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National Committee for Pig Production, Danish Bacon and Meat Council, Department of Veterinary Research and Development, DK-1609 Copenhagen V, Denmark
Received 13 September 2002/ Returned for modification 16 December 2002/ Accepted 5 March 2003
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ABSTRACT |
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INTRODUCTION |
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Results from a comprehensive longitudinal study in two multiple-site pig production systems in the United States revealed considerable temporal variability in Salmonella prevalence between cohorts of pigs (11). In a Danish study, the Salmonella status of sow herds was measured based upon (i) the prevalence of serotype Typhimurium bacteria among weaners and (ii) the seroprevalence among sows. Both factors were shown to constitute important risk factors for Salmonella infection in finisher herds, as measured by the high seroprevalence found by examining meat juice samples at slaughter (15).
We decided to conduct an investigation to study the complex nature of subclinical Salmonella infections at the levels of the herd and the individual pig. The objectives of the study were the following: (i) to describe the time of onset and duration of Salmonella shedding; (ii) to study the patterns of bacterial transmission between individual pigs until slaughter; (iii) to investigate the transmission between the different age groups, as well as the association between bacteriological shedding and serological response in cohorts of pigs from weaning to slaughter; (iv) to compare antemortem and postmortem findings in individual pigs and thereby focus on food safety; and (v) to provide input estimates for a quantitative risk analysis model, simulating Salmonella prevalence from the growing pig to the slaughtered carcass (1).
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MATERIALS AND METHODS |
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The participating farmers agreed not to introduce Salmonella-reducing measures during the 6-month study period. Among five farms potentially suited for the study, pooled pen fecal samples were collected from various rooms to ensure that Salmonella bacteria were present, preferably in most of these rooms. Three farms were chosen based on results from these preliminary samples and based on geographical convenience. Two of the farms, with 650 and 440 sows, were two-site operations. The remaining farm was a three-site, 300-sow operation. All three herds were self-supplying.
Sampling scheme. In each herd, 10 litters were randomly selected, and in each litter, the ears of six randomly selected piglets were tagged. To account for variations in Salmonella shedding over time, litters from each herd were divided into two groups of five litters that were raised at approximately 1-month intervals. Thus, on each farm there were two cohorts consisting of 30 pigs each, yielding a total of 180 piglets at the start of the study. All ear-tagged pigs from a given cohort were supposed to be raised together for the entire observation period.
Individual blood and fecal samples were collected from the sows at weaning. In addition, individual blood and fecal samples were collected from pigs on the following occasions: (i) just prior to weaning (feces only), (ii) midway through the period in the nursery, (iii) just before leaving the nursery, (iv) monthly in the finishing unit, and (v) just prior to slaughter. Due to different management practices, one herd was sampled on seven occasions and the other two herds were sampled on six occasions. The intervals between sampling occasions varied from 20 to 29 days between sampling occasions just prior to weaning, midway through the nursery period, and just before leaving the nursery. The intervals between sampling occasions in the finishing unit ranged from 21 to 35 days, except with cohort 1B, for which the interval was only 14 days because of an earlier slaughter than expected. The maximum difference between sampling occasions between cohorts within herds was 7 days.
All pigs within a cohort were slaughtered on the same day. At slaughter, the individual carcasses were identified and samples were collected from ileocecal lymph nodes, cecal contents, and the carcass surface. The carcass samples were taken by swabbing 100 cm2 of the ham, chest, and jaw regions, respectively. A template was placed on the surface of the carcass, and one cotton gauze swab was used to swab all three sites (8). Pigs were processed within 1 h after arrival. Abattoir data were collected to investigate the association between antemortem shedding, carrier state, and possible cross-contamination with Salmonella bacteria at slaughter.
Bacteriological analyses were carried out by using standard microbiological culturing methods, including nonselective preenrichment, selective enrichment, and serotyping. Blood samples were analyzed by using the Danish mix-enzyme-linked immunosorbent assay with a cutoff optical density percentage (OD%) of 20%.
Statistics. To assess the relationship between serology in sows and Salmonella shedding by their offspring, the GENMOD procedure in SAS was used (SAS/STAT user's guide, version 8, SAS Institute, Inc., Cary, N.C.). The proportion of culture-positive piglets per sow on the second sampling occasion was used as the observation or response variable. Herd and cohort were used as explanatory variables. The p-scale option in SAS was used to control for overdispersion originating from the herd and the cohort.
