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Graduate Student Literature Review: Farm management practices: Potential microbial sources that determine the microbiota of raw bovine milk*

Open AccessPublished:July 18, 2022DOI:https://doi.org/10.3168/jds.2021-21758

      ABSTRACT

      Environmental and herd-associated factors such as geographical location, climatic conditions, forage types, bedding, soil, animal genetics, herd size, housing, lactation stage, and udder health are exploited by farmers to dictate specific management strategies that ensure dairy operation profitability and enhance the sustainability of milk production. Along with milking routines, milking systems, and storage conditions, these farming practices greatly influence the microbiota of raw milk, as evidenced by several recent studies. During the past few years, the increased interest in high-throughput sequencing technologies combined with culture-dependent methods to investigate dairy microbial ecology has improved our understanding of raw milk community dynamics throughout storage and processing. However, knowledge is still lacking on the niche-specific communities in the farm environment, and on the factors that determine bacteria transfer to the raw milk. This review summarizes findings from the past 2 decades regarding the effects of farm management practices on the diversity of bacterial species that determine the microbiological quality of raw cow milk.

      Key words

      INTRODUCTION

      Bovine milk, a nutritious food commonly consumed worldwide, serves as the raw material for manufacturing a wide range of food products. However, the high nutrient and water contents of milk make it a prime habitat for beneficial, pathogenic, and spoilage bacteria (
      • Bernier Gosselin V.
      • Dufour S.
      • Adkins P.R.F.
      • Middleton J.R.
      Persistence of coagulase negative staphylococcal intramammary infections in dairy goats.
      ;
      • Yuan L.
      • Sadiq F.A.
      • Burmolle M.
      • Wang N.
      • He G.
      Insights into psychrotrophic bacteria in raw milk: A review.
      ) that can significantly influence its quality and safety, as well as deteriorate the shelf life of dairy products. The main microorganisms involved in the premature spoilage of dairy products have been extensively reviewed (
      • Martin N.H.
      • Torres-Frenzel P.
      • Wiedmann M.
      Invited review: Controlling dairy product spoilage to reduce food loss and waste.
      ). Psychrotrophic bacteria such as Pseudomonas spp. and spore-forming thermoduric bacteria such as Bacillus spp. and Paenibacillus spp., have been pointed out as the main microbial causes of food loss and waste in the dairy chain (
      • Martin N.H.
      • Torres-Frenzel P.
      • Wiedmann M.
      Invited review: Controlling dairy product spoilage to reduce food loss and waste.
      ). Moreover, it is well known that the abundance of psychrotrophic bacteria increases in raw milk during cold storage (
      • Rasolofo E.A.
      • St-Gelais D.
      • LaPointe G.
      • Roy D.
      Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk.
      ;
      • McHugh A.J.
      • Feehily C.
      • Fenelon M.A.
      • Gleeson D.
      • Hill C.
      • Cotter P.D.
      Tracking the dairy microbiota from farm bulk tank to skimmed milk powder.
      ), and that the occurrence of spore-forming bacteria in milk can cause cheese defects (
      • Martin N.H.
      • Torres-Frenzel P.
      • Wiedmann M.
      Invited review: Controlling dairy product spoilage to reduce food loss and waste.
      ). The microbiological quality of raw milk is therefore a key determinant of the quality of processed dairy products such as pasteurized milk, cheese, yogurt, or powdered milk (
      • McHugh A.J.
      • Feehily C.
      • Fenelon M.A.
      • Gleeson D.
      • Hill C.
      • Cotter P.D.
      Tracking the dairy microbiota from farm bulk tank to skimmed milk powder.
      ;
      • Martin N.H.
      • Torres-Frenzel P.
      • Wiedmann M.
      Invited review: Controlling dairy product spoilage to reduce food loss and waste.
      ).
      From the dairy farm to silos at processing facilities, the factors that drive the microbiological quality of raw milk include animal health and farm environment and management practices, as well as storage and transportation conditions. During the past 2 decades, several studies have provided new insights into our understanding of the occurrence and effects of the raw milk microbiota on dairy products. It has been clearly demonstrated that the microbial diversity of raw milk after milking varies in time and between farms (
      • Li N.
      • Wang Y.
      • You C.
      • Ren J.
      • Chen W.
      • Zheng H.
      • Liu Z.
      Variation in raw milk microbiota throughout 12 months and the impact of weather conditions.
      ;
      • McHugh A.J.
      • Feehily C.
      • Fenelon M.A.
      • Gleeson D.
      • Hill C.
      • Cotter P.D.
      Tracking the dairy microbiota from farm bulk tank to skimmed milk powder.
      ). Since the pioneering studies on the link between cow teat skin bacteria and the microbiota of raw milk (
      • Verdier-Metz I.
      • Michel V.
      • Delbès C.
      • Montel M.-C.
      Do milking practices influence the bacterial diversity of raw milk?.
      ;
      • Vacheyrou M.
      • Normand A.-C.
      • Guyot P.
      • Cassagne C.
      • Piarroux R.
      • Bouton Y.
      Cultivable microbial communities in raw cow milk and potential transfers from stables of sixteen French farms.
      ), contrasting microbial community compositions of the cow teat skin have been reported in the literature (
      • Parente E.
      • Ricciardi A.
      • Zotta T.
      The microbiota of dairy milk: A review.
      ). Beyond the biases introduced by the variety of microbial analysis techniques used to investigate the microbiota, variations in farming practices are an important cause of the observed differences in the raw milk community assembly (
      • Murphy S.I.
      • Kent D.
      • Martin N.H.
      • Evanowski R.L.
      • Patel K.
      • Godden S.M.
      • Wiedmann M.
      Bedding and bedding management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      ). In this regard, studies by
      • Vissers M.M.M.
      • Driehuis F.
      • Te Giffel M.C.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of butyric acid bacteria spores in farm tank milk.
      on the contamination of bulk tank milk by butyric acid bacteria and Bacillus cereus (2007b) spores were the first to advise the development of farming practices to limit the occurrence of spore-forming bacteria within the farm environment and ultimately in the bulk tank milk, thus helping to improve the quality of raw milk. This narrative review aims to highlight the findings from the past 2 decades on the interplay of farming practices and the quality of raw bovine milk.

