Phage typing of Salmonella Typhimurium - is it still a useful tool for surveillance and outbreak investigation?

Phage typing has for decades been useful as a phenotypical, definitive method for epidemiological characterisation of Salmonella Typhimurium. The system recommended by the World Health Organization (WHO) Collaborative Centre for phage typing of Salmonella has, however, become rather complex, and the present study illustrates the challenges of sufficient standardisation of the interpretation of lysis results to make sure that the same strain is assigned to the same phage type in different laboratories. Even though molecular typing methods will replace phenotypic characterisation methods in the future, it is our opinion that phage typing will remain for some time a useful tool to strengthen global Salmonella surveillance. Therefore, improved standardisation and quality assurance is essential to obtain a robust and harmonized method that allows comparison of results between laboratories.

Spatial scan statistics to assess sampling strategy of antimicrobial resistance monitoring program.

The collection and analysis of data on antimicrobial resistance in human and animal populations are important for establishing a baseline of the occurrence of resistance and for determining trends over time. In animals, targeted monitoring with a stratified sampling plan is normally used. However, to our knowledge it has not previously been analyzed whether animals have a random chance of being sampled by these programs, regardless of their spatial distribution. In this study, we used spatial scan statistics, based on a Poisson model, as a tool to evaluate the geographical distribution of animals sampled by the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP), by identifying spatial clusters of samples and detecting areas with significantly high or low sampling rates. These analyses were performed for each year and for the total 5-year study period for all collected and susceptibility tested pig samples in Denmark between 2002 and 2006. For the yearly analysis, both high and low sampling rates areas were significant, with two clusters in 2002 (relative risk [RR]: 2.91, p < 0.01 and RR: 0.06, p < 0.01) and one in 2005 (RR: < 0.01, p < 0.01). For the 5-year analysis, one high sampling rate cluster was detected (RR: 2.56, p = 0.01). These findings allowed subsequent investigation to clarify the source of the sampling clusters. Overall, the detected clusters presented different spatial locations over the years and we can conclude that they were more associated to temporary sampling problems than to a failure in the sampling strategy adopted by the monitoring program. Spatial scan statistics proved to be a useful tool for assessment of the randomness of the sampling distribution, which is important when evaluating the validity of the results obtained by an antimicrobial monitoring program.

Using data on resistance prevalence per sample in the surveillance of antimicrobial resistance.

OBJECTIVES: In most existing antimicrobial resistance monitoring programmes, one single bacterial colony from each collected sample is susceptibility tested against a panel of antimicrobials. Detecting the proportion of colonies resistant to different antimicrobials in each sample can provide quantitative data on antimicrobial resistance (resistance prevalence per sample). METHODS: In this study, a total of 98 faecal samples from slaughter pigs were tested for tetracycline and sulphonamide resistance in Escherichia coli using the single colony method, and these results were compared with the results obtained using the resistance prevalence per sample method. RESULTS: The results obtained by the resistance prevalence per sample method showed a lower occurrence of resistance. Tetracycline resistance in E. coli was found in 36.7% of the samples using the single colony method, while the mean tetracycline resistance prevalence was 22.5% using the resistance prevalence per sample method. Similarly, sulphonamide resistance was 32.7% using the single colony method and 19.6% when using the resistance prevalence per sample method. Although different estimates were obtained by each method, the correlation test and the regression model demonstrated that there is a significant association between the results obtained using both methods (P value <0.01) for both antimicrobials tested. CONCLUSIONS: To support risk assessment and analysis of the association between consumption of antimicrobials and occurrence of resistance, there is a need to move towards a more quantitative approach when dealing with antimicrobial resistance in a population, and the resistance prevalence per sample method can provide some of this additional information.

The effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in Campylobacter and Escherichia coli.

Modern food animal production depends on use of large amounts of antibiotics for disease control. This provides favourable conditions for the spread and persistence of antimicrobial-resistant zoonotic bacteria such as Campylobacter and E. coli O157. The occurrence of antimicrobial resistance to antimicrobials used in human therapy is increasing in human pathogenic Campylobacter and E. coli from animals. There is an urgent need to implement strategies for prudent use of antibiotics in food animal production to prevent further increases in the occurrence of antimicrobial resistance in food-borne human pathogenic bacteria such as Campylobacter and E. coli.

Development of a phage typing system for Staphylococcus hyicus.