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RESULTS |
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Bacteriological and serological results. At the time of weaning, none of the 30 sows were culture positive, but one sow in cohort 1B and all sows in cohort 3A were seropositive. In all three herds, only Salmonella serotype Typhimurium was isolated during the entire study period. At weaning, none of the piglets were found to be culture positive. Overall, Salmonella shedding reached a peak in the nursery (i.e., after 60 days, which corresponds to 9 weeks of age), and subsequently declined during the finishing period (Fig. 1). The serological response was observed approximately 30 days later and, on average, reached its peak in the mid-finishing period (i.e., after 120 days, which corresponds to 17 weeks of age) (Fig. 1).
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Relationship between Salmonella shedding and serology. The relationship between Salmonella shedding and serological response across all six cohorts is shown in Table 1. Only those pigs sampled on four or more occasions (n = 160) were included. As can be seen in Table 1, Salmonella was detected at least once in 53.1% of the pigs, while 62.4% of the pigs were seropositive more than once. Interestingly, 30.5% [(10.0% + 5.6% + 0.6%)/(35.6% + 13.8% + 3.1% + 0.6%)] of Salmonella-shedding pigs did not show an antibody response at any time during the study. In addition, Salmonella was never isolated from 40.9% [(11.0% + 6.3% + 4.4% + 3.8%)/(23.5% + 18.9% + 8.8% + 11.2%)] of the seropositive animals. Salmonella bacteria were found more than twice in only 3.7% of all pigs tested on at least four occasions.
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Correlations between sampling results. The correlations between results from individual pigs on different sampling occasions were assessed by calculating the ORs for bacteriological and serological results (Table 2). Regarding the bacteriological results, we found that the earlier in the study period and the shorter the time interval between sampling occasions, the greater the magnitude of the correlation (assessed by OR). For example, an individual pig that was culture positive on the second sampling occasion had a threefold-higher chance (OR = 3.22) of being culture positive on the third sampling occasion than that of a pig that was culture negative on the second sampling occasion and had a chance (OR = 1.02) of being culture positive on the fifth sampling occasion that was equal to that of a pig that was culture negative on the fifth sampling occasion. As all fecal samples were culture negative on the first sampling occasion, these samples were not included. Sampling on the seventh sampling occasion was carried out only with herd 1, and as none of these fecal samples were positive, the correlation between any other sampling occasion and the seventh sampling occasion was zero. The serological correlations (assessed with ORs) were higher than the bacteriological correlations. We found that there were significant correlations not only between subsequent sample results but also between sampling occasions at larger time intervals, especially late in the study during the finishing period.
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DISCUSSION |
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The decision to monitor more than one cohort per herd was inspired by experiences with bacteriological examinations in the Danish Salmonella control program. Repeated examinations of the same herd might produce variable results, e.g., between batches (within-herd variation) or monthly examinations (true variation over time). Our results show considerable variation in both Salmonella occurrence and shedding patterns between cohorts in the same herds, which shows that inclusion of more than one cohort per herd was justified.
Salmonella in the farrowing unit. Except for a few sows examined prior to the start of the study, none of the sows and none of the piglets in the cohorts were shedding Salmonella prior to weaning. This finding suggests that sows in subclinically infected herds play a less important role in Salmonella transmission, although the presence of some seroreacting sows indicated a certain level of Salmonella exposure in the sow units. The fact that none of the 30 sows shed Salmonella bacteria at weaning does not exclude the possibility that some sows shed intermittently in the farrowing pens or in the dry sow unit. However, this specific issue was not investigated in this study. Furthermore, other sows in the farrowing unit might have been shedding, which would enable transmission to the nursery through their piglets. This supposition seems to be supported by the results of Funk et al. (11), who found low levels of Salmonella shedding in both sows and piglets before weaning in three of five cohorts examined. Finally, the amount of feces collected from piglets at weaning was in many cases limited, which might result in an underestimation of the actual prevalence (10).
The observation that piglets nursed by seroreacting sows had a significantly lower prevalence of Salmonella in culture after weaning suggests a role for passively transferred protection against Salmonella infection. This possibility was unfortunately not investigated, but it deserves further study.
