Faecal shedding and strain diversity of Listeria monocytogenesin healthy ruminants and swine in Northern Spain

Listeria monocytogenes is a ubiquitous organism that is occasionally present in the intestinal tract of various animal species and can cause severe illness in humans after the ingestion of contaminated food products. Although the annual incidence of human listeriosis is quite low in the Basque Country (1.0 cases per 100,000 inhabitants in 2006) compared to salmonellosis (77.2 cases) or campylobacteriosis (114.5 cases) [1], L. monocytogenes is among the most important foodborne bacterial pathogens due to the high mortality rate (20–30% mortality) and severity of the disease particularly among pregnant women, neonates and immunosuppressed adults. In addition, L. monocytogenes febrile gastroenteritis can also affect healthy people, though many of these cases most probably go unreported.

Adult swine can be infected by L. monocytogenes but rarely develop disease [2] and the bacterium is not commonly isolated from swine faeces [3–5]. However, pork meat products have been linked to human infection [6, 7] and contamination of the slaughter and processing environment has been traced back to healthy carrier pigs [8]. In ruminants, L. monocytogenes can cause neurological disease and abortion, but in general, animals infected are asymptomatic carriers that shed the bacterium in their faeces [2]. Faecal contamination of the farm environment favours animal re-infection and persistence of the pathogen in the production units [9]. In addition, the widespread distribution of L. monocytogenes in nature and soil environments is favoured by its ability to grow in a wide range of temperature and pH [10]. This is particularly important in silage production since in many cases the pH reached in the fermentation process is not low enough to prevent growth of L. monocytogenes [11]. Therefore, ruminants fed on silage are at higher risk of getting L. monocytogenes infection [12].

Hence, animal production units may represent a reservoir for L. monocytogenes and source for human infection via faecal contamination of food products since certain L. monocytogenes types carried by farm animals have been associated with human infections [13, 14]. Though eradication from the farm is highly unlikely due to the ability of L. monocytogenes to survive and multiply in many habitats and hosts, transmission and contamination load could probably be reduced through the implementation of adequate intervention strategies. In this context, this study was aimed at determining the prevalence and strain diversity of L. monocytogenes in healthy ruminants and swine herds, as a first step before establishing efficient farm-based control measures.

This is the first study carried out in farms from the Basque Country to determine the prevalence of L. monocytogenes in healthy animals. Comparison of prevalence results among different studies can be influenced by variation in sampling strategies and season, and differences in detection methods used. In addition, most data on L. monocytogenes in ruminants are obtained from cases of clinical listeriosis. Although day-to-day variation in L. monocytogenes faecal shedding in dairy cattle has been demonstrated [14], single day sampling can provide an initial snap-shot image of the general situation with regard to the prevalence of L. monocytogenes in healthy herds in a region where no such data are available. In this manner, the proportion of faecal shedders observed in this study was similar to that reported by Nightingale et al. [9] among ruminants without clinical symptoms. Likewise, prevalence was higher in cattle (particularly dairy cattle) than sheep. Nightingale et al. [9] showed that contrary to sheep, cattle exposed to L. monocytogenes through contaminated silage amplify the pathogen to high levels subsequently increasing faecal shedding, and thus contributing to the maintenance and dispersal of L. monocytogenes into the farm environment. A pronounced seasonal variation in faecal shedding has been reported in cattle farms with peak prevalences during the colder months associated to increased silage feeding [15]. Quality of feed provided during the indoor season has also been considered an important risk factor for listeriosis in ruminants [15]. Samples for the Herd prevalence study were collected throughout the year, and no seasonal variation in herd prevalence was observed. However, it is noteworthy that the highest within-herd prevalence of L. monocytogenes shedders corresponded to a dairy cattle herd sampled in winter (dairy cattle herd E in Table 2). Nevertheless, in the Basque Country, sheep and beef cattle spend most of the year pasture-grazing outdoors, whereas dairy cattle are kept indoors throughout the year under a diet based on silage, which has been reported as a risk for L. monocytogenes infection [12]. Stress associated to lactation in dairy cattle might affect susceptibility to L. monocytogenes infection [2] and contribute to differences in shedding between beef and dairy cattle. Daily variability in the number of faecal shedders [14] could explain the wide differences in the within-herd prevalence values observed among the four dairy cattle herds, and the absence of shedders in two ovine herds when sampled individually. The higher prevalence of ruminant herds positive for L. monocytogenes in areas with larger animal census suggests a possible relation between animal density and infection risk, however, more extensive epidemiological data collection (herds contact at mountain pastures and animal trade) and environmental sampling are needed to confirm this link and identify sources of contamination and infection routes.

