Use of magnetic resonance imaging in the study of goat mammary glands in vivo

A technique is described for the in-vivo determination of mammary gland size and gross composition in goats by using nuclear magnetic resonance imaging (MRI). The volume of test objects determined with MRI had an error of +0.4 +/- 1.6% of the actual volume. In lactating goats the in-vivo MRI estimate of mammary parenchymal volume was significantly greater than, but highly significantly correlated with, the weight of parenchyma determined post mortem (for the whole udder, r = 0.88, P less than 0.001; for individual glands, r = 0.85, P less than 0.001). MRI-determined estimates of the volume of fluid within the mammary gland were within 1.2% of the volume of milk removed from the udders after imaging. The spin-lattice (T1) relaxation time of the whole udder correlated closely with the volume of fluid within the udder. The T1 relaxation time of parenchymal tissue measured in vivo did not differ significantly from that determined immediately after post-mortem excision.     

The local immune response in the mammary gland of the sow following infusion of a protein antigen

A study was made of the local immune response in the udder of the sow following infusion of a soluble antigen. Four mammary glands of each of four pregnant sows were infused with ferritin prepartum. Samples of blood, colostrum, and milk were collected during the following lactation; animals were slaughtered and mammary tissue removed for immunohistology. Blood, colostrum, milk, and mammary tissues were similarly collected from nonimmunized (control) sows. Colostral and milk whey from immunized sows contained higher levels of immunoglobulins than whey from control sows. There was an increase in numbers of IgA-containing plasma cells and total lymphoid cells in mammary tissue of immunized sows compared with controls. The results suggested that the local immune response was at least as great in non-infused glands as infused glands of immunized sows.     

Udder quarter milk composition at different levels of somatic cell count in cow composite milk

Automatic milking systems have made possible the separation of high- and low-quality milk at the udder quarter level during the milking process. The aim of this study was to investigate the composition and yield of milk from individual udder quarters to determine whether deteriorated milk composition occurs in udders that are assumed to be healthy and whether quarters with high-quality milk are found in udders with high milk somatic cell count (SCC). Milk samples were collected on one occasion from 90 cows at udder quarter level and cow composite level. The milk was analyzed for content of total protein, whey protein, casein, fat, lactose, citric acid and SCC; milk yield was registered. The cows were divided into three groups depending on the SCC of their composite milk. Cows in group 1, cow composite SCC < 100 000 cells/ml, were assumed to have healthy udders. However, instances of increased SCC and decreased milk quality were discovered in one or more udder quarters of approximately 30% of the group. Cows in group 2, cow composite SCC of 100 000 to 300 000 cells/ml, and group 3, cow composite SCC > 300 000 cells/ml, were assumed to have affected udders. However, the majority of these cows had one or more udder quarters in which increased SCC and deteriorated milk quality were not detected. Calculations of bulk-tank milk values, when separation of milk from affected udder quarters was performed, indicate that SCC changes to a much greater degree compared to the other milk components. These results show that milk from affected udder quarters suffers compositional changes, but calculations of simulated separation indicate that the compositional changes in bulk-tank milk are small. The effect of separation of milk from individual udder quarters on bulk-tank milk needs to be further studied.     

All three promoters of the acetyl-coenzyme A-carboxylase alpha-encoding gene are expressed in mammary epithelial cells of ruminants.

The activity of the enzyme acetyl-CoA-carboxylase alpha (ACC-alpha) is rate limiting for the de novo synthesis of fatty acids. The encoding gene is expressed from three promoters in ruminants (PI-PIII). Their individual contribution to the formation of milk fat is unknown. Promoter-specific molecular probes were hybridized in situ to serial sections of mammary glands from cows and sheep to determine their developmental and spatial expression profile in the udder. We show that all three promoters are active in mammary epithelial cells (MECs) of udders from both species. This implies that, in principle, none of these promoters can be singled out as the key element controlling the ACC-alpha-related contribution to establishment of milk fat content, although the activity of PIII only is known to be disproportionally stimulated by lactation in MECs. We propose that all three promoters may be relevant for milk fat synthesis in cattle, whereas PII and PIII are crucial for milk fat formation in sheep. We show also that ACC-alpha synthesis is not strictly coupled to casein synthesis, particularly during pregnancy and involution.     

