Isolation of Biofilm-Forming Staphylococci from the Bulk-Tank Milk of Small Ruminant Farms in Greece.

The objectives of this study were (i) to describe staphylococcal isolates recovered from bulk-tank raw milk collected from sheep and goat farms during a countrywide study performed in Greece, (ii) to study management factors potentially associated with their presence in bulk-tank milk and (iii) to provide evidence regarding their association with the quality of the milk. In total, 312 staphylococcal isolates, recovered from samples of bulk-tank raw milk from 444 small ruminant farms in Greece, were evaluated in this work. The in vitro formation of biofilm by the isolates was tested by combining the findings of (a) culture appearance on Congo Red agar plates and (b) results of a microplate adhesion test. The most frequently identified species was Staphylococcus aureus (75 isolates); other frequently recovered species were S. simulans (44 isolates), S. equorum (34 isolates) and S. haemolyticus (26 isolates); in total, 23 species were identified. In total, 224 (71.8%) isolates were biofilm-forming and were recovered from the bulk-tank milk samples of 148 sheep flocks (45.5%) and 55 goat herds (46.2%). There was evidence of seasonality in the isolation of staphylococci: during spring, mostly biofilm-forming isolates were recovered, whilst during summer, mostly non-biofilm-forming isolates were recovered. Among farms applying machine-milking, the proportion of farms from which biofilm-forming isolates were recovered was higher where water with temperature < 50 °C or ≥90 °C was used to clean the milking parlour. In the multivariable analyses, for farms applying machine-milking, the temperature of the water emerged as the only significant variable (p = 0.024), whilst in farms applying hand-milking, the only tendency that emerged was for the frequency of collection of milk from the farm tank (p = 0.08). In sheep flocks, recovery of biofilm-forming staphylococci from the bulk-tank milk was associated with higher somatic cell counts and higher total bacterial counts in the milk. The study identified abiotic factors related to the presence and isolation of these bacteria, specifically the temperature of water used for the cleaning of the milking parlour (in farms where machine-milking is applied) and the frequency of milk collection from the farm tank. These factors apply after the production of milk, and they could thus be regulated appropriately in order to reduce bacterial load and improve the quality of milk delivered to dairy plants. In sheep farms, an association was also seen between recovery of biofilm-forming staphylococci and high somatic cell counts in milk.

Isolation of Listeria ivanovii from Bulk-Tank Milk of Sheep and Goat Farms-From Clinical Work to Bioinformatics Studies: Prevalence, Association with Milk Quality, Antibiotic Susceptibility, Predictors, Whole Genome Sequence and Phylogenetic Relationships.

A cross-sectional study was performed in 325 sheep and 119 goat dairy farms in Greece. Samples of bulk-tank milk were examined by standard microbiological techniques for Listeria spp. Listeria monocytogenes was isolated from one (0.3%) and Listeria ivanovii from three (0.9%) sheep farms. No associations between the isolation of L. monocytogenes or L. ivanovii and milk quality were found. No resistance to antibiotics was identified. Three variables emerged as significant predictors of isolation of the organism: the presence of pigs, low average relative humidity and a high number of ewes on the farm. The three L. ivanovii isolates were assessed in silico for identification of plasmids, prophages, antibiotic resistance genes, virulence factors, CRISPRs and CAS genes. Phylogenetic analysis using the core genome revealed that the three strains belonged to the L. ivanovii subsp. ivanovii branch and were especially close to the PAM 55 strain. All strains of the branch appeared to be very similar, with the distance between them being small.

High Milk Somatic Cell Counts and Increased Teladorsagia Burdens Overshadow Non-Infection-Related Factors as Predictors of Fat and Protein Content of Bulk-Tank Raw Milk in Sheep and Goat Farms.

