Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing

Following the persistent detection of Listeria monocytogenes in raw bovine milk sold through a vending machine, the 120 lactating cows of the herd producing the milk were subjected to bacteriological investigation. A single cow with subclinical mastitis (1.2–1.3 × 10⁵ somatic cells/mL) and persistent L. monocytogenes excretion was detected. The cow was subjected to antimicrobial therapy, but L. monocytogenes excretion remained high (>3.0 × 10² cfu/mL). Following culling of the infected cow, L. monocytogenes disappeared from the tank milk, and further isolates were recovered from the mammary parenchyma and lymph nodes of the infected cow. To investigate the clonal nature of the contamination, all isolates recovered in the study (n = 13) were analyzed by serogroup PCR, pulsed-field gel electrophoresis, and whole-genome sequencing. Our results demonstrated the clonal nature of the contamination. All isolates belonged to lineage II, serogroup IIa, sequence type 37, clonal complex 37 and harbored some virulence determinants. This case showed that, although relatively rare, prolonged milk contamination by L. monocytogenes can originate from subclinical and persistently infected cows, posing a health risk to consumers.     

Associations between exposure to bovine herpesvirus 1 (BoHV-1) and milk production,reproductive performance,and mortality in Irish dairy herds

As cost-benefit analyses are required to prioritize and promote disease control and eradication programs within a jurisdiction, national data relating to disease-related production losses are particularly useful. The objectives of the current study were to use Irish bovine herpesvirus 1 (BoHV-1) prevalence data in dairy herds, obtained by bulk milk sampling on 4 occasions over the 2009 lactation, to document associations between milk production, fertility performance, mortality, and BoHV-1 herd status. Bulk milk (n = 305) antibody ELISA was used to classify farms as positive or negative in terms of endemic BoHV-1. Cow-level (milk parameters only) and herd-level performance data were sourced from the Irish Cattle Breeding Federation. Ordinary linear and negative binomial regressions were used to investigate associations between milk, fertility, and mortality performance and herd-level BoHV-1 results (both categorical and continuous variables). Only slight effects on the rates of carryover cows, nonpregnant cows, and total deaths were highlighted with increasing ELISA sample/positive (%) values (incidence rate ratio = 1.001). Multiparous cows in herds BoHV-1 bulk milk antibody positive recorded a reduction in milk yield per cow per year of 250.9 L in the multivariable linear model. Milk fat and protein yields were also affected by herd BoHV-1 status, again highlighting sub-optimal milk production in BoHV-1 bulk milk-positive herds. The current study has highlighted an economical method of investigating losses due to endemic infection using repeated bulk milk sampling over a single lactation. These data can contribute to analyzing the cost-benefit of applying BoHV-1 control strategies both on farm and at a national level.     

Peptidome comparison following gastrointestinal digesta of bovine versus caprine milk serum

Infant formula is used as a supplement for newborns. Although bovine milk-based infant formulas dominate the market, caprine milk-based infant formula has attracted increasing attention because of its lower allergenicity. This study compared the digestive peptidome of bovine and caprine milk serum proteins by using in vitro infant simulating conditions. The result showed that the degradation pattern of milk proteins was similar, whereas the digestive rates of milk proteins differed between bovine and caprine milks. Several proteins, such as α-lactalbumin (LALBA), β-lactoglobulin (LGB), serum amyloid A protein (SAA1), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactotransferrin (LTF), released more peptides during digestion of caprine milk serum than during digestion of bovine milk serum; however, more peptides derived from α_S1-casein (CSN1S1) were found in bovine digesta. In addition, antimicrobial-related peptides were mostly only found in caprine intestinal digesta. The results of this study may be useful in understanding the digestion characteristics of milk serum proteins and providing guidance on the improvement of infant formula.     