Bacteriophages were released by 98% of 100 Staphylococcus hyicus strains studied after treatment with mitomycin C. Twenty-three phages with different lytic spectra were included in a phage typing system and used for typing S. hyicus. On a test-set of 100 epidemiologically unrelated S. hyicus strains isolated from Danish pig herds, the phages were able to type 92% of the strains, producing 16 different phage types. Reproducibility of the phage typing system after subculture of the strains and using fresh phage stock was 96%. Typability ranged from 52 to 80% when typing porcine strains originating from other countries. Although phages were isolated from porcine skin strains exclusively, the system produced phage types in S. hyicus strains of bovine origin. Ten strains of S. aureus and S. chromogens were not typable by these phages. Strains belonging to one phage type (A/B/C/W) were isolated significantly more often from piglets with exudative epidermidis than from healthy piglets. The phage typing system described appears to be a valuable tool in diagnosis of exudative epidermidis in pigs, and furthermore, might be of value in epidemiological studies of S. hyicus.

Salmonella typhimurium phage types from human salmonellosis in Denmark 1988-1993.

A total of 989 isolates of Salmonella enterica ssp. enterica serovar Typhimurium from cases of human salmonellosis were investigated by phage typing. The isolates comprised all isolates recovered during the month of August in each of the years from 1988 to 1993. Phage typing assigned 82.6% of the strains to 36 different definitive types, 11.9% of the strains belonged to types of unknown lysis pattern (RDNC), and 5.5% could not be typed by the phages used (NT). Three phage types (12, 66 and 110) made up approximately 50% of the isolates in each of the years investigated. During the period in question these types showed major changes in prevalence: phage type 12 increased from 4.0% in 1988 to a maximum of 55.2% in 1992, and phage type 66 and phage type 110 were reduced from 40.1% and 27.8% to a minimum of 3.9% and 4.8% in 1993 and 1992, respectively. The increasing prevalence of phage type 12 among isolates from human salmonellosis most likely reflects the increasing significance of pork as a source of human salmonellosis. The reduction in phage types 66 and 110 is paralleled by a reduction in S. Typhimurium in Danish poultry. Some phage types were associated with travel, namely 17, 193 and 194. It is concluded that phage typing, although here performed retrospectively, produces valuable epidemiological information regarding changes in the relative importance of different sources of infection in humans. It is suggested that phage typing be performed prospectively on both human and animal S. Typhimurium isolates in Denmark.

Typing of Salmonella enterica serovar Saintpaul: an outbreak investigation.

During the summer of 1993 an outbreak of human salmonellosis caused by Salmonella serovar Saintpaul occurred in Denmark. A total of 35 isolates originating from pigs, turkeys and imported foodstuffs, and 10 human isolates were compared following their characterization by agglutination of the 0:5 factor, antibiogram typing, plasmid profiling, ribotyping and pulsed field gel electrophoresis, in order to identify the most probable source of infection. After typing, the source of the investigated outbreak remains obscure because so far no isolates with traits of the outbreak strain have been recovered from production animals. Presence of the 0:5 factor and absence of plasmids in human and porcine isolates pointed to pork as the source of infection, whereas human isolates and all Danish isolates from turkeys had the same ribotype, indicating that turkey was the infection source. A possible explanation for the failure to find isolates with traits of the outbreak strain could be the presence of a third, but so far unidentified, source. The present investigation illustrates the necessity of using more than one epidemiological typing method for outbreak investigation. This is especially important when the organism involved is relatively uncommon and little is known about its diversity and distribution.

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.

Glycopeptide resistance in Enterococcus faecium from broilers and pigs following discontinued use of avoparcin.

The use of the glycopeptide growth promoter avoparcin was discontinued in Denmark in 1995 following concerns that vancomycin-resistant Enterococcus faecium occurring as a result of its use could be transferred to humans via food. The present study is an analysis of results obtained by the continuous surveillance of an antimicrobial resistance in Denmark (DANMAP) with the aim of determining the effect of the ban on the occurrence of glycopeptide resistance among E. faecium isolated from broilers and pigs. Among isolates from broilers, the proportion that were resistant to glycopeptides has shown a statistically high significant decline between the end of 1995 and the first half of 1998, whereas in pigs the ban appears to have no such effect. One possible explanation is that the broiler industry generally uses all in-all out production compared with continuous production in pig herds. Alternatively, the results indicate that the different outcomes may result from different co-selection patterns in pigs and broilers. In pigs, the antimicrobials most commonly used favored co-selection of glycopeptide resistant strains of E. faecium while in broilers the antimicrobials most widely used selected for glycopeptide-susceptible strains. The results show that intervention to reduce antimicrobial resistance may not always be effective and preventing resistance problems therefore becomes essential.