Sequential pathological studies in the udder of goats intramammarily infected withAspergillus fumigatus

Intramammary inoculation of goats withAspergillus fumigatus spores resulted in the development of mastitis with characteristic gross and microscopic lesions. The mastitis and the lesions were restricted to the infected udder halves only and there was no dissemination of infection to other tissues of the body. The experiment was continued for 45 days. Gross changes in the infected udder were observed up to the 45th day post-infection. The lesions, in general, included variable sized abscesses in the first 15 days followed by development of varying sized greyish-white nodules in the infected udders. Microscopic changes consisted of granulomatous reaction with well developed granulomas in the infected udders. Hyphae and spores ofAspergillus fumigatus could be demonstrated in sections of the infected udders up to 45 days after infection. Reisolation of the fungus consistently was achieved up to 45 days. It is concluded that intramammary inoculation ofAspergillus fumigatus spores in goats leads to chronic granulomatous mastitis.     

Experimental candidal mastitis in goats: Clinical,haematological, biochemical and sequential pathological studies

The present study, first of its kind, was conducted with the objectives to understand hitherto little known aspects of candidal mastitis, like its sequential pathology, pathogenesis and clinico-biochemical changes. For this purpose, unilateral intramammary inoculation of 10 goats with Candida albicans (1.2 × 10⁷ yeast cells) resulted in the development of mastitis, with gross and microscopic lesions being restricted to the infected udder halves only and without dissemination of infection to the opposite uninfected udder halves as well as other organs of the body. The experiment was continued for 40 days and after infection, there was sharp fall in milk yield and Candida albicans was directly demonstrated in the milk and re-isolated from the milk and udder tissues up to 30th day after inoculation. An increase in total immunoglobulins in the milk and plasma along with increase in total plasma proteins were also observed. Haematology revealed leukocytosis and neutrophilia. Microscopically, there was acute purulent mastitis, which later became chronic, nonpurulent and interstitial with formation of granulomas. It was concluded that Candida albicans was highly pathogenic to the lactating goat mammary gland even without immunosuppression or antibiotic treatment, resulting in severe irreversible tissue damage and nearly complete agalactia. This revised version was published online in August 2006 with corrections to the Cover Date.     

Secretion and immunochemical properties of the trypsin inhibitor from bovine colostrum.

A trypsin inhibitor was isolated from bovine colostrum by affinity chromatography. Immunoelectrophoresis detected two immunogenic components in the isolated inhibitor, but only one of these was specific for the inhibitor; the other one was identical with an antigen present in liver, kidney, spleen, adrenal, thyroid, thymus, brain, ovarian, testicular and udder tissue and in bull seminal plasma. Using immunoabsorption and immunofluorescence it was shown that the antigens specific for the trypsin inhibitor of colostrum could be demonstrated only in the tissue of an udder that is secreting colostrum. The inhibitor is secreted by the secretory epithelium of the milk alveoli of the udder, during the period when the latter secretes colostrum. This inhibitor was not detected in the milk. Cross-reaction between antisera to colostral inhibitor and basic pancreatic inhibitor or seminal plasma inhibitors yielded negative results. Antiserum to bovine colostral inhibitor showed a positive reaction with inhibitor isolated from porcine colostrum.     

Epidemiological and Clinical Aspects of the First Outbreak of Bovine Mastitis Caused by Prototheca zopfii in Goiás State, Brazil

The purpose of this survey was to describe the occurrence of bovine mastitis caused by Prototheca zopfii in Goiás State, Brazil. Samples of milk, environment and udder were taken from a herd of 120 Holstein cows. Sabourauds dextrose agar plates were incubated under aerobic conditions at 37 °C/96 h, for microbiological analysis. Somatic cell count and milk composition were also determined. Histological sections from two udders were stained with HE and PAS. Prototheca zopfii was identified in six cows whose milk had a watery appearance. They also showed a pronounced decrease in milk yield, fat and lactose. Pronounced infiltration of mononuclear cells, atrophy of alveoli and fibrosis were observed. The presence of this agent in other herds in the State is highly likely.     

