Non-lactating Gland Research Articles

Staphylococcus aureus intramammary challenge in non-lactating mammary glands stimulated to rapidly grow and develop with estradiol and progesterone

Intramammary infections (IMI) are prevalent in non-lactating dairy cattle and their occurrence during periods of significant mammary growth and development (i.e. pregnant heifers and dry cows) is believed to interfere with growth, development, and subsequent milk production. However, direct study of IMI impacts on non-lactating but developing mammary glands is lacking. The objectives of this study were to (1) define how IMI affected total and differential mammary secretion somatic cell counts in mammary glands stimulated to rapidly grow using estradiol and progesterone, and (2) characterize changes in mammary morphology in response to IMI. Mammary growth was stimulated in 19 non-pregnant, non-lactating cows and 2 quarters of each cow were subsequently infused with either saline (n = 19) or Staphylococcus aureus (n = 19). Mammary secretions were taken daily until mammary tissues were collected at either 5 or 10 days post-challenge. Staph. aureus quarter secretions yielded greater concentrations of somatic cells than saline quarters and contained a greater proportion of neutrophils. Staph. aureus mammary tissues exhibited higher degrees of immune cell infiltration in luminal and intralobular stroma compartments than saline quarters. Infected tissues also contained reduced areas of epithelium and tended to have greater amounts of intralobular stroma. Results indicate that IMI in non-lactating glands that were stimulated to grow, produced immune cell infiltration into mammary tissues and secretions, which was associated with changes in mammary tissue structure. The observed reduction of mammary epithelium indicates that IMI impair mammary development in rapidly growing mammary glands, which may reduce future reduced milk yields.

Stem/Progenitor Cells in Non-Lactating Versus Lactating Equine Mammary Gland

The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation, and involution. Based on the facts that (i) mammary stem/progenitor cells (MaSC) are proposed to be the driving forces behind mammary growth and function and (ii) variation exists between mammalian species with regard to physiological and pathological functioning of this organ, we believe that studying MaSC from different mammals is of great comparative interest. Over the years, important data has been gathered on MaSC of men and mice, although knowledge on MaSC in other mammals remains limited. Therefore, the aim of this work was to isolate and characterize MaSC from the mammary gland of horses. Hereby, our salient findings were that the isolated equine cells met the 2 in vitro hallmark properties of stem cells, namely the ability to self-renew and to differentiate into multiple cell lineages. Moreover, the cells were immunophenotyped using markers for CD29, CD44, CD49f, and Ki67. Finally, we propose the mammosphere assay as a valuable in vitro assay to study MaSC during different physiological phases since it was observed that equine lactating mammary gland contains significantly more mammosphere-initiating cells than the inactive, nonlactating gland (a reflection of MaSC self-renewal) and, moreover, that these spheres were significantly larger in size upon initial cultivation (a reflection of progenitor cell proliferation). Taken together, this study not only extends the current knowledge of mammary gland biology, but also benefits the comparative approach to study and compare MaSC in different mammalian species.

P28. Primary mammary epithelial cells from the lactating epithelium have distinct growth and differentiation capacity

The mammary gland is a unique organ in its ability to undergo repetitive cycles of growth, differentiation, function and involution in the mature adult. These processes depend upon a pool of committed multipotent cells that undergo asymmetric division to give rise to more differentiated progeny that proliferate at the onset of gestation. It is normally not possible to access these cells from lactating women to generate primary mammary epithelial cultures for research as surgical intervention is rare during lactation. We have identified cells expressing markers for multipotent mammary epithelial cells (CD49f, CD24 and p63) in human breastmilk samples. These primary mammary epithelial cells (PMEC) can be expanded and manipulated in epithelial-specific monolayer culture to produce differentiated cells expressing luminal (CK18) and myoepithelial (CK14) markers. Using the immortalised non-tumorigenic mammary line MCF10A and tumorigenic mammary line MDA231 as a comparison, we show that PMEC have a distinctive response to extra-cellular matrix (ECM) and growth and functional regulators. In monolayer culture PMEC bypass replicative senescence at day 8 in response to ethanolamine and hormones prolactin, placental lactogen and RANKL, while immortal lines show no response in their rapid growth pattern. In three-dimensional ECM culture PMEC and MCF10A produce alveolus-like structures that maintain a basal layer of CD49f-positive cells and a luminal layer of CK18-positive cells, while MDA231 form disorganized invasive colonies. Uniquely, PMEC also produce hallmark milk proteins beta-casein and alpha-lactalbumin in response to prolactin stimulation in the luminal secretory layer. These data imply that the cell population expanded during lactation is receptive to distinct stimuli that results in differentiation in ways not observed in cells taken from the non-lactating gland. We demonstrate that human milk is a non-invasive source of these cells, providing a tool for further research into the specific pathways that regulate differentiation. Using such models may be useful to further understand mechanisms that underlie mammary gland pathologies.

