Seasonal Variability of Thermophilic Campylobacter Spp. in Raw Milk Sold by Automatic Vending Machines in Lombardy Region.

In temperate climates, a seasonal trend was observed in the incidence of human campylobacteriosis cases, with peaks reported in spring and autumn in some countries, or in summer in others; a similar trend was observed in Campylobacter spp. dairy cattle faecal shedding, suggesting that cattle may play a role in the seasonal peak of human infection. The objectives of this study were to assess if a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk exists and to evaluate a possible relation between this and the increase of human campylobacteriosis incidence in summer months. The results showed a mean prevalence of 1.6% of milk samples positive for thermophilic Campylobacter spp. with a wide range (0.0-3.1%) in different months during the three years considered. The statistical analysis showed a significant difference (P<0.01) of the prevalence of positive samples for thermophilic Campylobacter spp. between warmer and cooler months (2.3 vs 0.6%). The evidence of a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk sold for direct consumption, with an increase of the prevalence in warmer months, may represent one of the possible links between seasonal trend in cattle faecal shedding and seasonal trend in human campylobacteriosis.

Introduction

Thermophilic Campylobacter spp. strains are some of the most frequent etiological agents of bacterial gastroenteritis in humans in many developed countries. C jejuni, C coli, C lari, and C upsaliensis are considered to be thermophilic because of their ability to grow at 42°C. Most detection methods are designed for the human pathogens C jejuni and C coli, and the majority of the studies deal with C jejuni, a common commensal of the gastrointestinal tracts of wild and farm animals that is ubiquitous in the natural environment (Kwan et al., 2008). Thermophilic Campylobacter can cause severe post-infection neuropathies, including Guillan-Barré syndrome and Miller Fisher syndrome (Rapp et al., 2012). Campylobacteriosis is a commonly reported zoonosis in the European Union (EU) since 2005 and the number of reported confirmed cases of human campylobacteriosis in the EU in 2014 was 236,851, which registered an increase of 22,067 cases compared with 2013. The EU notification rate was 71.0 per 100,000 population in 2014, which showed an increase by 9.6% compared with 2013 (64.8 per 100,000 population) (EFSA, 2015). In temperate climates, a seasonal trend was observed in the incidence of human campylobacteriosis cases, with peaks reported in spring and autumn in some countries, or in summer in other countries (Stanley et al., 1998). A similar trend was observed in Campylobacter spp. dairy cattle faecal shedding (Stanley and Jones, 2003), suggesting that cattle may play a role in the seasonal peak of human infection (Kwan et al., 2008).

The significance of cattle as reservoir of human campylobacteriosis is related to food products contamination (meat and milk), to environmental and water contamination, and to a direct transmission to humans. Several studies have identified poultry meat as the main food vehicle associated with sporadic cases of campylobacteriosis, but there is also strong evidence that raw milk consumption represents a risk factor for campylobacteriosis. Raw cow’s milk has been reported to be a vehicle of human campylobacteriosis in outbreaks both in the United States and Europe (Claeys et al., 2013; Hauri et al., 2013; Longenberger et al., 2013;Taylor et al.,2013; EFSA, 2015). In the United States the expert consultation on the global view of campylobacteriosis reported that raw unpasteurised milk is the most common food vehicle for campylobacteriosis outbreaks, whereas the relative contribution of raw milk accounts for only a small fraction of sporadic cases (WHO, 2013). The European Union summary report on zoonoses, zoonotic agents and food-borne outbreaks in 2012 reports that milk was the second most frequently identified food vehicle in the strong-evidence Campylobacter outbreaks, 20.0% of which implicated milk, indicating a well-documented risk of campylobacteriosis (EFSA, 2015).

In Italy, the results of diagnostic activities performed in different surveys and official control data showed that the prevalence of C jejuni in raw milk samples collected by self-service vending machines varies from 0 to 1.5% (Giacometti et al., 2012, 2013; Bianchini et al., 2014). In addition, two outbreaks of C jejuni linked to the consumption of raw milk have been reported in Italy, one in Veneto Region and the other in Marche Region (Amato et al., 2007).

The objective of this study was to assess if a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk exists and to evaluate a possible link with the increase of human campylobacteriosis cases in summer months.

Materials and Methods

Data were collected from official microbiological records monitoring raw milk samples from self-service vending machines in Lombardy Region. All samples were analysed at the Institute for Experimental Veterinary Medicine of Lombardy and Emilia-Romagna, Brescia, following the ISO 10272-1:2006 method (ISO, 2006). Test procedures are accredited according to the International Organization for Standardization 17025:2005 (ISO, 2005) by ACCREDIA, the Italian accreditation body.

In this study a total of 1629 raw milk samples analysed during 2012-2014 was considered; statistical analysis was performed using SPSS software (SPSS Statistics ver. 21; IBM Corp., Chicago, IL, USA) by χ2 test comparing the prevalence of thermophilic Campylobacter contamination during warmer months (April-September) vs cooler months (October-March).

