Madelaine Norström

A space-time cluster investigation of an outbreak of acute respiratory disease in Norwegian cattle herds.

Outbreaks of acute respiratory disease occurred in several cattle herds in the south-east regions of Norway during the winter and spring of 1995. The present study was conducted to investigate the temporal and spatial dynamics of the occurrence of acute respiratory disease between January and April 1995 in the affected cattle herds, in two neighbouring counties in Norway. This was done to assess the possible role of an infectious agent in these outbreaks, as well as to investigate the possibility of transmission between neighbouring herds. During the study period, a total of 190 (19.8%) outbreaks occurred in 955 cattle herds located in the defined study area. Descriptive maps of the spatial and spatio-temporal patterns of the outbreak were generated, and two different statistical tests of space-time interaction (the Knox test and k-nearest neighbour test) and a cluster-detection test (the space-time scan statistic) were used. The results of all the space-time interaction tests strongly suggested that the case herds were clustered in time as well as in space. In addition, the space-time scan statistic defined the most-likely cluster to be located within the second veterinary district (Toten where the disease occurred) during 9-15 weeks in 1995. The relative risk of a herd being infected inside the most-likely cluster compared with the surrounding study area was estimated to be 3.59 (p=0.001). The transmission rate between neighbouring farms therefore appeared to have been higher within this cluster than in the surrounding study area. This study supports the hypothesis that one common source of infection was involved in the outbreaks during the study period.

Risk factors for epidemic respiratory disease in Norwegian cattle herds.

An epidemic of acute respiratory disease associated with bovine respiratory syncytial virus (BRSV) occurred during the winter and spring of 1995 in two neighbouring veterinary districts in the south-eastern part of Norway. The objective of this study was to describe the time course of the outbreak associated with BRSV in the cattle herds, and to determine the association between selected herd factors and the risk of experiencing a herd outbreak of acute respiratory disease. Data from 431 cattle herds on the dates of disease occurrence, location of the farms, herd size, age profile and production type were collected retrospectively for 1995. The risk of acute respiratory disease occurring in a cattle herd was related to the herd size as well as the type of production, with an expressed interaction between these two variables. From the Cox proportional-hazards model, the risk of a herd outbreak in a mixed herd of 20 animals was estimated to be 1.7-times greater than in a dairy herd and 3.3-times greater than a beef herd (reference category) of a comparable size. On increasing the herd size to 50 animals, the risk increased 1.3-fold for a mixed herd, 3.3-fold for a dairy herd, and 2.1-fold for a beef herd, compared to the risk for a corresponding type of herd of 20 animals.

Antimicrobial resistance in Campylobacter jejuni from humans and broilers in Norway

In this study comprising isolates from 2001 to 2003, resistance was considerably more widespread among Campylobacter jejuni from humans infected abroad than infected within Norway. The discrepancy was particularly notable for fluoroquinolone resistance (67.4% vs. 6.5%). This is probably a reflection of a low resistance prevalence in Norwegian broiler isolates (1.2% fluoroquinolone resistant).

Emergence of AmpC-producing Escherichia coli in the broiler production chain in a country with a low antimicrobial usage profile

The aim of this study was to estimate the prevalence of cephalosporin-resistant Escherichia coli at the different levels of the Norwegian broiler production pyramid and identify the mechanisms responsible for the resistance phenotype. Samples from all levels of the broiler production pyramid and retail chicken meat (fillets) were included (n=649). The occurrence of cephalosporin-resistant E. coli at the different production levels ranged from 8 to 43%. All these isolates had an AmpC-phenotype, and the majority carried the blaCMY-2 gene. In addition, a few isolates with up-regulated chromosomal ampC were identified. The results show that Norway has a relatively high prevalence of cephalosporin-resistant E. coli in the broiler production chain in spite of a very low consumption of antimicrobial agents. Cephalosporins have not been used in the Norwegian broiler production, and it has been hypothesised that import of breeding animals and hatching eggs may be the source of these resistant bacteria. We demonstrate that these bacteria are disseminated in the production pyramid despite the lack of selection pressure from antimicrobial agents.

Effect of climate and farm environment on Campylobacter spp. colonisation in Norwegian broiler flocks.

