Prevalence of methicillin-resistant Staphylococcus aureus carrying mecA or mecC in dairy cattle.

In the Netherlands, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been found in pigs, veal calves, horses and poultry. However, little is known about its prevalence in healthy dairy cattle. Recently, a new mec gene, called mecC, has been found in methicillin-resistant S. aureus (MRSA) isolates from humans and animals in several countries. The objective of our pilot study was to investigate the prevalence of MRSA (mecA and mecC) in dairy cows at a large slaughterhouse. Samples from the skin between the udder and hind leg were taken from 411 cows. The samples were incubated in Mueller-Hinton enrichment broth with 6.5% NaCl, followed by selective enrichment and plated onto Columbia agar with 5% sheep blood, Brilliance MRSA 2 agar and Baird-Parker agar. Suspected colonies were tested by PCR for a S. aureus specific DNA fragment, the mecA and mecC genes and the Panton-Valentine leucotoxin (PVL) genes. All MRSA isolates and methicillin-susceptible S. aureus (MSSA) isolates were typed by spa typing and MLVA typing. Sixteen of 411 (3.9%) cows, all originating from different farms, were found to be MRSA positive and this prevalence is lower than in Dutch pigs, veal calves and broilers. All MRSA isolates belonging to livestock-associated MLVA complex 398, were PVL-negative and spa type t011 predominated. MSSA isolates (n=39) were of many different MLVA types and spa type t543 was found most often. Four MSSA isolates belonging to MLVA clonal complex 398 and spa types t011 (n=2), t108 and t034 were isolated from different MRSA-negative animals. In conclusion, the prevalence of MRSA in dairy cows was low and isolates carrying the mecC gene were not found, indicating that it is absent or has a low prevalence (<0.73%) in Dutch dairy cows.

Prevalence of livestock-associated MRSA on Dutch broiler farms and in people living and/or working on these farms.

This study aimed to determine the prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) on 50 Dutch broiler farms. Of 145 persons living and/or working on these farms, eight tested positive for MRSA (5.5%). Investigation of 250 pooled throat samples of broilers and 755 dust samples resulted in four farms where MRSA-positive samples were present (8.0%). All isolates belonged to the CC398 complex. Living and/or working on a MRSA-positive farm was a risk for MRSA carriage; 66.7% of people on positive farms were MRSA positive vs. 1.5% on negative farms (P<0.0001). Due to the low number of positive farms and persons, and high similarity in farm management, it was impossible to draw statistically valid conclusions on other risk factors. For broiler farming, both farm and human MRSA prevalence seem much lower than for pig or veal farming. However, MRSA carriage in people living and/or working on broiler farms is higher compared to the general human population in The Netherlands (5.5% vs. <0.1%). As broiler husbandry systems are not unique to The Netherlands, this might imply that people in contact with live broilers are at risk for MRSA carriage worldwide.

Prevalence and risk factor analysis of livestock associated MRSA-positive pig herds in The Netherlands.

In 2005, methicillin-resistant Staphylococcus aureus was found in pig herds and in humans in contact with pigs. To determine the prevalence of, this now-called livestock-associated (LA) MRSA among pig herds in The Netherlands and to identify and quantify risk factors, an observational study of 202 pig herds was performed between 2007 and 2008. Five environmental wipes and 60 nasal swabs from each herd were collected, and microbiological analysis was performed on single environmental samples and pooled nasal samples. A herd was considered MRSA-positive if ≥1 sample tested positive. The prevalence of MRSA-positive herds was 67% in breeding herds and 71% in finishing herds. Multivariable logistic regression analysis was then performed on data from 171 breeding herds. The number of MRSA-positive herds increased from ∼30% at the start to ∼75% at the end of the study, most likely due to transmission between herds. The prevalence of MRSA increased with herd size, as ∼40% of smaller herds (<250 sows) were MRSA-positive compared to >80% of larger herds (>500 sows). Other risk factors (e.g. antimicrobial use, purchase of gilts and hygiene measures) were not significantly associated with MRSA, though associated with herd size. Herd size appeared to be a compilation of several factors, which made larger herds more often MRSA positive.

Comparison of sampling methods used for MRSA-classification of herds with breeding pigs.

