The role of domestic reservoirs in domestically acquired Salmonella infections in Norway: epidemiology of salmonellosis, 2000-2015, and results of a national prospective case-control study, 2010-2012.

In Norway, incidence of sporadic domestically acquired salmonellosis is low, and most frequently due to Salmonalla Typhimurium. We investigated the risk factors for sporadic Salmonella infections in Norway to improve control and prevention measures. Surveillance data for all Salmonella infections from 2000 to 2015 were analysed for seasonality and proportion associated with domestic reservoirs, hedgehogs and wild birds. A prospective case-control study was conducted from 2010 to 2012 by recruiting cases from the Norwegian Surveillance System for Communicable Diseases and controls from the Norwegian Population Registry (389 cases and 1500 controls). Univariable analyses using logistic regression were conducted and a multivariable model was developed using regularised/penalised logistic regression. In univariable analysis, eating snow, dirt, sand or playing in a sandbox (aOR 4.14; CI 2.15-7.97) was associated with salmonellosis. This was also the only exposure significantly associated with illness in the multivariable model. Since 2004, 34.2% (n = 354) of S. Typhimuirum cases had an MLVA profile linked to a domestic reservoir. A seasonal trend with a peak in August for all Salmonella types and in February for S. Typhimurium was observed. Indirect exposure to domestic reservoirs remains a source of salmonellosis in Norway, particularly for children. Information to the public about avoiding environmental exposure should be strengthened and initiatives to combat salmonellosis in the food chain should be reinforced.

The national web-based outbreak rapid alert system in Norway: eight years of experience, 2006-2013.

In 2005, the Norwegian Institute of Public Health established a web-based outbreak rapid alert system called Vesuv. The system is used for mandatory outbreak alerts from municipal medical officers, healthcare institutions, and food safety authorities. As of 2013, 1426 outbreaks have been reported, involving 32913 cases. More than half of the outbreaks occurred in healthcare institutions (759 outbreaks, 53·2%). A total of 474 (33·2%) outbreaks were associated with food or drinking water. The web-based rapid alert system has proved to be a helpful tool by enhancing reporting and enabling rapid and efficient information sharing between different authorities at both the local and national levels. It is also an important tool for event-based reporting, as required by the International Health Regulations (IHR) 2005. Collecting information from all the outbreak alerts and reports in a national database is also useful for analysing trends, such as occurrence of certain microorganisms, places or sources of infection, or route of transmission. This can facilitate the identification of specific areas where more general preventive measures are needed.

Two outbreaks of diarrhoea in nurseries in Norway after farm visits, April to May 2009.

During a 2009 nationwide outbreak of sorbitolfermenting Escherichia coli O157 in Norway, the Norwegian Institute of Public Health was notified of diarrhoea outbreaks in two nurseries. A link to the nationwide outbreak was suspected and investigated, including retrospective cohort studies. Both nurseries had recently visited farms. Faecal specimens were obtained from symptomatic children as well as from the farm animals and tested for Campylobacter, Salmonella, Yersinia, Shigella and pathogenic E. coli, and isolates were further characterised. Nursery A had 12 symptomatic children, and we found the same strain of C. jejuni in faeces from children and lambs. Nursery B had nine symptomatic children, including one child with bloody diarrhoea carrying enterohaemorrhagic E. coli (EHEC) O26. EHEC O26 with a similar multiple-locus variable number tandem repeat analysis (MLVA)-profile was found in sheep. Five children had enteropathogenic E. coli (EPEC) O76. Animals were not tested for EPEC O76. We found no significant association between illness and risk factors for either nursery. The isolated pathogens differed from the one involved in the nationwide outbreak. In each nursery outbreak, the pathogens isolated from children matched those found in farm animals, implicating animal faeces as the source. Hygiene messages are important to prevent similar outbreaks.

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization.