Expression and Regulation of Glucose Transporters in the Bovine Mammary Gland

Glucose is the primary precursor for the synthesis of lactose, which controls milk volume by maintaining the osmolarity of milk. Glucose uptake in the mammary gland plays a key role in milk production. Glucose transport across the plasma membranes of mammalian cells is carried out by 2 distinct processes: facilitative transport, mediated by a family of facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Transport kinetic studies indicate that glucose transport across the plasma membrane of the lactating bovine mammary epithelial cell has a Km value of 8.29mM for 3-O-methyl-d-glucose and can be inhibited by both cytochalasin-B and phloretin, indicating a facilitative transport process. This is consistent with the observation that in the lactating bovine mammary gland, GLUT1 is the predominant glucose transporter. However, the bovine lactating mammary gland also expresses GLUT3, GLUT4, GLUT5, GLUT8, GLUT12, and sodium-dependent SGLT1 and SGLT2 at different levels. Studies of protein expression and cellular and subcellular localizations of these transporters are needed to address their physiological functions in the mammary gland. From late pregnancy to early lactation, expression of GLUT1, GLUT8, GLUT12, SGLT1, and SGLT2 mRNA increases from at least 5-fold to several hundred-fold, suggesting that these transporters may be regulated by lactogenic hormones and have roles in milk synthesis. The GLUT1 protein is detected in lactating mammary epithelial cells. Its expression level decreases from early to late lactation stages and becomes barely detectable in the nonlactating gland. Both GLUT1 mRNA and protein levels in the lactating mammary gland are not significantly affected by exogenous bovine growth hormone, and, in addition, GLUT1 mRNA does not appear to be affected by leptin.

Secretory phenomena in the non-lactating human mammary gland

The normal non-lactating premenopausal human mammary gland has been shown by immunohistochemistry and transmission electron microscopy to secrete a number of antimicrobial peptides such as beta-defensins, the cathelicidin LL37, lactoferrin and adrenomedullin. In addition, the non-lactating gland elaborates a prominent glycocalyx at the apical membrane of the glandular epithelial cells, parts of which are shed into the lumen of endpieces and ducts. This glycocalyx includes the mucins MUC 1 and MUC 4, a strongly Alcian Blue positive palyanionic component and sulfated material stained with Aldehyde Fuchsin. MUC 1 and the Alcian Blue positive material are considered to play an antimicrobial role, too. Lactalbumin and lipid droplets also occur in the non-lactating gland. At the EM-level secretory phenomena operating by exocytosis and by means of the apocrine mechanism have been observed. Cytoskeletal components presumably play a role in apocrine secretion. Apart from secretion at the cellular apex, secretion at the cellular basis also occurs regularly, which may represent the production of para- or endocrine factors.

Coliform mastitis.

Gram-negative bacteria that commonly cause bovine mastitis are classified as environmental pathogens. The point sources of coliform bacteria that cause infections include bedding materials, soil, manure and other organic matter in the environment of cows. Rates of coliform mastitis increase during climatic periods that maximize populations in the environment. The portal of entry into the mammary gland for Gram-negative bacteria is the teat canal. Once in the gland, bacteria must utilize available substrates in the mammary secretion to replicate and evade host defenses. Rates of coliform mastitis are greater during the transitional phases of the non- lactating period than during lactation. The ability to infect the non-lactating gland is directly related to the ability of bacteria to acquire iron from the mammary secretion. The primary host defense against coliform mastitis during lactation is the elimination of bacteria by neutrophils migrating into the gland in response to inflammation. Damage to the host is mediated by the release of endotoxin. The severity and duration of clinical signs associated with coliform mastitis are reduced by the use of core-antigen bacterins.