Histone H3K14 and H4K8 hyperacetylation is associated with Escherichia coli-induced mastitis in mice

Mastitis is a multietiological complex disease, defined as inflammation of parenchyma of mammary glands. Bacterial infection is the predominant cause of mastitis, though fungal, viral and mycoplasma infections also have been reported. Based on the severity of the disease, mastitis can be classified into subclinical, clinical and chronic forms. Bacterial pathogens from fresh cow milk were isolated and classified by standard microbiological tests and multiplex PCR. Epidemiological studies have shown that Escherichia coli is the second largest mastitis pathogen after Staphylococcus aureus in India. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR profile and presence of virulence genes, a field isolate of E. coli was used for intramammary inoculation in lactating mice. Histopathological examination of hematoxylin and eosin stained sections showed severe infiltration of polymorphonuclear neutrophils, mononuclear inflammatory cells in the alveolar lumen and also in interstitial space, and necrosis of alveolar epithelial cells after 24 h. Western blot and immunohistochemical analysis of mice mammary tissues showed significant hyperacetylation at histone H3K14 residue of both mammary epithelial cells and migrated inflammatory cells. Quantitative real-time PCR and genome-wide gene expression profile in E. coli infected mice mammary tissue revealed differential expression of genes related to inflammation, immunity, antimicrobial peptide expression, acute phase response and oxidative stress response. Expression of milk proteins was also suppressed. ChIP assay from paraffinized tissues showed selective enrichment of acetylated histone H3K14 and H4K8 at the promoters of overexpressed genes. These data suggest that E. coli infection in mice mammary tissue leads to histone hyperacetylation at the promoter of immune genes, which is a pre-requisite for the expression of inflammatory genes in order to mount a drastic immune response.     

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.     

Tuberculosis in apparently healthy milch cows.

Screening for mycobacteria was carried out on samples of normal milk, supramammary lymph nodes (SLNS) and udder tissue from apparently healthy milch cows, including the milk from those suspected of tuberculous mastitis. Milk samples from the slaughtered and live Fulani cows which were apparently healthy yielded 5% and 7% of tubercle bacilli, respectively, while suspected tuberculous milk from slaughtered cows and live cows yielded 8% and 10% of the bacilli, respectively. Tubercle bacilli were isolated both from SLNS with and without lesions. The udder tissue was positive for mycobacteria even though visible lesions were absent.     

Membrane Filtration Technique for Isolating Organisms from Raw Milk of Normal Udders

The effectiveness of two methods for the isolation of bacteria from raw milk was investigated. Raw milk from normal udders was subjected to the direct membrane filtration technique and the 18-hr incubation technique. The results indicate that the membrane filtration technique gives a more complete picture of the flora of a normal udder than does the 18-hr incubation technique. Use of various methods for the isolation of organisms from milk samples may account for the extensive disagreement on the presence and types of organisms associated with normal and mastitic udders.     

Coccidia in the mammary glands of shrews (Order: Insectivora)

Three of 6 female long-clawed shrews, Sorex unguiculatus Dobson, 1890, collected on the island of Hokkaido, Japan, were found to have unsporulated oocysts and sexual stages (both macro- and microgamonts) in varying stages of development of an unidentified coccidium in both lactating and nonlactating mammary glands. Gamonts developed in the alveoli of the mammary glands, and oocysts were found in the lactiferous ducts and in pools of milk. Mature macrogamonts were 11.9 x 15.2 microm (10-14 x 14-20 microm), whereas completely developed microgamonts with many gametes were 14.8 x 16.8 microm (10-18 x 13-20 microm). Oocysts in tissue sections were 19.5 x 13.8 microm and had a smooth outer wall that was <1 microm thick. Little histopathology was associated with the infections. Infected cells were enlarged and appeared cloudy, and in some areas there was leucocytic infiltration by macrophages, small and large lymphocytes, neutrophils, and eosinophils. No basophil was seen. We also found sections of a nematode, probably a Mammanidula sp., in sections of an active mammary gland in 1 of the shrews not infected with the coccidium.     