Transmission in the nursery. A few studies have shown that it is possible to raise pigs free of Salmonella by strategic movement at or shortly after weaning (6, 9). These studies suggest that sow-offspring transmission plays a minor role and focus attention on the transmission occurring in the finishing unit. On the other hand, Kranker et al. (15) demonstrated that seropositivity of sows was significantly associated with the finding of Salmonella serotype Typhimurium in the nursery. However, that study included a larger number of herds, some with and some without Salmonella, and therefore its results cannot be directly compared with those of the present study, as weaners from negative herds were not at risk. Kjærsgaard et al. (14) found a significant association between the prevalence of exotic Salmonella serotypes in different batches of sows and that of their piglets after weaning. Funk et al. (11) detected fecal shedding of Salmonella in sows and piglets before weaning. Those authors concluded that the success of segregated early weaning as proposed by Dahl et al. (6) might be farm specific and that absolute exclusion of preweaning infection is unlikely to be achieved by contemporary early weaning alone. Funk et al. (11) furthermore found that even if sows were culture positive during gestation or lactation, the risk of their litters being culture positive did not significantly increase. Interestingly, these authors did not demonstrate a serotype-specific association between culture-positive sows and their piglets. However, this observation might be biased by the facts that many different serotypes were present and that only one isolate per pig was serotyped.
The rapid increase in Salmonella prevalence in the nursery might have been triggered by weaning stress. The most important stress factors are likely to be (i) a change in feed, (ii) the commingling of litters, and (iii) a piglet's being deprived of the antibodies found in sow's milk before the activation of its own immune response. The observations that no piglets were shedding Salmonella just before weaning but that 3 to 4 weeks later in the nursery between 5 and 50% of the piglets were culture positive strongly suggest that horizontal transmission occurred in the nursery. According to Berends et al. (5) and Funk et al. (11), residual infections in the nursing unit and in the transport vehicles seem to be the most plausible origins of infection of the piglets and can explain the sudden rise in prevalence in culture. Since no piglets were shedding Salmonella at weaning and residual infection was not measured in our study, a causal relationship between these factors and the sudden rise in prevalence in culture in the nursery could not be determined. Most likely, Salmonella shed by newly weaned piglets and residual infection act cooperatively, and stress is very likely to exacerbate the resulting infections.
Transmission patterns during the finisher period. Overall, the present study shows that Salmonella shedding decreased during the fattening period (Fig. 1) but with considerable variation in onset and duration of shedding between and within cohorts (Fig. 3). Some pigs that were found to be shedding in the nursery apparently cleared themselves of infection, whereas others continued shedding in the finishing units. The data also show that despite the exposure, certain pigs were not infected in the nursery but started shedding in the finishing unit. This finding was reflected in the bacteriological curves for both cohorts in herd 1 (Fig. 2) around week 13. Data in Fig. 3 suggest that a shedding pig might infect at-risk penmates at any stage. Residual infection in the finisher units might also play a role, since some of the pigs started shedding after moving to those units. The results from herd 3 suggest that, irrespective of previous exposure or shedding, finisher pigs are still susceptible to a sudden substantial increase in exposure (e.g., through slurry overflow).
Association between serology and the onset and duration of shedding. Experimental inoculations with Salmonella have shown that the onset of serological response and peak seroprevalence occur at approximately 7 and 30 days postinoculation, respectively (17). In our study there were somewhat longer time periods between peak prevalence in culture, the onset of a serological response, and peak seroprevalence (Fig. 1). The different course of the bacteriological and serological responses occurred because under natural conditions pigs are infected at different points in time, with variability in both exposure and host response (Fig. 3). The results from herds 1 and 2 indicate that even though Salmonella-infected herds might have a proportion of seroreactors sent for slaughter, most pigs have stopped shedding by the time of slaughter. Hence, unless they are carriers and resume shedding or are fecally cross-contaminated during transport or holding, these animals do not constitute a risk to human health with respect to Salmonella. This finding seems to be supported by the autoregressive pattern of the correlation structure for successive culture-positive events (see Table 2), for which self-cure is the most likely explanation. Progressive self-cure and carrier-state animals (19) minimize the amount of Salmonella shedding and consequently reduce the possibility of our detecting Salmonella by the culture technique. The corresponding correlation structure for successive seropositive events (Table 2) is not directly influenced by self-cure but might be due to continuous exposure and immunostimulation, to the half-life of serum antibodies, or to both.
