Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n=67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.

Diagnostic gap in Bovine viral diarrhea virus serology during the periparturient period in cattle

Detection of antibodies against Bovine viral diarrhea virus (BVDV) in serum and milk by enzyme-linked immunosorbent assay (ELISA) is a crucial part of all ongoing national schemes to eradicate this important cattle pathogen. Serum and milk are regarded as equally suited for antibody measurement. However, when retesting a seropositive cow 1 day after calving, the serum was negative in 6 out of 9 different ELISAs. To further investigate this diagnostic gap around parturition, pre- and postcalving serum and milk samples of 5 cows were analyzed by BVDV antibody ELISA and serum neutralization test (SNT). By ELISA, 3 out of the 5 animals showed a diagnostic gap in the serum for up to 12 days around calving but all animals remained positive in SNT. In milk, the ELISA was strongly positive after birth but antibody levels decreased considerably within the next few days. Because of the immunoglobulin G (IgG)1-specific transport of serum antibodies into the mammary gland for colostrum production, the IgG subclass specificity of the total and the BVDV-specific antibodies were determined. Although all 5 animals showed a clear decrease in the total and BVDV-specific IgG1 antibody levels at parturition, the precalving IgG1-to-IgG2 ratios of the BVDV-specific antibodies were considerably lower in animals that showed the diagnostic gap. Results showed that BVDV seropositive cows may become "false" negative in several ELISAs in the periparturient period and suggest that the occurrence of this diagnostic gap is influenced by the BVDV-specific IgG subclass response of the individual animal.

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

Colostrogenesis: IgG1 Transcytosis Mechanisms

Biological transport of intact proteins across epithelial cells has been documented for many absorptive and secretory tissues. Immunoglobulins were some of the earliest studied proteins in this category. The transcellular transport (transcytosis) of immunoglobulins in neonatal health and development has been recognized; the process is especially significant with ungulates because they do not transcytose immunoglobulins across the placenta to the neonate. Rather, they depend upon mammary secretion of colostrum and intestinal absorption of immunoglobulins in order to provide intestinal and systemic defense until the young ungulate develops its own humoral defense mechanisms. The neonatal dairy calf's ability to absorb immunoglobulins from colostrum is assisted by a ~24 h "open gut" phenomenon where large proteins pass the intestinal epithelial cells and enter the systemic system. However, a critical problem recognized for newborn dairy calves is that an optimum mass of colostrum Immunoglobulin G (IgG) needs to be absorbed within that 24 h window in order to provide maximal resistance to disease. Many calves do not achieve the optimum because of poor quality colostrum. While many studies have focused on calf absorption, the principal cause of the problem resides with the extreme variation (g to kg) in the mammary gland's capacity to transfer blood IgG1 into colostrum. Colostrum is a unique mammary secretory product that is formed during late pregnancy when mammary cells are proliferating and differentiating in preparation for lactation. In addition to the transcytosis of immunoglobulins, the mammary gland also concentrates a number of circulating hormones into colostrum. Remarkably, the mechanisms in the formation of colostrum in ungulates have been rather modestly studied. The mechanisms and causes of this variation in mammary gland transcytosis of IgG1 are examined, evaluated, and in some cases, explained

Short communication: Immunoglobulin variation in quarter-milked colostrum

Whereas whole first-milked colostrum IgG1 variation is documented, the IgG1 difference between the quarter mammary glands of dairy animals is unknown. First colostrum was quarter-collected from healthy udders of 8 multiparous dairy cows, all within 3h of parturition. Weight of colostrum produced by individual quarters was determined and a sample of each was frozen for subsequent analysis. Immunoglobulin G1 concentration (mg/mL) was measured by ELISA and total mass (g) was calculated. Standard addition method was used to overcome colostrum matrix effects and validate the standard ELISA measures. Analysis of the data showed that cow and quarter (cow) were significantly different in both concentration and total mass per quarter. Analysis of the mean IgG1 concentration of the front and rear quarters showed that this was not different, but the large variation in individual quarters confounds the analysis. This quarter difference finding indicates that each mammary gland develops a different capacity to accumulate precolostrum IgG1, whereas the circulating hormone concentrations that induce colostrogenesis reach the 4 glands similarly. This finding also shows that the variation in quarter colostrum production is a contributor to the vast variation in first milking colostrum IgG1 content. Finally, the data suggests other factors, such as locally acting autocrine or paracrine, epigenetic, or stochasticity, in gene regulation mechanisms may impinge on colostrogenesis capacity.