Short communication: Technological and seasonal variations of vitamin D and other nutritional components in donkey milk

Vitamin D is an essential nutrient that plays a crucial role in calcium homeostasis and bone metabolism and also acts as a hormone. Although several studies on the content of vitamin D in bovine milk have been conducted, little information is available regarding donkey milk. In the context of the nutritional assessment of donkey milk, the aim of this study was to assess the vitamin D content in donkey milk and its chemical profile, with particular reference to seasonal and technological modifications after pasteurization. The study was conducted on a dairy farm that produces donkey milk for human consumption located in central Italy. At sampling time, an aliquot of total bulk milk production was sampled before and after pasteurization (63°C for 30 min without homogenization) with a total of 20 raw and 20 pasteurized milk samples. The samples were collected for 10 mo, every 15 d, from May to February 2017. All the samples were analyzed for the chemical composition and vitamin D₂ and D₃ content by HPLC after saponification. The donkey milk analyzed showed a higher average vitamin D content (raw milk: vitamin D₂ = 1.68, vitamin D₃ = 0.60 μg/100 mL; pasteurized milk: vitamin D₂ = 1.38, vitamin D₃ = 0.30 μg/100 mL) than reported for bovine and human milk. The results of the effect of pasteurization on milk did not highlight significant differences in the total content of vitamin D. However, vitamin D₃ has a poor thermal stability, which led to a significant reduction in content in pasteurized milk compared with raw milk. The total vitamin D content of donkey milk did not show significant variations between seasons; however, a higher concentration of vitamin D₃ was found in spring and summer. In conclusion, raw and pasteurized donkey milk showed a high content of vitamin D, which could be useful in meeting the deficiencies of this vitamin in humans. Further investigations are needed to improve the vitamin D content in donkey milk by increasing its endogenous synthesis or its transfer in milk and to clarify other variability factors.     

Short communication: Milk ELISA status for bovine leukosis virus infection is not associated with milk production in dairy cows

The objective of this study was to assess whether the milk ELISA status for antibodies against bovine leukemia virus was associated with 305-d milk production in Canadian dairy cattle. Test results and test-day production data from 19,785 dairy cows were available for analysis. A linear mixed model was used with the estimated 305-d milk production as the outcome and lactation number, somatic cell count, calving season, days in milk, and breed as fixed effects. Herd nested in province was included as random effect. In conclusion, bovine leukemia virus antibody milk ELISA status was not associated with milk production.     

Short communication: Cold atmospheric plasma inactivation of Prototheca zopfii isolated from bovine milk

Mastitis is a serious bovine diseases that can be caused by Prototheca zopfii, yeast-like algae belonging to the family Chlorellaceae. The substantial economic losses and health damage associated with bovine mastitis emphasize the need to develop effective strategies aimed at control of the infection. Unfortunately, P. zopfii is highly resistant to most common antibacterial and antifungal agents, as well as to heat treatment. We report here the first attempt to use cold atmospheric plasma to inactivate this pathogen. We studied 20 strains of P. zopfii isolated from milk samples taken from cows with clinical or subclinical mastitis. The studies confirmed the high level of resistance of P. zopfii to typical antifungal agents, such as voriconazole, fluconazole, amphotericin B, caspofungin, anidulafungin, and micafungin. In contrast, each of the strains revealed high susceptibility to cold atmospheric plasma, >2-fold higher compared with a reference strain of Candida albicans. The obtained results are promising and open up a new approach in the fight against P. zopfii.     

Short communication: milk fat composition of 4 cattle breeds in the Netherlands

Milk fatty acid (FA) composition was compared among 4 cattle breeds in the Netherlands: Dutch Friesian (DF; 47 animals/3 farms), Meuse-Rhine-Yssel (MRY; 52/3), Groningen White Headed (GWH; 45/3), and Jersey (JER; 46/3). Each cow was sampled once between December 2008 and March 2009 during the indoor housing season, and samples were analyzed using gas chromatography. Significant breed differences were found for all traits including fat and protein contents, 13 major individual FA, 9 groups of FA, and 5 indices. The saturated fatty acid proportion, which is supposed to be unfavorable for human health, was smaller for GWH (68.9%) compared with DF (74.1%), MRY (72.3%), and JER (74.3%) breeds. The proportion of conjugated linoleic acid and the unsaturation index, which are associated positively with human health, were both highest for GWH. Differences in milk fat composition can be used in strategies to breed for milk with a FA profile more favorable for human health. Our results support the relevance of safeguarding the local Dutch breeds.     

