Microbiology of the Dairy Animal responsible for the pathological condition )-that are absorbed through the rumen wall for use by the animal as sources of energy and biosynthetic precursors Thus, the ruminant animal cannot directly use carbohydrates for energy, and it is absolutely dependent upon its microflora to, in effect, predigest its food. By virtue of its large size, the rumen has the function of slowing down the rate of passage of feed through the organ, which permits microbial digestion of essentially all of the nonstructural carbohydrate of the feed(starches and sugars) as well as over half of the more recalcitrant feed fiber(cellulose and hemicellu loses)(Van Soest, 1994). Rumen contents, which contain 6-18% dry matte are mixed by strong muscular movement and are periodically returned via the esophagus to the mouth for additional chewing(rumination). Despite this, the solids have a tendency to stratify, with some maintaining a suspension in the rumen liquor, some settling to the bottom of the rumen, and some being borne up by gas bubbles to form a floating mat at the liquid surface. Passage rates vary with intake, with the rates for solids averaging about twice of that for liquids From several published experiments, mean retention times for the rumen liquid range from 8 to 24 h, whereas that of the particulate phase range from 14 to 52 h(Broderick et al., 1991). The consequence of these long retention times for solids is that ruminant animals can use fibrous feeds(forages and certain agricul- tural byproducts) that are not usable by humans and other monogastric animals with the ultimate conversion of these feedstuffs to useful products In addition to VFAS, other products of the fermentation include microbial cells and fermentation gases. The microbial cells eventually pass through the omasum and into the abomasum( the acidic"true stomach"), where the microbial cell protein is hydrolyzed to amino acids that are available for subsequent intesti- nal absorption. This microbial protein is a major contributor to the protein re- quirements of the animal, and it acts to counterbalance somewhat the considerable loss of feed protein that occurs as a result of microbial proteolysis and amino acid fermentation that occurs in the rumen(see Sec. IV C.5). Fermentation gases include primarily carbon dioxide(50-70%)and meth- ane(30-40%). Rates of gas production immediately after a meal can exceed 30 L/h, and a typical cow may release 500 L of methane per day (Wolin, 1990) Although some gas is absorbed across the rumen wall and carried by the blood to the lungs for exhalation. most is eructated through the mouth A. Milk Composition In the United States, milk has a strict legal on:"the lacteal secretion practically free of colostrum, obtained by co milking of one or more healthy cows(Office of the Federal Register, 1995). Parallel definitions are
Microbiology of the Dairy Animal 5 responsible for the pathological condition.)—that are absorbed through the rumen wall for use by the animal as sources of energy and biosynthetic precursors. Thus, the ruminant animal cannot directly use carbohydrates for energy, and it is absolutely dependent upon its microflora to, in effect, predigest its food. By virtue of its large size, the rumen has the function of slowing down the rate of passage of feed through the organ, which permits microbial digestion of essentially all of the nonstructural carbohydrate of the feed (starches and sugars) as well as over half of the more recalcitrant feed fiber (cellulose and hemicelluloses) (Van Soest, 1994). Rumen contents, which contain 6–18% dry matter, are mixed by strong muscular movement and are periodically returned via the esophagus to the mouth for additional chewing (rumination). Despite this, the solids have a tendency to stratify, with some maintaining a suspension in the rumen liquor, some settling to the bottom of the rumen, and some being borne up by gas bubbles to form a floating mat at the liquid surface. Passage rates vary with intake, with the rates for solids averaging about twice of that for liquids. From several published experiments, mean retention times for the rumen liquid range from 8 to 24 h, whereas that of the particulate phase range from 14 to 52 h (Broderick et al., 1991). The consequence of these long retention times for solids is that ruminant animals can use fibrous feeds (forages and certain agricultural byproducts) that are not usable by humans and other monogastric animals, with the ultimate conversion of these feedstuffs to useful products. In addition to VFAs, other