Occurrence of Zearalenone and Its Metabolites in the Blood of High-Yielding Dairy Cows at Selected Collection Sites in Various Disease States.

Zearalenone (ZEN) and its metabolites, alpha-zearalenol (α-ZEL) and beta-zearalenol (ß-ZEL), are ubiquitous in plant materials used as feed components in dairy cattle diets. The aim of this study was to confirm the occurrence of ZEN and its selected metabolites in blood samples collected from different sites in the hepatic portal system (posthepatic-external jugular vein EJV; prehepatic-abdominal subcutaneous vein ASV and median caudal vein MCV) of dairy cows diagnosed with mastitis, ovarian cysts and pyometra. The presence of mycotoxins in the blood plasma was determined with the use of combined separation methods involving immunoaffinity columns, a liquid chromatography system and a mass spectrometry system. The parent compound was detected in all samples collected from diseased cows, whereas α-ZEL and ß-ZEL were not identified in any samples, or their concentrations were below the limit of detection (LOD). Zearalenone levels were highest in cows with pyometra, where the percentage share of average ZEN concentrations reached 44%. Blood sampling sites were arranged in the following ascending order based on ZEN concentrations: EJV (10.53 pg/mL, 44.07% of the samples collected from this site), ASV (14.20 pg/mL, 49.59% of the samples) and MCV (26.67 pg/mL, 67.35% of the samples). The results of the study indicate that blood samples for toxicological analyses should be collected from the MCV (prehepatic vessel) of clinically healthy cows and/or cows with subclinical ZEN mycotoxicosis. This sampling site increases the probability of correct diagnosis of subclinical ZEN mycotoxicosis.

Haematological findings in 158 cows with acute toxic mastitis with a focus on the leukogram.

BACKGROUND: In cows with acute toxic mastitis (ATM), the leukogram aids in the assessment of the severity of disease. The goal of our study was to compare the leukogram of 158 cows with ATM (cases) and 168 clinically healthy cows (controls). We hypothesised that the leukograms of surviving and non-surviving cows differ and that there are variables of the leukogram with sufficient prognostic potential to be used in the decision to treat or not to treat a cow with ATM. The cows were examined clinically and underwent haematological and biochemical examination of blood and bacteriological culture of milk samples. RESULTS: All cows with ATM had a poor appetite or anorexia, and 34 cows (21.5%) were recumbent. A single quarter was affected in 119 cows (75.3%), two quarters in 37 cows (23.4%) and three quarters in two cows (1.3%). Bacteriological culture showed Gram-negative pathogens in 100 cows (63.3%), Gram-positive in 15 (9.5%) and yeast in 4 (2.5%). The median total leukocyte count of cases was 4300 cells/µL (interquartile range = 2300-8200/µL), which was significantly lower than 8000 cells/µL (6525-9300/µL) in controls. Except for band neutrophils and metamyelocytes, the counts of all components of the leukogram were lower in cases compared with controls. Significantly more cows with ATM had leukopenia (60.1 vs. 4.1%) or leukocytosis (10.1 vs. 3.0%) than controls. Diseased cows had significantly lower segmented neutrophil counts than controls (860 vs. 2598 cells/µL), and 69.5 and 17.3%, respectively, had counts below the reference interval. Cases had increased band (77.3%) and metamyelocyte (25.0%) counts compared with controls (0.6 and 0%, respectively). In diseased cows, eosinopenia occurred in 66.4% (controls, 1.8%), monocytopenia in 40.6% (4.2%) and lymphopenia in 60.2% (1.8%). Twenty-one diseased cows (16.4%) had a regenerative and 57 (44.5%) had a degenerative left shift. The median neutrophil-to-lymphocyte ratio was 0.97 in diseased cows and 0.63 in controls. Toxic changes in neutrophils including cytoplasmic basophilia and vacuolisation were seen in 101 (91.8%) of 110 blood smears of diseased cows. The leukogram of the surviving and non-surviving cows did not differ significantly, and the hypothesis was rejected. CONCLUSIONS: ATM results in severe changes in the leukogram particularly leukopenia, lymphopenia, and degenerative left shift. The hypothesis that the leukograms of surviving and non-surviving cows differ was rejected. The leukogram has not sufficient prognostic potential to be used in the decision to treat or not to treat a cow with ATM.

Bovine serum miR-21 expression affected by mastitis.

