Anne Wingstrand

The occurrence and epidemiology of Salmonella in European pig slaughterhouses

This study was part of an international research project entitled SALINPORK (FAIR CT-950400) initiated in 1996. The objectives were to investigate the occurrence of Salmonella in pig slaughterhouses and to identify risk factors associated with the contamination of pig carcasses. Data was collected from 12 slaughterhouses in five European countries. Isolates were characterized by serotyping, phage typing and antimicrobial susceptibility. In one country, no Salmonella was found. Salmonella was isolated from 5.3% of 3485 samples of pork and from 13.8% of 3573 environmental samples from the seven slaughterhouses in the four remaining countries. The statistical analyses (multi-level logistic regression) indicated that the prevalence was significantly higher during the warmer months and that the environmental contamination increased during the day of slaughter. The polishing (OR 3.74, 95% CI 1.43-9.78) and pluck removal (OR 3.63, 95% CI 1.66-7.96) processes were found to contribute significantly to the total carcass contamination, the latter especially if the scalding water also was contaminated. To reduce carcass contamination, it is recommended to ensure sufficiently high temperatures of scalding water (62 degrees C) and appropriate cleaning and disinfection of the polishing equipment at least once a day in order to reduce the level of carcass contamination and consequently the prevalence of Salmonella in pork.

THE TIME COURSE OF THE SPECIFIC ANTIBODY RESPONSE BY VARIOUS ELISAS IN PIGS EXPERIMENTALLY INFECTED WITH TOXOPLASMA GONDII

With the aim of developing routine serological tests for monitoring the Toxoplasma infection status of Danish swine herds, four ELISAs based on tachyzoite antigen were set up: (1) an indirect ELISA for IgG-antibody; (2) a blocking ELISA for antibody to the membrane antigen, P30; (3) an indirect ELISA for IgM; (4) a reverse, antibody-catching IgM-ELISA. Groups of pigs (number between 6 and 10) were inoculated with tachyzoites of the RH-strain, tissue cysts of two complete strains, or oocysts in two doses (10(3) and 10(4). All inoculations were tolerated well. Irrespective of strain and stage used for inoculation, specific IgG and anti-P30 blocking activity appeared after 1-2 weeks, with OD-values stabilizing after 3-6 weeks and persisting throughout the study period (3-4 months). Specific IgM appeared quickly, but was short-lived (approximately 2 weeks). A cut-off OD-value of 0.36 for positive seroreaction in the indirect IgG-ELISA was determined on the basis of 69 sera from four herds, investigated in the dye-test (serum dilution 1:10) and ELISA. The chosen cut-off gave optimal combined sensitivity and specificity of 0.94 and 0.92, respectively, using the dye-test as a standard. Corresponding figures for the blocking ELISA were 37% inhibition as cut-off, with sensitivity and specificity of 0.94 and 0.94, respectively. Sera from a total of 87 pigs, experimentally infected with bacteria of the genera Salmonella, Yersinia or Actinobacillus and with the parasites Isospora suis, Trichinella spiralis or Ascaris suum, in no case produced cross-reactions in the IgG-ELISA. However, 3/9 pigs inoculated with 50 000 sporocysts of Sarcocystis miescheriana gave maximal OD-readings of 0.40-0.45 during the 13-15 weeks observation period. None of the sera from heterologously infected animals produced inhibitions in the anti-P30 blocking ELISA exceeding 36%.

Elimination of Salmonella typhimurium infection by the strategic movement of pigs

Three field investigations were carried out to assess the feasibility of raising salmonella-free finishers from pigs born in infected herds, by moving the pigs to clean and disinfected facilities before their expected exposure to the bacteria from the environment. Three herds with persistently high levels of subclinical infection with S typhimurium in the finishing pigs were used. They practised all-in all-out management in the nurseries and in the grower units. A total of 844 pigs were moved, either at weaning, from the nursery, or from the grower unit to newly built or rigorously cleaned and disinfected finishing units with no known history of salmonella infection. No detectable infection was observed at slaughter either serologically or bacteriologically by random testing of the pigs which had been moved, whereas a proportion of the pigs raised at the same time in the continuous systems on the farms were found to be infected.