      SOME BACTERIA OF INTEREST FROM RAW MILK

      Investigations of the raw milk microbiota have shown high complexity but somehow low variability in terms of α and β diversity metrics, probably owing to the large number of taxa commonly found in raw milk. However, various compositions of the core microbial community have been described. Upon comparing 5 studies that analyzed the microbiota of bulk tank milk by high-throughput sequencing,
      • Parente E.
      • Ricciardi A.
      • Zotta T.
      The microbiota of dairy milk: A review.
      reported that up to 2,000 taxa were identified at the genus level, of which Enterococcus, Facklamia, Lactobacillus, Lactococcus, Paeniclostridium, Pseudomonas, Psychrobacter, Rikenellaceae, Rombustia, Ruminococcaceae, Staphylococcus, Stenotrophomonas, Streptococcus, and Turicibacter were among the 25 most abundant and prevalent. From almost 1,000 tanker truck raw milk samples,
      • Kable M.E.
      • Srisengfa Y.
      • Laird M.
      • Zaragoza J.
      • McLeod J.
      • Heidenreich J.
      • Marco M.L.
      The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility.
      reported a core microbiota composed of 29 taxa, of which the most abundant included Streptococcus, Staphylococcus, and unidentified Clostridiales. In a study involving 472 bulk tank milk samples collected from 19 herds, Ruminococcaceae, Acinetobacter, Clostridiales, Bacteroidales, Pseudomonas, Staphylococcus, Lachnospiraceae, Corynebacterium, Planococcaceae, Bacillus, Thermoanaerobacterium, and 5–7N15 composed the core microbiota (
      • Rodrigues M.X.
      • Lima S.F.
      • Canniatti-Brazaca S.G.
      • Bicalho R.C.
      The microbiome of bulk tank milk: Characterization and associations with somatic cell count and bacterial count.
      ). It has recently been stated that Microbacterium, Pediococcus, Fusobacterium, Propionibacterium, Acinetobacter, Bifidobacterium, Pseudomonas, Staphylococcus, Streptococcus, Lachnospiraceae, Corynebacterium, Bacteroides, Enterococcus, Ruminococcaceae, Aerococcus, Jeotgalicoccus, Psychrobacter, and Enterobacter were frequently associated with bovine milk (
      • Oikonomou G.
      • Addis M.F.
      • Chassard C.
      • Nader-Macias M.E.F.
      • Grant I.
      • Delbès C.
      • Bogni C.I.
      • Le Loir Y.
      • Even S.
      Milk microbiota: What are we exactly talking about?.
      ). In addition to the core microbiota, most studies have evaluated the abundance of aerobic mesophilic, lactic acid, and psychrotrophic bacteria during the past 20 years (Table 1). These taxa commonly found in raw milk encompass cow-associated (gut and skin), disease-causing, spoilage, beneficial, and psychrotrophic bacteria. In this section only some of the bacteria taxa or phenotypic groups that have been recently mentioned as relevant for milk quality are emphasized. Readers are referred to the review by
      • Quigley L.
      • O'Sullivan O.
      • Stanton C.
      • Beresford T.P.
      • Ross R.P.
      • Fitzgerald G.F.
      • Cotter P.D.
      The complex microbiota of raw milk.
      for broader information on the microbiota of raw milk, or to those by
      • Oliveira R.B.A.
      • Margalho L.P.
      • Nascimento J.S.
      • Costa L.E.O.
      • Portela J.B.
      • Cruz A.G.
      • Sant'Ana A.S.
      Processed cheese contamination by spore-forming bacteria: A review of sources, routes, fate during processing and control.
      and
      • Boor K.J.
      • Wiedmann M.
      • Murphy S.
      • Alcaine S.
      A 100-Year Review: Microbiology and safety of milk handling.
      for a comprehensive overview of raw milk pathogens and subsequent implications on the safety of dairy products.
      Table 1Mean abundances of bacterial groups in raw milk as reported in the studies reviewed
      Number of herds × sampling periods
      When the samples were not bulk tank raw milk, the nature of the sample is given.
      Geographic siteMethod
      MPN = most probable number. Bactoscan: Foss Electric; Petrifilm: 3M.
      Targeted bacterial groupLog cfu/mL
      Dashes between values indicate viable counts range.
      Reference
      974 raw tanker milkCalifornia, USAQuantitative PCRAerobic mesophilic bacteria3.15
      • Kable M.E.
      • Srisengfa Y.
      • Laird M.
      • Zaragoza J.
      • McLeod J.
      • Heidenreich J.
      • Marco M.L.
      The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility.
      71 raw silo milkCalifornia, USAQuantitative PCRAerobic mesophilic bacteria4.2
      • Kable M.E.
      • Srisengfa Y.
      • Xue Z.
      • Coates L.C.
      • Marco M.L.
      Viable and total bacterial populations undergo equipment- and time-dependent shifts during milk processing.
      24 herds × 3Eastern CanadaViable countsLactic acid bacteria2.56
      • Gagnon M.
      • Ouamba A.J.K.
      • LaPointe G.
      • Chouinard P.Y.
      • Roy D.
      Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.
      84 herdsEastern CanadaViable countsAnaerobic spore-forming bacteria0.35
      • Gagnon M.
      • Hamelin L.
      • Fréchette A.
      • Dufour S.
      • Roy D.
      Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality.
      Aerobic mesophilic spore-forming bacteria0.38
      Aerobic thermophilic spore-forming bacteria0.35
      70 herds × 3Eastern CanadaBactoscanTotal bacteria4.26
      • Robles I.
      • Kelton D.F.
      • Barkema H.W.
      • Keefe G.P.
      • Roy J.P.
      • von Keyserlingk M.A.G.
      • DeVries T.J.
      Bacterial concentrations in bedding and their association with dairy cow hygiene and milk quality.
      33 herdsNew York state, USAViable countsAerobic mesophilic spores1.7
      • Miller R.A.
      • Kent D.J.
      • Boor K.J.
      • Martin N.H.
      • Wiedmann M.
      Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      Aerobic mesophilic bacteria3.8
      9 herds (472 samples)New York state, USAViable countsAerobic mesophilic bacteria3.47
      • Rodrigues M.X.
      • Lima S.F.
      • Canniatti-Brazaca S.G.
      • Bicalho R.C.
      The microbiome of bulk tank milk: Characterization and associations with somatic cell count and bacterial count.
      5 herdsNew York state, USAViable countsAerobic mesophilic spores0.3
      • Evanowski R.L.
      • Kent D.J.
      • Wiedmann M.
      • Martin N.H.
      Milking time hygiene interventions on dairy farms reduce spore counts in raw milk.
      Aerobic thermophilic spores0.3
      108 herds × 2Basse-Normandie, FranceViable countsAerobic mesophilic bacteria3.69
      • Mallet A.
      • Guéguen M.
      • Kauffmann F.
      • Chesneau C.
      • Sesboué A.
      • Desmasures N.
      Quantitative and qualitative microbial analysis of raw milk reveals substantial diversity influenced by herd management practices.
      Lactococci3.85
      Lactobacilli4.21
      Leuconostoc3.11
      Pseudomonas2.72
      Gram-negative bacteria2.88
      Yeasts1.92
      Molds0.41
      729 herdsDenmark, Germany, and the NetherlandsViable countsAerobic mesophilic bacteria3.9
      • de Koning K.
      • Slaghuis B.
      • van der Vorst Y.
      Robotic milking and milk quality: Effects on bacterial counts, somatic cell counts, freezing point and free fatty acids.
      13 herdsGalicia, SpainFlow cytometryTotal bacteria4.15–4.70
      • Castro Á.
      • Pereira J.M.
      • Amiama C.
      • Barrasa M.
      Long-term variability of bulk milk somatic cell and bacterial counts associated with dairy farms moving from conventional to automatic milking systems.
      105 herds × 52The NetherlandsBactoscanTotal bacteria3.9
      • Klungel G.H.
      • Slaghuis B.A.
      • Hogeveen H.
      The effect of the introduction of automatic milking systems on milk quality.
      24 herdsThe NetherlandsMPNButyric acid bacteria spores−0.3
      • Vissers M.M.M.
      • Driehuis F.
      • Te Giffel M.C.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of butyric acid bacteria spores in farm tank milk.
      ,
      • Vissers M.M.M.
      • Te Giffel M.C.
      • Driehuis F.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of Bacillus cereus spores in farm tank milk.
      Viable countsBacillus cereus spores−1.74
      45 farms × 3NorwayViable countsAerobic mesophilic bacteria4.27
      • Skeie S.B.
      • Håland M.
      • Thorsen I.M.
      • Narvhus J.
      • Porcellato D.
      Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control.
      1 herd × 6Northern ItalyMPNAnaerobic spore-forming bacteria1.48
      • Bava L.
      • Colombini S.
      • Zucali M.
      • Decimo M.
      • Morandi S.
      • Silvetti T.
      • Brasca M.
      • Tamburini A.
      • Crovetto G.M.
      • Sandrucci A.
      Efficient milking hygiene reduces bacterial spore contamination in milk.
      PetrifilmAerobic mesophilic bacteria3.7
      PetrifilmColiforms1.24
      Viable countsLactic acid bacteria3.52
      Viable countsPropionic bacteria1.02
      23 herdsNorthern ItalyMPNAnaerobic spore-forming bacteria2.76
      • Zucali M.
      • Bava L.
      • Colombini S.
      • Brasca M.
      • Decimo M.
      • Morandi S.
      • Tamburini A.
      • Crovetto G.M.
      Management practices and forage quality affecting the contamination of milk with anaerobic spore-forming bacteria.
      Viable countsLactic acid bacteria3.8
      Viable countsPropionic bacteria2
      PetrifilmAerobic mesophilic bacteria4.09
      PetrifilmColiforms2.15
      2 herds × 2Southern GermanyViable countsAerobic mesophilic bacteria4.5
      • Breitenwieser F.
      • Doll E.V.
      • Clavel T.
      • Scherer S.
      • Wenning M.
      Complementary use of cultivation and high-throughput amplicon sequencing reveals high biodiversity within raw milk microbiota.
      3 raw milk vending machines × 10Southern ItalyViable countsAerobic mesophilic bacteria5
      • Tremonte P.
      • Tipaldi L.
      • Succi M.
      • Pannella G.
      • Falasca L.
      • Capilongo V.
      • Coppola R.
      • Sorrentino E.
      Raw milk from vending machines: Effects of boiling, microwave treatment, and refrigeration on microbiological quality.
      Lactic acid bacteria4
      Enterococci2
      Enterobacteriaceae1.8
      Total coliforms2
      Fecal coliforms0.5
      Pseudomonas5
      Coagulase-negative cocci2
      Yeasts5
      1 herdCroatiaViable countsEnterococci<1–5
      • Dobranić V.
      • Kazazić S.
      • Filipović I.
      • Mikulec N.
      • Zdolec N.
      Composition of raw cow's milk microbiota and identification of enterococci by MALDI-TOF MS—Short communication.
      Enterobacteria<1–4.69
      Escherichia coli<1–3.3
      Staphylococci<1–4
      Aerobic mesophilic bacteria3–5.39
      Psychrophilic bacteria<2–5.17
      Lactic acid bacteria<2–4.77
      20 herds (150 samples)Czech RepublicViable countsAerobic mesophilic bacteria4.18
      • Mikulová M.
      Content of free fatty acids, lipolytic bacteria and somatic cells in relation to milking technology.
      Psychrophilic lipolytic bacteria2.8
      1 herdSlovakiaViable countsAerobic mesophilic bacteria6.71
      • Pukančíková L.
      • Lipničanová S.
      • Kačániová M.
      • Chmelová D.
      • Ondrejovič M.
      Natural microflora of raw cow milk and their enzymatic spoilage potential.
      Psychrophilic bacteria6.62
      Thermophilic bacteria3.11
      3 herds × 5Vologda district, RussiaViable countsAerobic mesophilic bacteria4.03
      • Tyapugin E.A.
      • Tyapugin S.E.
      • Simonov G.A.
      • Uglin V.K.
      • Nikiforov V.E.
      • Serebrova I.S.
      Comparative evaluation of technological factors affecting milk production and quality with various milking technologies.
      21 herdsTunisiaViable countsAerobic mesophilic bacteria6
      • Kmiha S.
      • Aouadhi C.
      • Klibi A.
      • Jouini A.
      • Béjaoui A.
      • Mejri S.
      • Maaroufi A.
      Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia.
      Viable countsAerobic mesophilic spores<1
      3 herds × 12Southeast Victoria, AustraliaViable countsAerobic mesophilic bacteria3.1–4.6
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      Psychrophilic bacteria2.1–3
      Thermoduric psychrophilic bacteria1–2
      168 herds × 217 states of USAViable countsColiforms1.43–2
      • Patel K.
      • Godden S.M.
      • Royster E.
      • Crooker B.A.
      • Timmerman J.
      • Fox L.
      Relationships among bedding materials, bedding bacteria counts, udder hygiene, milk quality, and udder health in US dairy herds.
      NAS1.25–1.8
      Streptococci or streptococci-like organisms2.8–3.16
      1 When the samples were not bulk tank raw milk, the nature of the sample is given.
      2 MPN = most probable number. Bactoscan: Foss Electric; Petrifilm: 3M.
      3 Dashes between values indicate viable counts range.