The expression levels of circulating microRNAs (miRNAs) can be affected by disease. The miRNA released from cells within exosomes can act as a remote communication tool and can participate in inflammatory response regulation. Therefore, circulating miRNA has the potential to be an indicator of local disease. The objective of this study was to investigate the serum level of bovine mastitis-related miRNAs. We found that miR-16 expression in serum was affected by hemolysis. The expression levels of miR-21 in serum were increased significantly in cows with mastitis compared with unaffected controls; however, the expression levels of miR-146a, miR-155, miR-222 and miR-383 in cows with mastitis were unchanged. We further verified the upregulation of miR-21 in the serum of cows with mastitis using a digital PCR system. Although the sensitivity and specificity of miR-21 in the serum to detect bovine mastitis was inferior to miRNA biomarkers in the milk, the significant increase of miR-21 in serum may reflect the impact of local inflammation on the systemic reaction.

Prognostic potential of pre-partum blood biochemical and immune variables for postpartum mastitis risk in dairy cows.

BACKGROUND: Mastitis is the most frequent diseases for transition cows. Identification of potential biomarkers for diagnosis of mastitis is important for its prevention. Thus, this study was conducted to investigate blood variables related to lipid metabolism, oxidative stress and inflammation, and serum variables that are related to health in postpartum cows. RESULTS: Seventy-six healthy Holstein dairy cows at week 4 before calving were selected to collect blood samples from weeks - 4 to 4 weekly relative to calving, respectively. Milk yield and composition were recorded weekly. According to the cut-off of somatic cell counts (SCC) for diagnosis of mastitis, 33 cows with SCC ≥ 500,000 cells ml- 1, 20 cows with 200,000 cells ≤ SCC < 500,000 cells ml- 1, and 23 cows with SCC < 200,000 cells ml- 1 were defined as high, middle, and low SCC, respectively. Serum concentrations of ß-hydroxybutyrate were higher (P < 0.01) during all weeks, and non-esterified fatty acids were higher in high SCC than in low SCC cows from weeks - 3 to 2 relative to calving. Higher serum concentrations of superoxide dismutase (P < 0.01) and lower malondialdehyde levels (P < 0.01) in low SCC than in high SCC cows indicate that the latter suffered from oxidative stress. The difference analysis of the three groups suggested that none of the above-mentioned variables can be used as potential prognostic candidates. On the other hand, high SCC cows exhibited higher blood neutrophil to lymphocyte ratio (NLR, P < 0.01) and platelet to lymphocyte ratio (PLR, P < 0.01) than low SCC cows, with a higher NLR (P < 0.01) in middle SCC than in low SCC cows. The high SCC cows had lower levels of anti-inflammatory factors including IL-10 (P = 0.05), but higher levels of proinflammatory factors such as IL-6 (P < 0.01), TNF-α (P < 0.05), and PSGL-1 (P < 0.01) than low SCC cows. CONCLUSIONS: The significantly different NLR and PLR pre-partum between the middle and low SCC cows suggest their prognostic potential for postpartum mastitis risk.

Changes in ionized calcium concentration in the blood of dairy cows with peracute coliform mastitis.

We determined the clinical signs and blood ionized calcium (iCa) levels in dairy cows with peracute coliform mastitis (PCM). The clinical scores at the onset of the disease (day 0) and on day 2 and subsequent days were significantly (P<0.01) higher than those of healthy cows. We found a positive correlation (r=0.894, P<0.01) between iCa and total calcium (TCa) concentrations in the blood of healthy cows ; however there was no correlation from day 0 to day 3 in the blood of PCM cows. Multiple regression analysis revealed that the concentration of iCa was correlated with rectal temperature, hematocrit value, platelet count, and albumin level of PCM cows at the onset of disease (r= -0.804, r=0.6576, r=0.6182, r=0.284, P<0.01, respectively). There was no correlation between the TCa concentration and these parameters for PCM cows at day 0. Low blood iCa concentration at day 0 for PCM cows was related to symptoms of septic shock involving hypothermia, activation of the blood coagulation system, and dehydration.

Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony-stimulating factor therapy during a chronic Staphylococcus aureus infection in lactating Holsteins.