Hyperketonemia during lipopolysaccharide-induced mastitis affects systemic and local intramammary metabolism in dairy cows

Hyperketonemia interferes with the metabolic regulation in dairy cows. It is assumed that metabolic and endocrine changes during hyperketonemia also affect metabolic adaptations during inflammatory processes. We therefore studied systemic and local intramammary effects of elevated plasma β-hydroxybutyrate (BHBA) before and during the response to an intramammary lipopolysaccharide (LPS) challenge. Thirteen dairy cows received intravenously either a Na-DL-β-OH-butyrate infusion (n = 5) to achieve a constant plasma BHBA concentration (1.7 ± 0.1 mmol/L), with adjustments of the infusion rates made based on immediate measurements of plasma BHBA every 15 min, or an infusion with a 0.9% NaCl solution (control; n = 8) for 56 h. Infusions started at 0900 h on d 1 and continued until 1700 h 2 d later. Two udder quarters were challenged with 200 μg of Escherichia coli LPS and 2 udder quarters were treated with 0.9% saline solution as control quarters at 48 h after the start of infusion. Blood samples were taken at 1 wk and 2h before the start of infusions as reference samples and hourly during the infusion. Mammary gland biopsies were taken 1 wk before, and 48 and 56 h (8h after LPS challenge) after the start of infusions. The mRNA abundance of key factors related to BHBA and fatty acid metabolism, and glucose transporters was determined in mammary tissue biopsies. Blood samples were analyzed for plasma glucose, BHBA, nonesterified fatty acid, urea, insulin, glucagon, and cortisol concentrations. Differences were not different for effects of BHBA infusion on the mRNA abundance of any of the measured target genes in the mammary gland before LPS challenge. Intramammary LPS challenge increased plasma glucose, cortisol, glucagon, and insulin concentrations in both groups but increases in plasma glucose and glucagon concentration were less pronounced in the Na-DL-β-OH-butyrate infusion group than in controls. In response to LPS challenge, plasma BHBA concentration decreased in controls and decreased also slightly in the BHBA-infused animals because the BHBA concentration could not be fully maintained despite a rapid increase in BHBA infusion rate. The change in mRNA abundance of citrate synthase in LPS quarters was significant between the 2 treatment groups. The results indicate that elevated circulating BHBA concentration inhibits gluconeogenesis before and during immune response to LPS challenge, likely because BHBA can replace glucose as an energy source.

Peripartal progesterone and prolactin have little effect on the rapid transport of immunoglobulin G into colostrum of dairy cows

Colostrum formation and lactogenesis in the mammary gland and the timing of parturition are regulated by endocrine signals. Changes in progesterone (P4) and prolactin (PRL) are considered key events that inhibit colostrum formation, trigger parturition, and signal the onset of lactation. The goal of our study was to determine if colostrum yield and composition and immunoglobulin transfer are affected by prepartum milking relative to the decrease in P4, peak of PRL, or occurrence of parturition. Twenty-three multiparous cows were randomly assigned to 1 of 2 groups: (1) control with first milking at 4h postcalving (CON, n=11), and (2) treatment group with first milking approximately 1d before calving and second milking at 4h after parturition (APM, n=12). Colostrum yields were recorded and proportional samples were analyzed for immunoglobulin G (IgG) concentration. Blood plasma samples for the analyses of P4 and PRL were collected 3 times daily at 8-h intervals for 4d prepartum and again taken at 4h after parturition. Total colostrum mass of APM cows was higher than that of CON cows. Immunoglobulin G concentration and protein content did not differ between antepartum milking in APM cows and postpartum milking in CON cows. Colostrum IgG concentration and protein content in APM cows at the postpartum milking were lower compared with the IgG concentration established at the prepartum (APM) and postpartum milkings of CON cows. Immunoglobulin G mass did not differ in first and second colostrum collection in APM cows but was lower compared with that of CON cows. The sum of IgG mass in APM cows (prepartum + postpartum collections) did not differ from that of CON cows. Lactose and fat in milk (concentration and mass) increased from first to second milking in APM cows. Total mass of lactose and fat in APM cows (prepartum + postpartum collections) was greater compared with that of CON cows. The finding that the time of milking relative to parturition, P4 decrease, and PRL peak slightly affected yield and quality of colostrum emphasizes the complex interactions of numerous endocrine and morphological changes occurring during colostrogenesis and lactogenesis in dairy cows. The considerably rapid transfer of immunoglobulins into colostrum of prepartum-milked cows within a few hours leads to the hypothesis that the transfer of IgG can be very fast and-contrary to earlier findings-persist at least until parturition.

