Experimental udder infections with Escherichia coli. 4. Discussion of the pathogenesis of acute mastitis

The correlations among findings obtained from experimental Escherichia coli udder infection and reported in the three previous communications are discussed in this paper. The defence capabilities elucidated included specific and unspecific activities of non-epithelial cells, primarily neutrophilic granulocytes, macrophages, lymphocytes, plasma cells, and mast cells, as well as the capability of epithelial cells proper of absorbing pathogenic material from lumens of lactiferous ducts and milk cisterns and of storing such material within intracytoplasmic vacuoles. Hence, alterations in the course of pathogenesis of acute coli mastitis were found to be of complex nature and could be properly followed up by their morphological patterns.     

Pathogenesis of experimental bovine mastitis following a small inoculum of Escherichia coli

Mastitis was produced in four quarters of two lactating cows by the inoculation of 50 or 200 viable Escherichia coli. Changes were investigated by light microscopy and scanning electron microscopy. After 10 hours the changes were confined to the superficial layer of the epithelium of the teat and lactiferous sinuses; single cells or small groups of cells were damaged and were being extruded from the tissue. By 14 hours there was extensive necrosis and sloughing of the epithelial cells which did not extend beyond the basement membrane, and an intense inflammatory response associated with the epithelial damage. The somatic cells in the milk, mainly polymorphonuclear leucocytes, increased 40 to 250 times and strongly inhibited the survival of E coli. Epithelial damage appeared first in the ventral portions of the gland and then extended through the lactiferous sinus to the large ducts and secreting tissue. This pattern of damage together with the failure to observe bacteria attached to the epithelia was consistent with the production of diffusible toxins by the organisms.     

Pathogenicity of Mycoplasma agalactiae subsp. bovis in goat mammary gland

Clinical mastitis was produced in three goats following inoculation into the mammary glands with 10⁵ colony-forming units (cfu)/ml of a local strain of Mycoplasma agalactiae subsp. bovis. The infection was characterized by pyrexia, reduction in milk yield and acute purulent inflammation of the lactiferous sinus and ducts, necrosis of the duct epithelium and by the 5th day, early proliferation of chronic inflammatory cells in the parenchyma.     

Experimental udder infection with Escherichia coli. 1. The behavior of selected clinical, hematological and other parameters concerned with milk composition

Four cows with clinically intact udders in high lactation were intracisternally infected by a pathogenic field strain of Escherichia coli. Infections were induced by quarters and with time intervals. Hence, when the animals were slaughtered six hours from beginning of the experiment, epithelial samples could be collected from cisternal walls and lactiferous ducts which, by the time of sampling, had been in contact with pathogens for one, four, and six hours. Highly acute mastitis began to develop two hours from experimental infection and exhibited typical clinical symptoms, including general disorders, unambiguous alterations to the white blood count, and changes in milk composition. These changes were typical of acute toxin action on the udder quarters. Concomitant reactions of non-infected control quarters, detectable by clinical manifestations or by laboratory diagnosis, were not observed.     

The effect of Escherichia coli endotoxin and culture filtrate on the lactating bovine mammary gland

The pathogenesis of coliform mastitis was studied by observing pathological changes in lactating glands after infusion of either endotoxin or the sterile culture filtrate (CCF) of the medium in which Escherichia coli strain B117 had been grown. Both infusions produced a rapid and intense inflammatory response by 4 h with a marked increase of serum proteins in the milk. Before dispersing into the milk, neutrophils were attached to the ductular epithelium; highest cell counts in the milk were recorded when the tissue reaction had waned. Oedema of the ductular epithelium occurred, particularly where neutrophils were actively migrating. The infusion of CCF produced, in addition to inflammation, degeneration and necrosis of ductular cells. The smallest lesions healed very rapidly. There was evidence of differing cell susceptibility to the necrotising toxin as well as uneven distribution over the epithelial surface. All changes observed were confined to the regions of the teat and lactiferous sinuses with little effect on the secreting tissue. The role of the necrotising toxin in the natural disease remains undetermined.     

A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders.     