Since the first report on methicillin resistant Staphylococcus aureus (MRSA) CC398 in pigs, several countries have determined the prevalence of MRSA-positive pig herds using different sampling and laboratory techniques. The objective of the study was to compare three sampling methods for MRSA-classification of herds. Therefore, nasal swabs of pigs and environmental wipes were collected from 147 herds with breeding pigs. Per herd, laboratory examination was done on 10 pools of 6 nasal swabs (NASAL), 5 single environmental wipes (ENVSINGLE) and one pool of 5 environmental wipes (ENVPOOL). Large differences in apparent prevalence of MRSA-positive herds between methods were found: 19.1% for ENVPOOL, 53.1% for ENVSINGLE, and 70.8% for NASAL. Pairwise comparisons of methods resulted in relative sensitivities of 26.9% (ENVPOOL vs. NASAL), 34.6% (ENVPOOL vs. ENVSINGLE), and 72.1% (ENVSINGLE vs. NASAL) with relative specificities of respectively 100%, 98.6% and 93.0%. Cohen's kappa was respectively 0.18, 0.32 and 0.55, thus varying between very poor and moderate agreement. Examination of environmental wipes is an easy and non-invasive method to classify herds for MRSA. The number of environmental wipes needed depends on e.g. required detection limits and within-herd prevalence. In low prevalent herds (e.g. herds with <3 positive pools of nasal swabs), 25 single environmental wipes are required to be 90% sure that MRSA is detected at a detection limit similar to analyzing 10 pools of nasal swabs. Individual analysis of environmental wipes is highly recommended, as pooling 5 environmental samples resulted in a substantial reduction of the apparent prevalence.

MRSA CC398 in the pig production chain.

In 2005, a distinct clone of methicillin resistant Staphylococcus aureus (MRSA CC398) was found in pigs and people in contact with pigs. The structure of the pig production chain in high technology pig husbandry enables pathogens to spread during animal trading, with an increasing prevalence in herds further down the chain. The objective of this study was to quantify the effect of the MRSA status of the supplying herd on the MRSA status of the receiving herd in order to gain more insight into the role of animal trading as a transmission route for MRSA CC398. Nasal samples (60-80 pigs per herd) were collected from 38 herds; in 20 herds, environmental samples were collected as well. Ten MRSA-positive herds (based on the results of nasal swabs of 10 individual pigs per herd) from a prior study were included in the data analysis. Herds were classified as MRSA positive if at least one sample tested positive. The 48 herds were part of 14 complete (40 herds) and 4 incomplete (8 herds) pig production chains. Fifty-six percent of the herds were classified as MRSA positive. MRSA-positive herds were observed at the start (breeding herds), middle (farrowing herds) and the end (finishing herds) of the pig production chain. All of the herds in 8 chains tested MRSA positive;, all of the herds in 5 chains tested MRSA negative and in the remaining 5 chains, MRSA-positive and MRSA-negative herds were detected. Seven spa types were found, which were all previously confirmed to belong to CC398. All of the isolates were susceptible to mupirocin, linezolid, rifampicin, fusidic acid and cotrimoxazole. Resistance against tetracycline, erythromycin and clindamycin was found in 100, 74 and 76% of the isolates, respectively. Seventy-nine percent of herds with a MRSA-positive supplier of pigs were MRSA positive, whereas 23% of herds with a MRSA-negative supplier were MRSA positive (OR=10.8; 95% CI: 1.5-110.1; P=0.011). The presence of entirely MRSA-positive and MRSA-negative chains and the strong association between the MRSA status of herds and their suppliers illustrates a large risk associated with purchasing pigs from MRSA-positive herds; a top-down strategy for future control programs is, therefore, a basic requirement. However, 23% of herds with a MRSA-negative supplier were MRSA positive and furthermore, 46% of the herds at the top of the pig production chain without a supplier tested MRSA positive. This underlined the need for the identification of additional risk factors for MRSA.

High prevalence of nasal MRSA carriage in slaughterhouse workers in contact with live pigs in The Netherlands.