Short communication: Analysis of mycotoxins in Spanish milk

We surveyed the presence of 22 mycotoxins in 191 Spanish cow milk samples. Mycotoxins could be carried over from diet into animal milk and have toxic effects on human and animal health. The interaction of different mycotoxins may be additive or synergetic. Therefore, surveillance of mycotoxin co-occurrence in milk is recommended. Aflatoxins M₁, B₁, B₂, G₁, and G₂, ochratoxins A and B, nivalenol, deoxynivalenol, deepoxy-deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, fusarenon X, T-2 and HT-2 toxins, fumonisins B₁, B₂, and B₃, sterigmatocystin, and zearalenone were analyzed. Samples were treated by liquid-liquid extraction with acidified acetonitrile, followed by an acetonitrile-water phase separation using sodium acetate. The analysis was carried out by HPLC coupled to a triple quadrupole mass spectrometer. None of the analyzed mycotoxins had a concentration level higher than their detection limit (0.05–10.1 µg/L). The aflatoxin M₁ in the samples never exceeded the level established by the European Union.     

Biologically active factors in bovine milk and dairy byproducts: Influence on cell culture

Substantial progress has been made in our knowledge of the biological properties of mammal milks. Many nutritional, biochemical, immunological, or other biological properties have been studied in mature or industrially processed bovine milk as well as in human milk and colostrum. This article is a critical review of selected publications covering (1) the use of bovine milk or dairy byproducts (processed acid and enzymatic whey fractions) as a serum substitute for cell cultures, (2) specific factors in bovine milk and industrially processed milk that affect cell proliferation, and (3) the known functional and biological roles of two whey proteins: β‐lactoglobulin and the PP3 component.     

Short communication: Outbreak of methicillin-resistant Staphylococcus aureus (MRSA)-associated mastitis in a closed dairy herd

Cows are probably the main source of contamination of raw milk with Staphylococcus aureus. Mammary glands with subclinical mastitis can shed large numbers of Staph. aureus in milk. Because of the risk of this pathogen to human health as well as animal health, the aim of this paper was to describe an outbreak of mastitis caused by methicillin-resistant Staph. aureus (MRSA), oxacillin-susceptible mecA-positive Staph. aureus (OS-MRSA), and methicillin-susceptible Staph. aureus (MSSA) on a dairy farm. Milk samples were obtained from all quarters, showing an elevated somatic cell count by the California Mastitis Test. The isolates were identified by phenotypic and genotypic methods. Staphylococcus spp. were isolated from 53% (61/115) of the milk samples, with 60 isolates identified as Staph. aureus (98.4%) and 1 isolate identified as Staphylococcus epidermidis (1.6%). The presence of the mecA gene was verified in 48.3% of Staph. aureus isolates. Of the Staph. aureus isolates, 23.3% were MRSA and 25.0% were OS-MRSA. The total of mastitis cases infected with MRSA was 12.2%. The detection of this large percentage of mastitis cases caused by MRSA and OS-MRSA is of great concern for the animals’ health, because β-lactams are still the most important antimicrobials used to treat mastitis. In addition, Staph. aureus isolates causing bovine mastitis represent a public health risk.     

Short communication: Temperature sensibility of Prototheca blaschkeae strains isolated from bovine mastitic milk

Dairy cow mastitis associated with microalgae of the genus Prototheca has been reported worldwide. This alga is extremely resistant to most antimicrobials commonly used in mastitis therapy. In milk processing, different thermal treatments are generally efficient at inactivating and eliminating microorganisms. Until recently, no reports on Prototheca blaschkeae susceptibility to heat treatment have been described. Thus, considering the potential zoonotic risk that Prototheca may represent, the objective of this study was to test the susceptibility of P. blaschkeae field isolates retrieved from bovine mastitis to different temperature/time ratios that are generally used in the milk processing industry: 62°C/15 min and 30 min; 70°C/20 s, 15 min, and 30 min; 75°C/20 s; 90°C/1 s; and 100°C/1 s. The results showed a growth reduction of all isolates after the heat treatments, but only at 100°C was a total growth inhibition observed.     