Defective localization of the Wilson disease protein (ATP7B) in the mammary gland of the toxic milk mouse and the effects of copper supplementation

Toxic milk (tx) is a copper disorder of mice that causes a hepatic accumulation of copper similar to that seen in patients with Wilson disease. Both disorders are caused by a defect in the ATP7B copper-transporting ATPase. A feature of the tx phenotype is the production of copper-deficient milk by lactating dams homozygous for the tx mutation; the milk is lethal to the pups. It has not been determined whether the production of copper-deficient milk is a direct consequence of impaired expression of ATP7B protein in the mammary gland. With the use of immunohistochemistry, our study demonstrated that the ATP7B protein was mislocalized in the lactating tx mouse mammary gland, which would explain the inability of the tx mouse to secrete normal amounts of copper in milk. Confocal microscopy analysis showed that, in the lactating tx mammary gland, ATP7B was predominantly perinuclear in comparison with the diffuse, cytoplasmic localization of ATP7B in the lactating normal mammary gland. Lactating tx mice showed impaired delivery of copper from the mammary gland to the milk and this was not ameliorated by dietary copper supplementation. In contrast, the normal mouse mammary gland responded to increased dietary copper by increasing the amount of copper in milk. A change in the distribution of the ATP7B protein from perinuclear in the non-lactating gland to a diffuse, cytoplasmic localization in the lactating gland of the normal (DL) mouse suggests that the relocalization of APT7B is a physiological process that accompanies lactation. We conclude that the impaired copper transport from the mammary gland into milk in lactating tx mice is related to the mislocalization of ATP7B.

Mesotocin receptors during pregnancy, parturition and lactation in the tammar wallaby.

Mestocin receptor concentrations in membrane preparations from reproductive tissues of the tammar Macropus eugenii throughout gestation and lactation were assessed using [3H]-oxytocin as the ligand. There was a single binding site which bound both mesotocin and oxytocin with high and similar affinities. Mesotocin receptor concentrations in the myometrium were low (708 +/- 199 fmol mg-1 protein) in early and middle gestation but increased significantly on day 23 of pregnancy of the 26-day gestation period to 1921 +/- 552 fmol mg-1 protein. Myometrial receptors reached a peak of 2483 +/- 575 fmol mg-1 protein on days 25 and 26 of gestation, but returned to basal levels about an hour after birth. Receptor concentrations in the contralateral non-gravid uterus were much lower (605 +/- 75 fmol mg-1) and did not significantly increase throughout the period of gestation but dropped one day before birth. Mesotocin receptors were undetectable in the endometrium, the yolk sac placenta and the lateral, median and anterior vagina of all animals tested. In the lactating mammary gland after birth mesotocin receptors were initially high (588 +/- 38 fmol mg-1) but decreased after 200 days and by late lactation were 224 +/- 55 fmol mg-1 protein on day 240, close to the time of weaning. Mesotocin receptors in the ipsilateral non-lactating gland were also high in early lactation (430 +/- 153 fmol mg-1) and declined in late lactation (62 +/- 20 fmol mg-1). The changing concentrations of mesotocin receptors in pregnancy and lactation demonstrate that they are specifically regulated in tammar reproductive tissues. The increase in mesotocin receptors in gravid, but not in the non-gravid myometrium three days before birth may make the uterus responsive to the surge of mesotocin at birth. Since this rise is unilateral and only occurs in the gravid myometrium it must be due to local effects from the ipsilateral ovary or the feto-placental unit. Likewise, the down-regulation of mesotocin receptors in the contralateral, non-gravid myometrium may be due to its proximity to the developing follicle. The changing concentrations in the lactating and the adjacent, non-lactating mammary gland also reflect a differential regulation of mesotocin receptors, probably mediated via the sucking stimulus. Thus, local influences appear to be of primary importance in the regulation of mesotocin receptors during reproduction in this marsupial.

