the consumption of the antimicrobial is discussed. The results show the present level of resistance to growth promoters in bacteria from food animals in Denmark. They will form the baseline for comparison with future prospective studies, thereby enabling the determination of trends over time.

Resistance to antimicrobial agents used for animal therapy in pathogenic-, zoonotic- and indicator bacteria isolated from different food animals in Denmark: a baseline study for the Danish Integrated Antimicrobial Resistance Monitoring Programme (DANMAP).

This study describes the establishment and first results of a continuous surveillance system of antimicrobial resistance among bacteria isolated from pigs, cattle and broilers in Denmark. The three categories of bacteria tested were: 1) indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium), 2) zoonotic bacteria (Campylobacter coli/jejuni, Salmonella enterica, Yersinia enterocolitica), and 3) animal pathogens (E. coli, Staphylococcus aureus, coagulase-negative staphylococci (CNS), Staphylococcus hyicus, Actinobacillus pleuropneumoniae). A total of 3304 bacterial isolates collected from October 1995 through December 1996 were tested for susceptibility to all major classes of antimicrobial agents used for therapy in Denmark. Bacterial species intrinsically resistant to an antimicrobial were not tested towards that antimicrobial. Acquired resistance to all antimicrobials was found. The occurrence of resistance varied by animal origin and bacterial species. In general, resistance was observed more frequently among isolates from pigs than from cattle and broilers. The association between the occurrence of resistance and the consumption of the antimicrobial is discussed, as is the occurrence of resistance in other countries. The results of this study show the present level of resistance to antimicrobial agents among a number of bacterial species isolated from food animals in Denmark. Thus, the baseline for comparison with future prospective studies has been established, enabling the determination of trends over time.

Surveillance of cattle herds for bovine virus diarrhoea virus (BVDV)-infection using data on reproduction and calf mortality.

The effect of bovine virus diarrhoea virus (BVDV)-infection on pregnancy rate, on stillbirths and mortality of neonatal calves and the size of newborn calves was evaluated in 8 herds in which persistently infected (PI)-animals had been identified. Data from 9 herds without PI-animals were used as controls. At the time of conception of the oldest PI-animal a significant drop in pregnancy rate to about half the herd average was found. About 6 months later a 3-fold rise in calf mortality was seen. This pattern was found in 4 of the herds. In the remaining 4 herds the pattern was less clear, probably reflecting different immune states of the herds. Rough estimation of the size of newborn calves showed that PI-animals were significantly smaller than normal animals. Monitoring of herds for the above-mentioned parameters may be a means of pointing out herds with PI-animals. Most of the data necessary for such surveillance schemes are already available and may readily be used.

Monoclonal blocking ELISA detecting serum antibodies to the glycoprotein gII of Aujeszky's disease virus.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Aujeszky's disease virus (ADV) in porcine serum was developed. This ELISA is based on the reaction between virus antigen immobilized in a microdilution plate and a monoclonal antibody (MAb) reactive with a highly stable epitope on a glycoprotein complex, gII, of ADV. The viral epitope was expressed by 18 European field, laboratory and vaccine strains of ADV. The MAb used in the test was selected among 15 MAbs all reactive with viral epitopes apparently recognized by the porcine immune system as well. Good agreement was found when serum samples from 375 pigs were tested in both a polyclonal and the monoclonal blocking ELISA.

Transmission of bovine virus diarrhoea virus (BVDV) by artificial insemination (AI) with semen from a persistently-infected bull.

Twelve heifers that did not have antibodies to bovine virus diarrhoea virus (BVDV) were inseminated with semen from a bull that was persistently infected with the virus and contained 10(4.0)-10(6.5) TCID50 0.1 ml-1. All 12 became infected, as indicated by seroconversion within 2 weeks of insemination. Four control heifers were inseminated with virus-free semen. The virus was not transmitted to these animals in spite of close contact with the heifers inseminated with the infected semen. All the heifers became pregnant and gave birth to clinically normal calves at term. However, one calf was born persistently infected with BVDV. After the birth of this persistently-infected calf the control heifers and their calves seroconverted. The study demonstrates that BVDV may be transmitted in cattle by artificial insemination (AI). Therefore entry of persistently-infected animals into AI centres should be prevented.

Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark.

The aim of the present study was to evaluate capsular-typing, plasmid-profiling, phage-typing and ribotyping for epidemiological studies of toxin-producing Pasteurella multocida ssp. multocida in Denmark. The evaluation of methods was based on 68 strains from nasal swabs and 14 strains from pneumonic lungs. Strains from lungs were all of capsular Type A, whereas strains from nasal swabs were of both capsular Types A and D. Only 9% of the strains contained plasmids, which could not be associated with antibiotic resistance. Phage-typing divided 61% of strains into 10 groups, while 39% were non-typable. CfoI ribotyping divided strains into four groups of which one type contained 94% of isolates. HindIII ribotyping divided strains into 18 types. A total of 18 strains from The Netherlands, UK and USA were subjected to HindIII ribotyping, resulting in 13 types of which six were identical to ribotypes of Danish strains. Phage-typing of isolates from an outbreak of atrophic rhinitis involving six herds in 1985 showed the existence of an epidemic strain. This type was recognised in the herd suspected of being the source of the infections and in four of the five infected herds. These findings were supported by HindIII ribotyping, as 85% of isolates from all herds were assigned to one ribotype. In conclusion, HindIII ribotyping seems to represent a useful tool for epidemiological studies of toxigenic P. multocida ssp. multocida.

A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycoplasma hyopneumoniae in porcine serum has been developed. The monoclonal antibody (mAb) reacts with an M. hyopneumoniae specific epitope on a molecule of approximately 74 kDa. Only sera from M. hyopneumoniae infected pigs were able to block the binding of the mAb although antibodies from M. flocculare infected pigs also recognized a 74 kDa molecule. Sera from experimentally infected pigs as well as field samples were compared by the ELISA and by an indirect hemagglutination assay (IHA). In experimental pigs, the earliest detectable antibody response was found to be almost identical for both assays, but for some of the pigs the time of detection was significantly earlier by blocking ELISA than by IHA. In naturally infected herds more samples were found to be positive by ELISA than by IHA. Furthermore, the results indicate that sera from naturally M. flocculare infected pigs may give rise to cross-reactions in the IHA. The blocking ELISA appears to be a valuable and reproducible tool in the surveillance and serodiagnosis of M. hyopneumoniae infections in pigs.

Epidemiology of bovine virus diarrhoea virus.

A better understanding of the epidemiology and pathogenesis of bovine virus diarrhoea virus (BVDV) has emerged in recent years. Fetal infections and in particular those resulting in birth of persistently infected calves are of central importance for the epidemiology of BVDV. A prevalence of persistently infected, viraemic animals of about 1% is found in Denmark and elsewhere by examination of randomly collected blood samples. A recent field study shows that 53% of randomly selected herds in an area in Denmark where BVDV is endemic had one or more persistently infected animals. Persistently infected cows may breed and will always transmit the infection to the calf. Such familial occurrence of persistent infection seems to be a fairly common phenomenon. Persistently infected cattle are important sources of infection to other cattle. Transiently infected cattle following experimental exposure will usually not transmit the infection by contact but this may not always apply to cattle after natural infection. Knowledge of the occurrence and potential for spread of virus from persistently infected bulls is reviewed. Virus is excreted with semen of both persistently and transiently infected bulls and BVDV may be transmitted by use of infected semen for insemination. The potential for spread of the infection through embryo transfer should be avoided by the use of adequate testing and controls.

Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle.

Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities.

The need for a veterinary antibiotic policy.

The international recognition of the 'stable to table' approach to food safety emphasises the need for appropriate and safe use of antibiotics in animal production. An appropriate use of antibiotics for food animals will preserve the long-term efficacy of existing antibiotics, support animal health and welfare and limit the risk of transfer of antibiotic resistance to humans. Furthermore, it may promote consumer confidence in the veterinary use of antibiotics. In advancing these arguments, the authors of this article argue that there is a need for a visible and operational policy for veterinary use of antibiotics, paying particular attention to the policies that are being developed in Denmark.