Human Milk Composition and Preservation: Evaluation of High-pressure Processing as a Nonthermal Pasteurization Technology

Human milk is seen not only as a food, but as a functional and dynamic biologic system. It provides nutrients, bioactive components, and immune factors, promoting adequate and healthy growth of newborn infants. When mothers cannot supply their children, donated breast milk is the nutrition recommended by the World Health Organization, as it is a better alternative than infant formula. However, because of the manner in which donor milk is handled in human milk banks (HMB) many of the properties ascribed to mother's own milk are diminished or destroyed. The major process responsible for these losses is Holder pasteurization. High-pressure processing (HPP) is a novel nonthermal pasteurization technology that is being increasingly applied in food industries worldwide, primarily as an alternative to thermal treatment. This is due to its capacity to inactivate microorganisms while preserving both nutritional and bioactive components of foods. This review describes human milk composition and preservation, and critically discusses HMB importance and practices, highlighting HPP as a potential nonthermal pasteurization technology for human milk preservation. HPP technology is described and the few currently existing studies of its effects in human milk are presented.     

Factors affecting the concentration of sphingomyelin in bovine milk

Sphingomyelin is a phospholipid located in the outer leaflet of the plasma membrane of most cells and is a component of the milk fat globule membrane. Sphingomyelin and its digestion products participate in several antiproliferative pathways that may suppress oncogenesis. Although milk and dairy products are important sources of sphingomyelin in the human diet, little is known about factors that influence sphingomyelin concentrations in milk fat or whether concentrations can be modified via the nutrition of cows. Sphingomyelin concentrations were determined in milk from Holstein and Jersey cows matched for parity and stage of lactation. Sphingomyelin was more concentrated in milk fat from Holstein cows than in milk fat from Jersey cows (1,044 vs. 839 μg/g of fat). Concentrations in whole milk did not differ because of greater milk fat content for milk from Jerseys. Differences between breeds may be related to the greater fat globule size in milk from Jerseys. Sphingomyelin content in whole milk increased with increasing days in milk because of associated increases in milk fat content. Regardless of breed, primiparous cows had greater amounts of stearic acid and less palmitic acid in sphingomyelin than did older cows. The sphingomyelin concentration in milk fat of cows in a commercial Jersey herd was lower for cows in their fourth or greater parity. Sphingomyelin content in whole milk was greater for cows in late lactation because of greater milk fat content. Feed restriction of multiparous Holstein cows to 37% of ad libitum dry matter intake increased milk fat content but did not affect milk sphingomyelin content or milk fat globule size. Supplementation of the diet with 4% soybean oil did not affect milk composition, sphingomyelin content, or milk fat globule size. Milk was sampled seasonally from 7 herds throughout Illinois during a 2-yr period. Sphingomyelin concentration in milk fat was greatest during summer and least during winter, but whole milk concentrations did not vary across seasons. We conclude that 1) sphingomyelin content of milk fat is greater in milk from Holsteins than that from Jerseys, 2) sphingomyelin content in whole milk increases with stage of lactation, and 3) sphingomyelin content of milk fat is greater during summer. However, efforts to produce milk with a greater sphingomyelin content through altering management and nutrition are unlikely to be successful.     

Use of MALDI-TOF MS technology to evaluate adulteration of small ruminant milk with raw bovine milk