Binding of host glycosaminoglycans and milk proteins: possible role in the pathogenesis of Streptococcus uberis mastitis

The role of indirect binding of host proteins through glycosaminoglycans (GAGs) on adherence and internalization of Streptococcus uberis to bovine mammary epithelial cells was evaluated. Preincubation of S. uberis with GAGs followed by incubation with fetal bovine serum (FBS), bovine milk or milk proteins resulted in greater adherence to and internalization of S. uberis into mammary epithelial cells than observed in untreated controls. Highest values were detected, when final incubation was done with milk. Greater adherence to and internalization into mammary epithelial cells were observed when heparin sulfate (HEP) and milk were used compared with any other GAG and FBS. When individual milk proteins were used, greatest adherence and internalization were observed when S. uberis strains were pretreated with HEP followed by treatment with beta-casein. The findings of this study illustrate a pathogenic strategy of S. uberis that may occur during the very early stages of infection.     

Cell types and their immunological functions in bovine mammary tissues and secretions--a review of the literature

In this work the literature concerning cells related to bovine mammary gland defence mechanisms has been reviewed. The cells considered in this review include leucocytes from mammary secretions, leucocytes located in the mammary tissues, and nonsecretory epithelial cells which line the teat and the lactiferous sinuses of the udder. The mammary secretions and tissues basically contain three types of cells: polymorphonuclear leucocytes, macrophages, and lymphocytes. The number of each type of cell varies, depending on the physiological and pathological states of the udder, and all cell functions are associated with the immuno-defence mechanisms of the mammary gland.     

Immunoglobulins, lysozyme and lactoferrin in the teat and udder of the dry cow during endotoxin-induced inflammation.

Immunoglobulins (Ig) and antibacterial proteins like lysozyme and lactoferrin are components of the humoral defence against infections. Changes in Ig, lysozyme and lactoferrin concentrations during endotoxin-induced inflammation in the test cistern and udder quarter of the dry cow were studied. Surgical closure of the passage between teat and udder cisterns enabled studies of reactions in the teat cistern without interference of the mammary gland. After endotoxin infusion, IgG1, IgG2, lysozyme, and to some extent IgM, increased in the teats and udder quarters, and were positively correlated with changes in somatic cell counts. No significant changes were observed in IgA or lactoferrin. The origin and significance of Ig, lysozyme and lactoferrin in the bovine teat and udder are discussed. Ig probably originated both from serum and from local plasma cells, while leukocytes appeared to be the source of lysozyme during inflammation. Secretory epithelium appeared to be the source of lactoferrin. Support for this theory was the almost total absence of lactoferrin in teat cistern samples.     

Pathogenesis of Staphylococcus aureus mastitis: bacteriologic, histologic, and ultrastructural pathologic findings

Three lactating cows were experimentally infected with Staphylococcus aureus ATCC 29740 (Newbould 305 strain). Cows were euthanatized 2 to 216 hours after inoculation. Bacteriologic and microscopic examinations showed that S aureus attached to epithelial cells of the mammary gland in vivo. The histopathologic changes observed were progressive swelling, vacuolar degeneration of epithelial layers, and multiple foci of epithelial erosions and ulcers throughout the ductal system. The cellular response of the infected glands was demonstrated by a rapid increase in the number of somatic cells in the secretion and by accumulation of neutrophils below, within, and on the epithelium of the teat and lactiferous sinuses. The inflammatory response did not prevent infection nor subsequent pathologic changes in the inoculated glands.     

Immunohistochemical detection of Mycoplasma agalactiae in formalin-fixed,paraffin-embedded tissues from naturally and experimentally infected goats

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin-fixed, paraffin-wax-embedded sections and labelled by the avidin-biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody-based immunohistochemical technique is an efficient and specific method for the post-mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.     

Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk

Adiponectin is an adipocyte-derived hormone, which circulates in the form of homo-multimers. The individual oligomers have a distinct profile of activity, playing crucial roles in several biological processes, including metabolism and inflammation. Adiponectin exerts many of its effects by interacting with the receptors, AdipoR1 and AdipoR2. In the present study, mRNA expression of adiponectin, AdipoR1 and AdipoR2 was evaluated by quantitative PCR in different areas of the mammary gland in healthy lactating cows. The adiponectin isoforms in milk and blood were investigated by Western blotting and 2D-electrophoresis, and the presence of adiponectin protein was determined by immunohistochemistry.Low level expression of adiponectin mRNA was found in all areas of bovine mammary gland tissues examined. AdipoR1 and AdipoR2 mRNAs were also detected in mammary tissues and their expression was particularly prominent in the parenchyma and cistern. Western blotting revealed a heterogeneous electrophoretic pattern, indicating that different adiponectin isoforms exist in milk, compared with blood. In particular, milk shows a low molecular weight isoform of adiponectin, corresponding to the globular domain. Adiponectin in milk is characterised by a more complex 2D electrophoretic pattern, compared with blood, as illustrated by the presence of proteins of different molecular weights and isoelectric points. Adiponectin protein was detected by immunohistochemistry in epithelial cells lining the secretory alveoli, in secretum within the alveolar lumen and in small peripheral nerves. The study findings support a role for adiponectin in regulating metabolism and immunity of the bovine mammary gland and potentially the calf intestine, following ingestion of milk.     