      Thermoduric Spore-Forming Bacteria

      Thermophilic, mesophilic, and psychrotrophic bacteria that can survive pasteurization and grow in pasteurized milk are called thermoduric, regardless of their capacity to produce spores (
      • Gleeson D.
      • O'Connell A.
      • Jordan K.
      Review of potential sources and control of thermoduric bacteria in bulk-tank milk.
      ). Reducing their occurrence in raw milk is therefore crucial. Spore contamination is a major and recurrent problem for the production of powdered milk (
      • Miller R.A.
      • Kent D.J.
      • Watterson M.J.
      • Boor K.J.
      • Martin N.H.
      • Wiedmann M.
      Spore populations among bulk tank raw milk and dairy powders are significantly different.
      ). The control of butyric acid-producing bacteria such as Clostridium tyrobutyricum is of great importance to minimize late blowing defects during cheese manufacture. The genus Bacillus is dominant among thermoduric spore-forming bacteria, and Bacillus licheniformis is one of the most ubiquitous (
      • Miller R.A.
      • Kent D.J.
      • Watterson M.J.
      • Boor K.J.
      • Martin N.H.
      • Wiedmann M.
      Spore populations among bulk tank raw milk and dairy powders are significantly different.
      ;
      • Kmiha S.
      • Aouadhi C.
      • Klibi A.
      • Jouini A.
      • Béjaoui A.
      • Mejri S.
      • Maaroufi A.
      Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia.
      ;
      • Gagnon M.
      • Ouamba A.J.K.
      • LaPointe G.
      • Chouinard P.Y.
      • Roy D.
      Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.
      ). Paenibacillus is also recognized as thermoduric (
      • Driehuis F.
      • Hoolwerf J.
      • Rademaker J.L.W.
      Concurrence of spores of Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus polymyxa in silage, dairy cow faeces and raw milk.
      ;
      • Skeie S.B.
      • Håland M.
      • Thorsen I.M.
      • Narvhus J.
      • Porcellato D.
      Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control.
      ;
      • Martin N.H.
      • Torres-Frenzel P.
      • Wiedmann M.
      Invited review: Controlling dairy product spoilage to reduce food loss and waste.
      ). Less common bacteria such as Methylonatrum, Cloacibacillus, and Planobacterium have been reported to show significant positive correlation with heat-resistant spore counts in raw milk (
      • Li N.
      • Wang Y.
      • You C.
      • Ren J.
      • Chen W.
      • Zheng H.
      • Liu Z.
      Variation in raw milk microbiota throughout 12 months and the impact of weather conditions.
      ). Thermoduric bacterial spores may grow and reach 20,000 cfu/mL in approximately 14 to 21 d following milk pasteurization (
      • Martin N.H.
      • Torres-Frenzel P.
      • Wiedmann M.
      Invited review: Controlling dairy product spoilage to reduce food loss and waste.
      ). Moreover, thermoduric bacteria of the genera Bacillus, Paenibacillus, Psychrobacillus, and Viridibacillus have shown lipolytic and proteolytic activity (
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      ;
      • Martin N.H.
      • Torres-Frenzel P.
      • Wiedmann M.
      Invited review: Controlling dairy product spoilage to reduce food loss and waste.
      ), especially in extended shelf life products.

      Psychrotrophs

      Psychrotrophic bacteria are among the most studied species of the raw milk microbiota, probably due to their spoilage potential and their enrichment under cold storage conditions, as shown for Pseudomonas and Acinetobacter (
      • Kable M.E.
      • Srisengfa Y.
      • Laird M.
      • Zaragoza J.
      • McLeod J.
      • Heidenreich J.
      • Marco M.L.
      The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility.
      ;
      • Skeie S.B.
      • Håland M.
      • Thorsen I.M.
      • Narvhus J.
      • Porcellato D.
      Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control.
      ). An increase from 5 to 8 log cfu/mL of Pseudomonas upon storing raw milk at 4°C for 72 h has been reported (
      • Tremonte P.
      • Tipaldi L.
      • Succi M.
      • Pannella G.
      • Falasca L.
      • Capilongo V.
      • Coppola R.
      • Sorrentino E.
      Raw milk from vending machines: Effects of boiling, microwave treatment, and refrigeration on microbiological quality.
      ). Likewise,
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      attributed a significant shift of the microbial structure of raw milk stored at 4°C for 4 d to the proliferation of psychrotrophs. The outgrowth of Pseudomonas fluorescens group and Bacillus spp., along with the emergence of Aeromonas, Listeria, and Stenotrophomonas, occurred at the expense of lactic acid bacteria (LAB) such as Streptococcus, Enterococcus, and Staphylococcus, as well as Enterobacteriaceae such as Hafnia, Rahnella, Klebsiella, Enterobacter, and Serratia. Psychrotrophic bacteria such as Pseudomonas can produce pigments and heat-stable enzymes that may affect the processability of raw milk. For instance, 50 to 75% of Pseudomonas and Bacillus isolates showed persistent proteolytic and lipolytic activity following heat treatment of raw milk at 142°C for 4 s (
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      ). A recent study by
      • Reichler S.J.
      • Trmčić A.
      • Martin N.H.
      • Boor K.J.
      • Wiedmann M.
      Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life.
      showed that species of the genus Pseudomonas still pose major threats to the shelf life of fluid milk.

      Lactic Acid Bacteria

      Within the order Lactobacillales, Lactococcus, Pediococcus, Enterococcus, Streptococcus, and lactobacilli are the most commonly reported genera in raw milk microbiota (
      • Bintsis T.
      Lactic acid bacteria as starter cultures: An update in their metabolism and genetics.
      ). Recently,
      • Zheng J.
      • Wittouck S.
      • Salvetti E.
      • Franz C.M.A.P.
      • Harris H.M.B.
      • Mattarelli P.
      • O'Toole P.W.
      • Pot B.
      • Vandamme P.
      • Walter J.
      • Watanabe K.
      • Wuyts S.
      • Felis G.E.
      • Gänzle M.G.
      • Lebeer S.
      A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae..
      proposed an important reclassification of the Lactobacillus genus into 25 genera, of which 23 are novel, along with the amendment of the family Lactobacillaceae to contain all previous members of the families Lactobacillaceae and Leuconostocaceae. Lactic acid bacteria are the most studied bacterial groups of the milk microbiota due to their technological relevance and health-promoting properties. Indeed, among multiple applications, LAB can be used as inoculants for ensiling (
      • Muck R.E.
      • Nadeau E.M.G.
      • McAllister T.A.
      • Contreras-Govea F.E.
      • Santos M.C.
      • Kung Jr., L.
      Silage review: Recent advances and future uses of silage additives.
      ) or starter and adjunct cultures for cheese manufacture (
      • Wilkinson M.G.
      • LaPointe G.
      Invited review: Starter lactic acid bacteria survival in cheese: New perspectives on cheese microbiology.
      ). However, heterofermentative LAB species can be associated with dairy product defects (
      • Gagnon M.
      • Ouamba A.J.K.
      • LaPointe G.
      • Chouinard P.Y.
      • Roy D.
      Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.
      ). Although LAB are generally considered as mesophilic, they can proliferate in raw milk during cold storage. It was previously found that 87% of LAB isolates from raw milk were able to grow at 6°C (
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      ). Moreover,
      • Kable M.E.
      • Srisengfa Y.
      • Laird M.
      • Zaragoza J.
      • McLeod J.
      • Heidenreich J.
      • Marco M.L.
      The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility.
      have demonstrated the enrichment of Lactobacillales in milk silos at dairy plants.
      The genus Enterococcus may be somewhat controversial. Although certain strains of the same species can be used as microbial additives in silage or as adjunct starter for cheese-making, some have exhibited resistance to numerous antibiotics including vancomycin (
      • Monticelli J.
      • Knezevich A.
      • Luzzati R.
      • Di Bella S.
      Clinical management of non-faecium non-faecalis vancomycin-resistant enterococci infection. Focus on Enterococcus gallinarum and Enterococcus casseliflavus/flavescens..
      ), as well as to heat treatments and salt, which can constitute potential threats to the safety of humans consuming raw milk (
      • Gagnon M.
      • Ouamba A.J.K.
      • LaPointe G.
      • Chouinard P.Y.
      • Roy D.
      Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.
      ). The most abundant enterococci in milk include Enterococcus faecalis, Enterococcus faecium, and Enterococcus durans (
      • Dobranić V.
      • Kazazić S.
      • Filipović I.
      • Mikulec N.
      • Zdolec N.
      Composition of raw cow's milk microbiota and identification of enterococci by MALDI-TOF MS—Short communication.
      ;
      • Gagnon M.
      • Ouamba A.J.K.
      • LaPointe G.
      • Chouinard P.Y.
      • Roy D.
      Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.
      ). It has been reported that their amount could reach 105 cfu/mL in raw milk (
      • Dobranić V.
      • Kazazić S.
      • Filipović I.
      • Mikulec N.
      • Zdolec N.
      Composition of raw cow's milk microbiota and identification of enterococci by MALDI-TOF MS—Short communication.
      ).