Neutrophils are principal host innate immune cell responders to mastitis infections. Thus, therapies have been developed that target neutrophil expansion. This includes the neutrophil-stimulating cytokine granulocyte colony-stimulating factor (gCSF). Pegylated gCSF (PEG-gCSF; Imrestor, Elanco Animal Health, Greenfield, IN) has been shown to reduce the natural incidence of mastitis in periparturient cows in commercial settings and reduce severity of disease against experimental mastitis challenge. Pegylated gCSF stimulates neutrophil expansion but also induces changes in monocyte and lymphocyte circulating numbers, surface protein expression changes, or both. We hypothesized that PEG-gCSF modulates surface expression of monocytes and neutrophils and facilitates their migration to the mammary gland. We challenged 8 mid-lactation Holsteins with approximately 150 cfu of Staphylococcus aureus (Newbould 305) in a single quarter via intramammary infusion. All animals developed chronic infections as assessed by bacteria counts and somatic cell counts (SCC). Ten to 16 wk postchallenge, 4 of the animals were treated with 2 subcutaneous injections of PEG-gCSF 7 d apart. Complete blood counts, SCC, bacterial counts, milk yield, feed intake, neutrophils extracellular trap analysis, and flow cytometric analyses of milk and blood samples were performed at indicated time points for 14 d after the first PEG-gCSF injection. The PEG-gCSF-treated cows had significantly increased numbers of blood neutrophils and lymphocytes compared with control cows. Flow cytometric analyses revealed increased surface expression of myeloperoxidase (MPO) on neutrophils and macrophages in milk but not in blood of treated cows. Neutrophils isolated from blood of PEG-gCSF-treated cows had decreased surface expression of CD62L (L-selectin) in blood, consistent with cell activation. Surprisingly, CD62L cell surface expression was increased on neutrophils and macrophages sourced from milk from treated animals compared with cells isolated from controls. The PEG-gCSF-treated cows did not clear the S. aureus infection, nor did they significantly differ in SCC from controls. These findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, although surface MPO+ cells accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggests an incomplete role for PEG-gCSF treatment against chronic S. aureus infection and possibly chronic mammary infections in general.

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species.

The aim of the study was to investigate the concentrations of acute-phase inter-α-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus spp. (STR) and coagulase-negative Staphylococcus spp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus spp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus spp. and 89% of animals infected with Staphylococcus spp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus spp. and Staphylococcus spp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 µg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus spp. and in 40% of animals infected with Staphylococcus spp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows.

Cell free mitochondrial DNA in serum and milk associated with bovine mastitis: a pilot study.

Mastitis is inflammation of mammary gland affecting all the species of domestic animals. Fragments of the mitochondrial genome released from dying cells are considered surrogate markers of mitochondrial injury. We hypothesized that bovine mastitis would be associated with increased cell free mitochondrial DNA (mtDNA) content in serum and milk. Milk and serum samples were collected from sub-clinical mastitic and normal animals. Mastitis was confirmed by California mastitis test and bacterial isolation. Oxidative stress, nitric oxide and inflammatory cytokines were also estimated. Real time polymerase chain reaction was conducted in serum and milk from sub-clinical mastitic animals and compared with healthy animals targeting the mtDNA genes cytochrome b. Mastitis animals showed higher oxidative stress markers and nitric oxide along with higher level of inflammatory cytokines. Cell free mtDNA was significantly higher in serum and milk of mastitic animals comparing to that of healthy control. The higher cell free relative mtDNA content in mastitis animals indicates injury to the mammary epithelial cells and thereby releasing the mtDNA in the milk and blood. This mtDNA may be a bio-marker of oxidative stress and tissue injury in bovine mastitis.

Tryptophan, kynurenine, kynurenic acid concentrations and indoleamine 2,3-dioxygenase activity in serum and milk of dairy cows with subclinical mastitis caused by coagulase-negative staphylococci.