Livestock-associated MRSA has been found in various animals, livestock farmers and retail meat. This study aimed to determine the prevalence and determinants of nasal MRSA carriage in pig slaughterhouse workers. Three large pig slaughterhouses in The Netherlands were studied in 2008 using human and environmental samples. The overall prevalence of nasal MRSA carriage in employees of pig slaughterhouses was 5.6% (14/249) (95% CI 3.4-9.2) and working with live pigs was the single most important factor for being MRSA positive (OR 38.2, P<0.0001). At the start of the day MRSA was only found in environmental samples from the lairages (10/12), whereas at the end of the day MRSA was found in the lairages (11/12), the dirty (5/12) and clean (3/12) areas and green offal (1/3). The MRSA status of the environmental samples correlated well with the MRSA status of humans working in these sections (r=0.75). In conclusion, a high prevalence of nasal MRSA carriage was found in pig-slaughterhouse workers, and working with live pigs is the most important risk factor. Exact transmission routes from animals to humans remain to be elucidated in order to enable application of targeted preventive measures.

Prevalence of livestock-associated MRSA in broiler flocks and risk factors for slaughterhouse personnel in The Netherlands.

To determine methicillin-resistant Staphylococcus aureus (MRSA) carriage in poultry and slaughterhouse personnel, 40 Dutch broiler flocks, in six slaughterhouses and 466 personnel were sampled. Of the employees, 26 were positive (5.6%), indicating a higher risk of exposure when compared to the general Dutch population (0.1%). This risk was significantly higher for personnel having contact with live animals (5.2%) - especially hanging broilers on the slaughterline (20.0%) - than for all other personnel (1.9%). Conventional electric stunning conferred a significantly higher risk of MRSA carriage for employees than CO2 stunning (9.7% vs. 2.0%). A total of 405 broilers were sampled upon their arrival at the slaughterhouse, of which 6.9% were positive. These broilers originated from 40 Dutch slaughter flocks of which 35.0% were positive. MRSA contamination in the different compartments of slaughterhouses increased during the production day, from 8% to 35%. Of the 119 MRSA isolates, predominantly livestock-associated MRSA ST398 was found, although 27.7% belonged to ST9 (spa type t1430). There is an increased risk of MRSA carriage in personnel working at broiler slaughterhouses, particularly those having contact with live animals.

Occurrence of methicillin-resistant Staphylococcus aureus in rats living on pig farms.

In The Netherlands, MRSA ST398 has emerged in hospitals and human carriers have been associated with exposure to pigs and cattle. High prevalences of MRSA ST398 in pigs and pig farmers have been determined and the transmission routes of MRSA on pig farms need to be elucidated. In the south of the Netherlands, in recent years, the black rat (Rattus rattus) has emerged as a prominent rodent on livestock farms. From March till May 2008, a survey on MRSA in rats living on livestock farms in the south of The Netherlands and the north of Belgium was conducted. In total, 40 black rats (R. rattus) and 3 brown rats (Rattus norvegicus) were collected on 12 farms including five pig farms, five poultry farms, one mixed pig and veal farm and one goat farm. MRSA ST398 was detected in black rats captured at two of the five pig farms as well as in a black rat living on the mixed pig and veal farm. From one black rat captured at another pig farm MRSA ST 97 was isolated. Considering the behaviour of rats on livestock farms, it is concluded that rats might play a role in the spread and persistence of MRSA on pig farms.

Methicillin-resistant Staphylococcus aureus in people living and working in pig farms.

We compared the prevalence of human and animal methicillin-resistant Staphylococcus aureus (MRSA) at pig farms in The Netherlands, and related this to individual and farm-level characteristics. More than half of the farms investigated (28/50) had MRSA in pigs or stable dust and about one third (15/50) of person(s) were identified as MRSA carriers. Human carriage was found only on farms with MRSA-positive pigs or dust. MRSA strains in human samples were the same spa-type as found in pigs and all were not typable by pulsed-field gel electrophoresis (NT-MRSA). Multivariate analyses showed that risk factors for human MRSA carriage were: working in pig stables (OR 40, 95% CI 8-209) and the presence of sows and finishing pigs (OR 9, 95% CI 3-30). Veterinary sample collectors sampling the pigs showed transient MRSA carriage only during the day of the farm visit. Working in pig stables with MRSA-positive pigs poses a high risk for acquiring MRSA, increasingly so when contact with live pigs is more intensive or long lasting.

Prevalence of VTEC O157 in dairy and veal herds and risk factors for veal herds.