      ON-FARM MANAGEMENT PRACTICES AND MILK MICROBIOTA

      Over the past few years, numerous studies have improved our knowledge on a variety of dairy farm management practices by concentrating on the factors (Table 2) that determine the microbiological quality of raw milk. In this section, advances from the past 20 years on feeding, bedding, milking methods, and seasonality (Figure 1) will be emphasized.
      Table 2Recent investigations on the effect of farming parameters on the milk microbiota
      ParameterReference
      Air quality
      • Du B.
      • Meng L.
      • Liu H.
      • Zheng N.
      • Zhang Y.
      • Guo X.
      • Zhao S.
      • Li F.
      • Wang J.
      Impacts of milking and housing environment on milk microbiota.
      Bedding
      • Miller R.A.
      • Kent D.J.
      • Boor K.J.
      • Martin N.H.
      • Wiedmann M.
      Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      ;
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      ;
      • Bradley A.J.
      • Leach K.A.
      • Green M.J.
      • Gibbons J.
      • Ohnstad I.C.
      • Black D.H.
      • Payne B.
      • Prout V.E.
      • Breen J.E.
      The impact of dairy cows' bedding material and its microbial content on the quality and safety of milk—A cross sectional study of UK farms.
      ;
      • Falardeau J.
      • Keeney K.
      • Trmčić A.
      • Kitts D.
      • Wang S.
      Farm-to-fork profiling of bacterial communities associated with an artisan cheese production facility.
      ;
      • Patel K.
      • Godden S.M.
      • Royster E.
      • Crooker B.A.
      • Timmerman J.
      • Fox L.
      Relationships among bedding materials, bedding bacteria counts, udder hygiene, milk quality, and udder health in US dairy herds.
      ;
      • Gagnon M.
      • Hamelin L.
      • Fréchette A.
      • Dufour S.
      • Roy D.
      Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality.
      ;
      • Robles I.
      • Kelton D.F.
      • Barkema H.W.
      • Keefe G.P.
      • Roy J.P.
      • von Keyserlingk M.A.G.
      • DeVries T.J.
      Bacterial concentrations in bedding and their association with dairy cow hygiene and milk quality.
      Feces
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      ;
      • Falardeau J.
      • Keeney K.
      • Trmčić A.
      • Kitts D.
      • Wang S.
      Farm-to-fork profiling of bacterial communities associated with an artisan cheese production facility.
      Feeding (concentrates)
      • Frétin M.
      • Martin B.
      • Rifa E.
      • Isabelle V.M.
      • Pomiès D.
      • Ferlay A.
      • Montel M.C.
      • Delbès C.
      Bacterial community assembly from cow teat skin to ripened cheeses is influenced by grazing systems.
      Feeding (grazing)
      • Vissers M.M.M.
      • Driehuis F.
      • Te Giffel M.C.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of butyric acid bacteria spores in farm tank milk.
      ;
      • Gleeson D.
      • O'Connell A.
      • Jordan K.
      Review of potential sources and control of thermoduric bacteria in bulk-tank milk.
      ;
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      Feeding (silage)
      • Vissers M.M.M.
      • Driehuis F.
      • Te Giffel M.C.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of butyric acid bacteria spores in farm tank milk.
      ,
      • Vissers M.M.M.
      • Te Giffel M.C.
      • Driehuis F.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of Bacillus cereus spores in farm tank milk.
      ;
      • Zucali M.
      • Bava L.
      • Colombini S.
      • Brasca M.
      • Decimo M.
      • Morandi S.
      • Tamburini A.
      • Crovetto G.M.
      Management practices and forage quality affecting the contamination of milk with anaerobic spore-forming bacteria.
      ;
      • Driehuis F.
      • Hoolwerf J.
      • Rademaker J.L.W.
      Concurrence of spores of Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus polymyxa in silage, dairy cow faeces and raw milk.
      ;
      • Gagnon M.
      • Ouamba A.J.K.
      • LaPointe G.
      • Chouinard P.Y.
      • Roy D.
      Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.
      Health status
      • Dobranić V.
      • Kazazić S.
      • Filipović I.
      • Mikulec N.
      • Zdolec N.
      Composition of raw cow's milk microbiota and identification of enterococci by MALDI-TOF MS—Short communication.
      ;
      • Rodrigues M.X.
      • Lima S.F.
      • Canniatti-Brazaca S.G.
      • Bicalho R.C.
      The microbiome of bulk tank milk: Characterization and associations with somatic cell count and bacterial count.
      Lactation stage
      • McHugh A.J.
      • Feehily C.
      • Fenelon M.A.
      • Gleeson D.
      • Hill C.
      • Cotter P.D.
      Tracking the dairy microbiota from farm bulk tank to skimmed milk powder.
      Milking system
      • Johansson M.
      • Lundh Å.
      • De Vries R.
      • Sjaunja K.S.
      Composition and enzymatic activity in bulk milk from dairy farms with conventional or robotic milking systems.
      ;
      • Skeie S.B.
      • Håland M.
      • Thorsen I.M.
      • Narvhus J.
      • Porcellato D.
      Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control.
      Season
      • Vissers M.M.M.
      • Te Giffel M.C.
      • Driehuis F.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of Bacillus cereus spores in farm tank milk.
      ;
      • Kable M.E.
      • Srisengfa Y.
      • Laird M.
      • Zaragoza J.
      • McLeod J.
      • Heidenreich J.
      • Marco M.L.
      The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility.
      ;
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      ;
      • Kmiha S.
      • Aouadhi C.
      • Klibi A.
      • Jouini A.
      • Béjaoui A.
      • Mejri S.
      • Maaroufi A.
      Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia.
      ;
      • Li N.
      • Wang Y.
      • You C.
      • Ren J.
      • Chen W.
      • Zheng H.
      • Liu Z.
      Variation in raw milk microbiota throughout 12 months and the impact of weather conditions.
      ;
      • Ruvalcaba-Gómez J.M.
      • Delgado-Macuil R.J.
      • Zelaya-Molina L.X.
      • Maya-Lucas O.
      • Ruesga-Gutiérrez E.
      • Anaya-Esparza L.M.
      • Villagrán-de la Mora Z.
      • López-de la Mora D.A.
      • Arteaga-Garibay R.I.
      Bacterial succession through the artisanal process and seasonal effects defining bacterial communities of raw-milk Adobera cheese revealed by high-throughput DNA sequencing.
      Soil
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      Teat microbiota
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      ;
      • Frétin M.
      • Martin B.
      • Rifa E.
      • Isabelle V.M.
      • Pomiès D.
      • Ferlay A.
      • Montel M.C.
      • Delbès C.
      Bacterial community assembly from cow teat skin to ripened cheeses is influenced by grazing systems.
      ;
      • Falardeau J.
      • Keeney K.
      • Trmčić A.
      • Kitts D.
      • Wang S.
      Farm-to-fork profiling of bacterial communities associated with an artisan cheese production facility.
      ;
      • Du B.
      • Meng L.
      • Liu H.
      • Zheng N.
      • Zhang Y.
      • Guo X.
      • Zhao S.
      • Li F.
      • Wang J.
      Impacts of milking and housing environment on milk microbiota.
      Teat preparation
      • Mallet A.
      • Guéguen M.
      • Kauffmann F.
      • Chesneau C.
      • Sesboué A.
      • Desmasures N.
      Quantitative and qualitative microbial analysis of raw milk reveals substantial diversity influenced by herd management practices.
      ;
      • Miller R.A.
      • Kent D.J.
      • Boor K.J.
      • Martin N.H.
      • Wiedmann M.
      Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      ;
      • Zucali M.
      • Bava L.
      • Colombini S.
      • Brasca M.
      • Decimo M.
      • Morandi S.
      • Tamburini A.
      • Crovetto G.M.
      Management practices and forage quality affecting the contamination of milk with anaerobic spore-forming bacteria.
      ;
      • Bava L.
      • Colombini S.
      • Zucali M.
      • Decimo M.
      • Morandi S.
      • Silvetti T.
      • Brasca M.
      • Tamburini A.
      • Crovetto G.M.
      • Sandrucci A.
      Efficient milking hygiene reduces bacterial spore contamination in milk.
      ;
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      ;
      • Evanowski R.L.
      • Kent D.J.
      • Wiedmann M.
      • Martin N.H.
      Milking time hygiene interventions on dairy farms reduce spore counts in raw milk.
      ;
      • Palii A.P.
      • Mihalchenko S.A.
      • Chechui H.F.
      • Reshetnichenko A.P.
      • Rozum Y.E.
      • Bredykhin V.V.
      • Bogomolov O.V.
      • Denicenko S.A.
      • Mitiashkina T.Y.
      • Sychov A.I.
      • Savchenko V.B.
      • Levkin D.A.
      • Pali A.P.
      Milking and udder health assessment in industrial farming.
      Figure thumbnail gr1
      Figure 1Origins of raw milk microbiota on dairy farms. Double arrows indicate microbial distribution among the 3 main sources (i.e., environment, housing, and milking). Microorganisms from sources (at the corners) are transferred to raw milk (in the center) during milking. Bacteria are cited for illustrative purposes. Taxa names are colored by associated sources.