Time course of the serological response to Yersinia enterocolitica O:3 in experimentally infected pigs

A total of 25 pigs inoculated with Yersinia enterocolitica serovar O:3 and 25 un-inoculated controls were followed weekly by sampling blood and faeces for 70 days post infection (p.i.). All inoculated pigs were faeces culture positive from day 5 to 21 p.i., whereafter shedding of bacteria declined to < 10% of the pigs at day 49 p.i. and to 0% at day 68 p.i. All control pigs remained Y. enterocolitica O:3 culture negative. When examined in an indirect ELISA using purified LPS from Y. enterocolitica 0:3, sera from all inoculated pigs showed significantly higher optical densities (OD) as compared to the control group. All inoculated pigs had seroconverted at day 19 p.i. and remained seropositive until slaughter at day 70 p.i. The maximum mean anti-LPS response was observed at day 33 p.i. with a positive/negative ratio of 780. No cross-reactions were observed with sera from 21 pigs, infected with Salmonella typhimurium. At necropsy at day 70 p.i., Y. enterocolitica O:3 was isolated from the tonsils of 20 inoculated pigs, whereas the rest of the gastrointestinal tract and associated lymph nodes were culture negative. The remaining inoculated pigs and all control pigs were culture negative at necropsy at day 70 p.i. The ELISA seems to be a promising alternative to bacteriological culture for detection of Y. enterocolitica O:3 infection in pig herds.

Clinical observations, pathology, bioassay in mice and serological response at slaughter in pigs experimentally infected with Toxoplasma gondii

Experimental infections of a total of 47 pigs with tachyzoites of the Toxoplasma gondii RH-strain, tissue cysts of the SSI-119 and R92 strains as well as oocysts of the SSI-119 strain were performed to determine the sensitivity of an indirect IgG-ELISA, using tachyzoite lysate of the RH-strain as antigen. The infections led to a dose dependent moderate clinical affection (inappetence, fever and poor general condition). Pigs infected with 10000 oocysts or with 1/2 mouse brain containing tissue cysts of the SSI-119 strain showed a significant decrease in weight gain compared to uninoculated pigs during the first 2 weeks p.i., followed, however, by compensatory growth during the next 6 weeks. At slaughter 3 to 4 months after inoculation 39/41 (95.1%) of pigs positive by bioassay in mice were seropositive in ELISA. Tissue cysts were not demonstrable by immunohistochemistry. ELISA OD-values obtained by analysis of meat juice from heart muscle and tongue (diluted 1:40) correlated strongly with OD-values by analysis of serum (diluted 1:400) (r heart juice = 0.942; r tongue juice = 0.915). Thus, meat juice samples were shown to provide a suitable alternative to serum for serological detection of Toxoplasma infection in pigs.

A European longitudinal study in Salmonella seronegative- and seropositive-classified finishing pig herds.

Surveillance and control are important aspects of food safety assurance strategies at the pre-harvest level of pork production. Prior to implementation of a Salmonella surveillance and control programme, it is important to have knowledge on the dynamics and epidemiology of Salmonella infections in pig herds. For this purpose, 17 finishing pig herds initially classified as seropositive and 15 as seronegative, were followed for a 2-year period through serological and bacteriological sampling. The study included 10 herds from Denmark, 13 from The Netherlands, 4 from Germany and 5 from Sweden and was performed between October 1996 and May 1999. The Salmonella status of finishing pig herds was determined by an initial blood sampling of approximately 50 finishing pigs close to market weight per herd. The development of the Salmonella status of the selected herds was assessed at seven subsequent sampling rounds of 25 blood samples from finishing pigs, 25 blood samples from grower pigs and 10 pen faecal samples each, approximately 3 months apart. The odds for testing finishers seropositive, given that growers were found seropositive previously were 10 times higher than if growers were seronegative (OR 10.0, 95% CI 3.2-32.8). When Salmonella was isolated from pen faecal samples, the herd was more likely to be classified seropositive in the same sampling round, compared to no Salmonella being detected (OR 4.0, 95% CI 1.1-14.6). The stability of an initially allocated Salmonella status was found to vary noticeably with time, apparently irrespective of a seropositive or seronegative classification at onset of the study. Given the measured dynamics in the occurrence of Salmonella in pig herds, regular testing is necessary to enable producers, advisors and authorities to react to sudden increases in the Salmonella prevalence in single herds or at a national level.