In addition to the marked differences in prevalence in culture between cohorts (Fig. 2), differences in pattern of horizontal transmission were observed (Fig. 3). In some cohorts horizontal transmission seemed to occur frequently (e.g., cohorts 1A and 3A), which is represented in Fig. 3 by many overlapping bars. Other cohorts showed a more stable infection pattern, with a low incidence of shedding (e.g., cohorts 1B and 2B), which is represented in Fig. 3 by few overlapping bars.
In an attempt to describe the dynamics of Salmonella infection in pigs, we adopted a novel approach to estimating the times and patterns of shedding. To the best of our knowledge, such an approach has not previously been published. Using our definitions of shedding time and animals at risk, the results suggest that the dynamics of Salmonella infections are complex in nature. Infection can probably be explained as a random process where a previously uninfected pig has a certain probability of getting infected each time it is exposed. The definition of animals at risk was considered valid. It seems unlikely that an individual pig shedding on the first sampling occasion, not shedding on the second occasion, but shedding again on the third occasion would have been found to have a true-negative culture on the second occasion. Even though the sampling protocol allowed misclassification of intermittent shedders, because of the 3- to 4-week sampling interval, the low sensitivity of the culture technique seems a more reasonable explanation. It is not practically possible to distinguish between intermittent shedding and low-level shedding (i.e., close to detection level). Despite this, an animal with either intermittent or low-level shedding must be considered infected.
All individuals were seronegative on the sampling occasions prior to shedding Salmonella, with the exception of three animals from herd 3, which were seropositive long before Salmonella was detected in their feces. This result further indicates that the sensitivity of the bacteriological culture technique was acceptable but by no means perfect.
We estimated the average shedding time to be 18 or 26 days, depending on the approach used. These shedding times are in accordance with results of Salmonella experts, who estimated the shedding time to range between 17 and 38 days (18a). The fact that some pigs in herd 3 became reinfected following a substantial increase in exposure by a slurry overflow demonstrates that it is possible to penetrate the immunologic barrier in seropositive individuals.
Salmonella findings at slaughter. Towards the end of the finishing period, cohorts 1A, 3A, and 3B showed a high seroprevalence and cohorts 3A and 3B also showed a high culture prevalence (Fig. 2 and Table 3). Interestingly, Salmonella was isolated most frequently at slaughter from these cohorts (Table 3). Although the data were limited in number, the results are in concordance with a large-scale study carried out by Sørensen et al. (18). Those authors demonstrated significant associations between serology, both at the individual level and at the herd level, and the occurrence of Salmonella in cecal lymph nodes, in cecal contents, and on carcasses. An approximately threefold increase in Salmonella prevalences between farm and slaughterhouse was found. This increase may be due to rapid cross-contamination during transport and lairage (1, 12). The fact that five of six pigs had culture-positive carcass swab samples and culture-negative lymph nodes and cecal contents suggests in-plant cross-contamination as a food safety risk.
Implications for assessment of preharvest prevalence. The observed variations in Salmonella bacteriology and serology between cohorts and over time indicate that none of these methods on their own were reliable for point estimates of preharvest prevalence in subclinically infected herds. Repeated sampling in different cohorts of animals is required to correctly assess the infection dynamics in the particular herd under study or surveillance. Serological testing is inexpensive, has a higher sensitivity than bacteriology, and has proven its usefulness in the monitoring of Salmonella in finisher pigs (2). However, if additional sampling for bacteriological analysis is required in a herd, the temporal variability in Salmonella levels should be taken into consideration by making the necessary adjustments in the sampling strategy. In three of the six cohorts, the seroprevalence dropped to acceptable levels (<40%). Given that the seroprevalence on the last sampling occasion also applied to the two preceding months, only herd 3 would be classified as other than level 1. After we consulted the central database (the Zoonosis Register), from which all data from the surveillance program are available, herds 1 and 2 were classified as level 1 the month after the last sampling occasion. Herd 3 was classified as level 2 the month after the last sampling occasion, and yet another month later, it was classified as level 3.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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