AIMS: To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1-3 and 8). METHODS AND RESULTS: We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over-representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic-uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2(EDL933) or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993-2008. CONCLUSION: We observed that the tir-255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of virulence clade-specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations.

Shigella sonnei infections in Norway associated with sugar peas, May-June 2009.

In May 2009, the Norwegian Institute of Public Health (NIPH) identified a possible outbreak of Shigella sonnei infection involving four cases. Additionally, five suspected cases in two separate households were reported. Inspectors from the Norwegian Food Safety Authority (NFSA) visited the two households and found an unopened package of sugar peas imported from Kenya in one of the households. One sample from the sugar peas was positive for Shigella sonnei by two PCR methods. Based on this result and information from patient interviews, the NFSA prohibited all sales of sugar peas imported from Kenya.

An outbreak of Salmonella Typhimurium infections in Denmark, Norway and Sweden, 2008.

In November-December 2008, Norway and Denmark independently identified outbreaks of Salmonella Typhimurium infections characterised in the multiple-locus variable number of tandem repeats analysis (MLVA) by a distinct profile. Outbreak investigations were initiated independently in the two countries. In Denmark, a total of 37 cases were identified, and multiple findings of the outbreak strain in pork and pigs within the same supply chain led to the identification of pork in various forms as the source. In Norway, ten cases were identified, and the outbreak investigation quickly indicated meat bought in Sweden as the probable source and the Swedish authorities were alerted. Investigations in Sweden identified four human cases and two isolates from minced meat with the distinct profile. Subsequent trace-back of the meat showed that it most likely originated from Denmark. Through international alert from Norway on 19 December, it became clear that the Danish and Norwegian outbreak strains were identical and, later on, that the source of the outbreaks in all three countries could be traced back to Danish pork. MLVA was instrumental in linking the outbreaks in the different countries and tracing the source. This outbreak illustrates that good international communication channels, early alerting mechanisms, inter-sectoral collaboration between public health and food safety authorities and harmonised molecular typing tools are important for effective identification and management of cross-border outbreaks. Differences in legal requirements for food safety in neighbouring countries may be a challenge in terms of communication with consumers in areas where cross-border shopping is common.

Assessment of the Potential Risks Associated with the Proposed Use of Composted Waste from the Production of Bacitracin as a Soil Additive

Bacitracin is a hexapeptide antibiotic, with a substituted thiazolidine nucleus, produced by some strains of B. licheniformis. It is mainly active against Grampositive bacteria, although many differences in susceptibility exist among the bacterial species. Alpharma A.S. Norway has produced bacitracin for use in human medicine since 1954. Until 1998, the fermentation waste from the production of bacitracin was added to animal feed in some European countries, including Norway, to promote growth of pigs and domestic fowl. In 1998, fermentation waste containing bacitracin as a food additive was banned by the EU to reduce the risk of developing bacitracin-resistant bacteria in animals, and the subsequent possible transfer of such bacteria to humans via the food chain. Use of fermentation waste containing bacitracin as a feed additive has not been officially banned in Norway, but it is no longer used for this purpose. Alpharma is therefore actively seeking alternative uses for their production waste. As the waste material is rich in nutrients, the company proposes that it could be developed as a soil additive by fermenting it with chipped bark and lime. The Norwegian Food Safety Authority (Mattilsynet) commissioned the Panel on Biological Hazards of the Norwegian Scientific Committee for Food Safety (Vitenskapskomitéen for mattrygghet) to develop a risk assessment regarding the use of composted waste material from Alpharma’s production of bacitracin, as a soil additive. In response, an ad hoc Working Group of experts was appointed with the mandate to draft a risk assessment which should include the following elements: assessment of risk to human health and/or the environment in relation to residual content of bacitracin in the finished soil additive product and assessment of the risk in relation to dissemination of the production strain and antimicrobial resistance genes. The Panel on Biological Hazards concludes that the risks to human health and the environment posed by residual bacitracin present in the finished product are minimal. Furthermore, as Bacillus licheniformis is considered essentially non-pathogenic, occurring rarely as an opportunistic pathogen, the risk posed by this bacterium to human health or the environment is very low. It is reasonable to assume that during the early composting process horizontal transfer of bacitracin and erythromycin resistance genes, from the B. licheniformis producer strain to environmental bacteria, will exceed background levels. However, this is considered to represent a low risk to human health and the environment.