Swine production in the Basque Country is not extensive and therefore, the number of swine herds analysed in this study was limited. However, L. monocytogenes was never detected in the pig herds analysed. Although L. monocytogenes occurs frequently in pork products, it is rarely isolated from swine faeces [3–5]. Higher infection rates are detected in skin swabs [16] or tonsils [3], and the prevalence of L. monocytogenes in swine generally increases from the farm to the manufacturing plants. Hence, the main source for contamination with L. monocytogenes appears to be the slaughter and processing environment where L. monocytogenes can survive for long periods [17]. Listeriosis is frequently traced to ready-to-eat (RTE) meat products, regardless of meat animal source, and some delicatessen RTE pork products have been involved in listeriosis outbreaks [6, 7]. Conversely, bovine or ovine meat products are rarely associated to human listeriosis, but ruminant healthy carriers may shed Listeria in faeces contaminating pastures or vegetables [18], surface waters [19] and milk [20]. In a study carried out in Navarra (Northern Spain) on food samples from different industries and markets [21], the incidence of L. monocytogenes in raw minced beef and pork meat was 34.9%, 5.4% in raw milk samples (6.8% in cattle milk and 3.0% in sheep milk), and 1.0% in soft cheese. Nevertheless, unpasteurised dairy products represent the major problem regarding human infection.

Strain typing can help to identify sources of infection and routes of transmission and in this sense, is commonly used for disease tracking in human infections. L. monocytogenes comprises a diversity of strains classified into 13 different serotypes, however, only three (1/2a, 1/2b and 4b) are commonly associated with human listeriosis [21, 22]. Food-stuffs are mainly contaminated by serogroup 1/2 isolates, whereas most human clinical isolates belong to serotype 4b [21, 22], and among these, a small subgroup with unique gene clusters represent the two major epidemic-associated clonal groups [23, 24]. In this study, serotype 4b complex was the most common in ruminants (84.2% of isolates and 81.7% of positive herds), followed by serotype 1/2a (13.2% of isolates and 14.0% of positive herds). Since each pool of faecal material can contain several L. monocytogenes strains with different serotype, these values cannot be interpreted as serotype prevalences but clearly indicate the predominance of serotype 4b in healthy ruminants from the Basque Country. As part of the USA National Animal Health Monitoring System Dairy 2002 survey, Van Kessel et al. [25] found a varied distribution of serotypes among L. monocytogenes isolated from cattle bulk tank milk in different regions of the country, with serotype 4b predominating in the Southeast and serogroup 1/2 elsewhere [25]. Serogroup 1/2 was predominant in beef and pork raw minced meat samples from different industries and markets in Navarra (Northern Spain); in cattle milk samples, serogroups 1/2 and 4 were similarly represented (47.8 and 39.1%, respectively), but in sheep milk 83.3% of the isolates were serotype 4b [21]. Conversely, in this study we detected serotype 1/2a at higher proportion in sheep than in cattle. Reporting of human listeriosis cases is compulsory in Spain and Microbiology laboratories at the hospital setting report isolations weekly. A 16-year survey carried out from 1990 to 2006 in one of the largest hospitals in the Basque Country identified 60 human clinical cases, with serotype 4b representing 78% of the cases [26], similar to other Spanish regions [27, 28]. Serotype 1/2b, only represented by two dairy cattle isolates in this study, was second (14%) among local human cases, whereas serotype 1/2a, second among animal samples, was the less common among human cases (6.8%) in the Basque Country [26]. In this context, serotyping is, however, of limited discriminatory value, and techniques like PFGE provide enhanced discrimination for outbreak investigations and surveillance purposes. Restriction analysis with ApaI performed on 68 isolates generated 40 patterns, and provided serotype-specific PFGE patterns that clearly separated 1/2a isolates from 4b isolates, and differentiated strains within serotypes. These results confirmed previously established relationships between serotype and PFGE patterns [22], and revealed that the L. monocytogenes population in Basque farms is genetically highly diverse. In general, isolates from different herds were very different, but occasionally identical or similar patterns were observed in different herds. Contact at communal mountain pastures could be an occasion for strain exchange among sheep and beef cattle herds. However, since dairy cattle are confined indoors, the identification of certain patterns in the three production systems or in dairy cattle herds distantly located, suggest other sources of infection. On the other hand, strains differing in more than seven bands, and therefore of limited genetic relatedness [29], were also identified within each herd suggesting multiple sources of contamination.

Listeriosis results in losses to the agricultural economy due to illness and increased infertility and abortion rates, but losses also occur when consumer confidence is undermined as a consequence of food-borne infections. Food safety programs that cover all aspects of food production (from farm to fork) are needed to provide a safe food supply and prevent foodborne illnesses. Identification of on-farm reservoirs is a pre-requisite for the implementation of farm-specific pathogen reduction programs. In this sense, this study showed a high prevalence of L. monocytogenes in ruminant herds compared to swine, suggesting that such herds may represent an important reservoir for L. monocytogenes in the Basque Country. The wide distribution and variability in L. monocytogenes shed within and among ruminant herds highlighted the complexity of pathogen control at the farm level. The ubiquitous nature of this pathogen hampers its total removal from the farm environment, but a reduction of the intestinal carriage rate in livestock herds would contribute to reduce the contamination pressure at the slaughterhouse and dairy production. In any case, since the relatively high prevalence of L. monocytogenes in ruminant herds does not correlate with the low incidence of human infections in the Basque Country, it can be speculated that control measures to avoid contamination of final food products are being efficient or, possibly, these could be animal-adapted strains with reduced ability to cause human infections. Continuous monitoring schemes and surveillance programs are needed to evaluate trends in the occurrence of L. monocytogenes in livestock and to prevent food contamination.