Pathological changes in the mammary gland and biochemical changes in milk of the goat following oral dosing with leaf of the Avocado (Persea americana)

Two varieties of avocado leaves (Persea americana var Guatemalan and var Mexican) were administered to lactating goats. The Mexican variety was without effect. The Guatemalan variety in doses exceeding 20 g fresh leaf per kg bodyweight, produced damage to the mammary gland with decreased milk production. The lesions were characterised by oedema and reddening, with clots in the large ducts. Microscopically, there was widespread degeneration and necrosis of the secretory epithelium, the necrotic cells sloughing into the lumen. There was no significant cellular inflammatory response. Concentrations of antitrypsin in the milk, indicating changes in vascular permeability, increased rapidly 15 h after a single high dose, coinciding with palpable oedema. Concentrations of NAGase, indicating cell damage, increased after 24 h. Goats given multiple doses followed a similar pattern but the initial response was delayed. The toxic principle, and its mode of action in selectively damaging mammary secretory cells, remains to be determined.     

Identification of IGF binding proteins in bovine milk and the demonstration of IGFBP-3 synthesis and release by bovine mammary epithelial cells.

Insulin-like growth factor system components are synthesized and secreted by mammary epithelial cells and multiple IGF binding proteins (IGFBP) are found in milk of various species. This study was conducted to identify the IGFBP in bovine milk, to compare them with those found in blood, and to identify the cell(s) responsible for mammary IGFBP synthesis. Bovine blood, milk, and cell culture-conditioned media were analyzed and characterized with Western ligand blot procedures for specific IGFBP. Electrophoresis and [125I]IGF-II ligand blot analyses of the samples indicated that, unlike serum and mammary primary cell culture-conditioned media, milk required removal of casein in order to accurately disclose all IGFBP. Immunoprecipitation studies identified IGFBP-2, -3, -4, and -5 in blood, milk, and primary cell culture conditioned media. The IGFBP were present at higher concentrations in serum than in milk, and milk concentrations were greater than that shown in conditioned media from primary cultures of bovine mammary cells. Northern analysis detected IGFBP-3 messenger RNA in extracts from fresh tissue and cells in culture, and in situ hybridization studies with fresh tissue utilizing probes for IGFBP-3 and alphaS1-casein showed that the mRNA for IGFBP-3 is predominant in the secretory epithelial cells, when compared to other tissue cell types.     

Effect of the intramammary device on milk infection status, yield, and somatic cell count and on the morphological features of the lactiferous sinus of the bovine udder

Twenty primiparous heifers were fitted intramammarily with polyethylene coils in both quarters of one random right or left udder half at 5 days after parturition. Foremilk samples were collected and udder-half milk yields were measured at the afternoon milking on days - 1, 3, 7, and 14 and on every 14th day for 8 months after the device was inserted. Three weeks after the heifers were fitted with the intramammary device, 6 were euthanatized for gross observation of devices and tissues and cytologic evaluation of the gland cistern epithelium. There were significantly fewer bacterial isolations (P less than 0.01) and less clinical mastitis (P less than 0.05) in treated quarters than in the control quarters. Coagulase-negative staphylococci were isolated at frequencies of 0 and 15.2% for treated and control quarters. The reduction in isolation frequency for treated, compared with control, quarters was less marked with other organisms. Intramammary devices in no way interfered with the milking process. Milk yields per milking were 4.2 kg for treated udder halves compared with 4.4 kg for control halves; however, this 0.2 kg difference was not significant. Mean milk somatic cell counts, as determined by electronic counter, were 34 X 10(3) and 81 X 10(3) cells/ml for control and treated quarters (P less than 0.05). Mean bovine serum albumin values were 0.160 and 0.175 mg/ml for control and treated udder halves (P less than 0.05), indicating an increased capillary permeability due to the device. Quantitative morphologic analysis of gland cisterns showed a significant (P less than 0.05) change toward a single layer of epithelial cells in treated quarters compared with a double layer in control quarters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody-containing cells and specialised epithelial cells in the bovine teat