products of the fermentation include microbial cells and fermentation gases. The microbial cells eventually pass through the omasum and into the abomasum (the acidic ‘‘true stomach’’), where the microbial cell protein is hydrolyzed to amino acids that are available for subsequent intestinal absorption. This microbial protein is a major contributor to the protein requirements of the animal, and it acts to counterbalance somewhat the considerable loss of feed protein that occurs as a result of microbial proteolysis and amino acid fermentation that occurs in the rumen (see Sec. IV.C.5). Fermentation gases include primarily carbon dioxide (50–70%) and methane (30–40%). Rates of gas production immediately after a meal can exceed 30 L/h, and a typical cow may release 500 L of methane per day (Wolin, 1990). Although some gas is absorbed across the rumen wall and carried by the blood to the lungs for exhalation, most is eructated through the mouth. III. MILK A. Milk Composition In the United States, milk has a strict legal definition: ‘‘the lacteal secretion, practically free of colostrum, obtained by complete milking of one or more healthy cows’’ (Office of the Federal Register, 1995). Parallel definitions are
Weimer provided for milk from goats and sheep (United States Public Health Service. 1993). Because of the central role of milk in the food supply and its ease of microbial contamination, production and processing of milk used for consump- tion is subject to tight regulation in most developed countries. In the United States, most milk is regulated according to the grade a Pasteurized Milk Ordi nance (United States Public Health Service, 1993), a document that sets the stan- dards for all aspects of milk production and processing. From a microbiological standpoint, the Pasteurized Milk Ordinance is important primarily in its setting the standards for acceptable numbers of viable microorganisms in milk before and after pasteurization. The ordinance sets limits for microbial counts in raw milk for pasteurization at 1 X 10/mL for milk from an individual producer and 3 X 10 /mL for commingled milk from multiple producers. The ordinance also establishes the permissible levels of antibiotic residues in milk, which affects the selection and implementation of antibiotic therapies to control infectious diseases dairy animals In addition to the direct contamination of milk with pathogens, many micro- organisms that are themselves not pathogenic can be responsible for altering the composition of milk after its synthesis. One example of a deleterious effect on milk is provided by mycotoxins. These compounds are secondary metabolites of fungi that can produce various toxic effects which can range from acute poisoning to carcinogenesis. The most widely known mycotoxins are the aflatoxins, which are produced by Aspergillus flavus, A. parasiticus, and A. nomius. Numerous structurally distinct aflatoxins have been identified(Fig. 3). The most notorious of these is aflatoxin B1, one of the most potent carcinogens known. Milk and dairy products may be contaminated by mycotoxins either directly(by contamination of milk or other dairy products with fungi followed by their growth) or indirectly (by contamination of animal feed with subsequent passage of the mycotoxin to milk)(van Egmond, 1989). In either event, contamination is largely dependent upon environmental conditions that determine the ability of the fungi to grow Two of the more potent aflatoxins, BI and B2, can be converted in the rumen to their respective 4-hydroxy derivatives, the somewhat less carcinogenic Mi and M2(see Fig 3). The extent of this conversion varies greatly among cows. For example, Patterson et al. (1980) reported that the Mi concentration in the milk of six cows fed approximately 10 ug aflatoxin B, /kg feed varied from 0.01 to 0.33 ug/L milk; on average 2.2%o of the ingested B, was converted to M, Applebaum et al. (1982)administered B, ruminally to 10 cows at higher doses (425-770 mg B/kg feed) and detected higher amounts of Bi in milk(1. 1-10.6 ug M,/L). Feeding of, or ruminal dosing with, high concentrations of Bl have significantly reduced feed intake and milk yield (Mertens, 1979). The effect is more powerful with impure B, than pure B1: suggesting the synergistic effects of other mycotoxins present in the impure preparation. Several other researchers have noted substantial differences in M, concentration among cows at similar o
