Andrea Serraino

Organophosphorus pesticides residues in Italian raw milk

Organophosphorus pesticides (OPPs), widely used in agriculture, can cause toxic effects to humans and animals. The main purpose of the present work was to determine the contamination in raw milk by the main organophosphorus pesticides used in Italy and to evaluate the opportunity to start specific procedures of risk management along the milk production chain. The samples, collected in 4 Italian dairy plants directly from the tank trucks during the delivering, were representative of 920 tonnes of raw milk. The isolation of the OPPs (acephate, chlorpyriphos, chlorpyriphos-methyl, diazinon, methamidophos, methidathion, phorate, pirimiphos-methyl) was performed by liquid partition followed by clean-up with solid phase extraction. The analyses were carried out by dual column gas chromatography using two nitrogen-phosphorus detectors. Among the 135 samples analysed, 37 were positive in traces and 10 showed an OPP contamination ranging from 5 to 18 microg/kg. The higher results were recorded in the samples collected during the autumn-winter period. The main pollutants detected were acephate and chlorpyriphos. In every positive sample found, the OPP contamination was lower than the maximum residue level (MRL) fixed by the European Commission.

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy.

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.

Sale of Raw Milk in Northern Italy: Food Safety Implications and Comparison of Different Analytical Methodologies for Detection of Foodborne Pathogens

The safety of raw milk sold in Northern Italy was investigated in relation to hygiene quality parameters and presence of Salmonella spp., Listeria monocytogenes, thermotolerant Campylobacter, and Verocytotoxin producing Escherichia coli O157:H7. The performance of different analytical methods used-official culture method (ISO), modified Bacteriological Analytical Manual cultural method (mBAM), and polymerase chain reaction (PCR)-was evaluated. The presence of Mycobacterium avium subsp. paratuberculosis (Map) was investigated only by PCR. All samples met regulations for alkaline phosphatase and inhibitory substance, while 18% and 44.8% of samples collected from vending machines had, respectively, somatic cell count (SCC) >300,000/mL and total bacterial count (TBC) >50,000 CFU/mL. The correlation between hygienic quality parameters in samples collected from bulk tank and vending machines showed a significant increase of TBC in vending machines meaning that raw milk was mishandled during distribution and sale. All pathogens investigated were detected in raw milk sold at vending machines; a total of five samples (5%) had at least one pathogen, of which two were detected by PCR and three by mBAM. None of the samples was positive by cultural ISO methods. Even if the comparison of analytical methods showed that none performs significantly better than the others, testing a higher volume of milk (25 versus 210 mL) affects significantly the detection rate of pathogens. Three samples (3%) were positive for Map, suggesting that raw milk is a significant source of Map exposure for consumers. The observed TBC increase and the detection of several pathogenic bacteria pose questions on the safety of raw milk; the use of ISO seems inefficient in detecting a low contamination level of pathogens in milk and consequently not appropriate as official method for testing. In order to ensure consumers safety, a new approach for the raw milk chain is required.

Quantitative risk assessment of verocytotoxin-producing Escherichia coli O157 and Campylobacter jejuni related to consumption of raw milk in a province in Northern Italy.

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the >5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

Field handling conditions of raw milk sold in vending machines: experimental evaluation of the behaviour of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Typhimurium and Campylobacter jejuni

The direct sale by farmers of raw milk for human consumption has been allowed in Italy since 2004. The aim of this study was to evaluate the behaviour of selected foodborne pathogens in raw milk sold in vending machines, in field handling conditions, and during shelf-life from production to consumption. Temperature of storage of raw milk in 33 farms authorized to produce and sell raw milk were investigated from farm to vending machine delivery, together with consumer habits in one province of the Emilia-Romagna region of northern Italy. Failure to maintain appropriate low temperatures during shelf-life was recorded and 43% of consumers did not boil milk before consumption. Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Typhimurium and Campylobacter jejuni strains were inoculated into raw milk samples, and the best (4°C as established by law) and worst temperature storage conditions detected (variable temperature) were simulated. Boiling tests were performed for each pathogen considered at high and low levels of contamination. Results showed an increase in L. monocytogenes in milk stored at 4°C and at variable temperatures recorded in shelf-life monitoring, an increase in E. coli O157:H7 and S. Typhimurium at variable temperatures but not at 4°C, and a decrease in C. jejuni in all storage conditions. Boiling milk is effective in making it safe for consumers. This study provides evidence that appropriate handling of raw milk, maintaining low temperatures, together with consumer education concerning boiling raw milk before consumption are key factors in preventing foodborne infections linked to raw milk consumption, and helps assess the risk of foodborne infection linked to raw milk consumption.

Occurrence and genetic diversity of Arcobacter butzleri in an artisanal dairy plant in Italy.

ABSTRACT The present study aimed to investigate the presence, distribution, and persistence of Arcobacter spp. in an artisanal dairy plant and to test the isolates to determine their different genotypes in the processing plant and in foods. Samples were collected in an artisanal cheese factory on four occasions between October and December 2012. Food samples (raw milk, ricotta cheese, mozzarella cheese, and conditioning liquid), water samples, and environmental samples were analyzed by the culture method; isolates were identified by multiplex PCR and genotyped by pulsed-field gel electrophoresis (PFGE) analysis. Arcobacter butzleri was isolated from 29 out of 59 samples (46.6%), 22 of which were from environmental samples and 7 of which were from food samples. Cluster analysis divided the strains into 47 PFGE patterns: 14 PFGE clusters and 33 unique types. Our findings indicate that the plant harbored numerous A. butzleri pulsotypes and that the manual cleaning and sanitation in the studied dairy plant do not effectively remove Arcobacter. The recurrent isolation of A. butzleri suggests that the environmental conditions in the dairy plant constitute a good ecological niche for the colonization of this microorganism. In some cases, the presence of indistinguishable strains isolated from the same facilities on different sampling days showed that these strains were persistent in the processing environment.