A long-lasting outbreak of Salmonella Typhimurium U323 associated with several pork products, Denmark, 2010

This paper shows that control of foodborne disease outbreaks may be challenging even after establishing the source of infection. An outbreak of Salmonella Typhimurium U323 infections occurred in Denmark from March to September 2010, involving 172 cases. Before the detection of human cases, several positive isolates of the outbreak strain had been found in a particular pig slaughterhouse and thus early traceback, investigation and control measures were possible. Several batches of pork and pork products were recalled and the slaughterhouse was closed twice for disinfection. No single common food item was identified as the outbreak source, but repeated isolation of the outbreak strain from the slaughterhouse environment and in pork and products as well as patient interviews strongly suggested different pork products as the source of infection. Furthermore, a matched case-control study identified a specific ready-to-eat spreadable pork sausage (teewurst) as the source of a sub-outbreak (matched odds ratio 17, 95% confidence interval 2·1-130).

Reappearance of Salmonella serovar Choleraesuis var. Kunzendorf in Danish pig herds

Salmonella enterica serovar Choleraesuis is a porcine adapted serovar which may cause serious outbreaks in pigs. Here we describe outbreaks of salmonellosis due to S. Choleraesuis in four Danish pig farms in 2012-2013 by clinic, serology, and microbiology and compare the isolates to those of a previous outbreak in 1999-2000. The infection was in some herds associated with high mortality and a moderate to high sero-prevalence was found. In 2012-2013 the disease contributed to increased mortality but occurred concomitant with other disease problems in the herds, which likely delayed the diagnosis by up to several months. Nine isolates from the four farms in 2012-2013 and 14 isolates obtained from the outbreak in Denmark in 1999-2000 were subjected to typing using pulsed-field gel electrophoresis (PFGE). Seven isolates were selected for whole genome sequencing (WGS). The PFGE results of 23 isolates displayed five different profiles. The isolates from 2012 to 2013 revealed two distinct profiles, both different from the isolates recovered in 1999-2000. Two of the 2012-2013 farms shared PFGE profiles and had also transported pigs between them. The profile found in the two other 2012-2013 farms was indistinguishable but no epidemiological connection between these farms was found. Analysis of the number of single nucleotide polymorphisms (SNPs) from the WGS data indicated that the isolates from the farms in 2012-2013 were more closely related to each other than to isolates from the outbreak in 1999. It was therefore concluded that the infection was a new introduction and not a persistent infection since the outbreak in 1999. It may further be suggested that there were two or three independent rather than a single introduction. The re-introduction of S. Choleraesuis in Denmark emphasizes the importance of strict hygiene measures in the herds. Further investigations using WGS are now in progress on a larger collection of isolates to study clonality at European level and trace the origin of the infections.

A proactive attempt to control an outbreak of foodborne disease in humans after detection of Salmonella during surveillance and control of pork in Denmark

Abstract: Based on Kuhn et al. (2012), this chapter describes the course and investigation of a large Danish foodborne outbreak in 2010, caused by a rare Salmonella Typhimurium phage type with the majority of patients residing in the eastern and southern parts of Denmark. It describes the interview studies conducted and the changes in the Salmonella subtypes and human demography during the outbreak. It also suggests interpretations of these changes. Comprehensive Danish register data on herds, slaughterhouses and Salmonella surveillance were utilized for the tracing of products and herds and for assessing the risks of herds, slaughterhouses and meat production plants being the source of infection in the outbreak.