Short communication: Timing of first milking affects serotonin (5-HT) concentrations

Hormonal signals differentially regulate the timing of parturition, as well lactogenesis and, potentially, colostrum formation in the mammary gland. Non-neuronal serotonin (5-HT) is a homeostatic regulator of the mammary gland. In the current study, we manipulated the timing of first milking to investigate its effects on serum 5-HT and calcium concentrations in the maternal and calf circulation, as well as in colostrum. Twenty-three cows were randomly assigned to a control (CON; n=10) group, milked for the first time at 4h postcalving, or a treatment (TRT; n=13) group, milked for the first time approximately 1 d before calving in addition to 4h postcalving. Maternal blood samples were collected for 4 d precalving, 3 times daily, and 1 blood sample was taken 4h postcalving. Calf blood samples were collected 4 (before first colostrum feeding) and 12h after birth, and at 3 wk of age. Calves from both treatments were fed colostrum from their respective mothers. Serum 5-HT concentrations were greater in CON cows and decreased significantly in TRT cows after milking was initiated precalving (951 vs. 524 ± 111 ng/mL, respectively). Cow serum calcium concentrations were affected by time, beginning to decrease 1 d precalving until 4h postcalving, but this drop in serum calcium was more pronounced in TRT cows. Serum 5-HT and calcium concentrations were negatively correlated (r=-0.57) for the CON cows and positively correlated (r=0.6) for the TRT cows. Maternal calcium and 5-HT decreased similarly due to precalving milking. Calcium and 5-HT concentrations were greater in colostrum collected from TRT cows milked precalving. Overall, calves had higher circulating 5-HT concentrations than cows, and calves born to TRT cows had increased 5-HT concentrations compared with the CON. Precalving milking could affect 5-HT synthesis within the mammary gland and therefore affect maternal 5-HT and calcium concentrations. Further research is needed in ruminants to assess the extent of 5-HT placental transfer, its role on pre- and postnatal development of the calf, the importance of its presence in colostrum, and potential long-term effects on calf health

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

Cryopreservation and transplantation of ovarian tissue exclusively to postpone menopause: technically possible but endocrinologically doubtful.

Transplantation of cryopreserved ovarian tissue has been shown to induce pregnancies and puberty successfully. Therefore, using cryopreserved ovarian tissue to postpone menopause (tissue hormone therapy [THT]) seems to be an interesting option to avoid conventional menopause hormone therapy (MHT). Pregnancy induction and replacing MHT by THT, however, are completely different topics as different requirements need to be met. First, MHT requires long-lasting and continuous hormone production. It still needs to be proven if the transplanted tissue is active for at least 5 years with a continuous follicle growth to avoid phases with low oestrogen production, which would otherwise cause menopausal symptoms and could reduce the postulated benefit for women's health. Second, the advantage of a physiological hormone production over a non-physiological MHT is still hypothetical. Third, women who have undergone hysterectomies who do not need progesterone for endometrial protection would only require oestrogens, imposing more health benefits (cardiovascular system, mammary gland) than oestrogen and progesterone production or replacement. Therefore, transplanting ovarian tissue exclusively to postpone menopause is endocrinologically doubtful and should only be carried out within clinical trials.

The role of coactivator associated arginine methyltransferase 1 (CARM1) in nuclear receptor mediated transcription

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARMI/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we generated and characterized CARM1 knockout mice. Embryos with a targeted disruption of CARM1 are 35% smaller in size than the wild-type littermates and die perinatally. We also generated Carm1-/- and Carm1+/+ mouse embryonic fibroblasts and tested gene expression in response to estrogen. Estrogenresponsive gene expression was aberrant in Carm1-/- fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. We subsequently studied the role of CARM1 in estrogen signaling in viva in the mammary gland. Conditional knockout of CARM1 in mammary gland and Carml-1-embryonic mammary anlagen transplant experiments did not show any defects in growth and development of the glands. To further dissect the role of CARM1 in estrogen receptor mediated transactivation, we performed cDNA microarray and serial analysis of gene expression on Carm1-/- and Carm1+/+ embryos treated with the estrogen analog, DES. Our results indicate global changes in estrogen regulated genes as well as genes involved in lipid homeostasis. Marker genes for Peroxisome Proliferator Activated Receptor γ (PPARγ) activity, adipsin and aP2, are downregulated in the Carm1-/- embryos. Furthermore, OCT frozen sections of 18.5dpc embryos, processed simultaneously for oil red O staining to look for neutral fat, reveals greatly reduced brown fat accumulation in the Carm1-/- embryos in contrast to wild-type and gain-of-function Carm1 transgenic (ubiquitous) embryo. We used a well-established 3T3-L1 preadipocyte cell line to knockdown CARM1 by short hairpin RNA. 3T3-L1 cells with CARM1 knockdown showed greatly reduced potential to differentiate into mature lipid accumulating adipocytes upon administration of adipogenic stimuli. Ligand-dependent activation of reporter genes by the PPARγ receptor showed that PPRE-luciferase reporter activity was enhanced in the presence of CARM1, additionally, luciferase activity was reduced to background levels when enzyme dead CARM1 (CARM1-VLD) was used. Thus, in this study, we have identified novel pathways that use CARM1 as coactivator and showed that CARM1 functions as a key component of PPARγ receptor mediated gene expression. ^