Visual evaluation of cattle cleanliness and correlation to carcass microbial contamination during slaughtering

The aim of the study was to establish whether the visual cleanliness of cattle slaughtered was correlated to hide and carcass contamination as indicated by aerobic colony count (ACC), Enterobacteriaceae count (EC) and Escherichia coli count (ECC). Cattle in a slaughterhouse were visually inspected and assigned to a category from 1 (very clean) to 5 (very dirty) based on cleanliness. Fifteen animals for each category were randomly selected, hide and carcass sampled and analyzed for ACC, EC and ECC. Results showed that increasing dirt on cattle was associated with higher ACC, EC and ECC on hide and carcasses. Carcass ACC and ECC belonging to animals classified in cleanliness categories 3, 4 or 5 have a higher probability of exceeding the limits set by the Reg. EU 2073/2005. The study supports the conclusion that the pre-slaughter visual evaluation of animal cleanliness and application of corrective actions can be an effective aid to reduce carcass contamination.

Foodborne pathogens in in-line milk filters and associated on-farm risk factors in dairy farms authorized to produce and sell raw milk in northern Italy.

All dairy farms authorized to produce and sell raw milk in a province of Northern Italy were investigated to determine the presence of Campylobacter spp., verocytotoxin-producing Escherichia coli (VTEC), Listeria monocytogenes, and Salmonella spp. in in-line milk filters and to assess their association with suspected risk factors on farms. A logistic regression model was used to analyze data collected describing the characteristics and management practices of 27 farms and the microbiological status of 378 in-line milk filters by both culture-based and molecular methods. Thermotolerant Campylobacter, VTEC, and L. monocytogenes were detected in 24 (6.45%), 32 (8.4%), and 2 (0.5%) samples, respectively. No Salmonella spp. were detected. For risk analysis, data of L. monocytogenes and Salmonella spp. were not included in the model because of the low prevalence or absence of these organisms. The univariate analysis disclosed that the presence of VTEC and/or Campylobacter spp. in milk filters was associated with lack of cleanliness of bedding, water trough, and feed trough; nonevaluation of water hardness; lack of cleanliness of milk tank; and nonapplication of forestripping. After multivariate analysis, an association was observed with inadequate cleanliness of bedding and milk tank and the nonapplication of forestripping. PCR analysis of milk filters was a rapid and sensitive method for the microbiological evaluation of herd contamination status and should be included among the registration requirements for the authorization to produce and sell raw milk. Specific control actions must be incorporated into the farmers daily practices to ensure the low-risk production of raw milk.

Isolation of Arcobacter Species in Water Buffaloes (Bubalus bubalis)

This is the first report of Arcobacter spp. in rectal fecal samples from healthy water buffaloes (Bubalus bubalis) reared on a dairy farm. Arcobacter species were isolated after enrichment, and isolates were identified at species level by multiplex-polymerase chain reaction assay. Thirty samples were examined and Arcobacter spp. were isolated from 96.7% of water buffaloes tested: 38 Arcobacter spp. isolates were obtained, with A. cryaerophilus as the dominant species followed by A. butzleri and A. skirrowii. Nine animals (31%) were colonized by more than one Arcobacter species. The present study indicates that water buffaloes can harbor a variety of Arcobacter spp. and that healthy buffaloes may act as hosts. Water buffalo fecal shedding of Arcobacter spp. may be of significance to human health, considering the potential fecal contamination during harvesting of raw milk and slaughtering.

Four-Year Monitoring of Foodborne Pathogens in Raw Milk Sold by Vending Machines in Italy

Prevalence data were collected from official microbiological records monitoring four selected foodborne pathogens (Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, and Campylobacter jejuni) in raw milk sold by self-service vending machines in seven Italian regions (60,907 samples from 1,239 vending machines) from 2008 to 2011. Data from samples analyzed by both culture-based and real-time PCR methods were collected in one region. One hundred raw milk consumers in four regions were interviewed while purchasing raw milk from vending machines. One hundred seventy-eight of 60,907 samples were positive for one of the four foodborne pathogens investigated: 18 samples were positive for Salmonella, 83 for L. monocytogenes, 24 for E. coli O157:H7, and 53 for C. jejuni in the seven regions investigated. No significant differences in prevalence were found among regions, but a significant increase in C. jejuni prevalence was observed over the years of the study. A comparison of the two analysis methods revealed that real-time PCR was 2.71 to 9.40 times more sensitive than the culture-based method. Data on consumer habits revealed that some behaviors may enhance the risk of infection linked to raw milk consumption: 37% of consumers did not boil milk before consumption, 93% never used an insulated bag to transport raw milk home, and raw milk was consumed by children younger than 5 years of age. These results emphasize that end-product controls alone are not sufficient to guarantee an adequate level of consumer protection. The beta distribution of positive samples in this study and the data on raw milk consumer habits will be useful for the development of a national quantitative risk assessment of Salmonella, L. monocytogenes, E. coli O157, and C. jejuni infection associated with raw milk consumption.