The aim of the study was to investigate serum and milk concentrations of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), and indoleamine 2,3-dioxygenase (IDO) activity in cows suffering from subclinical mastitis caused by coagulase-negative staphylococci (MSCNS). TRP and kynurenines were determined by high-performance liquid chromatography (HPLC), and IDO activity was calculated as the KYN/TRP ratio. The blood and milk samples were collected from 40 midlactation Holstein-Fresian cows from two herds in the Lublin region in Poland. In the milk samples from 20 cows with subclinical mastitis, coagulase-negative staphylococci were isolated and in the milk obtained from healthy cows growth of microorganisms was not detected. TRP, KYN and KYNA concentrations were significantly lower in milk of cows with MSCNS compared to control animals (4.47 vs. 7.24 µM, 0.14 vs. 0.21 µM, 1.58 vs. 2.18 nM, respectively). There was no statistically significant difference in TRP and KYNA concentrations in serum between the studied animal groups (32.97 vs. 39.29 µM, 31.3 vs. 26.5 nM, respectively). In turn, the level of KYN was lower in the serum (0.81 vs. 1.13 µM) of cows with mastitis compared to healthy ones. No statistically significant differences in IDO activity, both in serum and in milk (25.24 and 27.55, 28.56 and 27.17, respectively) was revealed between the studied groups. These findings may have potential implications for diagnosis of mastitis in cows because reduction of these parameters in milk might be a marker predicting the occurrence of the disease.

Comparative plasma and interstitial fluid pharmacokinetics and tissue residues of ceftiofur crystalline-free acid in cattle with induced coliform mastitis.

Ceftiofur (CEF) is a third-generation cephalosporin that is the most widely used antimicrobial in the dairy industry. Currently, violative meat residues in cull dairy cattle are commonly associated with CEF. One potential cause for violative residues is altered pharmacokinetics of the drug due to disease, which could increase the time needed for the residue to deplete. The objectives of this study were (a) to determine the absolute bioavailability of CEF crystalline-free acid (CFA) in healthy versus diseased cows; (b) to compare the plasma and interstitial fluid pharmacokinetics and plasma protein binding of CEF between healthy dairy cows and those with disease; and (c) to determine the CEF residue profile in tissues of diseased cows. For this trial, disease was induced through intramammary Escherichia coli infusion. Following disease induction and CEF CFA administration, for plasma concentrations, there was not a significant effect of treatment (p = 0.068), but the treatment-by-time interaction (p = 0.005) was significant. There was a significantly greater concentration of CEF in the plasma of the DIS cows at T2 hr (p = 0.002), T8 hr (p < 0.001), T12 hr (p = 0.001), and T16 hr (p = 0.002). For PK parameters in plasma, the slope of the terminal phase of the concentration versus time curve was significantly lower (p = 0.007), terminal half-life was significantly longer (p = 0.014), and apparent volume of distribution during the elimination phase was significantly higher (p = 0.028) diseased group. There was no difference in plasma protein binding of CEF and interstitial fluid pharmacokinetics. None of the cows had kidney CEF residues above the US tolerance level following observation of the drug's withdrawal period, but one cow with a larger apparent volume of distribution and longer terminal half-life had tissue residues slightly below the tolerance. Whereas these findings do not support the hypothesis that severely ill cows need longer withdrawal times, alterations in the terminal half-life suggest that it is theoretically possible.

Identification of bta-miR-15a∼16a cluster expression, localization and regulated target in Holsteins.

Bovine mastitis is an inflammation response of the mammary gland tissues caused mainly by pathogenic bacteria in cows. Previous studies showed that bta-miR-15a and bta-miR-16a modulate immunity and inflammation responses. In this study, we investigated the expression pattern and tissue localization of bta-miR-15a and bta-miR-16a. The expression levels of bta-miR-15a and bta-miR-16a were significantly upregulated in mammary tissues and blood neutrophils of mastitis-infected cows, compared with those of healthy cows (P < 0.05). Through in situ hybridization, we examined the tissue localization of bta-miR-15a and bta-miR-16a and found that they were expressed in the ductal and acinar cells of mammary gland tissues, where they had a stronger expression signal in the mammary tissues of cows with mastitis than that in healthy cows' tissues. Moreover, we identified CD163 as the target gene of bta-miR-15a and bta-miR-16a. Luciferase assay indicated that bta-miR-15a, bta-miR-16a, and bta-miR-15a∼16a cluster led to the significant reduction in the luciferase activity of CD163 3'UTR vector (P < 0.05). Meanwhile, the luciferase activity had a significantly low value compared with that of single bta-miR-15a or bta-miR-16a plasmid (P < 0.05) in the presence of bta-miR-15a∼16a cluster. The bta-miR-15a∼16a cluster may function more effectively in inhibiting luciferase reporter gene activity of target gene CD163 than single miRNA. Our study provides an insight into the relationship between bovine mastitis and gene expression of bta-miR-15a/16a, which suggested that bta-miR-15a∼16a cluster may play a role against mastitis by binding to target CD163 gene in Holstein dairy cattle.