      Feeding

      In addition to hay and concentrates, silage is a key element for dairy cow feeding (
      • Schingoethe D.J.
      A 100-year review: Total mixed ration feeding of dairy cows.
      ). Researchers have extensively studied the microbial ecology of silage (
      • Muck R.E.
      • Nadeau E.M.G.
      • McAllister T.A.
      • Contreras-Govea F.E.
      • Santos M.C.
      • Kung Jr., L.
      Silage review: Recent advances and future uses of silage additives.
      ), but few studies have investigated the link between silage and the microbiota of raw milk. Evidence from the literature suggests that spores from soil contaminate forage plants and remain viable in silage (
      • Oliveira R.B.A.
      • Margalho L.P.
      • Nascimento J.S.
      • Costa L.E.O.
      • Portela J.B.
      • Cruz A.G.
      • Sant'Ana A.S.
      Processed cheese contamination by spore-forming bacteria: A review of sources, routes, fate during processing and control.
      ). Spores that survive the cow gastrointestinal tract may be found in feces, from where they may reach the teat skin and finally enter the milk during milking (
      • Driehuis F.
      • Hoolwerf J.
      • Rademaker J.L.W.
      Concurrence of spores of Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus polymyxa in silage, dairy cow faeces and raw milk.
      ). In a study by
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      , feces and teat skin swab samples from cows housed indoors (fed with silage) or outdoors (fed by grazing) clustered separately from each other following principal coordinate analysis, indicating different community structures. The authors also identified fecal material as a major source of contamination of raw milk. Ensiled forages, particularly corn silage, may harbor high numbers of bacterial spores (
      • Bava L.
      • Colombini S.
      • Zucali M.
      • Decimo M.
      • Morandi S.
      • Silvetti T.
      • Brasca M.
      • Tamburini A.
      • Crovetto G.M.
      • Sandrucci A.
      Efficient milking hygiene reduces bacterial spore contamination in milk.
      ). Several studies have demonstrated that on dairy farms where cows were fed high-quality silage having low content of bacterial spores, low numbers of spores were found in raw milk (
      • Vissers M.M.M.
      • Driehuis F.
      • Te Giffel M.C.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of butyric acid bacteria spores in farm tank milk.
      ,
      • Vissers M.M.M.
      • Te Giffel M.C.
      • Driehuis F.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of Bacillus cereus spores in farm tank milk.
      ;
      • Zucali M.
      • Bava L.
      • Colombini S.
      • Brasca M.
      • Decimo M.
      • Morandi S.
      • Tamburini A.
      • Crovetto G.M.
      Management practices and forage quality affecting the contamination of milk with anaerobic spore-forming bacteria.
      ). However, for non-spore-forming bacteria, little is known about the identity of those that can be transferred into raw milk during milking. Producing high-quality silage is thus critical to minimize the occurrence of spoilage bacteria in raw milk. Chemical (acids, salts) and microbial (mostly LAB) additives have long been used for this purpose. Currently, silage microbiology research is concentrated on the generation of new additives or their combinations that ensure the production of high-quality silage with improved aerobic stability and enhanced animal performance and productivity (
      • Muck R.E.
      • Nadeau E.M.G.
      • McAllister T.A.
      • Contreras-Govea F.E.
      • Santos M.C.
      • Kung Jr., L.
      Silage review: Recent advances and future uses of silage additives.
      ;
      • Nair J.
      • Huaxin N.
      • Andrada E.
      • Yang H.E.
      • Chevaux E.
      • Drouin P.
      • McAllister T.A.
      • Wang Y.
      Effects of inoculation of corn silage with Lactobacillus hilgardii and Lactobacillus buchneri on silage quality, aerobic stability, nutrient digestibility, and growth performance of growing beef cattle.
      ;
      • Carvalho B.F.
      • Sales G.F.C.
      • Schwan R.F.
      • Ávila C.L.S.
      Criteria for lactic acid bacteria screening to enhance silage quality.
      ). Finding new microbial inoculants with high efficiency is achieved through a series of selection criteria encompassing strain isolation and characterization, safety evaluation, metabolic efficiency, antagonistic properties, fermentation performance, evaluation of silage aerobic stability, and assessment of animal performance (
      • Carvalho B.F.
      • Sales G.F.C.
      • Schwan R.F.
      • Ávila C.L.S.
      Criteria for lactic acid bacteria screening to enhance silage quality.
      ). As reported by
      • Drouin P.
      • Tremblay J.
      • Chaucheyras-Durand F.
      Dynamic succession of microbiota during ensiling of whole plant corn following inoculation with Lactobacillus buchneri and Lactobacillus hilgardii alone or in combination.
      and
      • Nair J.
      • Huaxin N.
      • Andrada E.
      • Yang H.E.
      • Chevaux E.
      • Drouin P.
      • McAllister T.A.
      • Wang Y.
      Effects of inoculation of corn silage with Lactobacillus hilgardii and Lactobacillus buchneri on silage quality, aerobic stability, nutrient digestibility, and growth performance of growing beef cattle.
      , the intensive assessment of the properties of a new combination inoculant containing strains of Lentilactobacillus buchneri NCIMB 40788 and Lentilactobacillus hilgardii CNCM-I-4785 showed promising results for ensiling whole-crop corn. From our analysis of these publications in addition to the meta-analysis from
      • Oliveira R.B.A.
      • Margalho L.P.
      • Nascimento J.S.
      • Costa L.E.O.
      • Portela J.B.
      • Cruz A.G.
      • Sant'Ana A.S.
      Processed cheese contamination by spore-forming bacteria: A review of sources, routes, fate during processing and control.
      on the effects of inoculants on silage quality and the performance of dairy cows, the review from
      • Carvalho B.F.
      • Sales G.F.C.
      • Schwan R.F.
      • Ávila C.L.S.
      Criteria for lactic acid bacteria screening to enhance silage quality.
      that discusses the steps and criteria of the selection process for silage inoculants, as well as several other references not cited in this review, none included an assessment of their ability to interfere with the milk fermentation process or alter the quality of dairy products. Recently,
      • Gagnon M.
      • Ouamba A.J.K.
      • LaPointe G.
      • Chouinard P.Y.
      • Roy D.
      Prevalence and abundance of lactic acid bacteria in raw milk associated with forage types in dairy cow feeding.
      focused on variations in the LAB profile from raw milk according to forage types, including either hay or grass/legume and corn silage prepared with and without inoculants for herd feeding. Although LAB composition significantly differed between forages, similar LAB profiles were observed in the associated milk samples. Interestingly, by typing LAB isolates, the authors found that only ∼6% of LAB occurring in raw milk might originate from forage types or commercial inoculants. However, heat-resistant LAB from silage may pose threats to milk processing. To prevent such issues, LAB strains not commonly found in raw milk and that are not heat-resistant could be selected as inoculants for ensiling, although silage was identified as a minor contributor to the milk microbiota on dairy farms (
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      ;
      • Du B.
      • Meng L.
      • Liu H.
      • Zheng N.
      • Zhang Y.
      • Guo X.
      • Zhao S.
      • Li F.
      • Wang J.
      Impacts of milking and housing environment on milk microbiota.
      ).
      In addition to providing cows with fresh forage, access to pasture changes several parameters in farm management, including, among others, bedding, air quality, and temperature. As reported by
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      , microbial communities in ryegrass and soil were more similar to that of bulk tank milk when cows were grazed, whereas the microbiota of silage showed a greater level of similarity to that of the bulk tank milk when cows were fed indoors. Greater relative abundances of cow-associated bacteria such as Eremococcus, Alloiococcus, Trichococcus, Prevotella, and Psychrobacter have been observed in the milk produced by cows housed indoors. In contrast, for grazing cows, greater relative abundances of environmental bacteria such as Corynebacteriales, Pseudomonas, Acinetobacter, and Lactococcus have been noted in raw milk. In the study by
      • Vissers M.M.M.
      • Driehuis F.
      • Te Giffel M.C.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of butyric acid bacteria spores in farm tank milk.
      , a reduction in the number of butyric spores in milk was attributed to the removal of silage from the diet. For high-moisture soils with elevated spore content, access to grazing can increase the number of spores in milk, due to direct contacts of cow teats with soil (
      • Gleeson D.
      • O'Connell A.
      • Jordan K.
      Review of potential sources and control of thermoduric bacteria in bulk-tank milk.
      ).
      • Frétin M.
      • Martin B.
      • Rifa E.
      • Isabelle V.M.
      • Pomiès D.
      • Ferlay A.
      • Montel M.C.
      • Delbès C.
      Bacterial community assembly from cow teat skin to ripened cheeses is influenced by grazing systems.
      reported that extensive grazing (grassland only) and semi-extensive grazing (grassland and concentrates) had similar effects on the milk bacterial community assembly across 2 sampling periods. However, they observed significant differences in the teat microbial composition. Feeding cows with concentrates altered the microbiota of the rumen and gastrointestinal tract, therefore modifying that of feces. As mentioned previously, fecal bacteria may contaminate the teat, inducing a community shift, but not sufficiently to affect the milk microbiota. Likewise, in another study in which cows had open access to a clover pasture that was separate from the barn, which itself was separate from the milking room,
      • Falardeau J.
      • Keeney K.
      • Trmčić A.
      • Kitts D.
      • Wang S.
      Farm-to-fork profiling of bacterial communities associated with an artisan cheese production facility.
      found that the microbiota of teat milk samples was compositionally similar to those from the pasture. Raw milk after 24-h exposure to air in the bulk tank room was similar to air, bulk and truck tanks, cheese plants, and Gruyère cheese. Finally, bulk tank milk samples showed similarity to cow-associated microbiota.

