Effect of chronic oxytocin-treatment on the bovine mammary gland immune system

The aim of this study was to evaluate the effect of chronic oxytocin (OT) treatment on the mammary gland immune system. In Experiment I fourteen healthy cows were used to study the effect of chronic intramuscular (im) OT administration on concentration of milk somatic cells and white blood cells (WBC). Cows in the OT-group (6) were im injected with 50 IU OT (5 ml) whereas animals of the C-group (6) were im injected with 5 ml of saline (9 g/l) for eight days (Day 1–8) before each milking. Milk samples were taken during normal milking time on Day 0–3, 5, 7, 9–11 and 18. Blood samples were taken immediately after each milking and analysed for WBC count, polymorphonuclear neutrophils, potassium (K), sodium (Na) and chloride (Cl) ions, and blood lactose. All milk samples were analysed for somatic cell counts (SCC), lactose, Na, Cl and electrical conductivity (EC). Furthermore mRNA expression of tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, IL-8 and cyclooxygenase-2 (COX2) in milk cells were measured via real-time RT-PCR. None of the investigated milk and blood parameters changed significantly in response to the OT treatment. The mRNA-expression of TNFα decreased (P < 0.05) to a minimum on Day 3 in response to OT administration. IL-1β and IL-6-mRNA expression decreased (P < 0.05) to a minimum within three day. IL-8 and COX2 expression did not change in response to OT treatment. In Experiment II twelve cows, randomly divided into two groups of six, were used to investigate the effect of chronic im OT administration on mammary tissue. Cows were im administered 50 IU OT (OT-group) or 5 ml saline (9 g/l; C-group) before each milking during eight days. Biopsy samples were taken after every morning milking. The mRNA expression of various inflammatory factors and the tight junction (TJ) proteins occludin (OCLN) and zonula occludens (ZO)-1, ZO-2 and ZO-3 were measured via real-time RT-PCR. TNFα-mRNA expression decreased (Day 2 with P < 0.05) within the first four days of OT administration and increased (P < 0.05) in the C-group on Day 2. IL-1β expression levels of the OT-group increased transiently and decreased on Day 3 and in the C-group values increased significantly on Day 3 as compared to Day 0. IL-6 expression in the OT-group decreased (P < 0.05) to a minimum on Day 1 and increased (P < 0.05) as compared to Day 0 on Day 7 and increased significantly on Day 1 and Day 5 compared to Day 0 in group C. IL-8 and COX2 expression did not change in response to OT administration. The mRNA-expression of OCLN and ZO-3 decreased (P < 0.05) as compared to Day 0 with a minimum on Day 7. ZO-1 and ZO-2 expression did not change due to OT administration. ZO-2-mRNA expression in C-group decreased significantly on Day 2 compared to Day 0. In conclusion, chronic OT administration induced increasing SCC and EC levels in milk as well as K and lactose in blood while nearly all investigated cytokines in milk cells and mammary tissue were down regulated. The mRNA expressions of the TJ proteins OCLN and ZO-3 were down-regulated in response to the OT treatment what indicates an increasing TJ permeability. Besides the effect on TJ proteins there was no obvious change of the immunological competence of the mammary gland in response to OT. However, a more complete milk ejection should help to remove pathogens during milking.

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Dose-dependent immune response in milk cells and mammary tissue after intramammary administration of lipopolysaccharide in dairy cows

Veterinární Medicína ◽

10.17221/1877-vetmed ◽

2008 ◽

Vol 52 (No. 6) ◽

pp. 231-244 ◽

Cited By ~ 9

Author(s):

C. Werner-Misof ◽

M.W. Pfaffl ◽

R.M. Bruckmaier

Keyword(s):