Polycomb repressive complex 2 and induced basal-like breast cancer phenotype mediated by cyclin E/Cdk2

Despite of much success of breast cancer treatment, basal-like breast cancer subtype still presented as a clinical challenge to mammary oncologist for its lack of available targeted therapy owing to their negative expression of targeted molecules, such as PgR, ERα and Her2. These molecules are all critical regulators in mammary gland development. EZH2, a histone methyltransferase, by forming Polycomb Repressive Complex 2(PRC2) can directly suppress a large array of developmental regulators. Overexpression of cyclin E has also been correlated with basal-like (triple-negative) breast cancer and poor prognosis. We found an important functional link between these two molecules. Cyclin E/Cdk2 can enhance PRC2 function by phosphorylating a specific residue of EZH2, threonine 416 and increasing EZH2's ability to complex with SUZ12. This regulation would further recruit whole PRC2 complex to core promoter regions of these developmental regulators. The local enrichment of PRC2 complex would then trimethylate H3K27 around the core promoter regions and suppress the expression of targeted genes, which included PgR, ERα, erbB2 and BRCA1. This widespread gene suppressive effect imposed by highly active PRC2 complex would then transform the lumina) type cell to adopt a basal-like phenotype. This finding suggested deregulated Cdk2 activity owing to cyclin E overexpression may contribute to basal phenotype through enhancing epigenetic silencing effects by regulating PRC2 function. Inhibition of Cdk2 activity in basal-like cancer cells may help release the suppression, reexpress the silenced genes and become responsive to existing anti-hormone or anti-Her2 therapy. From this study, the mechanisms described here provided a rationale to target basal-like breast cancer by new combinational therapy of Cdk2 inhibitors together with Lapatinib, or Aromatin. ^

The increased level of β1,4-galactosyltransferase required for lactose biosynthesis is achieved in part by translational control

β1,4-Galactosyltransferase (β4GalT-I) participates in both glycoconjugate biosynthesis (ubiquitous activity) and lactose biosynthesis (mammary gland-specific activity). In somatic tissues, transcription of the mammalian β4GalT-I gene results in a 4.1-kb mRNA and a 3.9-kb mRNA as a consequence of initiation at two start sites separated by ≈200 bp. In the mammary gland, coincident with the increased β4GalT-I enzyme level (≈50-fold) required for lactose biosynthesis, there is a switch from the 4.1-kb start site to the preferential use of the 3.9-kb start site, which is governed by a stronger tissue-restricted promoter. The use of the 3.9-kb start site results in a β4GalT-I transcript in which the 5′- untranslated region (UTR) has been truncated from ≈175 nt to ≈28 nt. The 5′-UTR of the 4.1-kb transcript [UTR(4.1)] is predicted to contain extensive secondary structure, a feature previously shown to reduce translational efficiency of an mRNA. In contrast, the 5′-UTR of the 3.9-kb mRNA [UTR(3.9)] lacks extensive secondary structure; thus, this transcript is predicted to be more efficiently translated relative to the 4.1-kb mRNA. To test this prediction, constructs were assembled in which the respective 5′-UTRs were fused to the luciferase-coding sequence and enzyme levels were determined after translation in vitro and in vivo. The luciferase mRNA containing the truncated UTR(3.9) was translated more efficiently both in vitro (≈14-fold) and in vivo (3- to 5-fold) relative to the luciferase mRNA containing the UTR(4.1). Consequently, in addition to control at the transcriptional level, β4GalT-I enzyme levels are further augmented in the lactating mammary gland as a result of translational control.

Hormonal prevention of breast cancer: Mimicking the protective effect of pregnancy

Full term pregnancy early in life is the most effective natural protection against breast cancer in women. Rats treated with chemical carcinogen are similarly protected by a previous pregnancy from mammary carcinogenesis. Proliferation and differentiation of the mammary gland does not explain this phenomenon, as shown by the relative ineffectiveness of perphenazine, a potent mitogenic and differentiating agent. Here, we show that short term treatment of nulliparous rats with pregnancy levels of estradiol 17β and progesterone has high efficacy in protecting them from chemical carcinogen induced mammary cancers. Because the mammary gland is exposed to the highest physiological concentrations of estradiol and progesterone during full term pregnancy, it is these elevated levels of hormones that likely induce protection from mammary cancer. Thus, it appears possible to mimic the protective effects of pregnancy against breast cancer in nulliparous rats by short term specific hormonal intervention.