Campylobacteriosis is the most frequently reported zoonosis in the EU. A recent report states that between 50% and 80% of the human campylobacteriosis cases could be attributed to broiler as a reservoir. The current study was conducted to investigate associations between the presence of Campylobacter spp. in Norwegian broiler flocks and factors related to the climate and the farm environment. Data from 18,488 broiler flocks from 623 different farms during 2002-2007 were included in the study. A logistic regression analysis was conducted where Campylobacter spp. status of a broiler flock at the time of slaughter was defined as the dependent variable and farm was modelled as a random effect. The following factors were found to increase the probability for a broiler flock to test positive for Campylobacter spp.: daily mean temperature above 6°C during the rearing period, private water supply, presence of other livestock farms within a distance of 2 km, presence of other broiler farms within a distance of 4 km with flocks positive for Campylobacter spp. within 30 days prior to slaughter, heavy rainfall 11-30 days prior to slaughter, region and year. Daily mean temperature below 0°C reduced the probability. The study emphasises the importance of the farm environment and the climate for the occurrence of Campylobacter spp. in broiler flocks. The farm environment is probably a part of the Campylobacter spp. pathway into and between broiler flocks where farmyard run-off and humans or flies entering the houses might constitute vehicles transporting the organism. Fly activity is temperature-driven and flies might be a part of the explanation of the increased risk for Campylobacter spp. related to increased temperature demonstrated in the study.

Antimicrobial Resistance in Staphylococcus pseudintermedius in the Norwegian Dog Population

The occurrence of antimicrobial resistance in Staphylococcus pseudintermedius from skin and ear infections in a representative sample of dogs unexposed to antimicrobial treatment before sampling was examined. The obtained isolates were further examined for genetic polymorphism and genetic background of resistance. A total of 59 isolates of S. pseudintermedius originating from 96 samples of 91 dogs in five different regions in Norway were included in this study. Susceptibility testing was performed using a broth dilution method. Resistant isolates were subjected to polymerase chain reaction for detection of resistance genes. All isolates were subjected to pulsed-field gel electrophoresis to examine the genetic polymorphism. In total, 19% of the isolates were susceptible to all antimicrobial agents included. Resistance to penicillin was most prevalent (70%), followed by resistance to fusidic acid (49%) and oxytetracycline (42%). Resistance to quinolones or cephalosporins was not observed. Resistance to penicillin, tetracycline, and erythromycin was mediated by the blaZ beta-lactamase gene, the tetM gene, and the ermB gene, respectively. One of the fusidic acid-resistant isolates harbored a fusC gene, whereas the mechanisms involved in resistance in the other fusidic acid-resistant isolates remained unknown. Pulsed-field gel electrophoresis showed a high genetic polymorphism of S. pseudintermedius. This study indicates that the occurrence of antimicrobial resistance is common among S. pseudintermedius from dogs unexposed to antimicrobial treatment before sampling, and that there is a high genetic polymorphism among S. pseudintermedius.

Analysis of simultaneous space-time clusters of Campylobacter spp. in humans and in broiler flocks using a multiple dataset approach

BackgroundCampylobacteriosis is the most frequently reported zoonosis in the EU and the epidemiology of sporadic campylobacteriosis, especially the routes of transmission, is to a great extent unclear. Poultry easily become colonised with Campylobacter spp., being symptom-less intestinal carriers. Earlier it was estimated that internationally between 50% and 80% of the cases could be attributed to chicken as a reservoir. In a Norwegian surveillance programme all broiler flocks under 50 days of age were tested for Campylobacter spp. The aim of the current study was to identify simultaneous local space-time clusters each year from 2002 to 2007 for human cases of campylobacteriosis and for broiler flocks testing positive for Campylobacter spp. using a multivariate spatial scan statistic method. A cluster occurring simultaneously in humans and broilers could indicate the presence of common factors associated with the dissemination of Campylobacter spp. for both humans and broilers.ResultsLocal space-time clusters of humans and broilers positive for Campylobacter spp. occurring simultaneously were identified in all investigated years. All clusters but one were identified from May to August. Some municipalities were included in clusters all years.ConclusionsThe simultaneous occurrence of clusters of humans and broilers positive for Campylobacter spp. combined with the knowledge that poultry meat has a nation-wide distribution indicates that campylobacteriosis cases might also be caused by other risk factors than consumption and handling of poultry meat.Broiler farms that are positive could contaminate the environment with further spread to new broiler farms or to humans living in the area and local environmental factors, such as climate, might influence the spread of Campylobacter spp. in an area. Further studies to clarify the role of such factors are needed.

Escherichia coli of animal origin in Norway contains a blaTEM-20-carrying plasmid closely related to blaTEM-20 and blaTEM-52 plasmids from other European countries