Immune Response ◽

Mrna Expression ◽

Real Time ◽

Polymorphonuclear Neutrophils ◽

Mammary Tissue ◽

Mrna Levels ◽

Rt Pcr ◽

Lps Challenge ◽

Cell Counts ◽

Cox 2

The immune response in milk cells and the status of mammary tight junctions (TJ) in response to intramammary (IM) infusion of different doses of Escherichia coli lipopolysaccharide (LPS) was investigated. Experiment I: Seven German Braunvieh cows were IM infused into one quarter with 1 μg (LPS-1) and 3 μg (LPS-3) of LPS, respectively, and the contralateral control quarter with saline (9 g/l; C). Milk samples were taken immediately before and 12, 24, 36, 48, 60, 84 and 108 h after infusion and analysed for somatic cell counts (SCC), lactose, sodium (Na) and chloride (Cl) ions, and electrical conductivity (EC). Milk cell mRNA expression of various inflammatory factors was quantified by real-time RT-PCR. Blood samples were taken immediately after milking for the analysis of leukocytes (WBC), polymorphonuclear neutrophils (PMN), Na and Cl. Milk SCC, lactose, Na, Cl and EC did not differ significantly between LPS-1 and C quarters after the challenge. In LPS-3 quarters SCC levels increased within the first 12 h, reached peak levels between 12 and 36 h (P ≤ 0.001) and decreased (P ≤ 0.05) thereafter to reach baseline at 108 hours. Lactose in LPS-3 quarters decreased (P ≤ 0.05) to a minimum at 24 h and increased slightly thereafter while EC, Na, and Cl increased transiently in response to LPS-3. WBC and PMN levels in both groups decreased numerically within 24 h after LPS administration. In LPS-1, WBC at 24, 48 and 108 h were significantly lower whereas in LPS-3 they were significantly higher than at time 0. TNFα-mRNA expression in both groups did not change in response to IM LPS-challenge. IL-1β-mRNA expression at 12, 24 and 36 h in LPS-1 quarters increased significantly as compared to time 0. In LPS-3 quarters the mRNA expression values of all tested ILs increased significantly as compared to time 0 within 12 h after LPS-challenge. IL-1β-mRNA expression decreased (P ≤ 0.05) at 48 and 84 h in LPS quarters. IL-8 mRNA was significantly decreased at 84 h after challenge in LPS-3 quarters. COX-2-mRNA expression in LPS-1 quarters decreased significantly as compared to time 0 at 48, 84 and 108 h, with a minimum at 84 h (P ≤ 0.05). In LPS-3 quarters COX-2-mRNA levels increased (P ≤ 0.05) within 48 h after the LPS-challenge. Experiment II: Six cows (5 German Braunvieh, 1 Brown Swiss) were injected in one quarter with 100 μg LPS and in the contralateral quarter with saline (9 g/l; C). Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of TJ proteins occludin (OCLN) and zonula occludens (ZO-) 1, 2 and 3 were quantified by real-time RT-PCR. OCLN-mRNA expression did not change in response to the IM infusion while that of ZO-1, ZO-2 and ZO-3 decreased significantly within six hours. In conclusion, a dose of 1 μg LPS did not initiate a immune response in the mammary gland. Furthermore the dose of 100 μg of LPS enhanced TJ permeability by reducing TJ plaque proteins density.

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Rapid Detection of Mycoplasma bovis, Staphylococcus aureus and Streptococcus agalactiae in Cattle Bulk Tank Milk in Cyprus and Relations with Somatic Cell Counts

Pathogens ◽

10.3390/pathogens10070841 ◽

2021 ◽

Vol 10 (7) ◽

pp. 841

Author(s):

Maria Liapi ◽

George Botsaris ◽

Costas Arsenoglou ◽

Nikolas Markantonis ◽

Christodoulos Michael ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Somatic Cell ◽

Real Time ◽

Streptococcus Agalactiae ◽

Mycoplasma Bovis ◽

Bulk Tank Milk ◽

Somatic Cell Counts ◽

Milk Samples ◽

Cell Counts ◽

Bulk Tank

One hundred and seventy-seven (177) bulk tank milk samples were analyzed with a commercially available real-time polymerase chain reaction kit and 11 (6.21%), 41 (23.16%), and 58 (32.77%) tested positive for Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae, respectively. Statistical analysis revealed a significant relationship between the presence of S. aureus and S. agalactiae. Enumeration of somatic cells was performed in the same samples by flow cytometry. The somatic cell counts were found higher in S. aureus and S. agalactiae positive samples. No association was found between M. bovis presence and somatic cells counts. Low internal assay control Ct values were found to be related with high somatic cell counts. Noticeably, this is the first report for the presence of M. bovis in Cyprus. Therefore, its presence was confirmed by bulk tank milk culture, conventional PCR, and next generation sequencing. Furthermore, M. bovis was typed with multilocus sequencing typing and was allocated to sequence type 29 (ST 29). Real-time PCR in bulk tank milk samples is a useful tool to detect mammary infections, especially for neglected pathogens such as M. bovis.

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Quantitative real-time RT-PCR analysis of NY-ESO-1 and LAGE-1a mRNA expression in normal tissues and tumors, and correlation of the protein expression with the mRNA copy number

International Journal of Oncology ◽

10.3892/ijo.26.1.57 ◽

2005 ◽

Author(s):

Shuichiro Sato ◽

Yuji Noguchi ◽

Hisashi Wada ◽

Shoichiro Fujita ◽

Shinichiro Nakamura ◽

...

Keyword(s):

Protein Expression ◽

Mrna Expression ◽

Real Time ◽

Copy Number ◽

Pcr Analysis ◽

Rt Pcr ◽

Mrna Copy Number ◽

Normal Tissues

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P1139EXPRESSION OF TOLL LIKE RECEPTORS, CELL ADHESION MOLECULES AND PRO-INFLAMMATORY CYTOKINES IN ESRD PATIENTS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p1139 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Mantabya Singh ◽

Narayan Prasad ◽

Nida Fatima ◽

Amit Gupta ◽

Chinmoy Sahu

Keyword(s):