Spontaneous mouse mammary tumours: incidence and cytokeratin expression

The purpose of this study was to advance our knowledge of the histogenesis of spontaneous mammary tumours in laboratory mice. Normal mammary tissue and 19 spontaneous mammary tumours from adult female mice Were examined using monoclonal and polyclonal antibodies differing in their recognition of various cytokeratin intermediate filament proteins ( CKs). All neoplasms were intraductal and were invasive carcinomas with a tubular, papillary, cystic or solid growth pattern. CK8-positive reactions Were detected in the normal alveolar and ductal epithelia and CK5- and CK14-positive reactions were seen in myoepithelial cells of nonlactating mammary glands. Positive staining for CK5 and CK8 was detected in all tumours and CK14 was expressed in those with a papillary pattern. Comparisons between non-lactating glands and tumours indicated that the neoplasms were well or moderately differentiated, there was no squamoid differentiation and that they arose from the alveoli and duct system, not the myoepithelial cells.

Thymidine Incorporation by Lactating Mammary Epithelium During Compensatory Mammary Growth in Beef Cattle

Ten Hereford cows, 100 d into first lactation, were assigned to treatment or control groups to study compensatory growth of mammary glands. The right udder half of treatment cows was covered to prevent suckling by the calf, whereas control cows were suckled on all quarters. Milk production was estimated the day treatment began and 4 d later by machine milking following removal of calves for 12h and i.v. injection of oxytocin. Five to 7 d after beginning treatment, cows were killed and mammary tissue was obtained from three regions within left and right glands for in vitro incubation with [3H]thymidine. Deoxyribonucleic acid of lactating udder halves did not increase in response to treatment although RNA: DNA ratio and milk production tended to increase. Incorporation of 3Hthymidine was greater in lactating quarters of treated cows than control cows (35,000 vs. 19,000 cpm/mg of DNA) with greatest incorporation in the basal regions of each gland. Furthermore, greatest incorporation of [3H]thymidine occurred in nonsuckled glands. Autoradiographic analyses confirmed incorporation data and indicated that 81% of proliferating cells were epithelial. Data suggest that proliferation of mammary epithelial cells, within both the lactating and nonlactating glands, occurred in response to milk stasis.

Lectin histochemical characteristics of the canine female mammary gland

SUMMARY Twelve biotinylated lectins and an avidin-biotin-peroxidase method were used to detect and localize specific carbohydrate residues on formalin-fixed, paraffin-embedded female canine mammary gland sections. Histologic sections from 3 lactating and 7 nonlactating mixed-breed dogs (age 5.6 ± 0.35 years) were incubated with Arachis hypogea agglutinin (peanut agglutunin; pna), Concanavalia ensiformis agglutinin (conA), Dolichos biflorus agglutinin (dba), Glycine max agglutinin (sba), Griffonia simplicifolia agglutinin-I (gs-I), Lens culinaris agglutinin (lca), Lycopersicon esculentum agglutinin (lea), Phytolacca americana mitogen (pokeweed mitogen; pwm), Ricinus communis agglutinin-I and-II (rca-I and -II), Triticum vulgaris (wga), and Ulex europaeus agglutinin-I (uea-I). Each lectin had a specific binding pattern, except sba and dba. In nonlactating glands, pna, conA, lea, and uea-I stained duct cells in a linear-binding pattern, with a mean percentage of positive ducts per section of 28.7 (± 0.6), 65.7 (± 0.3), 100 (± 0), and 8.4 (± 0.2), respectively. Strong apical, lateral, basal, and cytoplasmic positivity on duct cells was seen after incubation of the sections with rca-I, rca-II, and wga in all ducts. In acinar cells, the binding pattern and the staining distribution of all the lectins studied were similar to those in duct cells. However, for pna, conA, and uea-I, the mean percentage of positive lobules per section was 33.7 (± 0.9), 62 (± 0.5), and 10.5 (± 0.2), respectively. In glands from lactating dogs, conA and uea-I did not stain. The cytoplasm of all myoepithelial cells was moderately stained with rca-I, rca-II, and wga. Endothelial cells stained with gs-I, pwm, rca-I, rca-II, wga, conA, and lca. The extracellular matrix, especially the periacinar and periduct regions, and the interstitial fibroblasts were positive for lca, rca-I, rca-II, and wga. Peripheral unmyelinated nerve fibers of the nipple were strongly positive for GS-I, pwm, rca-I, rca-II, and wga. Some of the lectins used (ie, pna, conA, uea-I, gs-I, pwm, and lea) appear to have selective staining of mammary gland structures that seems to be correlated with various physiologic functions. The contrasting binding pattern of lectins specific for the same sugar indicates a lack of knowledge of interactions between lectins and carbohydrate residues in tissue sections.