The aim of this study was to determine the herd prevalence of veal and dairy herds and to identify risk factors for VTEC O157 positive veal herds. The study was based on monitoring data from November 1996 through July 2005 of 1051 dairy herds and 930 veal herds. The herd level prevalence (95% CI) was 8.0% (6.4-9.6) for dairy herds and 12.6% (10.5-14.7) for veal herds. Within the population of veal herds, a prevalence of 39.8% (33.9-45.6) was found for pink veal herds (n = 269) and 1.5% (0.7-2.8) for white veal herds (n = 661). Multivariable logistic regression showed that the type of veal (pink vs. white; OR = 21.6; 95% CI: 10.4-45.0), ventilation (mechanical vs. natural; OR = 0.4; 95% CI: 0.2-0.8), time between arrival in the herd and sampling (3-5 months vs. 0-2 months: OR = 2.33; 95% CI: 1.1-5.1, > or = 6 months vs. 0-2 months: OR = 4.11; CI: 1.9-8.9), other feed than the 7 most common (yes vs. no; OR = 2.1; 95% CI: 1.2-3.7) and at least one dog present in the stable (yes vs. no; OR = 2.6; 95% CI: 1.5-4.6) were significantly (P < 0.05) associated with the presence of VTEC O157. The large difference in the VTEC O157 prevalences for pink veal and white veal production might have been caused by a very different management of these type of herds. However, this could not be studied with the data collected.

High prevalence of methicillin resistant Staphylococcus aureus in pigs.

Recently methicillin resistant Staphylococcus aureus (MRSA) was isolated from pigs and pig farmers in The Netherlands. In order to assess the dissemination of MRSA in the Dutch pig population, we screened 540 pigs in 9 slaughterhouses, where a representative portion of Dutch pigs (63%) was slaughtered in 2005. We found 209 (39%) of the pigs to carry MRSA in their nares. Forty-four of 54 groups of 10 consecutive pigs (81%), each group from a different farm, and all slaughterhouses were affected. All MRSA isolates belonged to 1 clonal group, showing Multi-Locus Sequence Type 398 and closely related spa types (mainly t011, t108 and t1254). Three types of the Staphylococcal Chromosome Cassette (SCCmec) were found: III (3%), IVa (39%) and V (57%). All 44 tested isolates (1 isolate per group) were resistant to tetracycline, reflecting the high and predominant use of tetracyclines in pig husbandry. Twenty-three percent of the isolates were resistant to both erythromycin and clindamycin and 36% to kanamycin, gentamicin and tobramycin but only a single isolate was resistant to co-trimoxazole and none to ciprofloxacin and several other antibiotics. The percentage of MRSA positive pigs was significantly different among slaughterhouses and among groups within slaughterhouses, indicating a high prevalence of MRSA in pigs delivered from the farms as well as cross contamination in the slaughterhouses.

A beef-associated outbreak of Salmonella Typhimurium DT104 in The Netherlands with implications for national and international policy.

A ten-fold increase in Salmonella Typhimurium DT104 cases in The Netherlands during September-November 2005 prompted an outbreak investigation. A population-based matched case-control study included 56 cases and 100 controls. Risk factors for infection were consumption of a pre-processed raw beef product (odds ratio 4.2, 95% confidence interval 1.5-12.0) and of food from mobile caterers (odds ratio 4.9, 95% confidence interval 1.1-22.1). Bacterial molecular typing established a link with another DT104 outbreak in Denmark caused by beef from a third European country. The incriminated beef was traced in The Netherlands and sampling yielded DT104 of the outbreak-associated molecular type. We concluded that this outbreak was caused by imported contaminated beef. Consumers should be informed about presence of raw meat in pre-processed food products. Optimal utilization of international networks and testing and traceability of foodstuffs has the potential to prevent foodborne infections.

Surveillance of Salmonella spp. and Campylobacter spp. in poultry production flocks in The Netherlands.