Mastitis in the transition period: identification of potential blood markers / Mastite no período de transição: identificação de potenciais marcadores no sangue

This study identified potential blood markers associated with mastitis in dairy cows under different dry therapies during the transition period, using a logistic regression model. Thirty-four Holstein dairy cows were divided into three groups: untreated controls (13 cows, 42 quarters); animals that received an antimicrobial at drying-off (10 cows, 40 quarters); and animals that were administered an internal teat sealant at drying-off (11 cow, 44 quarters). Blood and quarter milk samples were collected 60 days before the expected day of calving, on the day of calving, and three, seven, 15, 21, and 30 days after calving. Milk samples were submitted for bacteriological analysis and somatic cell count. Blood samples were collected for analyses of the following: the erythrogram and leucogram; plasm fibrinogen concentration; hepatic and renal functions; metabolic profile; serum calcium and phosphorous levels; total serum protein and albumin concentrations. The concentration of total serum proteins was associated with a high somatic cell count. Similarly, the concentrations of total serum proteins and triglycerides were associated to milk bacteriological positive samples during the transition period. Thus, the occurrence of mastitis in dairy cows during the transition period was greater in animals that showed higher concentrations of serum total proteins and triglycerides, in contrast to the use of dry cow therapy.(AU)

O presente estudo identificou potenciais marcadores sanguíneos associados tanto à mastite, durante o período de transição em vacas leiteiras submetidas à antimicrobianoterapia, quanto à utilização do selante interno de teto na secagem por modelo de regressão logística. Trinta e quatro vacas da raça Holandesa foram divididas em três grupos experimentais, a saber: 13 animais (42 quartos mamários) que não receberam tratamento na secagem; 10 animais (40 quartos mamários) tratados por via intramamária com uma bisnaga do antimicrobiano para vaca seca à base de cefalônio anidro após a última ordenha em cada quarto mamário; e 11 animais (44 quartos mamários) que receberam, por via intramamária, uma bisnaga de selante à base de subnitrato de bismuto após a última ordenha em cada quarto mamário. As amostras de leite e sangue foram coletadas 60 dias antes da data prevista do parto, na secagem, e no dia do parto e após três, sete, 15 e 21 dias após o parto. As amostras de leite foram utilizadas para o exame microbiológico e a determinação da contagem de células somáticas. As amostras de sangue foram utilizadas para determinação do eritrograma, leucograma, fibrinogênio plasmático, funções hepáticas e renais, perfil metabólico, proteína total e albuminas séricas e concentração sérica de cálcio e fósforo. A concentração total de proteínas séricas foi associada à alta contagem de células somáticas. Similarmente, a concentração total de proteínas séricas e triglicérides foi associada ao isolamento de patógenos causadores de mastite nas amostras de leite durante o período de transição. Desse modo, conclui-se que vacas leiteiras com concentrações séricas maiores de proteína total e triglicérides têm maior chance de apresentar mastite durante o período de transição; em contraste, o uso da antimicrobianoterapia de vaca seca reduz esse risco.(AU)

Using milk leukocyte differentials for diagnosis of subclinical bovine mastitis.

This research study aimed to evaluate the use of the milk leukocyte differential (MLD) to: (a) identify quarter milks that are culture-positive; and (b) characterize the milk leukocyte responses to specific groups of pathogens causing subclinical mastitis. The MLD measures the absolute number and relative percentage of inflammatory cells in milk samples. Using the MLD in two dairy herds (170 and 172 lactating cows, respectively), we studied all lactating cows with a most recent monthly Dairy Herd Improvement Association somatic cell count (SCC) >200 × 103 cells/ml. Quarter milk samples from 78 cows meeting study criteria were analysed by MLD and aseptically collected milk samples were subjected to microbiological culture (MC). Based upon automated instrument evaluation of the number and percentage of inflammatory cells in milk, samples were designated as either MLD-positive or - negative for subclinicial mastitis. Positive MC were obtained from 102/156 (65·4%) of MLD-positive milk samples, and 28/135 (20·7%) of MLD-negative milk samples were MC-positive. When MC was considered the gold standard for mastitis diagnosis, the calculated diagnostic Se of the MLD was 78·5% (IC(95%) = 70·4 to 85·2%) and the Sp was 66·5% (IC(95%) = 58·6 to 73·7%). [corrected]. Quarter milks positive on MC had higher absolute numbers of neutrophils, lymphocytes and macrophages, with higher neutrophils% and lymphocytes% but lower macrophages%. The Log10 (N/L) ratios were the most useful ratio to differentiate specific subclinical mastitis quarters from healthy quarters. Use of the MLD on cows with monthly composite SCC > 200 × 103 cells/ml for screening at quarter level identified quarters more likely to be culture-positive. In conclusion, the MLD can provide an analysis of mammary quarter status more detailed than provided by SCC alone; however, the MLD response to subclinical mastitis was not found useful to specifically identify the causative pathogen.