Infection following challenge of the lactating and dry udder of dairy cows with Actinomyces pyogenes and Peptostreptococcus indolicus

Challenge of 12 mammary glands of cows in mid-lactation with 10 7 colony forming units (cfu) of Peptostreptococcus indolicus on two occasions led to clinical mastitis in only four quarters. The bacteria were rarely recovered and disappeared from the secretion within 14 days. In challenges 7 days prior to drying off eight of 12 quarters became infected and at drying off all quarters challenged became infected. The infections established at drying off persisted well into the dry period. P. indolicus infection was also established in all of 12 dry glands challenged, but usually eliminated at calving or early in the next lactation. Isolation of P. indolicus was accompanied in about one-third of cases by changes in the appearance of the secretion. Intramammary challenge with Actinomyces (formally Corynebacterium) pyogenes led to clinical and subclinical infections in nine of 12 lactating glands and in all of six dry glands. Dry period infections with A. pyogenes were more severe and rarely eliminated even by antibiotic therapy. Infections during lactation were often eliminated either naturally or by antibiotic therapy. Intermittent recovery of A. pyogenes from the lactating mammary gland, without clinical signs of infection, was possible for up to 90 days after challenge. Combined infections with A. pyogenes and P. indolicus were clinically more severe with a higher frequency of systemic involvement. It was shown that in the non-lactating gland an acute mastitis, similar to ‘summer mastitis’ could be established either by simultaneous inoculation with A. pyogenes and P. indolicus or by subsequent inoculation of quarters excreting P. indolicus with A. pyogenes.

Kinetics of endotoxin-induced inflammation in ovine mammary gland.

The inflammatory response to stimulation with endotoxin of lactating and nonlactating mammary glands of sheep was examined. Similar numbers of neutrophils and mononuclear cells were recovered from nonlactating glands sampled every 2 h for 8 h and in glands sampled once at 8 h. Thus the inflammatory response was initiated by 2 h and repeated sampling did not modify the time course of the response. In contrast, in lactating ewes, fewer cells were recovered from glands sampled every 2 h than from glands sampled once at 8 h. Fewer neutrophils were also recovered when glands were serially sampled from 4 h to 8 h. Thus, removal of milk and inflammatory exudate modified the time course of the leukocyte influx into lactating glands. Significant accumulation of neutrophils occurred by 2 h in dose-response experiments in nonlactating glands. Peak accumulation of neutrophils occurred between 2 and 6 h, and a marked decline occurred after 8 h. In lactating glands, a slower onset and longer duration of neutrophil accumulation occurred. Twenty- to 30-fold more neutrophils were recovered by 8 h in lactating than nonlactating glands. This difference was not due to a lower threshold of sensitivity to endotoxin. Infusion of milk into nonlactating glands did not modify the intensity or time course of the inflammatory response to endotoxin. Thus, the physiological state of resident cells within the lactating gland, rather than the interaction of inflammatory exudate with milk, can account for the different reaction pattern in lactating glands. Inflammation in the nonlactating gland closely resembles inflammatory responses in skin and provides a convenient model for investigating the initiation and regulation of inflammatory processes.

Studies on the inflammatory response during involution of the ovine mammary gland.

Cessation of lactation, initiated by the removal of lambs from their mothers, induced an inflammatory response with elevated numbers of neutrophil leucocytes in mammary secretions being detected within 16 h. Leucocyte numbers were still elevated 7 d later. Infusion of whole milk into non-lactating glands caused no change in leucocyte numbers after 24 h. Involution secretions, however, had the capacity to induce an inflammatory response when infused into non-lactating glands. Daily infusion of endotoxin (100 ng) into non-lactating glands resulted in a marked attenuation of the inflammatory response. Such desensitization did not accompany reinfusion of involution secretion. Responses to involution secretion were enhanced in glands desensitized to endotoxin. The failure of desensitization to develop in glands receiving repeated infusions of involution secretion and the lack of cross-desensitization between involution secretion and endotoxin indicate that a novel mediator of inflammation may be produced during mammary involution in sheep.