EU conference 'The Microbial Threat'.

A global or European strategy should be developed to deal with increasing antimicrobial resistance. This strategy includes surveillance of antimicrobial resistance and monitoring of the use of antimicrobial agents in animals and humans. In animals, surveillance should be focussed on potential transfer of resistant, zoonotic, food-born pathogens and resistance genes to humans. In humans the surveillance should be clinically relevant. Guidelines for rational therapy should be implemented and 'antibiotic teams' should be installed in each hospital to evaluate the prescription of antibiotics and its compliance with guidelines. Keeping animals for food production involves the responsibility for their well being. This includes treatment of infections. However, the use of feed additive, growth-promoting antimicrobials related to therapeutics in human medicine, should be banned immediately. Research aimed at intervention strategies for antimicrobial resistance should be given a high priority with adequate financing both nationally and in Europe. Well co-ordinated European research programmes should have priority; this includes the need to install a European multidisciplinary scientific advisory group.

Investigation of an outbreak of human salmonellosis caused by Salmonella enterica ssp. enterica serovar Infantis by use of pulsed field gel electrophoresis.

Analysis of chromosomal DNA restriction patterns produced by pulsed field gel electrophoresis (PFGE) was used to investigate an outbreak of human salmonellosis caused by Salmonella enterica ssp. enterica serovar Infantis (S. Infantis) involving more than 500 registered human cases. The outbreak had been tentatively traced back to a single pig slaughterhouse. A total of 135 isolates from various sources produced 21 different PFGE patterns with the restriction endonuclease XbaI. All human isolates from the outbreak belonged to a single type, the 'EPI-type', whereas human isolates recovered before and after the outbreak belonged to several different types. All isolates investigated from the suspect pig slaughterhouse and its supplier pig herds belonged to the EPI-type. Isolates from pork from the central meat market in Copenhagen, which received most of the carcasses from the suspect slaughterhouse, also belonged to the EPI-type. This was furthermore, the case for isolates from beef from the same market, indicating that cross-contamination had taken place. All isolates from pork and some, but not all, isolates from beef, collected in butchers' shops during the outbreak belonged to the EPI-type. The typing results supported that the outbreak was a common source outbreak, probably originating from a limited number of supplier pig herds supplying animals to a single slaughterhouse.

Isolation of vancomycin resistant Enterococcus faecium from food.

In a survey of vancomycin resistant Enterococcus faecium (VREF) in Danish meat products, VREF could be detected in 16% of 160 samples of broilers collected at slaughterhouses and in 15% of 26 samples of pork collected from the retail trade. VREF were isolated by enrichment for 24 h in nutrient broth supplemented with vancomycin (50 micrograms/ml) prior to plating on Slanetz and Bartley agar. Using direct plating on Slanetz and Bartley agar, VREF could be isolated from only 1.7% of 540 samples of broilers from slaughterhouses and 2.2% of 90 samples of broilers from retail outlets. VREF was not detected in 124 samples of pork and 128 samples of beef from retail outlets by the direct plating method. An additional enrichment step in nutrient broth supplemented with vancomycin enhanced the detection rate of VREF by approximately three times compared to the direct plating method when investigating the same 160 samples of broilers by the two methods. The implications and public health aspects of VREF in food is discussed.

Listeria monocytogenes in bovine mastitis. Possible implication for human health.

During the 23-year period 1972 through 1994 quarter milk samples from 1,132,958 cows originating from 36199 herds were examined for the presence of Listeria monocytogenes. Through the period the reference population amounted to 12,742,600 cow years and 401,682 herd years. The percentage of cows infected with L. monocytogenes varied from 0.01 to 0.1% (mean 0.04%) and of herds with an infected cow from 0.2 to 4.2% (mean 1.2%) through the period, showing a low but constant level of infection. A comparison of 33 isolates from bovine mastitis and 27 human clinical isolates was made by sero- and ribotyping. Serotyping showed that all bovine and 17 (63%) of the human isolates belonged to serogroup 1, whereas 10 (37%) of the human isolates belonged to serogroup 4. Ribotyping using EcoRI as restriction enzyme divided the 60 isolates into 16 different types, 7 of which were found among both the bovine and human types. The combination of the typing methods showed that 26 (79%) bovine and 13 (48%) human isolates shared common types. This study showed that a low but constant percentage of Danish dairy herds have cows infected with L. monocytogenes and that some of the bovine types could be found among types causing human infections.