Results

Twenty-six out of 1629 raw milk samples examined resulted positive. All isolates were identified as C jejuni. Results showed a mean prevalence of 1.6% of samples positive for thermophilic Campylobacter spp. with a range of 0.0-3.1% in different months during the three years considered. Mean prevalence per month of samples positive for thermophilic Campylobacter spp. is reported in Figure 1.

Figure 1.

Monthly distribution of the prevalence of raw milk samples positive for thermophilic Campylobacter spp. during 2012-2014. *No samples were tested in August.

Most of the samples were analysed by the official veterinary services in the periods between May and November. One month (January) showed a noticeable lower number of samples tested by official authorities and in one month (August) no sample was performed.

The statistical analysis showed a significant difference (P<0.01) of the prevalence of samples positive for thermophilic Campylobacter spp. between warmer and cooler months (2.3 vs 0.6%).

Discussion

A total of 1629 milk samples collected during 2012-2014 in Lombardy Region were analysed for the presence of thermophilic Campylobacter spp.; twenty-six out of 1629 examined (1.6%) resulted positive. The overall prevalence of milk samples positive for thermophilic Campylobacter spp. is in agreement with previous studies performed in Italy on raw milk sold for direct consumption, which ranged between 0 and 2.2% in different regions (Giacometti et al., 2012, 2013). The observed prevalence is also in agreement with other investigations performed in different countries (Desmasures et al., 1997; Dontorou et al., 2003; Meyer-Broseta et al., 2003; Oliver et al., 2005, 2009; Wysok et al., 2011).

Most of the samples were analysed by official veterinary services in warmer months; this is in agreement with the principle that sampling should be planned according to the microbiological risk and should follow the regulatory guidelines of the Italian Ministry of Health (2007) on raw milk. Regional rules specify that every year at least two samplings must be performed for each farm authorised to sell raw milk and that at least one sampling must be performed during summer. In the 3-year-period of this study, a mean of 230 farms was authorised to sell raw milk; the total number of samples tested (1629) shows that the mean value of milk samples collected from each farm was 2.3/year.

A seasonal trend in thermophilic Campylobacter spp. contamination of raw milk samples was observed in this study, with higher prevalence in warmer months (April-September), corresponding to the higher prevalence rates of faecal shedding observed in cattle (Grove-White et al., 2010) even in Italy (Merialdi et al., 2015) and in humans (Stampi et al., 1992). Different reasons for seasonal increase of human campylobacteriosis were proposed, including increase shedding of animal reservoirs and higher levels of the food chain contamination, changes in human behaviour and the abundance of invertebrate vectors. However, the driver of this trend is still unknown (Spencer et al., 2012). Despite the most important risk factors for campylobacteriosis are recognised to be handling and consumption of poultry meat and cross-contamination of uncooked products, there is now a growing evidence, based on molecular typing of isolates, that non-poultry sources of human infection has been underestimated. Epidemiological data suggest that the vehicles of common sources outbreaks are different from sporadic infections (Stanley and Jones, 2003): in fact the most frequent food vehicle reported in campylobacteriosis outbreaks is represented by raw unpasteurised milk, whereas for sporadic cases poultry represents the main risk factor (Taylor et al., 2013). Also, a rural-urban association of strains was demonstrated, with the majority of children infections caused by cattle strains in rural areas and by poultry strains in urban areas. Cattle products, the environment contamination, and direct contact between cattle and humans are well-recognised sources of infection of pathogenic agents (Ellis-Iversen et al., 2009). The transmission pathways of thermophilic Campylobacter spp., and in particular C jejuni, from dairy cows to humans may include direct contact with dairy animals, consumption of untreated water contaminated with animal effluents and consumption of contaminated milk (Gilpin et al., 2013; Taylor et al., 2013).

High rates of campylobacteriosis have been reported in high-density dairying areas (Gilpin et al., 2013) and in particular rural residence was identified as a recognised risk factor for children (Bessel et al., 2010). Recently, in Italy, multi locus sequence typing of C. jejuni isolates showed a correspondence between milk and cattle isolates and five of the most frequently detected clinical human CCs isolates (CC-21, CC-45, CC-48, CC-61, CC206) in Europe, emphasising the role of cattle as a major source of food contamination and human infection (Bianchini et al., 2014).

Conclusions

Common sources of campylobacteriosis outbreaks are rare but show a marked seasonality trend and in particular contaminated water, milk or cattle represent the most commonly implicated vehicle or risk factor (Stanley et al., 1998; Kwan et al., 2008). In their study, Gilpin et al. (2013) attributed the significant higher number of campylobacteriosis cases in summer months to an increase of food-related cases. The evidence of a seasonal trend in thermophilic Campylobacter spp. contamination of raw milk sold for direct consumption with an increase of the prevalence in warmer months may represent one of the possible links between seasonal trend in cattle faecal shedding and seasonal trend in human campylobacteriosis. Bacteriological surveillance of raw milk samples may help to understand the role of Campylobacter spp. in foodborne and/or zoonotic infections transmitted to humans.