In The Netherlands, a national programme for the surveillance of zoonotic bacteria in farm animals has been operative since 1997. We describe the results of the surveillance of Salmonella spp. in flocks of laying hens and broilers and of Campylobacter spp. in broiler flocks in the period 1999-2002. The prevalence of Salmonella spp. in laying-hen flocks has significantly decreased from 21.1% in 1999 to 13.4% in 2002. This decreasing trend might indicate that the control measures taken by the poultry industry were effective. S. Enteritidis was the predominant serovar in laying hens accounting for one third of the positive flocks. Although prevalence estimates for Salmonella spp. in broiler flocks did not yield a significant decreasing trend in 1999-2002, a decrease in Salmonella prevalence to 11% was measured in 2002. During the study period, S. Paratyphi B var. Java emerged in broilers to become the predominant serovar in 2002 accounting for one third of the positive flocks. The prevalence of Campylobacter spp. in broiler flocks did not increase nor decrease continuously between 1999 and 2002, which roughly corresponds with the monitoring results from the poultry industry. In this period, the estimated flock prevalence roughly averaged around 20%, with C. jejuni being the predominant species. The approach of monitoring presented in this paper can serve as a blueprint for monitoring schemes in farm animal populations to be developed in the context of the EC Zoonoses Directive.

Escherichia coli O157 prevalence in Dutch poultry, pig finishing and veal herds and risk factors in Dutch veal herds.

In the period October 1996 through December 2000, a total of 7163 pooled faecal samples of laying hen and broiler flocks, finishing-pig herds and veal herds were examined for the presence of Salmonella spp., Campylobacter spp. and verocytotoxin-producing Escherichia coli O157 as part of a national monitoring programme in The Netherlands. Isolates were tested for eae and VT genes. Risk factors for Dutch veal herds were quantified. For all herd/flock types, faecal samples were cultured for E. coli O157. Of broiler flocks, laying flocks and finishing pig herds, respectively, 1.7%, 0.5% and 0.4% were E. coli O157 positive. In total, 42 of the 454 veal herds (9.3%) showed at least one positive pooled sample. E. coli O157-positive herds were compared (with logistic regression) to negative herds, regarding variables obtained from the questionnaire taken from the farm manager. To account for season, a sine function was included in the logistic regression as offset variable. In the final model, 'pink-veal production' (compared to white-veal production), 'group housing of the sampled herd' (compared to individual housing), 'more than one stable present' (compared to one stable present), 'hygienic measures regarding visitors' (compared to no hygienic measures), 'interval arrival-sampling of a herd of >20 weeks' (compared to < or =10 weeks), and 'presence of other farms within 1 km' (compared to no presence of farms <1 km) showed associations (P<0.05) with the presence of E. coli O157. These results need careful interpretation; they should be considered as indications for further (experimental or cohort-based) research rather then causal associations.

A longitudinal study of Escherichia coli O157 in cattle of a Dutch dairy farm and in the farm environment.

From July 1999 till November 2000, a longitudinal study was conducted on a dairy farm in The Netherlands to study within herd prevalence and types of verocytotoxin producing Escherichia coli (VTEC) of serogroup O157 over time, and determine environmental reservoirs and possible transmission routes. Faeces, blood, milk and environmental samples were collected 14 times with intervals varying from 4 to 10 weeks during the study period. Faecal samples were selectively cultured for Escherichia coli O157. Isolates were tested by PCR for the most common virulence genes, VTI, VTII and eae, and typed by pulsed field gel electrophoresis. In total, 71 isolates were obtained, of which 49 from dairy cows, 8 from young stock, 5 from other animals and 9 from the environment. Positive samples were all detected in summer and early fall. VT- and eae-genes were found in all tested isolates, except in one. DNA typing showed that three clusters of O157 isolates could be identified. One of these clusters contained samples of two shedding seasons, indicating persistence on the farm during winter and spring. Repeated measures analysis of variance showed that cows with O157 VTEC infection had higher daily milk production in the period preceding sampling (p = 0.0055). There was no significant association between the results of the LPS-ELISA on serum samples from dairy cows and their O157 status.

[Sharp increase of infections with Salmonella serotype Typhimurium DT104 in The Netherlands]. / Sterke toename van infecties met Salmonella serotype Typhimurium DT104 in Nederland.

Salmonella remains an important source of food-related outbreaks of gastro-enteritis. In The Netherlands, regional laboratories send cultured Salmonella isolates to the National Institute of Public Health and the Environment (RIVM) for sero- and phage typing. Abnormal increases in the incidence of Salmonella infections are monitored by means of surveillance. Since the middle of September 2005, there has been a sharp increase in Salmonella Typhimurium DT104 (Dutch phage type 506 and 401) isolates, from all parts of the country and mainly from 6- to 20-year-olds. Since a first series of telephone interviews did not yield a clear source of the outbreak, a more extensive, written study has been started in a larger group of patients and corresponding controls.