Relations between the occurrence of resistance to antimicrobial growth promoters among Enterococcus faecium isolated from broilers and broiler meat.

From 1995 to 2001, Enterococcus faecium isolates were collected from broiler flocks at slaughter and broiler meat products at retail outlets and were tested for susceptibility to classes of antimicrobials used for growth promotion in broilers in Denmark, namely: evernimicin, glycopeptide, macrolide and streptogramin. By February 1998, all antimicrobial growth promoters (AGPs) were withdrawn from the Danish broiler production. The present study investigates, by logistic regression analyses, the (1) changes in the occurrence of AGP resistance among E. faecium from broilers and broiler meat from the fourth quarter of 1995 to the fourth quarter of 2001 and (2) relations between the occurrence of AGP resistance among E. faecium isolates from Danish broilers and AGP resistance among E. faecium isolates from the broiler meat of Danish and unknown origin collected in the same quarter within the year. In the present study, we showed that after the AGP withdrawal, a significant decline in resistance to avilamycin, erythromycin, vancomycin and virginiamycin was observed among E. faecium from broilers and broiler meat. In addition, a decline in the occurrence of AGP resistance among E. faecium from Danish broilers was associated with a decrease in the predicted probability of isolating an AGP-resistant E. faecium isolate from a randomly selected broiler meat product. In the analyses "relations between the occurrence of AGP resistance among E. faecium isolated from broilers and broiler meat collected in the same quarter" errors in the explanatory variable were expected. Therefore, a simulation study was performed to validate the results from logistic regression analyses. The results obtained by the two methods were similar.

Antibiotic-resistance and plasmids in Staphylococcus hyicus isolated from pigs with exudative epidermitis and from healthy pigs.

A total of 100 S. hyicus strains isolated from healthy piglets and piglets with exudative epidermitis originating from 100 different herds was examined for drug-resistance and prevalence of plasmids. Resistance to macrolide/linosamide antibiotics could be related to plasmids in 55 (93%) of the 59 resistant strains: A plasmid of 2.4 kb mediating resistance to macrolides and lincosamides was observed in 25 strains, and a plasmid of 11.5 kb mediating resistance to both macrolides/lincosamides and tetracycline was observed in 30 strains. A plasmid with a molecular weight of 4.5 kb was shown by curing experiments to be associated with resistance to tetracycline in 12 strains. All together, 47 strains were resistant to tetracycline. In 42 (89%) of these strains tetracycline-resistance was found to be encoded by plasmids. Fifty six strains were resistant to streptomycin, and resistance was associated with the presence of a 4.4 kb plasmid in 17 strains studied. Resistance to penicillin, observed in 44 strains, and resistance to kanamycin, observed in 15 strains, could not be related to plasmids in any of these strains. The 11.5 kb plasmid was observed in 39% of the strains isolated from piglets with EE, and in 7% of the strains isolated from healthy piglets. Despite its higher prevalence in strains from piglets with EE, the 11.5 kb plasmid could not be shown to encode production of capsule or exfoliative substances: factors which might play a role in the development of exudative epidermitis in piglets.

Evaluation of phenotypic and genotypic methods for epidemiological typing of Staphylococcus aureus isolates from bovine mastitis in Denmark.

The value of five different typing methods (antibiogram typing, biotyping, phage typing, plasmid profiling and restriction fragment length polymorphism of the gene encoding 16S and 23S ribosomal RNA (ribotyping)), in discriminating 105 Staphylococcus aureus strains from bovine milk samples obtained from 105 different Danish dairy herds was investigated. A total of 85 strains (81%) proved susceptible to all of the 11 antibiotics tested, and the remaining 20 strains could be divided into 5 different antibiogram patterns. The predominant resistance pattern, penicillin resistance, was observed in 15 (75%) of the 20 antibiotic resistant strains. Biotyping assigned the strains to 14 different types, with the most common type accounting for 25.7% of the strains. Ninety eight (93.3%) strains could be typed by phages, assigning them to 19 different phage types. The predominant phage type accounted for 31.4% of the strains. Eight different plasmid profiles was observed among 24 (23%) strains harbouring plasmids. Ribotyping yielded 30 different types, with the most common accounting for 29.5% of the strains. The single most discriminatory typing method was ribotyping (0.863) followed by biotyping (0.842) and phage typing (0.795). Plasmid profiling (0.395) and antibiogram typing (0.327) had low discriminatory indices. Correspondence among ribotypes and the presence or absence of plasmids were observed, as was some degree of correspondence between ribotype, phage type and biotype. In general the correspondence between phage type and ribotype were stronger than between biotype and ribotype and between biotype and phage type. All combinations of two or more methods led to an improved index of discrimination compared to the individual methods indicating, that some subdivision of types had taken place. The combination of phage, bio- or ribotyping or all three methods in combination are considered to be an efficient combination of typing methods for epidemiological investigation of S. aureus mastitis.