GC-MS Metabolomics Identifies Metabolite Alterations That Precede Subclinical Mastitis in the Blood of Transition Dairy Cows.

The objectives of this study were to determine alterations in the serum metabolites related to amino acid (AA), carbohydrate, and lipid metabolism in transition dairy cows before diagnosis of subclinical mastitis (SCM), during, and after diagnosis of disease. A subclinical mastitis case was determined as a cow having somatic cell count (SCC) > 200 000/mL of milk for two or more consecutive reports. Blood samples were collected from 100 Holstein dairy cows at five time points at -8 and -4 weeks before parturition, at the week of SCM diagnosis, and +4 and +8 weeks after parturition. Twenty healthy control cows (CON) and six cows that were diagnosed with SCM were selected for serum analysis with GC-MS. At -8 weeks a total of 13 metabolites were significantly altered in SCM cows. In addition, at the week of SCM diagnosis 17 metabolites were altered in these cows. Four weeks after parturition 10 metabolites were altered in SCM cows and at +8 weeks 11 metabolites were found to be different between the two groups. Valine (Val), serine (Ser), tyrosine (Tyr), and phenylalanine (Phe) had very good predictive abilities for SCM and could be used at -8 weeks and -4 weeks before calving. Combination of Val, isoleucine (Ile), Ser, and proline (Pro) can be used as diagnostic biomarkers of SCM during early stages of lactation at +4 to +8 weeks after parturition. In conclusion, SCM is preceded and followed by alteration in AA metabolism.

Particle-Induced X-ray Emission Analysis of Zierum Trace and Major Elements in Cattle with Acute Coliform Mastitis.

The aim of the present study was to examine the applicability of the direct determination of trace and major element concentrations in serum samples collected from Holstein dairy cattle with acute coliform mastitis (n = 53) compared with a healthy control group (n = 39). Twenty-eight elements (Na, Mg, Al, Si, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ce, Ni, Cu, Zn, Ga, As, Se, Br, Rb, Sr, Y, Zr, Nb, Mo, and Pb) were detected by particle-induced X-ray emission (PIXE). Significant differences were observed in serum K, Fe, Zn, and Br concentrations, but not in those of the remaining twenty-four elements. Furthermore, serum Fe concentrations (0.751 ± 0.583 µg/ml, n = 18) were significantly lower in dairy cattle with a poor prognosis than in those with a good prognosis (0.945 ± 0.393 µg/ml, n = 35, P < 0.05) and healthy controls (1.458 ± 0.391 µg/ml, n = 39, P < 0.01). We proposed a diagnostic cut-off point for serum Fe concentrations of <0.82 µg/ml based on receiver operating characteristic (ROC) curves in order to identify cattle with a poor prognosis. The results of the present study indicated that assessing the elemental composition of serum, particularly iron, is a promising prognostic tool for determining the outcomes of cattle with severe acute coliform mastitis.

Expression patterns of miR-146a and miR-146b in mastitis infected dairy cattle.

This study reports a significant up-regulation of bta-miR-146a and bta-miR-146b expression levels in bovine mammary tissues infected with subclinical, clinical and experimental mastitis. Potential target genes are involved in multiple immunological pathways. These results suggest a regulatory function of both miRNAs for the bovine inflammatory response in mammary tissue.

Identification of CD14 transcript in blood polymorphonuclear neutrophil leukocytes and functional variation in Holsteins.