Detection of adulteration of small ruminant milk is very important for health and commercial reasons. New analytical and cost-effective methods need to be developed to detect new adulteration practices. In this work, we aimed to explore the ability of the MALDI-TOF mass spectrometry to detect bovine milk in caprine and ovine milk using samples from 18 dairy farms. Different levels of adulteration (0.5, 1, 5, 10, 20, 40, 60, and 80%) were analyzed during the lactation period of goat and sheep (in May, from 60 to 90 d in milk, and in August, from 150 to 180 d in milk). Two different ranges of peptide-protein spectra (500–4,000 Da; 4–20 kDa) were used to establish a calibration model for predicting the concentration of adulterant using partial least squares and generalized linear model with lasso regularization. The low molecular weight part of the spectra together with the generalized linear model with lasso regularization regression model appeared to have greater potential for our aim of detection of adulteration of small ruminants' milk. The subsequent prediction model was able to predict the concentration of bovine milk in caprine milk with a root mean square error of 11.4 and 17.0% in ovine milk. The results offer compelling evidence that MALDI-TOF can detect the adulteration of small ruminants' milk. However, the method is severely limited by (1) the complexity of the milk proteome resulting from the adulteration technique, (2) the potential degradation of thermolabile proteins, and (3) the genetic variability of tested samples. Additionally, the root mean square error of prediction based only on one individual sample adulteration series can drop down to 6.34% for quantification of adulterated caprine milk and 6.28% for adulterated ovine milk for the full set of concentrations or down to 2.33 and 4.00%, respectively, if we restrict only to low concentrations of adulteration (0, 0.5, 1, 5, 10%).     

Short communication: Volatiles in microfluidized raw and heat-treated milk

As innovative processing equipment is introduced to milk processing, it is essential to determine its effect on milk aroma, a critical factor in consumer acceptance of the final dairy product. Microfluidization is known to cause severe high-pressure homogenization of milk fat and, although severe processing is known to release undesired aromas, no information is available on the levels of the volatile compounds in milk immediately after microfluidization. We hypothesized that microfluidization would alter levels of volatile compounds in milk that may affect aroma. The concentration of 11 selected volatile compounds in raw, thermized, pasteurized, and UHT 3.0% fat milk samples were compared before and after microfluidization at 170 MPa and common 2-stage homogenization at 15 MPa. Overall, the different milk samples had similar trends in response to homogenization, although UHT milk started with lower values of nonanoic acid, and acetone and higher levels of hexanal and heptanol. In many cases, microfluidization did not significantly alter volatile levels compared with the starting milk. Heptanal was the only compound observed to increase in thermized and UHT milk, whereas nonanoic acid and acetone decreased in raw, thermized, and pasteurized milks and octanoic acid decreased in thermized and UHT milks. The highest levels of almost all of the volatiles were found in the 2-stage homogenized milk. Overall, microfluidization had minimal effect on the volatile compound profiles of milk, although sensory evaluation is needed to confirm effects on aroma and flavor.     

Short communication: Snapshot of industry milk hauling practices in the western United States

The Pasteurized Milk Ordinance (PMO) mandates milk hauling sanitation and operational practices; however, the use of vague language (i.e., “as needed”) and gaps in processes lead to variability in industry practices. Our aim was to characterize industry milk hauling practices and identify areas that may be an unexplained source of contamination in the dairy processing continuum, and communicate this information with industry to cultivate best practices. The objectives of this study were to (1) survey industry hauling sanitation and operation practices in the Pacific Northwest region of the United States, and (2) quantify microbial populations [aerobic plate count (APC), lactic acid bacteria, coliforms] on the internal surfaces of transfer hoses (tanker and receiving bay) to determine their potential contribution to the microbiological quality of raw milk. Eleven facilities (78% response rate) participated in our survey. All facilities surveyed were compliant with the PMO; however, overall milk reception layout, sanitation practices, and routine maintenance greatly varied between facilities. Farm hose samples (n = 115) had significantly higher microbial loads (APC: mean 4.7 log cfu/100 cm²; median 5.1 log cfu/cm²) than receiving hose samples (n = 57; APC: mean: 2.1 log cfu/100 cm²; median 1.9 log cfu/100 cm²). Microbial populations on transfer hose surfaces did not correlate with time since last cleaning for either tanker or receiving bay hoses. Microbial content of farm hoses is likely to reflect the microbial quality of the previous milk transferred through the hose, making on-farm management practices the primary consideration to maintain low microbiological counts downstream. Upon arrival at the processor, 10% of farm hoses were missing caps. Although this did not correlate with elevated microbiological counts, uncapped farm hoses are exposed to the farm environment, provide opportunity for contamination, and are in violation of the PMO. Through observations made during our studies, manual cleaning procedures appear to be a major weakness in hauling practices and need more attention. Recognizing and communicating variability and areas of weakness allows industry to elevate their hauling sanitation and operational practices to maintain optimum milk microbiological quality.     

