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.

Expression of Foxi3 is regulated by ectodysplasin in skin appendage placodes

BACKGROUND Foxi3 is a member of the large forkhead box family of transcriptional regulators, which have a wide range of biological activities including manifold developmental processes. Heterozygous mutation in Foxi3 was identified in several hairless dog breeds characterized by sparse fur coat and missing teeth. A related phenotype called hypohidrotic ectodermal dysplasia (HED) is caused by mutations in the ectodysplasin (Eda) pathway genes. RESULTS Expression of Foxi3 was strictly confined to the epithelium in developing ectodermal appendages in mouse embryos, but no expression was detected in the epidermis. Foxi3 was expressed in teeth and hair follicles throughout embryogenesis, but in mammary glands only during the earliest stages of development. Foxi3 expression was decreased and increased in Eda loss- and gain-of-function embryos, respectively, and was highly induced by Eda protein in embryonic skin explants. Also activin A treatment up-regulated Foxi3 mRNA levels in vitro. CONCLUSIONS Eda and activin A were identified as upstream regulators of Foxi3. Foxi3 is a likely transcriptional target of Eda in ectodermal appendage placodes suggesting that HED phenotype may in part be produced by compromised Foxi3 activity. In addition to hair and teeth, Foxi3 may have a role in nail, eye, and mammary, sweat, and salivary gland development.

Concomitant lipopolysaccharide-induced transfer of blood-derived components including immunoglobulins into milk

During a mammary immune response, the integrity of the blood-milk barrier is negatively affected and becomes leaky. The aim of the present study was to demonstrate the blood origin, and to investigate changes in the concentration, of various constituents including immunoglobulins in blood and milk during the early phase of lipopolysaccharide (LPS)-induced mastitis. Five lactating dairy cows received continuous β-hydroxybutyrate (BHBA) clamp infusions to maintain elevated BHBA blood concentrations (1.5 to 2.0 mmol/L) from 48 h before and 8h after LPS administration. One udder quarter was infused with 200 μg of Escherichia coli LPS. A second quarter served as control. Milk and blood samples were taken hourly for 8h postchallenge (PC). The somatic cell count in LPS-challenged quarters was increased from 4h PC to the end of the experiment compared with control quarters. In LPS-challenged quarters, l-lactate, BHBA, lactate dehydrogenase (LDH), IgG(1), and IgG(2) were increased at 3h PC and remained elevated until the end of experiment (8h PC) compared with control quarters. In addition, the optical density values in milk in a nonquantitative ELISA for antibodies directed against bluetongue virus (used as a measure of nonspecific antibody transfer; all animals were vaccinated) increased and, thus, indicates an increase in these antibodies in response to LPS treatment. l-Lactate concentration also increased in blood 2h PC and in the milk of control quarters during the experiment from 3h PC. A second experiment was conducted in vitro to investigate a possible contribution from destructed milk cells to l-lactate concentration and activity of LDH in milk. Aliquots of milk samples (n=8) were frozen (-20°C) or disrupted with ultrasound, respectively. Freeze thawing and ultrasound treatment increased LDH in milk samples, but had no effect on l-lactate concentrations. Results suggest that intramammary infusion of LPS induces a systemic response, as evidenced by an elevation of blood l-lactate concentration. The concomitant changes of all investigated components suggest that they were blood derived. However, the increase in blood components in the milk is not necessarily supportive of the mammary immune system, and likely a side effect of reduced blood-milk barrier integrity.