The inflammatory activity of activated complement in ovine and bovine mammary glands.

Infusion of zymosan-activated plasma (ZAP), a source of activated complement, induced inflammation in non-lactating mammary glands but not in lactating glands of ewes and cows. Interstitial injection of ZAP in lactating ovine glands also failed to induce inflammation as assessed by the neutrophil content of milk. The inflammatory activity of ZAP in non-lactating glands was inhibited by infusion of whole milk but not by skimmed milk. These results suggest that components of whole milk mask the inflammatory activity of activated complement. In addition, lactating mammary tissue of ewes and cows may lack sensitivity to the inflammatory effect of activated complement. Thus, activated complement cannot be an important mediator of inflammation in the lactating gland.

In Vitro Growth of Mastitis Associated Streptococci in Bovine Mammary Secretions1,2

Cell-free, fat-free mammary secretions were tested in vitro for ability to support growth of streptococci associated with mastitis. Secretions were obtained prior to drying off, during the dry period, at calving, and during lactation from four cow treatment groups. Treatment groups were 1) dry cow therapy, 2) dry cow therapy and mammary glands subjected to induced inflammation 7 d postdrying-off, 3) no dry cow therapy and no induced inflammation, 4) no dry cow therapy but mammary glands subjected to induced inflammation. Growth of Streptococcus uberis, Streptococcus faecalis, and Streptococcus agalactiae in secretions from nonlactating glands was unaffected by induced inflammation. Growth of Streptococcus bovis was significantly inhibited in secretion obtained 14 d after induced inflammation. Dry cow therapy had no effect on streptococcal growth in secretion obtained 7 d after therapy. Streptococcal growth was greatest in secretions from involuted glands, and there was little or no evidence for growth inhibitory factors in cell-free, fat-free secretions obtained during the dry period. Milk from lactating glands inhibited streptococcal growth, and the inhibitory factor was presumptively identified as lactoperoxidase. Apolactoferrin, immunoglobulin, or both had little effect on streptococcal growth.

Durikainema macropi gen. et sp. nov. (Muspiceoidea: Robertdollfusidae). A remarkable nematode from Macropodidae (Marsupialia).

Durikainema macropi gen. et sp. nov. (Muspiceoidea: Robertdollfusidae) is described from the mesenteric and hepatic portal veins of Macropus giganteus Shaw 1790, M. agilis (Gould 1842) and M. rufogriseus (Desmarest 1817) (Marsupialia: Macropodidae) from Queensland, Australia. It is also known from histological sections of hepatic portal veins of M. robustus Gould 1841, M. fuliginosus (Desmarest 1817) and Lagorchestes conspicillatus Gould 1842. The new genus resembles the Enoplina in cephalic and caudal characters and the Dorylaimina in other characters. Durikainema resembles Robertdollfusa Chabaud and Campana 1950 in its small form, absence of mouth oesophagus and anus, atrophied digestive tube, reduction of female genital apparatus to a uterine pouch, viviparity and cephalic cuticular inflation in larvae. It differs from this genus in its complex and well developed cephalic structures and its well developed body musculature in both sexes. Durikainema is tentatively placed in the Robertdollfusidae, Muspiceoidea. Larvae develop beyond firststage in the uterus of the female. They have been found in the non-peripheral blood of male and female M. giganteus, the lactating mammary gland of female M. agilis but not the non-lactating glands of the same female and in the deep capillaries of thigh skin of male M. agilis. Transmission of the parasite may be direct by a percutaneous or milk route, or indirect by a haemophagous arthropod.