This paper presents the results of an extensive countrywide investigation performed in 325 dairy sheep flocks and 119 goat herds throughout Greece. The objectives of the study were (a) to investigate fat and protein content in the bulk-tank raw milk of small ruminant farms in Greece and (b) to identify factors potentially influencing that content and factors that can contribute to increased content. The mean fat/protein contents in bulk-tank raw milk of sheep and goats were 6.16 ± 0.05%/4.43 ± 0.01% and 4.77 ± 0.44%/3.23 ± 0.30%, respectively. Significant differences were seen in protein content between farms in the various parts of the country. For sheep, multivariable analyses revealed breed and age of lamb removal from dams as significant for fat content, and somatic cell counts, management system in the farm, administration of anthelmintic treatment during the last stage of pregnancy, and farmer education as significant for protein content. For goats, significant factors were month into lactation period, age of kid removal from dams, and breed for fat content, and somatic cell counts, month into lactation, grazing duration, and % Teladorsagia larvae in faecal samples for protein content. For concurrently high fat and protein content, in multivariable analyses, the following emerged as significant factors: somatic cell counts in milk, numbers of parasite eggs in faeces, and veterinary collaboration (sheep), and month into lactation and somatic cell counts in milk (goats). The results indicate that high somatic cell counts in milk (reflecting the presence of mastitis) and gastrointestinal parasitic infections (mainly Teladorsagia infection) appear to exert a more significant influence on fat and protein content of milk, in comparison to non-infection-related factors.

Ochratoxin A in Slaughtered Pigs and Pork Products.

Ochratoxin A (OTA) is a mycotoxin that is produced after the growth of several Aspergillus and Penicillium spp. in feeds or foods. OTA has been proved to possess nephrotoxic, hepatotoxic, teratogenic, neurotoxic, genotoxic, carcinogenic and immunotoxic effects in animals and humans. OTA has been classified as possibly carcinogenic to humans (Group 2B) by the IARC in 2016. OTA can be mainly found in animals as a result of indirect transmission from naturally contaminated feed. OTA found in feed can also contaminate pigs and produced pork products. Additionally, the presence of OTA in pork meat products could be derived from the direct growth of OTA-producing fungi or the addition of contaminated materials such as contaminated spices. Studies accomplished in various countries have revealed that pork meat and pork meat products are important sources of chronic dietary exposure to OTA in humans. Various levels of OTA have been found in pork meat from slaughtered pigs in many countries, while OTA levels were particularly high in the blood serum and kidneys of pigs. Pork products made from pig blood or organs such as the kidney or liver have been often found to becontaminated with OTA. The European Union (EU) has established maximum levels (ML) for OTA in a variety of foods since 2006, but not for meat or pork products. However, the establishement of an ML for OTA in pork meat and meat by-products is necessary to protect human health.

Effect of modified atmosphere packaging on physicochemical and microbiological characteristics of Graviera Agraphon cheese during refrigerated storage.

The aim of this work was to examine the effect of modified atmosphere packaging on the physicochemical and microbiological changes of Graviera Agraphon cheese during refrigerated storage. Blocks of Graviera Agraphon cheese weighing around 200 g were packaged under natural (control) or modified atmosphere packaging (MAP) conditions (50% N2 - 50% CO2) and stored at 4 °C or 10 °C for up to 85 d. Prior to packaging, groups of cheese blocks were inoculated with one each of the following foodborne pathogens at around 104 log cfu/g: Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli O157:H7 or Staphylococcus aureus, whilst further groups of cheese blocks were not inoculated. The protein, fat, moisture and salt contents as well as the pH of control and MAP cheese samples did not change significantly (P > 0.05) throughout 4 °C storage, while the pH values of control and MAP cheese samples were significantly (P < 0.05) reduced at 10 °C storage. At 10 °C storage, yeasts and molds, psychrotrophs and lactic acid bacteria (LAB) were significantly higher (P < 0.05) for the normal atmosphere than the MAP cheese samples after the 4th, 8th and 4th days, respectively. At 4 °C storage, the yeasts and molds or psychrotrophs were significantly higher (P < 0.05) than those of control after the 6th and 15th days, respectively at 4 °C storage. All foodborne pathogens showed a higher decrease (P < 0.05) at 10 °C than 4 °C storage. S. aureus proved more sensitive in inactivation in the MAP conditions than atmospheric conditions. L. monocytogenes and S. aureus presented a higher decrease than that of E. coli O157:H7 and S. Typhimurium. In conclusion, MAP proved efficient in retarding the growth of yeasts, molds, psychrotrophs and E. coli O157:H7, L. monocytogenes, S. Typhimurium and S. aureus in Graviera Agraphon cheese during refrigerated storage at 4 and 10 °C.