6 Weimer provided for milk from goats and sheep (United States Public Health Service, 1993). Because of the central role of milk in the food supply and its ease of microbial contamination, production and processing of milk used for consumption is subject to tight regulation in most developed countries. In the United States, most milk is regulated according to the Grade A Pasteurized Milk Ordinance (United States Public Health Service, 1993), a document that sets the standards for all aspects of milk production and processing. From a microbiological standpoint, the Pasteurized Milk Ordinance is important primarily in its setting the standards for acceptable numbers of viable microorganisms in milk before and after pasteurization. The ordinance sets limits for microbial counts in raw milk for pasteurization at 1 105/mL for milk from an individual producer and 3 105 /mL for commingled milk from multiple producers. The ordinance also establishes the permissible levels of antibiotic residues in milk, which affects the selection and implementation of antibiotic therapies to control infectious diseases in dairy animals. In addition to the direct contamination of milk with pathogens, many microorganisms that are themselves not pathogenic can be responsible for altering the composition of milk after its synthesis. One example of a deleterious effect on milk is provided by mycotoxins. These compounds are secondary metabolites of fungi that can produce various toxic effects which can range from acute poisoning to carcinogenesis. The most widely known mycotoxins are the aflatoxins, which are produced by Aspergillus flavus, A. parasiticus, and A. nomius. Numerous structurally distinct aflatoxins have been identified (Fig. 3). The most notorious of these is aflatoxin B1, one of the most potent carcinogens known. Milk and dairy products may be contaminated by mycotoxins either directly (by contamination of milk or other dairy products with fungi followed by their growth) or indirectly (by contamination of animal feed with subsequent passage of the mycotoxin to milk) (van Egmond, 1989). In either event, contamination is largely dependent upon environmental conditions that determine the ability of the fungi to grow and produce toxins. Two of the more potent aflatoxins, B1 and B2, can be converted in the rumen to their respective 4-hydroxy derivatives, the somewhat less carcinogenic M1 and M2 (see Fig. 3). The extent of this conversion varies greatly among cows. For example, Patterson et al. (1980) reported that the M1 concentration in the milk of six cows fed approximately 10 µg aflatoxin B1/kg feed varied from 0.01 to 0.33 µg/L milk; on average 2.2% of the ingested B1 was converted to M1. Applebaum et al. (1982) administered B1 ruminally to 10 cows at higher doses (425–770 mg B1/kg feed) and detected higher amounts of B1 in milk (1.1–10.6 µg M1/L). Feeding of, or ruminal dosing with, high concentrations of B1 have significantly reduced feed intake and milk yield (Mertens, 1979). The effect is more powerful with impure B1 than pure B1; suggesting the synergistic effects of other mycotoxins present in the impure preparation. Several other researchers have noted substantial differences in M1 concentration among cows at similar or
Microbiology of the Dairy Animal OcH Aflatoxin B1 Aflatoxin M OCH Aflatoxin B2 Aflatoxin M 2 Figure 3 Bioconversion of aflatoxins B, and B2 to M, and M2, respectively different stages of milk production and milk yield and between milkings of the same cow(Kiermeier et al., 1977: Lafont et al 1980) B. Milk Biosynthesis In evaluating the microbial role in providing the animal with milk precursors, it is useful briefly to describe the biosynthesis of milk. a more detailed treatment of the process is provided by Bondi(1983) Although the mammary gland comprises only 5-7%o of the dairy cows ody weight, it represents perhaps the animals highest concentration of meta- bolic activity. Careful breeding and ad vances in nutrition over the years have resulted in the annual production of milk nutrients from a single cow sufficient to provide the nutrients required by 50 calves Milk is produced in secretory cells clustered in groups known as alveoli These cells feed milk through an arborescent duct system that collects milk into the udder. Production of milk is strongly controlled by endocrine hormones. Fol lowing parturition, the cells secrete antibody-rich colostrum for several days until