      Bedding

      In addition to providing comfort to dairy cows, bedding materials harbor variable microbial community structures, of which the richness and diversity vary depending on bedding types and associated management practices. Using a culture-dependent approach, high numbers of anaerobic spores, mainly from C. tyrobutyricum, Clostridium butyricum, Clostridium beijerinckii, and Clostridium sporogenes have been found in straw bedding (
      • Bava L.
      • Colombini S.
      • Zucali M.
      • Decimo M.
      • Morandi S.
      • Silvetti T.
      • Brasca M.
      • Tamburini A.
      • Crovetto G.M.
      • Sandrucci A.
      Efficient milking hygiene reduces bacterial spore contamination in milk.
      ). Greater amounts of Bacillus spp., coliforms, Klebsiella spp., streptococci, and streptococci-like organisms have been found in used compared with unused bedding samples of new sand, reclaimed sand, manure solids, and organic non-manure materials (
      • Patel K.
      • Godden S.M.
      • Royster E.
      • Crooker B.A.
      • Timmerman J.
      • Fox L.
      Relationships among bedding materials, bedding bacteria counts, udder hygiene, milk quality, and udder health in US dairy herds.
      ). Investigations using culture-dependent methods confirmed a shift in the microbiota of used bedding materials but highlighted contrasting results about specific taxa (
      • Beauchemin J.
      • Fréchette A.
      • Thériault W.
      • Dufour S.
      • Fravalo P.
      • Thibodeau A.
      Comparison of microbiota of recycled manure solids and straw bedding used in dairy farms in eastern Canada.
      ;
      • Ray T.
      • Gaire T.N.
      • Dean C.J.
      • Rowe S.
      • Godden S.M.
      • Noyes N.R.
      The microbiome of common bedding materials before and after use on commercial dairy farms.
      ). Although cows may spend much of their time lying down, enabling direct contact of the udder or the teat skin with bedding materials, bedding has been identified as a minor source of raw milk contamination (
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      ;
      • Falardeau J.
      • Keeney K.
      • Trmčić A.
      • Kitts D.
      • Wang S.
      Farm-to-fork profiling of bacterial communities associated with an artisan cheese production facility.
      ). Recycled manure solids (RMS) are an ecological alternative to conventional bedding materials that have been attracting increasing interest from farmers. However, few studies have focused on microbial communities populating RMS, and conflicting results have been reported regarding the effects of this bedding type on the raw milk microbiota. In a cross-sectional study involving 125 farms on which lactating cows were bedded on either sawdust (35%), sand (33%), or RMS (32%),
      • Bradley A.J.
      • Leach K.A.
      • Green M.J.
      • Gibbons J.
      • Ohnstad I.C.
      • Black D.H.
      • Payne B.
      • Prout V.E.
      • Breen J.E.
      The impact of dairy cows' bedding material and its microbial content on the quality and safety of milk—A cross sectional study of UK farms.
      found that despite observing greater bacterial counts in RMS, no subsequent greater bacterial load occurred in the associated milk compared with that of cows bedded on sawdust and sand. Conversely, in a study enrolling 168 dairy farms on which cows were bedded on either RMS (19.6%), new sand (27.4), reclaimed sand (18.5%), or organic non-manure bedding (34.5%),
      • Patel K.
      • Godden S.M.
      • Royster E.
      • Crooker B.A.
      • Timmerman J.
      • Fox L.
      Relationships among bedding materials, bedding bacteria counts, udder hygiene, milk quality, and udder health in US dairy herds.
      found that coliform counts in bulk tank milk seemed to be greater on dairy farms using RMS or organic non-manure bedding compared with reclaimed sand. They also found that streptococci and streptococci-like organism counts in bulk tank milk were greater on farms using RMS compared with new sand or organic non-manure bedding.
      • Robles I.
      • Kelton D.F.
      • Barkema H.W.
      • Keefe G.P.
      • Roy J.P.
      • von Keyserlingk M.A.G.
      • DeVries T.J.
      Bacterial concentrations in bedding and their association with dairy cow hygiene and milk quality.
      investigated the influence of 4 types of bedding, including sand used on 12 farms, straw and other dry forage on 33 farms, wood products comprising shavings and sawdust on 17 farms, and RMS on 8 farms. The use of RMS as bedding resulted in significantly greater bacterial counts in the bulk tank milk.
      • Gagnon M.
      • Hamelin L.
      • Fréchette A.
      • Dufour S.
      • Roy D.
      Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality.
      reported, as have others before (
      • Miller R.A.
      • Kent D.J.
      • Boor K.J.
      • Martin N.H.
      • Wiedmann M.
      Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      ;
      • Bradley A.J.
      • Leach K.A.
      • Green M.J.
      • Gibbons J.
      • Ohnstad I.C.
      • Black D.H.
      • Payne B.
      • Prout V.E.
      • Breen J.E.
      The impact of dairy cows' bedding material and its microbial content on the quality and safety of milk—A cross sectional study of UK farms.
      ), that the concentration of bacterial spores in milk was not affected by the bedding material, including RMS. This contrasts with findings from other studies that provided evidence of a direct influence of bedding type, including RMS, on the spore content in raw milk (
      • Martin N.H.
      • Kent D.J.
      • Evanowski R.L.
      • Zuber Hrobuchak T.J.
      • Wiedmann M.
      Bacterial spore levels in bulk tank raw milk are influenced by environmental and cow hygiene factors.
      ;
      • Murphy S.I.
      • Kent D.
      • Martin N.H.
      • Evanowski R.L.
      • Patel K.
      • Godden S.M.
      • Wiedmann M.
      Bedding and bedding management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      ). Milk samples associated with RMS harbored greater amounts of heat-resistant Streptococcus and E. faecalis, whereas straw-associated milk samples showed greater abundance of heat-resistant Kocuria (
      • Gagnon M.
      • Hamelin L.
      • Fréchette A.
      • Dufour S.
      • Roy D.
      Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality.
      ). The scarce literature currently available reports several procedures for making RMS and differences in sampling protocols and study experimental designs, as well as differences in other factors related to farming practices (
      • Miller R.A.
      • Kent D.J.
      • Boor K.J.
      • Martin N.H.
      • Wiedmann M.
      Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      ;
      • Bradley A.J.
      • Leach K.A.
      • Green M.J.
      • Gibbons J.
      • Ohnstad I.C.
      • Black D.H.
      • Payne B.
      • Prout V.E.
      • Breen J.E.
      The impact of dairy cows' bedding material and its microbial content on the quality and safety of milk—A cross sectional study of UK farms.
      ;
      • Murphy S.I.
      • Kent D.
      • Martin N.H.
      • Evanowski R.L.
      • Patel K.
      • Godden S.M.
      • Wiedmann M.
      Bedding and bedding management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      ;
      • Gagnon M.
      • Hamelin L.
      • Fréchette A.
      • Dufour S.
      • Roy D.
      Effect of recycled manure solids as bedding on bulk tank milk and implications for cheese microbiological quality.
      ;
      • Robles I.
      • Kelton D.F.
      • Barkema H.W.
      • Keefe G.P.
      • Roy J.P.
      • von Keyserlingk M.A.G.
      • DeVries T.J.
      Bacterial concentrations in bedding and their association with dairy cow hygiene and milk quality.
      ), which could be linked to the variation in effects of bedding types including RMS on the microbiota of raw milk. Based on published studies dealing with the influence of RMS on the quality and safety of raw milk and dairy products, it is difficult to draw objective conclusions. However, links between bedding material and cow cleanliness have been reported (
      • Robles I.
      • Kelton D.F.
      • Barkema H.W.
      • Keefe G.P.
      • Roy J.P.
      • von Keyserlingk M.A.G.
      • DeVries T.J.
      Bacterial concentrations in bedding and their association with dairy cow hygiene and milk quality.
      ), and larger stalls have been associated with lower bacterial loads in raw milk (
      • Robles I.
      • Kelton D.F.
      • Barkema H.W.
      • Keefe G.P.
      • Roy J.P.
      • von Keyserlingk M.A.G.
      • DeVries T.J.
      Bacterial concentrations in bedding and their association with dairy cow hygiene and milk quality.
      ). Additional research is therefore needed to provide new knowledge and sufficient proof-of-concept demonstrations of possible risks associated with the use of RMS on dairy farms. Future studies implementing culture-independent methods to analyze the relationships between bedding materials and the microbial quality of raw milk should distinguish viable versus dead bacterial cells. Polyphasic approaches combining culture-based and DNA-based methods should be encouraged when specific taxa groups are targeted. Besides bedding types, providing detailed sample metadata such as status (unused, used), physicochemical characteristics, depth, and bedding management operations when analyzing bedding microbiota would guide objective comparisons of results between published data (
      • Ray T.
      • Gaire T.N.
      • Dean C.J.
      • Rowe S.
      • Godden S.M.
      • Noyes N.R.
      The microbiome of common bedding materials before and after use on commercial dairy farms.
      ).