Sir, The situation regarding antimicrobial resistance in bacteria from food-producing animals in Norway is, in an international perspective, favourable. The resistance frequencies are moderate and the situation has been stable since the start of the Norwegian monitoring programme in the veterinary sector (NORM-VET) (www. vetinst.no) in the year 2000. We report the first bacterial isolate of animal origin detected in Norway with reduced susceptibility to cephalosporins. The isolate (Escherichia coli 2006-01-1248, hereafter termed E. coli 1248) originated from a broiler domesticated in a cephalosporin-free environment and was included in the NORM-VET 2006 programme. MICs of cefotaxime, ceftiofur and ampicillin were 1, 4 and .32 mg/L, respectively. The cefotaxime MIC of 1 mg/L is close to the clinical breakpoint recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (susceptible 1 mg/L, resistant .2 mg/L). However, this value is considerably higher than those observed for susceptible E. coli strains (wild-type population) having MICs of 0.25 mg/L (www.eucast.org). MICs of cefalotin, ceftazidime and cefepime, determined using Etest (AB Biodisk, Solna, Sweden) with E. coli ATCC 25922 as susceptible control, were 64, 0.5 and 0.25 mg/L, respectively. E. coli 1248 was positive in the double-disc synergy test and in the confirmatory test. The tests were carried out as recommended by the manufacturer using discs containing cefotaxime (30 mg), ceftriaxone (30 mg), ceftazidime (30 mg), cefepime (30 mg), amoxicillin/clavulanic acid (30 mg/15 mg), and ceftazidime and cefepime with and without clavulanic acid (Rosco, Taastrup, Denmark) (User’s guide NEO-SENSITABS susceptibility testing 19th Edn, www.rosco.dk). PCR was performed for the detection of blaCTX-M, blaSHV and blaTEM 1,2 using the following control strains: E. coli K4-23 (blaCTX-M-9), E. coli Dak2 (blaSHV) and E. coli 76-33094-7 (blaTEM). Amplicons were produced with the blaTEM-specific primers only. Sequencing showed that a blaTEM-20 gene variant 3 was present. Conjugation showed that blaTEM-20 was located on a self-transferable plasmid. The transconjugant had the following MICs of b-lactams: ampicillin, .32 mg/L; cefotaxime, 0.5 mg/L; and ceftiofur, 1 mg/L. The blaTEM-20 sequence contained three silent mutations at positions 346, 682 and 925 when compared with a previously published sequence (accession number Y17581). The other blaTEM-20 gene variant, conferring high-level resistance to thirdgeneration cephalosporins, showed a 135 bp deletion in the promoter and a G!T mutation at position 162. These mutations were not identified in the sequence from E. coli 1248, and this may explain the lower MICs exhibited. The blaTEM-20 gene has previously been detected in Salmonella Paratyphi B dTþ from poultry in the Netherlands. The strain Salmonella Paratyphi B dTþ 63.48, kindly donated to us for further investigation, was compared with E. coli 1248. The blaTEM-20 gene was also carried by a conjugative plasmid in the Salmonella Paratyphi B dTþ strain, and the two blaTEM-20 sequences were 100% identical. The transconjugant was resistant to b-lactams only with the same MICs as the E. coli 1248 transconjugant. Both blaTEM-20 plasmids were assigned to the incompatibility (Inc) group I1 by PCR-based replicon typing. Plasmid multilocus sequence typing (pMLST) for subtyping IncI1 plasmids was applied to the blaTEM-20 plasmids. 5 The following alleles (accession numbers) were obtained: repI1-1 (EU370458), ardA-2 (EU370453), trbA-pndC-2 (EU40466), sogS-3 (EU70463) and pilL-3 (EU370457). These alleles corresponded to sequence type 5, previously assigned to an IncI1 plasmid carrying blaTEM-52 identified in a Salmonella Infantis strain isolated in Belgium in 2005 (the alleles showed 100% nucleotide identity except for one nucleotide in the ardA locus). This blaTEM-52 plasmid is reported to be widely disseminated among different Salmonella serovars from poultry and humans in Belgium and France. The blaTEM-20 plasmids were large (.100 kb) and showed profiles that seemed to be similar when comparing the PstI and EcoRI restriction patterns with published restrictions patterns of the blaTEM-52 plasmid. 5–7

Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production

Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the blaCMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare blaCMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying blaCMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying blaCMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of blaCMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing blaCMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.

Space-time patterns of Campylobacter spp. colonization in broiler flocks, 2002-2006.

This study was performed to investigate space-time patterns of Campylobacter spp. colonization in broiler flocks in Norway. Data on the Campylobacter spp. status at the time of slaughter of 16 054 broiler flocks from 580 farms between 2002 and 2006 was included in the study. Spatial relative risk maps together with maps of space-time clustering were generated, the latter by using spatial scan statistics. These maps identified the same areas almost every year where there was a higher risk for a broiler flock to test positive for Campylobacter spp. during the summer months. A modified K-function analysis showed significant clustering at distances between 2.5 and 4 km within different years. The identification of geographical areas with higher risk for Campylobacter spp. colonization in broilers indicates that there are risk factors associated with Campylobacter spp. colonization in broiler flocks varying with region and time, e.g. climate, landscape or geography. These need to be further explored. The results also showed clustering at shorter distances indicating that there are risk factors for Campylobacter spp. acting in a more narrow scale as well.