Microbiology of the Dairy Animal 7 Figure 3 Bioconversion of aflatoxins B1 and B2 to M1 and M2, respectively. different stages of milk production and milk yield and between milkings of the same cow (Kiermeier et al., 1977; Lafont et al., 1980). B. Milk Biosynthesis In evaluating the microbial role in providing the animal with milk precursors, it is useful briefly to describe the biosynthesis of milk. A more detailed treatment of the process is provided by Bondi (1983). Although the mammary gland comprises only 5–7% of the dairy cow’s body weight, it represents perhaps the animal’s highest concentration of metabolic activity. Careful breeding and advances in nutrition over the years have resulted in the annual production of milk nutrients from a single cow sufficient to provide the nutrients required by 50 calves. Milk is produced in secretory cells clustered in groups known as alveoli. These cells feed milk through an arborescent duct system that collects milk into the udder. Production of milk is strongly controlled by endocrine hormones. Following parturition, the cells secrete antibody-rich colostrum for several days until
milk secretion begins. Continued production of milk is stimulated by sucklin or by milking through the stimulation of several hormones, particularly prolactin. Nutrients for milk synthesis are provided to the udder through the blood via a pair of major arteries. The ability of the mammary gland to capture milk precursors effectively from the arterial blood supply-expressed as a"per cent extraction"calculated from the difference of precursor concentrations in arterial and venous blood-is truly impressive(Table 2)when one considers the rapid flow of arterial blood through the udder, which in dairy cows can approach 20 L/min. Production of 1 L of milk requires approximately 500 L of arterial blood flow through the udder. Milk is predominantly(80-87%)water. The majorcomponents of milk solids are lactose, protein, and fats. The composition of milk varies with feeding regimens, individual animals and breed. Marked differences are also noted among different ruminant species as well, with sheep's milk having substantially greater content of protein and fat than the milk of cows or goats (Table 3). Much of the energy re- quired for biosynthesis of milk in the udder is produced by oxidation of glucose (30-50%)or acetate(20-30%). In the ruminant animal, glucose is not derived directly from dietary carbohydrate, but is instead produced by gluconeogenic path ways, primarily using propionate, a major product of the ruminal fermentation. Lactose, a disaccharide of D-glucose and D-galactose linked by an a-1,4- glycosidic bond, is synthesized by a series of reactions using D-glucose as the starting substrate. Approximately 60% of the glucose consumed in the mammary gland is used for lactose synthesis. Lactose concentration in milk is relatively invariant with diet and stage of lactation, although its concentration declines sub- stantially in mastitic cows(see Sec. VIA) Table 2 Arterial Concentrations of milk Precursors and the Efficiency of Their Extraction in the Udder of goats Arterial Extraction efficiency Bloo 119 Glucose Acetate Lactate ource: Bondi. 1983
8 Weimer milk secretion begins. Continued production of milk is stimulated by suckling or by milking through the stimulation of several hormones, particularly prolactin. Nutrients for milk synthesis are provided to the udder through the blood via a pair of major arteries. The ability of the mammary gland to capture milk precursors effectively from the arterial blood supply—expressed as a ‘‘per cent extraction’’ calculated from the difference of precursor concentrations in arterial and venous blood—is truly impressive (Table 2) when one considers the rapid flow of arterial blood through the udder, which in dairy cows can approach 20 L/min. Production of 1 L of milk requires approximately 500 L of arterial blood flow through the udder. Milk ispredominantly (80–87%)water. The majorcomponents ofmilk solids are lactose, protein, and fats. The composition of milk varies with feeding regimens, individual animals, and breed. Marked differences are also noted among different ruminant species as well, with sheep’s milk having substantially greater content of protein and fat than the milk of cows or goats (Table 3). Much of the energy required for biosynthesis of milk in the udder is produced by oxidation of glucose (30–50%) or acetate (20–30%). In the ruminant animal, glucose is not derived directly from dietary carbohydrate, but is instead produced by gluconeogenic pathways, primarily using propionate, a major product of the ruminal fermentation. Lactose, a disaccharide of D-glucose and D-galactose linked by an α-1,4- glycosidic bond, is synthesized by a series of reactions using D-glucose as the starting substrate. Approximately 60% of the glucose consumed in the mammary gland is used for lactose synthesis. Lactose concentration in milk is relatively invariant with diet and stage of lactation, although its concentration declines substantially in mastitic cows (see Sec. VI.A). Table 2 Arterial Concentrations of Milk Precursors and the Efficiency of Their Extraction in the Udder of Goats Arterial Extraction concentration efficiency Precursor (mg/L) (%) Blood: O2 119 45 Glucose 445 33 Acetate 89 63 Lactate 67 30 Plasma: 3-Hydroxybutyrate 58 57 Triglycerides 219 40 Source: Bondi, 1983