Evaluation and application of ribotyping for epidemiological studies of Actinobacillus pleuropneumoniae in Denmark.

The aim of the present study was to evaluate ribotyping as an epidemiological tool for Actinobacillus pleuropneumoniae and apply the method in studies of A. pleuropneumoniae infections in Danish pig herds. The evaluation of ribotyping was based on the 13 international reference strains and 106 epidemiologically unrelated Danish field strains representing the nine serotypes of biotype 1 (1, 2, 5A/B, 6, 7, 8, 10, 12, and K2:O7) and one serotype 14 of biotype 2. Enzymes CfoI and HindIII were chosen for generation of ribotype patterns. Ribotyping of the reference strains resulted in 10 CfoI types and 11 HindIII types. Ribotyping of the Danish strains resulted in 17 different CfoI ribotypes and 24 different HindIII ribotypes. Combining HindIII- and CfoI-ribotyping divided the Danish strains into 26 different types. The stability, reproducibility and typability of ribotype patterns were good, and the discriminatory power was between 0.85-0.89. The relatively low discriminatory power was caused by four predominant types, containing 61% of the isolates. The typing system was applied in studies of routes of infection of specific pathogen-free (SPF) pig herds and included 112 strains of A. pleuropneumoniae. Airborne transmission from neighboring conventional pig farms was investigated in 12 cases of infected SPF herds. Transmission via vehicles transporting pigs between SPF herds was investigated in nine cases while transmission by trading of pigs between SPF herds was investigated in two cases. Serotype 2 was isolated from all SPF herds included in this study, except one, emphasizing the high prevalence of this serotype in Denmark. By ribotyping, airborne transmission was indicated in five of 12 cases, transmission via pig transporting vehicle was indicated in six of nine cases, and transmission via trading was indicated in one of two cases. In many cases findings of predominant ribotypes made interpretations of suspected routes of transmission difficult. The relationship of strains based on ribotypes was calculated using Dices coefficient and clustered by UPGMA. HindIII ribotypes of serotype 2 strains were closely related, though only showing 43% similarity to HindIII ribotypes of remaining serotypes.

Comparison of conventional culture methods and two commercial enzyme immunoassays for detection of Salmonella in porcine fecal samples and cecal contents.

Two commercial enzyme immunoassays, designated EIA-1 and EIA-2, for the detection of salmonella in feces and cecal contents were compared to conventional culture methods. Out of 362 cecal content samples, 35 were positive by EIA-1 and 30 were positive by EIA-2 and conventional methods. Out of 189 fecal samples, 41 were positive by EIA-1, 30 were positive by EIA-2, and 24 were positive by conventional culture methods. The EIA-1 assay method detected more positive samples overall, although only comparison of the fecal samples was statistically significant. The results indicate that the EIA-1 method is rapid, requiring only 24 hours, and may be more sensitive than the EIA-2 assay and conventional culture techniques. Enzyme immunoassays for the detection of salmonella in fecal samples or cecal contents provide a reasonable alternative to conventional culture techniques.

The effect of discontinuing the use of antimicrobial growth promoters on the productivity in the Danish broiler production.

On 15 February 1998, the Danish poultry industry voluntarily decided to discontinue the use of all antimicrobial growth promoters (AGPs). To investigate how the removal of AGPs influenced the broiler productivity in Denmark, data from 6815 flocks collected from November 1995 to July 1999 by the Danish Poultry Council were analysed. The three flock parameters were: kilogram broilers produced per square meter (per rotation), feed-conversion ratio (total kilogram feed used per rotation/total kilogram live weight per rotation) and total percent dead broilers ((number of dead broilers during the rotation/number of broilers put in the house per rotation)x100). Data were analysed using a mixed model, allowing the correlation structures in the data to be taken into account. The analyses showed that kilogram broilers produced per square meter and percent dead broilers in total were not affected by the discontinued use of AGPs. However, the feed-conversion ratio increased marginally 0.016 kg/kg and has remained at this level throughout the rest of the study period.