Short communication: differential immunoglobulin transfer during mastitis challenge by pathogen-specific components

Mastitis induced by Escherichia coli is often characterized by severe clinical signs, indicating a more powerful combat of the immune system against the pathogen compared with Staphylococcus aureus infections, which are often represented by chronic and subclinical diseases. The aim of this study was to test the major pathogenic component lipopolysaccharide (LPS) from E. coli and lipoteichoic acid (LTA) from Staph. aureus for their effects on blood-milk barrier integrity and the related transfer of immunoglobulins and lactate from blood into milk. A similar somatic cell count (SCC) increase was achieved by intramammary challenge of 1 quarter of 5 cows with 20 µg of LTA, and 8 cows with 0.2 µg of LPS (maximum log SCC/mL: 7). Milk IgG(1) concentrations increased in LPS- but not in LTA-challenged quarters. Milk IgG(2) concentrations were increased in treated quarters at 3h after LPS, and 6h after LTA challenge. Higher maximum levels of IgG(2) were reached in milk of LPS-treated quarters (173 ± 58 μg/mL) than of LTA-challenged quarters (62 ± 13 μg/mL). Immunoglobulin G(1) and IgG(2) levels did not change in control quarters. l-Lactate concentrations in milk increased 4h after LPS and 5h after LTA challenge and reached higher maximum levels in LPS- (221 ± 48 mg/L) than in LTA-treated quarters (77 ± 18 mg/L). In conclusion, a mammary inflammation on a quantitatively similar level based on SCC increase achieves a more efficient transfer of blood components such as IgG(2) via the blood-milk barrier if induced by LPS from E. coli than by LTA from Staph. aureus. This pathogen-specific difference may play an important role in the cure rate of the respective intramammary infection, which is usually lower in Staph. aureus- than in E. coli-induced mastitis.

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.

Three-dimensional printing creates models for surgical planning of aortic valve replacement after previous coronary bypass grafting.

PURPOSE Resternotomy for aortic valve replacement in patients with previous coronary artery bypass grafting and an internal mammary artery graft may be a surgical problem. Thus, we are exploring the effect of using rapid prototyping techniques for surgical planning and intraoperative orientation during aortic valve replacement after previous coronary artery bypass grafting (CABG). DESCRIPTION As a proof of concept, we studied a patient who had undergone CABG 5 years earlier. At that time the patient received a left internal mammary artery graft to the left anterior descending artery and a venous graft to the right coronary artery. Now the patient required aortic valve replacement due to symptomatic aortic valve stenosis. The left internal mammary artery bypass and the right coronary artery bypass were patent and showed good flow in the angiography. The patient was examined by 128-slice computed tomography. The image data were visualized and reconstructed. Afterwards, a replica showing the anatomic structures was fabricated using a rapid prototyping machine. EVALUATION Using data derived from 128-slice computed tomography angiography linked to proprietary software, we were able to create three-dimensional reconstructions of the vascular anatomy after the previous CABG. The models were sterilized and taken to the operating theatre for orientation during the surgical procedure. CONCLUSIONS Stereolithographic replicas are helpful for choosing treatment strategies in surgical planning and for intraoperative orientation during reoperations of patients with previous CABG.

Periodontitis and Gingivitis in Inflammatory Bowel Disease

BACKGROUND: The oral cavity is frequently affected in patients with inflammatory bowel disease (IBD), especially in patients with Crohn's disease (CD). Periodontitis is thought to influence systemic autoimmune or inflammatory diseases. We aimed to analyze the relationship of periodontitis and gingivitis markers with specific disease characteristics in patients with IBD and to compare these data with healthy controls. METHODS: In a prospective 8-month study, systematic oral examinations were performed in 113 patients with IBD, including 69 patients with CD and 44 patients with ulcerative colitis. For all patients, a structured personal history was taken. One hundred thirteen healthy volunteers served as a control group. Oral examination focussed on established oral health markers for periodontitis (bleeding on probing, loss of attachment, and periodontal pocket depth) and gingivitis (papilla bleeding index). Additionally, visible oral lesions were documented. RESULTS: Both gingivitis and periodontitis markers were higher in patients with IBD than in healthy control. In univariate analysis and logistic regression analysis, perianal disease was a risk factor for periodontitis. Nonsmoking decreased the risk of having periodontitis. No clear association was found between clinical activity and periodontitis in IBD. In only the CD subgroup, high clinical activity (Harvey-Bradshaw index > 10) was associated with 1 periodontitis marker, the loss of attachment at sites of maximal periodontal pocket depth. Oral lesions besides periodontitis and gingivitis were not common, but nevertheless observed in about 10% of patients with IBD. CONCLUSIONS: IBD, and especially perianal disease in CD, is associated with periodontitis. Optimal therapeutic strategies should probably focus on treating both local oral and systemic inflammation.