Tissue from the ends of teats of dry, periparturient and lactating cows were studied using light and electron microscopy. Accumulations of infiltrating leucocytes mainly in the folds of the distal rosette of the teat cistern (Furstenberg's rosette) were detected; plasma cells predominated. The latter were classified by the type of immunoglobulin (Ig) which they synthesised. Plasma cells synthesising IgG1 were found to be the major antibody producing cell type of the teat. Neither the number of stromal plasma cells present nor the class of Ig which they synthesised was significantly altered by changes in mammary gland secretory activity. Scanning electron microscopy revealed areas of epithelium of Furstenberg's rosette that contained cells differing in surface characteristics from epithelial cells of adjacent areas of the teat cistern.     

B-mode ultrasonography of the bovine udder and teat

The udders and teats of cows were examined by brightness mode (B-mode) ultrasonography. A 5-MHz linear array transducer and a 5-MHz or 10-MHz mechanical sector transducer were used in the examinations. In lactating animals, the teat sinus, gland sinus, and lactiferous ducts were imaged easily. The scans also allowed visualization of the layers of the teat wall. The annular fold and the folding of the mucosa at the junction of the teat sinus and the papillary duct were seen best with the 5-MHz and 10-MHz mechanical sector transducers. In one cow lacking milk flow from one quarter, a mass at the junction of the papillary duct and the teat sinus was observed ultrasonographically. Surgical removal of the mass resulted in a return of milk flow from that quarter.     

Immunohistochemical Detection of Mycoplasma agalactiae in Formalin‐Fixed,Paraffin‐Embedded Tissues from Naturally and Experimentally Infected Goats

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin‐fixed, paraffin‐wax‐embedded sections and labelled by the avidin–biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody‐based immunohistochemical technique is an efficient and specific method for the post‐mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.     

Cytologic observations of the bovine teat end

Cells infiltrating from the vasculature and histologic components of internal tissues of teats (mammary papilla) from noninfected udder quarters were studied, using light and electron microscopy. Morphometric analysis demonstrated a progressive increase in number of infiltrating cells from the distal teat cistern (sinus papillaris) to the junction of the Furstenberg's rosette (distal termination and convergence of mucosal folds lining the teat cistern) and the streak canal (ductus papillaris). Plasma cells contributed to cellular increases in subepithelial connective tissue and were the most prevalent infiltrating cell type. Plasma cells also penetrated the basal epithelial lining of the rosette area and occasionally migrated to the luminal surface near the squamocolumnar junction. Neutrophils and monocytes contributed to the increase in cells infiltrating the epithelial lining. Few infiltrating cells were observed in epithelium and underlying stroma of the streak canal. Cytologic comparison demonstrated a reduction in all cell types from lactating to involuting phases of lactation. Greater numbers of plasma cells, lymphocytes, and monocytes were observed in teat end tissues from quarters previously infected with Staphylococcus aureus.     

CD44 is highly expressed on milk neutrophils in bovine mastitis and plays a role in their adhesion to matrix and mammary epithelium

Mastitis, inflammation of the mammary gland, is a common and economically important disease in dairy animals. Mammary pathogenic organisms, such as Escherichia coli, invade the teat canal,milk ducts, and mammary alveolar space, replicate in mammary secretions, and elicit a local inflammatory response characterized by massive recruitment of blood polymorphonuclear neutrophil leukocytes (PMN) into the alveoli and milk ducts. CD44 is a trans-membrane glycoprotein previously shown to play a role in mediation and control of blood PMN recruitment in response to inflammatory signals. Here we show, for the first time, increased expression of CD44 on recruited milk PMN in bovine mastitis and the expression of a CD44 variant, CD44v10, on these PMN. Furthermore, we demonstrate that CD44 mediates specific adhesion of bovine blood PMN to hyaluronic acid and mammary epithelial cells. Our results suggest that in mastitis CD44 plays a role in recruiting blood PMN into the mammary glands, the exact nature of this role needs to be elucidated.