      Milking

      The milking environment provides ideal conditions for bacterial growth, as it is composed of several microbial niches that can directly or indirectly share their contents. The maintenance of proper hygiene in this operation is therefore essential for controlling environmental pathogens or spoilage bacteria and reducing milk contamination rates. As mentioned earlier, the amount of dirt on the udder or teat has been positively correlated with the level of raw milk contamination (
      • Miller R.A.
      • Kent D.J.
      • Boor K.J.
      • Martin N.H.
      • Wiedmann M.
      Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.
      ;
      • Zucali M.
      • Bava L.
      • Colombini S.
      • Brasca M.
      • Decimo M.
      • Morandi S.
      • Tamburini A.
      • Crovetto G.M.
      Management practices and forage quality affecting the contamination of milk with anaerobic spore-forming bacteria.
      ;
      • Palii A.P.
      • Mihalchenko S.A.
      • Chechui H.F.
      • Reshetnichenko A.P.
      • Rozum Y.E.
      • Bredykhin V.V.
      • Bogomolov O.V.
      • Denicenko S.A.
      • Mitiashkina T.Y.
      • Sychov A.I.
      • Savchenko V.B.
      • Levkin D.A.
      • Pali A.P.
      Milking and udder health assessment in industrial farming.
      ). The degree of cleanliness of the milker's hands also affects the microbiological quality of raw milk, and recommendations have been made for proper hand hygiene or wearing gloves (
      • Palii A.P.
      • Mihalchenko S.A.
      • Chechui H.F.
      • Reshetnichenko A.P.
      • Rozum Y.E.
      • Bredykhin V.V.
      • Bogomolov O.V.
      • Denicenko S.A.
      • Mitiashkina T.Y.
      • Sychov A.I.
      • Savchenko V.B.
      • Levkin D.A.
      • Pali A.P.
      Milking and udder health assessment in industrial farming.
      ). Implementing a combination of interventions, including training milking staff to efficiently clean teat-ends and to use a laundering approach with a specific recipe of detergent and chlorine bleach for cleaning towels, resulted in 37 and 40% decreases in the mesophilic and thermophilic contents, respectively, in bulk tank raw milk compared with those before the interventions (
      • Evanowski R.L.
      • Kent D.J.
      • Wiedmann M.
      • Martin N.H.
      Milking time hygiene interventions on dairy farms reduce spore counts in raw milk.
      ). However, a previous study by
      • Doyle C.J.
      • Gleeson D.
      • O'Toole P.W.
      • Cotter P.D.
      Impacts of seasonal housing and teat preparation on raw milk microbiota: A high-throughput sequencing study.
      demonstrated that teat preparation before milking (including water wash, forestripping, disinfection, and drying with a paper towel) did not show a major effect on milk microbiota compared with untreated teats. According to the authors, this contrasting result might be explained by high standards of hygiene measures implemented in the experimental farm in which the study was carried out.
      • Zucali M.
      • Bava L.
      • Colombini S.
      • Brasca M.
      • Decimo M.
      • Morandi S.
      • Tamburini A.
      • Crovetto G.M.
      Management practices and forage quality affecting the contamination of milk with anaerobic spore-forming bacteria.
      showed that the implementation of milking practices including use of gloves, drying teats, forestripping, pre-dipping, and post-dipping on farms resulted in reduced milk contamination by anaerobic spore-forming bacteria. Pre-dipping has been associated with low abundance of aerobic mesophilic bacteria, Lactococcus, yeasts, and molds, whereas post-dipping has been correlated with low levels of presumed cheese-ripening bacteria and Leuconostoc (
      • Mallet A.
      • Guéguen M.
      • Kauffmann F.
      • Chesneau C.
      • Sesboué A.
      • Desmasures N.
      Quantitative and qualitative microbial analysis of raw milk reveals substantial diversity influenced by herd management practices.
      ). Similarly, comparing methods of teat preparation,
      • Bava L.
      • Colombini S.
      • Zucali M.
      • Decimo M.
      • Morandi S.
      • Silvetti T.
      • Brasca M.
      • Tamburini A.
      • Crovetto G.M.
      • Sandrucci A.
      Efficient milking hygiene reduces bacterial spore contamination in milk.
      demonstrated that the application of pre-dipping, wiping, forestripping, and post-dipping as a milking routine considerably reduced the amount of aerobic spore-forming bacteria, total aerobic bacteria, and propionibacteria in milk compared with the application of forestripping only, or the combination of forestripping with post-dipping. However, these treatments did not affect the occurrence of LAB and coliforms in raw milk. Based on the authors' observations, it seems that implementing the complete milking routine of teat hygiene prevented the occurrence of C. tyrobutyricum in milk. Although several protocols of teat preparation are being implemented on dairy farms, particular attention should be paid to this critical phase of milk production to limit the bacterial load of milk. For this purpose, achieving a consensus of the effects of teat hygiene procedures on the raw milk microbiota remains a challenge for researchers, given that most studies reporting theses effects have examined a limited number of samples or herds and showed inconsistency in methodological approaches. The development of standardized methods and the availability of qualified staff may facilitate large-scale investigations of the effects of teat sanitation on the quality of raw milk.
      The types of milking system and housing are important factors that shape the milking environment. Automatic milking systems (AMS) have gained in frequency of installation, and it has been estimated that about 38,000 milking robots are currently operational around the world (
      • Hogenboom J.A.
      • Pellegrino L.
      • Sandrucci A.
      • Rosi V.
      • D'Incecco P.
      Invited review: Hygienic quality, composition, and technological performance of raw milk obtained by robotic milking of cows.
      ). Although the high milking frequency achieved with AMS may possibly reduce the time bacteria stay in the teat, it may concomitantly favor bacterial access to the teat canal, which stays open after milking (
      • Hogenboom J.A.
      • Pellegrino L.
      • Sandrucci A.
      • Rosi V.
      • D'Incecco P.
      Invited review: Hygienic quality, composition, and technological performance of raw milk obtained by robotic milking of cows.
      ). Moreover, stagnant fluids such as residual milk in the milking robot may result in greater numbers of spoilage bacteria in raw milk (
      • Hogenboom J.A.
      • Pellegrino L.
      • Sandrucci A.
      • Rosi V.
      • D'Incecco P.
      Invited review: Hygienic quality, composition, and technological performance of raw milk obtained by robotic milking of cows.
      ). In a recent study,
      • Skeie S.B.
      • Håland M.
      • Thorsen I.M.
      • Narvhus J.
      • Porcellato D.
      Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control.
      collected the bulk tank raw milk from 45 dairy farms implementing 3 types of housing and milking systems, including freestall with milking parlor, freestall with AMS, and tiestall with pipeline milking. They showed that milk samples from AMS had greater total mesophilic bacteria count than the 2 other milking systems and attributed the observed difference to poor teat cleaning and absence of teat drying with AMS. However, the wider range of viable counts reported from farms implementing stall housing with pipeline milking systems might reflect greater variation in milking routines in conventional systems.
      • Skeie S.B.
      • Håland M.
      • Thorsen I.M.
      • Narvhus J.
      • Porcellato D.
      Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control.
      also reported significant effects of the types of housing and milking systems on milk microbiota at the genus level, along with significant variations in the occurrence of Pseudomonas, Lactococcus, Acinetobacter, Facklamia, and Psychrobacter sequence variants. Comparing bulk tank raw milk samples from farms using AMS or conventional milking systems,
      • Johansson M.
      • Lundh Å.
      • De Vries R.
      • Sjaunja K.S.
      Composition and enzymatic activity in bulk milk from dairy farms with conventional or robotic milking systems.
      revealed greater proteolytic activity that was not associated with enzymes naturally present in milk (plasmin and plasminogen) on AMS farms. Recently, a systematic review by
      • Cogato A.
      • Brščić M.
      • Guo H.
      • Marinello F.
      • Pezzuolo A.
      Challenges and tendencies of automatic milking systems (AMS): A 20-years systematic review of literature and patents.
      examined the tendencies and gaps associated with AMS in industrial and scientific research. They revealed that interests were more focused on animal welfare and productivity than on milk quality and therefore highlighted the need for more research on cleaning operations and animal health to improve milk quality. This feature supports concerns about the microbiological quality of raw milk produced using AMS as discussed previously.
      Regardless of the milking system, biofilm formation on milking equipment is of major concern for animal and human health, as well as for milk quality. However, few studies have been devoted to biofilms formed on the inner surfaces of the milking tools, and consequently their real influence on milk and dairy product quality is not well known.
      • Weber M.
      • Liedtke J.
      • Plattes S.
      • Lipski A.
      Bacterial community composition of biofilms in milking machines of two dairy farms assessed by a combination of culture-dependent and -independent methods.
      recently investigated the living biomass associated with biofilms on the milking machines on 2 farms. They found that Actinobacteria could be among the primary colonizers of stainless-steel surfaces inside the milking equipment, and that the genera Bacillus, Kocuria, Microbacterium, Staphylococcus, Acinetobacter, Chryseobacterium, and Pseudomonas were frequently detected.
      Seasonality in the diversity of raw milk microbiota has been widely demonstrated (
      • Kable M.E.
      • Srisengfa Y.
      • Laird M.
      • Zaragoza J.
      • McLeod J.
      • Heidenreich J.
      • Marco M.L.
      The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility.
      ;
      • Li N.
      • Wang Y.
      • You C.
      • Ren J.
      • Chen W.
      • Zheng H.
      • Liu Z.
      Variation in raw milk microbiota throughout 12 months and the impact of weather conditions.
      ;
      • Ruvalcaba-Gómez J.M.
      • Delgado-Macuil R.J.
      • Zelaya-Molina L.X.
      • Maya-Lucas O.
      • Ruesga-Gutiérrez E.
      • Anaya-Esparza L.M.
      • Villagrán-de la Mora Z.
      • López-de la Mora D.A.
      • Arteaga-Garibay R.I.
      Bacterial succession through the artisanal process and seasonal effects defining bacterial communities of raw-milk Adobera cheese revealed by high-throughput DNA sequencing.
      ). In a longitudinal study involving 10 dairy farms,
      • Li N.
      • Wang Y.
      • You C.
      • Ren J.
      • Chen W.
      • Zheng H.
      • Liu Z.
      Variation in raw milk microbiota throughout 12 months and the impact of weather conditions.
      showed that milk microbial communities sampled throughout 12 mo clustered into 2 groups, one comprising fall and spring, and the other summer and winter samples.
      • Kable M.E.
      • Srisengfa Y.
      • Laird M.
      • Zaragoza J.
      • McLeod J.
      • Heidenreich J.
      • Marco M.L.
      The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility.
      have estimated that seasonal variation could account for 5% of the variation in raw tanker milk (sampled consecutively during fall, spring, and summer across 2 yr) microbial composition and structure. Greater viable counts of raw milk mesophilic and thermoduric bacteria were obtained in summer compared with winter and spring (
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      ;
      • Kmiha S.
      • Aouadhi C.
      • Klibi A.
      • Jouini A.
      • Béjaoui A.
      • Mejri S.
      • Maaroufi A.
      Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia.
      ), whereas for psychrotrophs, greater viable counts were obtained in winter (
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      ). Bacteria that correlated with warmer temperatures included Bacillus thuringiensis, B. licheniformis, Bacillus pumilus, Bacillus subtilis, and Paenibacillus, whereas Pseudomonas, Acinetobacter, Psychrobacter, B. cereus, Bacillus weihenstephanensis, Bacillus circulans, Actinobacteria, and Propionibacterium were found to be associated with cooler temperatures (
      • Vissers M.M.M.
      • Te Giffel M.C.
      • Driehuis F.
      • De Jong P.
      • Lankveld J.M.G.
      Minimizing the level of Bacillus cereus spores in farm tank milk.
      ;
      • Vithanage N.R.
      • Dissanayake M.
      • Bolge G.
      • Palombo E.A.
      • Yeager T.R.
      • Datta N.
      Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential.
      ;
      • Kmiha S.
      • Aouadhi C.
      • Klibi A.
      • Jouini A.
      • Béjaoui A.
      • Mejri S.
      • Maaroufi A.
      Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia.
      ;
      • Li N.
      • Wang Y.
      • You C.
      • Ren J.
      • Chen W.
      • Zheng H.
      • Liu Z.
      Variation in raw milk microbiota throughout 12 months and the impact of weather conditions.
      ). In addition to temperature, humidity was revealed as a key factor that determines the seasonal variation in microbial diversity. Accordingly, although the abundances of Chitinophaga and Niastella were associated with low humidity, greater temperature and humidity levels generally favored the proliferation of most bacteria (
      • Li N.
      • Wang Y.
      • You C.
      • Ren J.
      • Chen W.
      • Zheng H.
      • Liu Z.
      Variation in raw milk microbiota throughout 12 months and the impact of weather conditions.
      ).
      • Ruvalcaba-Gómez J.M.
      • Delgado-Macuil R.J.
      • Zelaya-Molina L.X.
      • Maya-Lucas O.
      • Ruesga-Gutiérrez E.
      • Anaya-Esparza L.M.
      • Villagrán-de la Mora Z.
      • López-de la Mora D.A.
      • Arteaga-Garibay R.I.
      Bacterial succession through the artisanal process and seasonal effects defining bacterial communities of raw-milk Adobera cheese revealed by high-throughput DNA sequencing.
      analyzed the microbial composition of raw milk samples across dry (from November to May) and rainy (from June to October) seasons and found greater abundances of Streptococcaceae and Lactococcus in the dry season, whereas Aeromonadaceae and Acinetobacter were more abundant in the rainy season. Although weather conditions, including temperature and humidity, seem to consistently modulate bacterial groups such as mesophilic or psychrotrophic bacteria, a core microbiota may exist for raw milk.
      • Li N.
      • Wang Y.
      • You C.
      • Ren J.
      • Chen W.
      • Zheng H.
      • Liu Z.
      Variation in raw milk microbiota throughout 12 months and the impact of weather conditions.
      reported that the genera Acinetobacter and Pseudomonas occurred in 112 raw bulk tank milk sampled over 12 mo. In a study by
      • Kable M.E.
      • Srisengfa Y.
      • Laird M.
      • Zaragoza J.
      • McLeod J.
      • Heidenreich J.
      • Marco M.L.
      The core and seasonal microbiota of raw bovine milk in tanker trucks and the impact of transfer to a milk processing facility.
      , a wider core microbiota of 29 taxa, among which the more abundant included unidentified Clostridiales, Peptostreptococcaceae, Ruminococcaceae, Staphylococcus, Streptococcus, Turicibacter, unidentified Lachnospiraceae, and Corynebacterium, was detected in 899 raw milk tanker trucks sampled over fall, spring, and summer. However, gaps still exist in the knowledge of the core microbiota at the species or strain level.