Stem Cell Research: A Novel Boulevard towards Improved Bovine Mastitis Management

The dairy industry is a multi-billion dollar industry catering the nutritional needs of all age groups globally through the supply of milk. Clinical mastitis has a severe impact on udder tissue and is also an animal welfare issue. Moreover, it significantly reduces animal value and milk production. Mammary tissue damage reduces the number and activity of epithelial cells and consequently contributes to decreased milk production. The high incidence, low cure rate of this highly economic and sometimes deadly disease is an alarming for dairy sector as well as policy makers. Bovine mammary epithelial cells (MECs) and their stem cells are very important in milk production and bioengineering. The adult mammary epithelium consists of two main cell types; an inner layer of luminal epithelial cells, which produce the milk during lactation, and an outer layer of myoepithelial cells resting on a basement membrane, which are responsible for pushing the milk through the ductal network to the teat cistern. Inner layer of columner/luminal cells of bovine MECs, is characterized by cytokeratin18, 19 (CK18, CK19) and outer layer such as myoepithelial cells which are characterized by CK14, α-smooth muscle actin (α-SMA) and p63. Much work has been done in mouse and human, on mammary gland stem cell research, particularly in cancer therapy, but stem cell research in bovine is still in its infancy. Such stem/progenitor cell discoveries in human and mouse mammary gland bring some hope for application in bovines. These progenitors may be therapeutically adopted to correct the structural/cytological defects in the bovine udder due to mastitis. In the present review we focused on various kinds of stem/progenitor cells which can have therapeutic utility and their possibilities to use as a potential stem cell therapy in the management of bovine post-mastitis damage in orders to restore milk production. The possibilities of bovine mammary stem cell therapy offers significant potential for regeneration of tissues that can potentially replace/repair diseased and damaged tissue through differentiation into epithelial, myoepithelial and/or cuboidal/columnar cells in the udder with minimal risk of rejection and side effects.     

Mastitis by Aspergillus fumigatus in sheep

Several outbreaks of sheep mastitis by Aspergillus fumigatus in Castilla y Leon (Spain), were studied. Only sheep that were treated intramammarily with antibacterial antibiotics during the dry period suffered this mastitis. Mastitis was acute with a morbidity up to 14 % and mortality near 100 %. The udder was markedly enlarged in size, fibrotic, haemorrhagic and with multiple compact nodules, some with purulent material inside; after 30-50 days postpartum, cheesy abscess of several centimetres in diameter were present. Some sheep had granulomatous nodules in the lung. Microscopy and culture shown the presence of A. fumigatus in milk, udder and lung. The route of infection was by intramammary via as a consequence of unhygienic intramammary treatment in the dry period.     

Uptake of free plasma amino acids by the lactating cow''s udder and amino acid composition of udder lymph

1. Total α-amino N and the amounts of 24 ninhydrin-positive substances were determined in several samples of plasma and lymph from the cow's udder. The arteriovenous differences of these substances across the mammary glands were measured in several experiments performed on lactating cows and in one experiment on a `dry' cow. Udder lymph obtained from live lactating cows by a lymph fistula and taken after killing lactating cows was analysed. 2. The concentrations of the individual free amino acids in udder lymph obtained from the live cow were similar to those found in cow's plasma. The concentrations of many amino acids in udder lymph taken immediately after death were two- to four-fold higher than those of the corresponding amino acids in udder lymph obtained from the live cow. 3. Most amino acids of the blood showed a considerable decrease in concentration by passage across the lactating mammary gland. Ornithine, a non-casein amino acid, showed arteriovenous differences of up to 60% of the arterial plasma concentration. No substantial amino acid uptake by the udder could be demonstrated in the experiment on the non-lactating cow. 4. The arteriovenous differences obtained for arginine, glutamine, isoleucine, leucine, lysine, valine, threonine and histidine were probably large enough to provide all the respective amino acid residues in milk protein. 5. The uptake of aspartic acid, asparagine, glutamic acid, serine and proline by the lactating cow's udder was not sufficient to account for all these respective amino acid residues found in milk protein.