Descriptive epidemiology of infections due to thermotolerant Campylobacter spp. in Norway, 1979-1988.

This report reviews the first ten years of Campylobacter surveillance in Norway. During 1979-1988, a total of 3,545 isolates of thermotolerant Campylobacter spp. were reported. The isolation rate increased from 1.8 per 100,000 persons per year in 1979 to 13.1 in 1988. The highest isolation rate for both sexes occurred during the first five years of life (31.0 per 100,000). A smaller second peak was detected in the age group 15-24 years (11.1 per 100,000). The male-to-female ratio was 1.52:1 for infants less than five years of age, compared with a ratio of 1.35:1 for all ages combined. Thirty-eight percent of the infections had most probably been acquired abroad. The isolation rate in urban areas (12.4 per 100,000) was over twice that observed in rural agricultural municipalities (5.5 per 100,000). However, this difference was largely due to a higher proportion of imported cases in urban areas, only small variations in isolation rate were observed when imported cases were excluded. The seasonal distribution of Campylobacter isolates showed a peak during the warm months of the year. Travel activity during summer holidays did not account for this trend, since the summer peak became even more pronounced when imported cases were excluded. A north-south gradient in the seasonality was observed; when domestic cases were considered, the summer peak became more accentuated with increasing latitude reaching a maximum in subarctic areas. This might be explained by corresponding variations in occurrence of campylobacters in surface water sources.

Geographically dependent distribution of gyrA gene mutations at codons 83 and 87 in Salmonella Hadar, and a novel codon 81 Gly to His mutation in Salmonella Enteritidis.

In all, 90 nalidixic acid-resistant clinical strains of Salmonella Hadar and Salmonella Enteritidis isolated in Norway but of predominantly foreign origin were subjected to sequencing of the gyrA, gyrB, parC and parE genes. All the isolates contained at least one mutation in gyrA codon 83 or codon 87. A highly significant correlation between mutations in gyrA codon 83 and strains originating from Southeast Asia was found in S. Hadar but not in S. Enteritidis. A novel gyrA codon 81 Gly to His mutation was discovered in one S. Enteritidis isolate. One amino-acid (aa) changing mutation was found outside the quinolone resistance-determining region (QRDR) of S. Hadar parC at codon 57, which has previously only been observed once in Salmonellae.

Isolation of Campylobacter spp. and Yersinia enterocolitica from domestic animals and human patients in Kenya.

Rectal swabs or faecal samples from 992 domestic animals and 97 human patients in the Nairobi region were cultured for thermophilic Campylobacter species and Yersinia enterocolitica. The highest isolation rate of campylobacters was obtained from diarrhoeic pigs (55.1%), followed by healthy chicken (51.5%), diarrhoeic dogs (47.2%), healthy pigs (44.0%), healthy ducks (29.4%), healthy goats (6.3%), healthy cattle (5.8%), diarrhoeic humans (3.1%), and healthy sheep (2.0%). Only one strain of Y. enterocolitica was obtained. This isolate, which conformed to Nilehn's biotype 1, was recovered from one (0.7%) of the 150 healthy pigs examined. Out of 317 thermophilic campylobacters isolated, 163 (51.4%) were classified as C. jejuni, whereas 127 (40.1%) belonged to C. coli. The remaining 27 strains fell into three categories which did not conform to any defined species. Of the total number of isolates, 74.1% were resistant to metronidazole, 90.9% were resistant to triphenyltetrazolium chloride (TTC), and 50.2% reduced selenite. The results indicate that domestic animals may play a significant role in the epidemiology of human campylobacteriosis in the Nairobi region by serving as reservoirs. Y. enterocolitica seems to be rare among man and animals in this area.