Q fever and seroprevalence of Coxiella burnetii in domestic ruminants.

Q fever is a zoonosis caused by Coxiella burnetii, an obligate intracellular gram­negative bacterium. Infection by C. burnetii has been demonstrated in many animal species, but ruminants are the major reservoirs and the main sources of human infection. In ruminants, C. burnetii infection is often asymptomatic, but it has been also associated with infertility and abortions. In humans, Q fever was considered predominately an occupational hazard due to close contact with infected ruminants by means of their contaminated birth products, urine, feces or milk. Q fever has recently gained renewed attention after the large outbreak in the Netherlands in 2007­2009, indicating its importance as an emerging public health threat. The seroprevalence of C. burnetii in ruminants is commonly detected by various tests but no official standard technique is still available. According to surveys conducted in many countries of the five continents, a relatively high proportion of farm ruminants are found seropositive to C. burnetii. The only country with an apparent zero prevalence is New Zealand. The seroprevalence in goats and sheep is usually higher than cattle.

Fate of enterotoxigenic Staphylococcus aureus and staphylococcal enterotoxins in Feta and Galotyri cheeses.

In this study the fate of enterotoxigenic Staphylococcus aureus and staphylococcal enterotoxins in Feta and Galotyri cheeses were studied. Initially, the enterotoxigenic abilities of four Staph. aureus LHA, LHB, LHC and LHD strains isolated from raw ovine milk were examined in both BHI broth and ovine milk. In BHI broth, the Staph. aureus LHA, LHB, LHC and LHD strains were found toxigenic at 37 °C producing the staphylococcal enterotoxins (SEs) serotypes SEA, SEB, SEC and SED, respectively, whereas in ovine milk at 37 °C, Staph. aureus LHD was found to produce only SED, while no SE production was observed for the other examined strains. Thus, the fate of only Staph. aureus LHD and SED were examined in Feta and Galotyri cheeses. The cheeses were made from raw ovine toxic milk with preformed SED or raw ovine milk contaminated with high (ca 6 log cfu/ml) and low inocula (ca 3 log cfu/ml) of Staph. aureus LHD. Results showed that the pathogen was eliminated at slower rate in Galotyri cheese than in Feta cheese, for the high (5 d vs. 16 d) or the low (1 d vs. 12 d) inoculum trials. In both cheeses produced from the toxic milk, SED was detected during manufacturing and storage. SED was also detected in the curd (2 h), when Staph. aureus LHD populations had reached ca 7 log cfu/g, and up to the end of storage for the high inoculum trials of both cheeses. No SED was observed for the low inoculum trials of either cheese.

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece.

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx(2)-positive, five of which were also stx(1)-positive. The remaining isolate was positive only for the stx(1) gene. All stx-positive isolates (whether positive for stx(1), stx(2) or stx(1) and stx(2)) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.

Influence of oestrus on the heat stability and other characteristics of milk from dairy goats.

We examined the heat stability, somatic cell count (SCC), pH, fat, protein and lactose content of milk from goats during the oestrous period, in order to investigate evidence of possible oestrus effects on milk physical and chemical properties. Goats free from mammary infections were ranked on average SCC from three tests so that they could be stratified randomly in pairs to synchronized oestrus or left as unsynchronized non-oestrus controls. The synchronisation consisted of insertion of an intravaginal progesterone-releasing device for 17 d, and introduction of the bucks the day of the device removal (D0). The repeated measurements analysis of variance model included the fixed effects of the experimental group (oestrus or control) and day and the corresponding interaction and also the random effect of doe. Reduced milk-heat stability, increased SCC, increased protein content and reduced pH were found in the milk samples of the oestrus group on D1, 2 and 3. The fat and lactose content of the milk was not affected by oestrus. These data indicate that the milk of goats during the mating period has reduced heat stability and, therefore, that dilution into bulk tanks should be recommended to avoid clotting when milk is intended for high thermal treatment.