      CONCLUSIONS

      The research outcomes reviewed herein clearly show that the complexity of raw milk microbiota reflects the farm environment from which it originates. Current knowledge on the routes of raw milk contamination provides evidence of the interplay among farming conditions, management practices, and the quality of raw milk before transport. Therefore, improving the quality and safety of raw milk and dairy products requires a better understanding of the multitude of ecological niches that make up the farm microbial environment, not to mention subsequent transport and handling equipment, as well as other factors such as stocking density, water, housing styles, and geographical location that deserve specific attention. This can be achieved by employing integrated high-throughput approaches that target both the microbiota and the metabolome of the analyzed matrix. As for the RMS reviewed in this analysis, more in-depth investigation of microbial dynamics during processing and of the final microbiota is needed to fully assess the associated risks for herd health and milk safety from a human perspective. Our current understanding of the pattern of raw milk contamination on farm is challenged by the limitations of bacterial species or strain-level identification using affordable amplicon sequencing technology. Pending cost reductions of metagenomic (or metataxonomic based on full-length sequencing of the 16S rRNA gene pool) studies that allow finer resolution, the culturomics approach (culture-based method) can be used to uncover and characterize the hidden microbiota (uncultured, undetected, or ambiguously classified microorganisms) associated with milk and sources of contamination. Silage exemplifies an important source of microorganisms with high fermentative potential. Promising inoculants for ensiling should be screened for their possible interference with milk processing. More research is needed to fill the gaps of bacteria interactions in silage as well as in other microbial sources on the farm. Future research should exploit network-based analysis techniques to decipher interconnections among these niches in relation to raw milk microbiota. This would likely generate relevant knowledge to recommend new intervention strategies or farming decisions to ensure the production of high-quality milk and dairy products.

      ACKNOWLEDGMENTS

      The authors thank the Natural Sciences and Engineering Research Council of Canada (Ottawa; Canada Research Chair on Lactic Cultures Biotechnology for Dairy and Probiotic Industries, 2003–2017), Novalait (Québec, Canada), Agriculture and Agri-Food Canada (Ottawa), and the Fonds de recherche du Québec—Nature et technologies for their financial contributions. They also especially thank the Op+Lait research group (Saint-Hyacinthe, Canada) for a scholarship award to A. J. K. Ouamba and M. Gagnon. The authors have not stated any conflicts of interest.

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