A variation of the amplified-fragment length polymorphism (AFLP) technique using three restriction endonucleases, and assessment of the enzyme combination BglII-MfeI for AFLP analysis of Salmonella enterica subsp. enterica isolates.

We have performed amplified-fragment length polymorphism (AFLP) fingerprinting on a collection of Salmonella enterica subsp. enterica serovar typhimurium strains with a restriction endonuclease combination (BglII and MfeI) that has previously been used successfully for typing Campylobacter jejuni isolates with high resolution. Additionally, a variation of the AFLP assay in which two rare cutting restriction enzymes (XbaI and BsrGI) in combination with the frequent cutter (HinP1I) was examined. The BglII and MfeI enzyme combination offered low resolution for genotyping Salmonella typhimurium isolates and is not recommended for this common serovar. The three-enzyme combination gave a higher discrimination, and is thus a new alternate way of performing AFLP fingerprinting of S. typhimurium.

Yersinia enterocolitica in food hygiene.

Yersinia enterocolitica and Yersinia enterocolitica-like bacteria constitute a fairly heterogenous group of bacteria which includes both well-established pathogens and a range of environmental strains which are ubiquitous in terrestrial and freshwater ecosystems. Pathogenic significance in man is mainly associated with a few serogroups (O:3, O:9, O:8, O:5,27). The pathogenic serogroups show different geographical distributions. The development of isolation procedures which clearly differentiate pathogenic from non-pathogenic variants has been difficult. Of special significance in food hygiene is the ability of Y. enterocolitica to grow in refrigerated foods. There is strong indirect evidence that pigs and food products of porcine origin are the major sources for human infection with Y. enterocolitica serogroups O:3 and O:9, the dominant human pathogens in most parts of the world. The reservoir(s) for serogroup O:8, which prevails in the U.S.A., is uncertain. The pig is the only animal consumed by man which regularly harbours pathogenic Y. enterocolitica. Improved isolation methods and DNA colony hybridization using genetic probes has indicated that the prevalence of pathogenic Y. enterocolitica in pork products is substantially higher than previously suggested. Prevention and control measures should focus on information of people involved in food processing and preparation and on the improvement of hygiene during slaughtering of swine. Important critical control points at the stage of slaughter are: (i) circumanal incision and removal of intestines, (ii) excision of the tongue, pharynx, and particularly the tonsils, (iii) post-mortem meat inspection procedures which involve incision of the mandibular lymph nodes, and (iv) deboning of head meat.

Survey of thermotolerant Campylobacter spp. and Yersinia spp. in three surface water sources in Norway.

We investigated the occurrence of thermotolerant Campylobacter and Yersinia spp. in three surface water sources in Norway which represented different levels of pollution and eutrophication. Samples were collected every fortnight during a 14-month period. In addition, samples from 100 private wells were examined for campylobacters only. Campylobacter was recovered from 42 (43.8%) of the 96 samples of surface water, whereas Yersinia spp. were isolated from four (4.2%) of the samples. Campylobacter was not isolated from the well water samples. The highest isolation rate of Campylobacter was obtained from the two most polluted water sources. The proportion of positive samples was significantly higher in the autumn (71.4%) than in the spring (36.4%) or summer (22.2%). The highest overall isolation rate was obtained at water temperatures ranging from 2.1 to 8.0 degrees C, and the lowest at temperatures greater than 15 degrees C. Logistic regression analysis showed a highly significant relationship between the prevalence of Campylobacter and the number of three types of indicator bacteria: faecal coliforms, faecal streptococci and sulphite-reducing clostridia. Of the 60 Campylobacter isolates obtained, 51.7% belonged to C. jejuni biotype 1, 20.0% belonged to C. jejuni biotype 2, 21.7% to C. coli, 3.3% to C. lari and 3.3% were non-typable. All four Yersinia isolates were non-pathogenic variants.