Incomplete Distal Renal Tubular Acidosis from a Heterozygous Mutation of the V-ATPase B1 Subunit

Congenital distal renal tubular acidosis (dRTA) from mutations of the B1 subunit of the V-ATPase is considered an autosomal recessive disease. We analyzed a dRTA kindred with a truncation-mutation of B1 (p.Phe468fsX487) previously shown to have failure of assembly into the V1 domain of the V-ATPase. All heterozygous carriers in this kindred have normal plasma bicarbonate concentrations, thus evaded the diagnosis of RTA. However, inappropriately high urine pH, hypocitraturia, and hypercalciuria are present either individually or in combination in the heterozygotes at baseline. Two of the heterozygotes studied also have inappropriate urinary acidification with acute ammonium chloride loading and impaired urine-blood pCO2 gradient during bicarbonaturia indicating presence of H+ gradient and flux defects. In normal human renal papillae, wild type B1 is located primarily on the plasma membrane but papilla from one of the heterozygote who had kidney stones had renal tissue secured from surgery showed B1 in both plasma membrane as well as a diffuse intracellular staining. Titrating increasing amounts of the mutant B1 subunit did not exhibit negative dominance over the expression, cellular distribution, or H+-pump activity of the wild type B1 in mammalian HEK293 cells and in V-ATPase-deficient S. cerevisiae. This is the first demonstration of renal acidification defects and nephrolithiasis in heterozygous carriers of mutant B1 subunit; which cannot be attributable to negative dominance. We propose that heterozygosity may lead to mild real acidification defects due to haploinsufficiency. B1 heterozygosity should be considered in patients with calcium nephrolithiasis and urinary abnormalities such as alkalinuria or hypocitraturia.

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

Monitoring of tumor response to Cisplatin using optical spectroscopy

INTRODUCTION Anatomic imaging alone is often inadequate for tuning systemic treatment for individual tumor response. Optically based techniques could potentially contribute to fast and objective response monitoring in personalized cancer therapy. In the present study, we evaluated the feasibility of dual-modality diffuse reflectance spectroscopy-autofluorescence spectroscopy (DRS-AFS) to monitor the effects of systemic treatment in a mouse model for hereditary breast cancer. METHODS Brca1(-/-); p53(-/-) mammary tumors were grown in 36 mice, half of which were treated with a single dose of cisplatin. Changes in the tumor physiology and morphology were measured for a period of 1 week using dual-modality DRS-AFS. Liver and muscle tissues were also measured to distinguish tumor-specific alterations from systemic changes. Model-based analyses were used to derive different optical parameters like the scattering and absorption coefficients, as well as sources of intrinsic fluorescence. Histopathologic analysis was performed for cross-validation with trends in optically based parameters. RESULTS Treated tumors showed a significant decrease in Mie-scattering slope and Mie-to-total scattering fraction and an increase in both fat volume fraction and tissue oxygenation after 2 days of follow-up. Additionally, significant tumor-specific changes in the fluorescence spectra were seen. These longitudinal trends were consistent with changes observed in the histopathologic analysis, such as vital tumor content and formation of fibrosis. CONCLUSIONS This study demonstrates that dual-modality DRS-AFS provides quantitative functional information that corresponds well with the degree of pathologic response. DRS-AFS, in conjunction with other imaging modalities, could be used to optimize systemic cancer treatment on the basis of early individual tumor response.