A large increase of Salmonella infections in 2003 in The Netherlands: hot summer or side effect of the avian influenza outbreak?

In June 2003, the Dutch National Salmonella Centre reported a significant excess isolation rate of Salmonella Enteritidis when compared with earlier years in most regional public health laboratories. By the end of 2003, this amounted to an extra 540 laboratory confirmed cases for the whole of the Netherlands, which implies an estimated 7500 extra cases of gastroenteritis caused by S. Enteritidis in the general population, an increase of 50% on previous years. The hot summer could not explain the findings. Strong evidence has been found to suggest that the increase in importation of salmonella contaminated eggs, as a side effect of a concurrent avian influenza outbreak, was the most probable reason for this excess.

Prevalence estimation and risk factors for Escherichia coli O157 on Dutch dairy farms.

To estimate the prevalence of Escherichia coli O157 on Dutch dairy herds, faecal samples were collected once from 678 randomly selected dairy farms in the period October 1996-December 2000. Samples were cultured for E. coli O157. Thirty-eight isolates were tested for virulence genes (eae, VT1 and VT2). A questionnaire about farm characteristics was taken from the farm manager, resulting in variables that could be analysed to identify and quantify factors associated with presence of E. coli O157. In total, 49 of the 678 herds (7.2%) showed at least one positive pooled sample. E. coli O157 was not isolated from herds sampled in December-April in consecutive years (except for one isolate found in March, 2000). VT- and eae-genes were found in 37 and 38 isolates, respectively. Logistic regression was performed on variables obtained from the questionnaire, comparing E. coli O157-positive herds to negative herds. To account for season, a sine function was included in the logistic regression as an offset variable. In the final model, the presence of at least one pig at the farm (OR = 3.4), purchase of animals within the last 2 years before sampling (OR = 1.9), supply of maize (OR = 0.29) to the cows, and sampling a herd in the year 1999 or 2000 (compared to sampling in 1998; OR = 2.1 and 2.9, respectively) had associations with the presence of E. coli O157.

Risk factors for the presence of Campylobacter spp. in Dutch broiler flocks.

The campylobacter status of 495 broiler flocks sampled in The Netherlands between April 1997 and December 2000 was related to farm- and flock-specific information obtained from questionnaires to identify potential risk factors for campylobacter presence. Approximately 30% of the flocks tested positive for Campylobacter spp. in at least one pooled faecal sample. Multivariable logistic regression showed significant risk increments for: ages 29-35 days (OR = 2.34) and 36-42 days (OR = 3.96) compared to 22-28 days; > or =5 broiler houses on the premises (OR = 3.02); the presence of other farm animals on the farm (OR = 1.88); the presence of animals on farms within 1 km (OR = 9.56); and summer (OR = 3.48) and fall (OR = 2.59) compared to winter. Furthermore, hatcheries differed (ORs from 5.42 to 20.2), although this variable showed strong collinearity with the variables explaining feed mill and integrated poultry operations. We also identified interaction in which an increased risk from children entering the broiler house (OR = 28.0) was diminished by the use of broiler-specific workclothes. Population attributable fractions (PAFs) suggested that animals on farms within 1 km (PAF = 0.76) and hatchery (PAF = 0.67) had the highest impacts on campylobacter presence in Dutch broiler flocks. These factors, however, lack an easy interpretation of the mechanism behind the suggested effect.

A large increase of Salmonella infections in 2003 in the Netherlands: hot summer or side effect of the avian influenza outbreak?

In June 2003, the Dutch national Salmonella centre reported a significant excess isolation rate of Salmonella Enteritidis when compared with earlier years in most regional public health laboratories. By the end of 2003, this amounted to an extra 540 laboratory confirmed cases for the whole of the Netherlands, which implies an estimated 7500 extra cases of gastroenteritis caused by S. Enteritidis in the general population, an increase of 50% on previous years. The hot summer could not explain the findings. Strong evidence has been found to suggest that the increase in importation of salmonella contaminated eggs, as a side effect of a concurrent avian influenza outbreak, was the most probable reason for this excess.