Identification of thermotolerant Campylobacter species from poultry using an enzyme-labelled oligonucleotide DNA probe.

A commercially available enzyme-labelled DNA probe for human Campylobacter strains has been tested and also found to hybridize with DNA from C. jejuni and C. coli isolates from poultry. DNA from 11 enteric, non-campylobacter organisms, included in the test as negative controls, failed to hybridize with the probe indicating that the probe might be used for identification of campylobacter from poultry too.

Pathways of Listeria monocytogenes contamination in the meat processing industry.

One hundred and thirty-three isolates of Listeria monocytogenes from deboned fresh meat, production environment, cold cuts from five meat processing plants and from one plant producing cured dried sausages, were characterized using multilocus enzyme electrophoresis. On the basis of electrophoretically demonstrable allelic variation at 21 enzyme loci, 21 electrophoretic types (ETs) were distinguished. Analysis of the genetic relationships among the 21 ETs revealed two distinct clusters: Cluster A and Cluster B. With the exception of two isolates from one plant, all isolates from deboned fresh meat belonged to Cluster B. During processing of cold cuts, however, isolates belonging to Cluster A became more frequent, and only one of the 37 isolates from cold cuts belonged to Cluster B. In contrast, six of the nine isolates from cured dried sausages had ETs in Cluster B. One clone of Cluster A, ET-6 was isolated from cold cuts in four of six plants. This is one of the ETs most frequently recovered from patients in Norway. Isolates of ET-6 were further characterized using restriction fragment length polymorphism (RFLP) analysis of chromosomal DNA. Six distinct restriction patterns were distinguished among the 44 ET-6 strains. In one plant, four different RFLP patterns could be identified. Two clone variants seemed to have colonized different areas in this plant for at least four years. However, in each of the other plants, all ET-6 isolates had the same RFLP patterns.

Occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products determined by a PCR method and a traditional culturing method.

From October 1997 to April 1998, a survey was conducted to assess the occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products, using a traditional culturing method and a PCR assay. A total of 300 pork samples was examined. Five slaughterhouses in the Norwegian Meat Cooperative were represented with 249 samples and another 51 samples were obtained from retail outlets in the city of Oslo. Using the NMKL method, Y. enterocolitica 0:3 was isolated from six (2%) of the samples, while the PCR method indicated presence of pathogenic Y. enterocolitica in 50 (17%) of the samples. The results indicate that a reduction has occurred in the prevalence of pathogenic Y. enterocolitica in Norwegian pork products, as compared to a previous Norwegian study conducted in 1988-1989. The study also highlights the need for further development and improvement of methods applied for the detection of pathogenic Y. enterocolitica in foods.

Risk factors for the presence of antibodies to Toxoplasma gondii in Norwegian slaughter lambs.

In a study of lambs from 194 sheep flocks from different areas of Norway, 44.3% of the flocks were found seropositive against Toxoplasma gondii using an ELISA test, with antibodies found in 16.2% of the 1940 individual animals. The risk factors for the occurrence of antibodies to Toxoplasma gondii found in the multivariate logistic regression were: daily presence of a young cat in the sheep house (Odds ratio, OR = 4.11, 95% CI = 1.01-19.7); 'atypical' grazing of lambs (OR = 6.35, CI = 2.36-17.11); use of mouse poison in the sheep house (OR = 2.26, CI = 1.02-5.00); farm situated at an altitude > 100 meters above seal level (101-250 m: OR = 1.20, CI = 0.49-2.92; 251-500 m: OR = 4.97; CI = 2.04-12.0; > 500 m: OR = 3.66, CI = 1.33-10.1). A lower risk was found for flocks with perforated metal floors in the sheep house (OR = 0.47, CI = 0.23-0.96) and timber construction of the sheep house (OR = 0.34, CI = 0.15-0.80). Based on these findings it was recommended that farmers avoid keeping young cats in the sheep houses, that close-to-farm grazing be kept to a minimum and that perforated metal floors be used in the sheep houses. However, with such a high seroprevalence, the proposed measures alone would not reduce the occurrence of Toxoplasma in lambs to a level where undercooked lamb can be consumed without posing an unacceptable risk for some consumer groups.