Microbiology of the Dairy Animal Table 3 Mean Composition of Milk from Domestic ruminants by weight in milk Component cow goat shee Prote Lactose Ca 0.12 0.13 0.20 P 0 0.11 0.16 Source: Bondi. 1983 Milkfat is a heterogeneous combination of triglycerides with very few (<2%)phospholipid or sterols. Triglycerides are composed of glycerol esterified to three molecules of fatty acids having 4-20 carbon atoms (almost exclusively even numbered). In all mammalian species, the fatty acids are derived in part from circulatory lipoproteins produced from dietary or body fat. These lipoproteins are hydrolyzed at the endothelial capillary wall and are subsequently recombined to produce milk triglycerides. In ruminant animals, almost half of the fatty acids are synthesized from acetate produced in the ruminal fermentation and from 3- hydroxybutyrate produced in the rumen wall from butyrate, another ruminal fer- mentation product Milkfat content is subject to variations in diet; because milkfat is an important determinant of selling price, diets which depress milkfat yield are avo even if they provide good milk yields. The Pasteurized Milk Ordinance 28.25%"milk solids not fatand 23.25 fat (United States Public Health Service, 1993) Protein in milk is predominantly (82-86%)casein with smaller amounts of globulins. Milk proteins are synthesized from amino acids extracted from the arterial blood supply. These amino acids, in turn, are derived from several sources: synthesis by the animal, dietary protein that escapes the rumen, and microbial protein produced in the rumen and hydrolyzed to amino acids and pep- tides by passage through the abomasum(see Sec. IVC5) IV. MICROBIOLOGY OF THE RUMEN A. Methods Rumen microbiology is of historical importance in that the rumen was the first anaerobic habitat whose microbiology was systematically investigated. Many of techniques for study of strictly anaerobic microbes were developed in thes
Microbiology of the Dairy Animal 9 Table 3 Mean Composition of Milk from Domestic Ruminants % by weight in milk Component cow goat sheep Fat 3.5 4.5 7.4 Protein 2.9 2.9 5.5 Lactose 4.9 4.1 4.8 Ca 0.12 0.13 0.20 P 0.10 0.11 0.16 Source: Bondi, 1983. Milkfat is a heterogeneous combination of triglycerides with very few (2%) phospholipid or sterols. Triglycerides are composed of glycerol esterified to three molecules of fatty acids having 4–20 carbon atoms (almost exclusively even numbered). In all mammalian species, the fatty acids are derived in part from circulatory lipoproteins produced from dietary or body fat. These lipoproteins are hydrolyzed at the endothelial capillary wall and are subsequently recombined to produce milk triglycerides. In ruminant animals, almost half of the fatty acids are synthesized from acetate produced in the ruminal fermentation and from 3- hydroxybutyrate produced in the rumen wall from butyrate, another ruminal fermentation product. Milkfat content is subject to variations in diet; because milkfat is an important determinant of selling price, diets which depress milkfat yield are avoided even if they provide good milk yields. The Pasteurized Milk Ordinance stipulates that whole milk in its final packaged form for beverage use shall contain 8.25% ‘‘milk solids not fat’’ and 3.25% fat (United States Public Health Service, 1993). Protein in milk is predominantly (82–86%) casein with smaller amounts of globulins. Milk proteins are synthesized from amino acids extracted from the arterial blood supply. These amino acids, in turn, are derived from several sources: synthesis by the animal, dietary protein that escapes the rumen, and microbial protein produced in the rumen and hydrolyzed to amino acids and peptides by passage through the abomasum (see Sec. IV.C.5). IV. MICROBIOLOGY OF THE RUMEN A. Methods Rumen microbiology is of historical importance in that the rumen was the first anaerobic habitat whose microbiology was systematically investigated. Many of the techniques for study of strictly anaerobic microbes were developed in these