Cell Adhesion ◽

Mrna Expression ◽

Inflammatory Cytokines ◽

Fungal Pathogens ◽

Tnf Alpha ◽

Rt Pcr ◽

Cell Counts ◽

Co Morbidity ◽

Esrd Patients ◽

Pro Inflammatory Cytokines

Abstract Background and Aims CAPD is well established modality of treatment for patients with end stage renal disease (ESRD). Peritonitis is a leading cause of technique failure and death in patients on CAPD. Studies on expressions of host factors like TLRs, CAMs and their relationship with inflammatory cytokines (IL-6, IL-12, TNF-alpha and IL-1 beta) involved in peritonitis and other co-morbidity and functional status are lacking throughout the world. Hence the present study has to be done to determine the expressions of TLR2 and TLR4, and CAMs in ESRD patients. To compare the expression of TLR2, TLR4, ICAM 1 and Pro-inflammatory cytokines (IL-6, IL-12, TNF-alpha and IL-1-beta) in Peritonitis, CAPD and CRF group patients. Method A total of 85 ESRD patients recruited and sub-divided into 3 groups. Group1- CAPD patient (n=25), Group 2- Peritonitis patient (n=30), and Group 3- CRF (n=30 patients). mRNA expression of TLR-2, TLR-4 were examined at gene levels by RT PCR and cell adhesion molecule (ICAM-1) were examined at gene and protein levels by RT PCR and ELISA respectively in Serum and Pro-inflammatory cytokines level were also examined by ELISA in serum. We performed microbiological culture for bacterial and fungal pathogens using automated BACTEC culture system. Cell counts were routinely done on every dialysate. Results Out of 30 samples of peritonitis group 15 were culture positive and 15 were culture negative. We found that in peritonitis group the mRNA expression of TLR-2 and TLR-4 was higher as compared to CRF (4.183±2.857vs 3.633±2.41) (p=0.049), (4.314±2.91vs 4.14±1.99) (p=0.015) and CAPD (4.183±2.857vs3.683±2.85) (p=0.041), (4.314±2.91vs 3.88±1.91) (p=0.009) respectively. At gene and protein level ICAM-1 was higher in peritonitis patient compared to CAPD (mRNA expression 4.76±2.64vs 4.36±3.48) (level in sera 660±201.2vs 514±157) (p=0.003). The IL-6 and IL-12 expression was higher in Peritonitis group as compared to CAPD (66.87±64.51vs214.35±220.05) (p=0.04) and (230.17±153.45vs417.04±302.96) (p=0.028) respectively. The TNF-alpha and IL-1-beta expression was not significant among the groups. Conclusion TLRs activation by bacterial molecules leads to the induction of cytokines (IL-6 and IL-12) and chemokine through the activation of NF-ķB pathway and may be responsible for atherosclerosis, morbidity and mortality in ESRD patients. Elevated level of ICAM-1, IL-6 and IL-12 may be responsible for chronic inflammation in Peritonitis group patients.

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PMCA1 mRNA Expression in Rat Aortic Myocytes: A Real-Time RT–PCR Study

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2000.3578 ◽

2000 ◽

Vol 276 (3) ◽

pp. 1024-1027 ◽

Cited By ~ 10

Author(s):

Tanya M. Mamic ◽

Nicola A. Holman ◽

Sarah J. Roberts-Thomson ◽

Gregory R. Monteith

Keyword(s):

Mrna Expression ◽

Real Time ◽

Rt Pcr

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Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution

Journal of Endocrinology ◽

10.1677/joe.0.1770305 ◽

2003 ◽

Vol 177 (2) ◽

pp. 305-317 ◽

Cited By ~ 52

Author(s):

D Schams ◽

S Kohlenberg ◽

W Amselgruber ◽

B Berisha ◽

MW Pfaffl ◽

...

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Mrna Expression ◽

Western Blotting ◽

Mammary Gland Development ◽

Mammary Tissue ◽

Positive Staining ◽

Bovine Mammary Gland ◽

Total Rna ◽

Gland Development

It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28, 96-108 h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.

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Quantification of SPS mRNA expression in banana fruit ripened under different conditions using real-time RT-PCR

Food Science and Biotechnology ◽

10.1007/s10068-011-0207-2 ◽

2011 ◽

Vol 20 (6) ◽

pp. 1495-1500

Author(s):

Wen Li ◽

Xue-ping Li ◽

Yuan-zhi Shao ◽

Jiang-hui Xie ◽

Wei-xin Chen ◽

...

Keyword(s):

Mrna Expression ◽

Real Time ◽

Banana Fruit ◽

Rt Pcr

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Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing

Journal of Dairy Research ◽

10.1017/s0022029910000725 ◽

2010 ◽

Vol 78 (1) ◽

pp. 49-55 ◽

Cited By ~ 18

Author(s):

Ricardo Bexiga ◽

Mikko T Koskinen ◽

Jani Holopainen ◽

Carla Carneiro ◽

Helena Pereira ◽

...

Keyword(s):

Somatic Cell ◽

Real Time ◽

Real Time Pcr ◽

Gram Positive ◽

Milk Samples ◽

Negative Results ◽

Streptococcus Uberis ◽

Cell Counts ◽

Significant Difference ◽

Culture Negative

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at −20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500 000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

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