Immune Response in the Mammary Gland

1.Three classes of immunoglobulins have been identified in lacteal secretions. IgG1 appears to be selectively transferred through the glandular epithelium into the udder. Presumably any method of immunization that stimulates circulating antibodies of the IgG1 class will be beneficial, resulting in IgG1 antibodies in the milk.2.IgA class immunoglobulins are also found in the milk, however, in lower concentration than IgG1 immunoglobulins. IgA is produced locally in the gland since an increase is seen in antigen-infused quarters over noninfused quarters. A factor that might be important is that probably the total amount of any immunoglobulin in lacteal secretions will not be increased by systemic immunization whereas local immunization does increase the amount of IgA.3.The method and route of immunization is possibly very important. Intramuscular, subcutaneous, or other routes that result in systemic stimulation of antibodies, to be effective, must for the most part stimulate class IgG1 immunoglobulins. Hammer, Kickhofen, and Henning (7) have recently published that there is a quantitative shift of antibody activity from IgG1 to IgG2 between the first and third courses of immunization of cattle (7). These IgG2 antibodies would be of little value for mammary gland immunity insofar as antibodies in the milk are concerned. However, since the tissues of the mammary gland are involved in a mastitis infection, specific antibodies of all immunoglobulin classes may be beneficial. Immunization by inoculation of a Streptococcus agalactiae, type II preparation into the area of the supramammary lymph node stimulates protective antibodies in the milk. These antibodies have been shown to be of IgG class principally, with less IgA. Finally, by intramammary infusion of antigen into one or more quarters of a nonlactating gland, an increase in specific antibodies of only the IgA class was noted.4.Progress in the determination of the immunogenic substance necessary to stimulate resistance to staphylococcal mastitis has been slow. Serological typing of staphylococci is not available; however, on the basis of phage typing, most mastitis staphylococci may fall into a dozen or so types (14). Serological typing and the immunogenic response of the group B streptococci are much further advanced. The immunology of the group C and group E streptococci is also in a primitive state. It probably is not necessary to separate and purify each immunogen that is required in a preparation; however, it is necessary that inactivated bacteria of each different antigenic type be present in a bacterin in order to stimulate all the various antibodies necessary.One can speculate that a combination of immunization routes that result in the stimulation of several of the classes of immunoglobulins might produce sufficient protective antibodies in the milk and in the mammary gland tissues to increase resistance. Local stimulation would increase specific IgA while systemic stimulation would result in specific IgG1, IgG2 and IgM. Local stimulation would presumably have to occur in the nonlactating gland with a nonirritating or nontissue damaging antigen. The antigen may be a bacterin consisting of a combination of each of the types of staphylococci and streptococci and be inactivated with formalin or some other inactivant. The bacterin should also contain staphylococcal toxoids.

Lactoferrin and IgG Immunoglobulins from Involuted Bovine Mammary Glands

Lactoferrin and IgG were major proteins in whey from involuted bovine mammary glands. Thirty to 50% of the protein with gamma electrophoretic mobility (pH 8.9) was lactoferrin. IgG and lactoferrin were not separated by electrophoresis or filtration through Sephadex G-200. Lactoferrin can be separated from fast-IgG, but not slow-IgG, by ion-exchange chromatography on DEAE Sephadex A-50. Separation of slow-IgG and lactoferrin can be achieved by ion-exchange chromatography on DEAE Sephadex A-50 followed by filtration through Sephadex G-200. These results suggest that lactoferrin, as it exists in whole whey, is in the form of a complex which is broken down by ion-exchange chromatography. While the ratio of fast-IgG to slow-IgG in blood serum is near unity, the concentration of fast-IgG is 3- to 5-fold greater than slow-IgG in the secretion from nonlactating glands. This finding suggests that the mechanism for selective transport of fast-IgG from blood serum to lacteal fluid may be operative in the nonlactating gland.

TRNA and aminoacyl-tRNA synthetases during differentiation and various functional states of the mammary gland

tRNA and aminoacyl-tRNA synthetases were obtained from undifferentiated virginal and differentiated (lactating and non-lactating) bovine and rabbit mammary glands. Comparative study indicates that the quantitative ratio of some individual tRNA's and possibly aminoacyl-tRNA synthetases in total preparations changes during differentiation in the mammary gland. The ability of IO − 4 to inactivate uncharged tRNA was used to examine charging levels of some tRNA's in vivo. The pool of aminoacyl-tRNA's in the lactating mammary gland proved to correspond to the amino acid composition of the main secretory proteins. The results of the study on the heterogeneity of leucyl-tRNA show that lactating cells synthesize leucine tRNA's of which one species is either absent in the non-lactating gland or present in undetectable concentration. Considerable difference was found in the ability of enzymes of virginal, lactating and non-lactating mammary glands to cross-react with heterologous tRNA.