Survey for toxoplasmosis in wild and domestic animals from Norway and Sweden.

Fifty-nine of 1250 (4.7%) wild and domestic animals from Norway and Sweden had positive dye-test titers (greater than or equal to 1:8) for antibody against Toxoplasma gondii. A dye-test titer of 1:8 (30-40 i.u.) or higher was detected in 3 of 732 small rodents (0.4%), 21 of 87 domestic cats (24%), 9 of 29 red foxes (Vulpes vulpes) (31%), 2 of 2 domesticated arctic foxes (Alopex lagopus), 12 of 99 red deer (Cervus elaphus) (12%), 5 of 8 roe deer (Capreolus capreolus) (63%) and in 7 of 34 wild rabbits (Oryctolagus cuniculus) (21%). Antibodies were not found in 29 shrews (Sorex spp.), 127 gulls (Larus spp.), 4 terns (Sterna sp.), 10 skuas (Stercorarius sp.) 68 domestic reindeer (Rangifer tarandus) and 21 wild reindeer. Histologic examination of brain tissue from another 51 wild rabbits on which serological data were not available, did not reveal cysts. Sero-conversion was not observed in laboratory mice inoculated with the same material. Infection with T. gondii was confirmed in two of the three sero-positive small rodents using a FA-technique. Cysts were not detected in the brains of another 55 rodents, of which 26 were sero-negative and 29 were not tested serologically.

A survey of tularemia in wild mammals from fennoscandia.

A total of 2696 wild mammals from Fennoscandia were surveyed for tularemia. Francisella tularensis was not detected in livers/spleens or kidneys from any of the 1992 small rodents captured in Norway and Denmark as judged by one or more of the following methods: cultivation, immunofluorescence microscopy and inoculation in laboratory mice. Serologic examination of 704 wild mammals from Norway, Finland and Sweden demonstrated 11 cases of antibody titers. Agglutinating antibodies were demonstrated in 2 of 565 small rodents (titer 1:160), 2 of 26 wild rabbits (titer 1:80) and in 7 of 60 red deer (titer 1:20-1:40). The titers in red deer were low and could be due to cross reactions. No agglutinating antibodies were demonstrated in the sera of 53 domestic reindeer.

The effect of lactic acid sprays on Campylobacter jejuni inoculated onto poultry carcasses.

Spraying poultry carcasses with 1% lactic acid 10 min after inoculation with Campylobacter jejuni, resulted in a significant reduction in the number of the bacteria after 4 h at 4 degrees C. Some of the inoculated cells, however, survived for at least 144 h. Spraying 10 min after inoculation with 2% lactic acid, totally eliminated all inoculated C. jejuni within 24 h. On the other hand, spraying 24 h after inoculation, with either 1% or 2% lactic acid did not eliminate all the bacteria. Inoculated C. jejuni on poultry carcasses not sprayed with lactic acid, survived at 4 degrees C throughout the sampling period (up to 144 h) and showed little tendency to decrease in number even when the carcasses started to deteriorate. Resident campylobacters on poultry carcasses were significantly reduced by the lactic acid treatment. Frozen and thawed chickens appeared to show a graying of the skins immediately after spraying with lactic acid, slightly stronger with 2% lactic acid, but the colour reverted to normal after 24 h. We were not able to observe any colour change on the fresh broiler chickens after lactic acid treatment. Our results indicated that lactic acid had a significant bactericidal effect on C. jejuni on both naturally and artificially contaminated poultry carcasses. This effect, however, became manifest only several hours after acid treatment.