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.

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.

Design of a system for monitoring antimicrobial resistance in pathogenic, zoonotic and indicator bacteria from food animals.

DANMAP is a Danish programme for integrated monitoring of and research on antimicrobial resistance in bacteria from food animals, food and humans. The paper describes how bacteria from broilers, pigs, and cattle are collected, as well as the procedures for data handling and presentation of results. The bacteria from animals include certain pathogens, selected so that they are representative for submissions to Danish diagnostic laboratories, as well as zoonotic bacteria (Campylobacter, Salmonella and Yersinia) and indicator bacteria (E. coli, E. faecium and E. faecalis), from samples collected at abattoirs. The latter samples are selected so that they are representative of the respective animal populations. Therefore, the apparent prevalence of antimicrobial resistance in the populations may be calculated. The isolates are identified to species level and the results of susceptibility testing are stored as continuous variables. All isolates are maintained in a strain collection so that they are available for subsequent research projects. The data handling facilities makes it possible to present results as percent resistant isolates or as the apparent prevalence of resistance in the population, or alternatively as graphical distributions of mm inhibition zones or MIC values. Computer routines have been established that make it possible to detect specific phenotypic expressions of resistance that may be of particular interest.

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 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.

Molecular characterization of Escherichia coli strains isolated from pigs with edema disease.

The present study was conducted to investigate the epidemiological relationship of isolates of Escherichia coli causing edema disease. Classical edema disease has not previously been described in Denmark, but between February 1994 and November 1995 cases appeared in 51 pig herds, among which direct or indirect trading contacts were confirmed for 36 of the herds. A total of 213 isolates from pigs with edema disease in Denmark and other countries and 23 E. coli O139 isolates from pigs with diarrhea or healthy pigs were analyzed to characterize their O serogroups, HindIII ribotypes, and pulsed-field gel electrophoresis (PFGE) types, and 183 of the isolates were also analyzed for their plasmid profiles. The resulting PFGE types of the isolates from pigs with edema disease were examined by cluster analysis. Ten isolates from three herds could not be typed with the available O antisera, whereas all other isolates were of serotype O139. However, all isolates from pigs with edema disease belonged to the same HindIII ribotype, which was not observed among the isolates from pigs with diarrhea or healthy pigs. All isolates from Danish pigs with edema disease except for three isolates originating from two herds belonged to the same or closely related XbaI PFGE types; the other three isolates were assigned to possibly related types. Isolates from pigs with edema disease in different countries belonged to different PFGE types. All isolates from Danish pigs with edema disease grouped together in one cluster, in contrast to isolates from other countries, which did not form any clusters. E. coli strains of serogroup O139 from pigs with diarrhea or isolated from the feces of healthy Danish pigs were very different. Plasmid profiles differed largely among isolates. However, among the isolates from Danish pigs with edema disease, one type predominated within herds. The present study indicated that most, if not all, of the observed cases of edema disease in Denmark were part of the same outbreak. The combination of PFGE typing and ribotyping was useful for studying the possible clonal relationship among strains, whereas plasmid profiling was less informative.

Detection of fimbrial and toxin genes in Escherichia coli and their prevalence in piglets with diarrhoea. The application of colony hybridization assay, polymerase chain reaction and phenotypic assays.

Two different genotypic methods, colony hybridization and polymerase chain reaction (PCR), were applied to detect enterotoxin, verotoxin and fimbrial genes in 708 Escherichia coli (E. coli) strains from piglets with diarrhoea. The results were compared with those obtained by phenotypic methods. DNA fragments specific for each of the enterotoxins LT, STa and STb, the verotoxins VT1, VT2 and VT2v, and for each of the fimbrial genes K88 (F4), K99 (F5), 987P (F6), F41 and F107, respectively, were used as probes in a colony hybridization assay of the E. coli strains. A PCR assay was used as genotypic test for the verotoxin gene. An Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was performed to test for the presence of K88 and K99 fimbriae, and a Vero cell test was performed to test for verotoxin production. Toxin detection kits were applied to detect the E. coli heat-labile enterotoxin (LT) and the heat-stable (STa) enterotoxin. The correlation between the results obtained by genotypic and phenotypic methods was 97.7-100%. The prevalence of the different fimbrial and toxin genes in E. coli strains from piglets with neonatal and postweaning diarrhoea were recorded. The verotoxin and the fimbrial F107 genes were found to be more frequent in postweaning E. coli strains than in neonatal E. coli strains.

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.

A field study of Mycoplasma bovis infection in cattle.

After an outbreak of mastitis in cattle caused by Mycoplasma bovis a study was made in 5 herds with recent cases (principal herds) and in 4 control herds. In the principal herds, M. bovis was isolated from milk samples, nasal swabs, and from one vaginal swab. M. bovis was also isolated from nasal swabs of calves in 2 of the 4 control herds, whereas all milk samples and vaginal swabs from the control herds were negative. Evaluation of serum antibody titres to M. bovis among non-mastitic animals of 3 principal herds and 1 control herd showed no difference in distribution of the titre values, which generally were low. However, cows excreting M. bovis in the milk had high antibody titres. The way of introduction to the herds and the spread of the infection within the herds could not be established by the study, which was supplemented by a DNA restriction fragment analysis of a number of M. bovis isolates.

Comparison of flow cytometry and virus isolation in cell culture for identification of cattle persistently infected with bovine viral diarrhea virus.

Detection of bovine viral diarrhea virus in 143 blood samples by virus isolation in cell culture and flow cytometry was performed. The material included 37 samples later shown to originate from persistently infected cattle. Thirty-three samples were positive by virus isolation, and all 37 samples were positive by the flow cytometric assay.

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.

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.

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.

Detection by enzyme-linked immunosorbent assay of Aujeszky's disease virus in tissues of infected pigs.

An enzyme-linked immunosorbent assay was developed for the detection of Aujeszky's disease virus antigen in tissue extracts and in nasal swabs. The enzyme-linked immunosorbent assay is based on two different monoclonal antibodies with specificity for the gII glycoprotein of Aujeszky's disease virus. Viral antigen was detected in 81 of 93 tissue extracts prepared from virus-infected organs. Fifteen outbreaks of Aujeszky's disease were analyzed in this study, and they were all identified by the gII antigen enzyme-linked immunosorbent assay.

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.

Optimized enzyme-linked immunosorbent assay for detection of human and bovine rotavirus in stools: Comparison with electron-microscopy, immunoelectro-osmophoresis, and fluorescent antibody techniques.

Detection of human and bovine rotavirus in stools is described using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) with poly-styrene microtest plates as solid phase, immunoglobulin fraction of rabbit antiserum to rotavirus (human) as catching antibody, and the same reagent labelled with horseradish peroxidase as conjugate. The ELISA has been optimized with regard to simplicity, rapidity, sensitivity, and specificity. In a comparative study, stool specimens from 81 infants and children and 92 neonatal calves with diarrhoea were tested for rotavirus by ELISA, electron microscopy (EM), immunoelectro-osmophoresis (IEOP), and fluorescent antibody technique (FA). The relative sensitivity of the different assays for human and bovine rotavirus was: EM 68%, 76%; IEOP 80%, 76%; FA not determined, 85%; and ELISA 86%, 98%, respectively. Less than 1 ng of purified human rotavirus could be detected in ELISA, whereas 100 ng was the minimal amount detected by IEOP. It is concluded that the developed ELISA is a simple, rapid, reliable, and sensitive method for the diagnosis of human and bovine rotavirus infections.