Polymorphonuclear neutrophil (PMN) leukocytes are primary phagocytic cells of the bovine mammary gland and a first line of defense against invading pathogens during bovine mastitis infection. Cluster of differentiation 14 (CD14) is mainly expressed in macrophages and neutrophils and acts as a co-receptor that binds bacterial lipopolysaccharide (LPS) and recruits PMNs to CD14-LPS complexes in mammary epithelial cells. In this study, we identified a novel splice variant in PMNs, named CD14-SV, characterized by a deleted region from c.143-579 nt compared to the CD14 reference mRNA sequence. Moreover, a single nucleotide polymorphism (c.523 A>G) in exon 2 of CD14 was identified and found to modify the secondary structure and hydrophilicity of the CD14 protein. Association analysis also showed that the milk somatic cell score, an indicator of mastitis, of cows with the GG genotype was lower than that of cows with the AA and AG genotypes. Our findings suggest that the expression of CD14 in bovine blood PMNs is regulated by alternative splicing, and that CD14-SV is a candidate functional marker that may influence mastitis-resistance in dairy cows.

15-F2t-Isoprostane Concentrations and Oxidant Status in Lactating Dairy Cattle with Acute Coliform Mastitis.

BACKGROUND: Severe mammary tissue damage during acute coliform mastitis in cattle is partially caused by oxidative stress. Although considered a gold standard biomarker in some human conditions, the utility of 15-F2t-Isoprostanes (15-F2t-Isop) in detecting oxidative stress in dairy cattle has not been validated. HYPOTHESIS: Concentrations of 15-F2t-Isop in plasma, urine, and milk correlate with changes in oxidant status during severe coliform mastitis in cattle. ANIMALS: Eleven lactating Holstein-Friesian dairy cows in their 3rd-6th lactation. METHODS: A case-control study using cows with acute coliform mastitis and matched healthy controls were enrolled into this study. Measures of inflammation, oxidant status, and redox status in plasma and milk samples were quantified using commercial assays. Plasma, urine, and milk 15-F2t-Isop were quantified by liquid chromatography/tandem mass spectrometry (LC-MS/MS) and ELISA assays. Data were analyzed by Wilcoxon rank sum tests (α = 0.05). RESULTS: Plasma 15-F2t-Isop quantified by LC-MS/MS was positively correlated with systemic oxidant status (r = 0.83; P = .01). Urine 15-F2t-Isop quantified by LC-MS/MS did not correlate with systemic oxidant status, but was negatively correlated with redox status variables (r = -0.83; P = .01). Milk 15-F2t-Isop quantified by LC-MS/MS was negatively correlated (r = -0.86; P = .007) with local oxidant status. Total 15-F2t-Isop in milk quantified by a commercial ELISA (cbELISA) was positively correlated with oxidant status in milk (r = 0.98; P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Free plasma 15-F2t-Isop quantified by LC-MS/MS and total milk 15-F2t-Isop quantified by cbELISA are accurate biomarkers of systemic and mammary gland oxidant status, respectively. Establishing reference intervals for free and total 15-F2t-Isops for evaluating oxidative stress in dairy cows should currently be based on the LC-MS/MS method.

Different chronological patterns of appearance of blood derived milk components during mastitis indicate different mechanisms of transfer from blood into milk.

This study aimed to describe chronological patterns of changes of various candidate blood components in milk during the acute phase of a mammary immune response in detail. Eight dairy cows were challenged with Escherichia coli lipopolysaccharide in one udder quarter. Milk from challenged and control quarters and blood samples were taken before, and 1 and 2 h after challenge and then every 15 min until 5 h after challenge. The SCC, serum albumin, immunoglobulin (Ig)G1, IgG2, lactate dehydrogenase (LDH), and L-lactate in milk and blood, and α-lactalbumin in blood were analysed. All selected parameters in milk increased in challenged quarters but did not increase in control quarters. Milk IgG1, IgG2, serum albumin, and LDH were already significantly increased at 2 h after challenge whereas a significant increase of SCC was detectable at 2.75 h and L-lactate was increased at 2.25 h after challenge. In blood L-lactate was increased at 3.75 h after challenge, however, other factors in blood did not change significantly within the 5 h of experiment. In conclusion, the increase of blood components in milk during inflammation follows two different patterns: There is a rapid increase for IgG1, IgG2, or LDH, before the increase of SCC, and their concentrations reach a plateau within 3 h. On the other hand, SCC and L-lactate show a slower but consistent increase not reaching a plateau within 5 h after LPS challenge. L-lactate increases to higher concentrations in milk than in blood. This clearly shows that the increase of blood components follows different patterns and is therefore a controlled and compound-specific process and not exclusively an unspecific type of leakage.