Short communication: Parapoxvirus and Orthopoxvirus coinfection in milk of naturally infected cows

Several studies have shown the occurrence of poxvirus infections associated with exanthematic lesions in cattle from many Brazilian states. Coinfection between viruses belonging to 2 genera, Orthopoxvirus (OPXV) and Parapoxvirus (PPV), was already identified from the lesions of affected cows and humans. The DNA and infectious viral particles of Vaccinia virus, an OPXV, have been detected in milk of naturally and experimentally infected cows. However, to date no reports have described the detection of Pseudocowpox virus, a PPV, in milk. Thus, we investigated the presence of PPV and OPXV in milk samples obtained from dairy cows from a Brazilian region with exanthematic disease outbreaks. From 2011 to 2014, 6 dairy farms with exanthematic disease outbreaks involving dairy cows, calves, and humans were visited. Twelve crusts of cows' teat lesions and 60 milk samples were collected. The crusts and milk samples were analyzed by PCR to detect OPXV or PPV DNA. According to the analyzed crusts, we detected PPV infection in 4 of the 6 visited farms, from which we investigated the PPV contamination in milk. From the 40 milk samples tested, PPV DNA was detected in 12 samples. Of these milk samples, 8 were positive for both PPV and OPXV. This is the first report of PPV DNA detection in milk samples from affected cows, indicating that the virus may be present in milk and potentially contaminating dairy products associated or not with OPXV. In addition to the lesions caused by direct contact, the presence of 2 or more poxvirus species in milk showed that the effect of zoonotic exanthematic diseases on public health and animal husbandry is relevant and cannot be overlooked.     

Considerable variation in the concentration of osteopontin in human milk, bovine milk, and infant formulas

Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes such as bone remodeling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. In the present study, ELISA for human and bovine milk OPN were developed and OPN concentration in human breast milk, bovine milk, and infant formulas was measured and compared. The OPN concentration in human milk was measured to approximately 138 mg/L, which corresponds to 2.1% (wt/wt) of the total protein in human breast milk. This is considerably higher than the corresponding OPN concentrations in bovine milk (∼18 mg/L) and infant formulas (∼9 mg/L). Moreover, bovine milk OPN is shown to induce the expression of the T helper 1 cytokine IL-12 in cultured human lamina propria mononuclear cells isolated from intestinal biopsies. Finally, the OPN concentration in plasma samples from umbilical cords, 3-mo-old infants, and pregnant and nonpregnant adults was measured. The OPN level in plasma from 3-mo-old infants and umbilical cords was found to be 7 to 10 times higher than in adults. Thus, the high levels of OPN in milk and infant plasma suggest that OPN is important to infants and that ingested milk OPN is likely to induce cytokine production in neonate intestinal immune cells.     

Short communication: extraction of beta-casein from goat milk

Peptides derived from milk β-casein have potential biological activities, such as antihypertensive and immunostimulating properties. These biological properties increase the demand for the production of specific bioactive peptides. β-Casein can be isolated directly from renneted skim milk, based on the preferential solubilization of β-casein at low temperature. This study was conducted to compare the recovery and purity of β-casein extracted from goat and cow milks. Rennet casein was prepared from both milks, heat treated, and dispersed in demineralized water at various temperatures. β-Casein recovery in the soluble phase increased with decreasing incubation temperature. Concentration of β-casein was 43% higher in goat milk than in cow milk, which had a direct effect on β-casein recovery. Furthermore, β-casein was extracted more efficiently from goat rennet casein. As a result, the extraction yield of β-casein was 53% higher in goat milk than in cow milk. The purity of β-casein extracted from both milks reached approximately 90% after incubation at 0°C.