Short communication: Association between prepartum subclinical magnesium imbalance and postpartum diseases in grazing dairy cows in Southern Chile.

This study aimed to evaluate the relationship between prepartum subclinical hypomagnesemia (pre-SHMg) and the occurrence of dystocia, metritis, clinical mastitis, lameness, and subclinical hypomagnesemia postpartum (post-SHMg) in pasture-based dairy cows. Also, the difference in means of prepartum magnesium (Mg) concentration by postpartum health events was evaluated. A total of 890 dairy cows from 32 commercial farms located in southern Chile were enrolled. Cows were examined twice, once between 30 and 3 days before and once between 3 and 30 days after calving. Blood samples were collected on both assessments, and cows were considered as having SHMg if serum total Mg < 0.65 mmol/L. On the postpartum visit, cows were evaluated for metritis and lameness. Information about clinical mastitis and dystocia was collected from on-farm records. Data were analyzed using multivariable mixed linear models and multivariable mixed logistic regression models. The overall prevalence of pre-SHMg was 9.9%, and its presence was associated with the occurrence of post-SHMg (odd ratio [OR] = 5.7; P < 0.0001) and metritis (OR = 3.1; P = 0.04). However, we did not detect an association between pre-SHMg and dystocia, clinical mastitis, or lameness after calving. Prepartum serum Mg concentrations were lower in cows that developed post-SHMg than those that did not (LSM ± SE = 0.75 ± 0.02 mmol/L vs. 0.83 ± 0.02 mmol/L; P < 0.0001). In conclusion, pre-SHMg was associated with a higher risk of post-SHMg and metritis in grazing dairy cows but not other postpartum health events.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS-induced mastitis in dairy cows.

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long-term manipulated glucose and insulin concentrations in combination with a LPS-induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT-qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha-lactalbumin, insulin-induced gene 1, κ-casein and acetyl-CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS-induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.

Molecular epidemiology of recurrent clinical mastitis due to Streptococcus uberis: evidence of both an environmental source and recurring infection with the same strain.

This study was undertaken because clinicians and farmers have observed that a considerable number of cows diagnosed with Streptococcus uberis mastitis have recurrences of mastitis in the same or a different quarter. The study was an attempt to answer whether these recurring cases were due to treatment failure (in which case a search would have begun for a better treatment for Strep. uberis mastitis) or due to reinfection with a different strain of Strep. uberis. Using pulsed-field gel electrophoresis (PFGE), we determined that the majority of recurrences (20 of 27) were caused by a new strain of Strep. uberis, indicating that treatment of the initial infection had been successful. A small number of recurrences (5 of 27) were caused by the initial strain, indicating persistence. The remaining 2 recurrences occurred in a new quarter but with the initial strain of Strep. uberis, indicating either spread between quarters or reactivation of a previous subclinical infection. Analysis of the PFGE profiles failed to reveal any strain-specific propensity to persist, because strains causing recurrences occurred in most of the major clusters.

Induced hyperketonemia affects the mammary immune response during lipopolysaccharide challenge in dairy cows.

Metabolic adaptations during negative energy and nutrient balance in dairy cows are thought to cause impaired immune function and hence increased risk of infectious diseases, including mastitis. Characteristic adaptations mostly occurring in early lactation are an elevation of plasma ketone bodies and free fatty acids (nonesterified fatty acids, NEFA) and diminished glucose concentration. The aim of this study was to investigate effects of elevated plasma ß-hydroxybutyrate (BHBA) at simultaneously even or positive energy balance and thus normal plasma NEFA and glucose on factors related to the immune system in liver and mammary gland of dairy cows. In addition, we investigated the effect of elevated plasma BHBA and intramammary lipopolysaccharide (LPS) challenge on the mammary immune response. Thirteen dairy cows were infused either with BHBA (HyperB, n=5) to induce hyperketonemia (1.7 mmol/L) or with a 0.9% saline solution (NaCl, n=8) for 56 h. Two udder quarters were injected with 200 µg of LPS after 48 h of infusion. Rectal temperature (RT) and somatic cell counts (SCC) were measured before, at 48 h after the start of infusions, and hourly during the LPS challenge. The mRNA abundance of factors related to the immune system was measured in hepatic and mammary tissue biopsies 1 wk before and 48 h after the start of the infusion, and additionally in mammary tissue at 56 h of infusion (8h after LPS administration). At 48 h of infusion in HyperB, the mRNA abundance of serum amyloid A (SAA) in the mammary gland was increased and that of haptoglobin (Hp) tended to be increased. Rectal temperature, SCC, and mRNA abundance of candidate genes in the liver were not affected by the BHBA infusion until 48 h. During the following LPS challenge, RT and SCC increased in both groups. However, SCC increased less in HyperB than in NaCl. Quarters infused with LPS showed a more pronounced increase of mRNA abundance of IL-8 and IL-10 in HyperB than in NaCl. The results demonstrate that an increase of plasma BHBA upregulates acute phase proteins in the mammary gland. In response to intramammary LPS challenge, elevated BHBA diminishes the influx of leukocytes from blood into milk, perhaps by via modified cytokine synthesis. Results indicate that increased ketone body plasma concentrations may play a crucial role in the higher mastitis susceptibility in early lactation.

Serum iron as an indicator of acute inflammation in cattle.

The aim of this study was to assess the value of serum iron concentration in the diagnosis of acute inflammation in cattle. The diagnostic value of this approach was compared with that of various other hematological tests, including commonly used techniques that measure the levels of various other acute-phase proteins. The study population comprised 10 cows with acute traumatic reticuloperitonitis (RPT group) and 10 cows with acute mastitis (mastitis group) admitted to the Veterinary Teaching Hospital at Firat University (Elazig, Turkey). Ten cows from local barns, kept and fed under same conditions as the diseased animals, were used as controls. After the clinical examination, blood samples were collected for biochemical, hematological, and acute-phase protein (haptoglobin, serum amyloid A, α-1 acid glycoprotein, and fibrinogen) analyses. The mean levels of serum iron in the RPT, mastitis, and control groups were 6.00, 7.82, and 26.78 µmol/L, respectively. Serum iron level was significantly reduced in the RPT and mastitis groups. The results of this study indicate that serum iron analysis, preferably in combination with other markers of inflammation, may be a useful diagnostic tool for acute inflammation in cattle. Because serum iron measurement is individually available and easily applicable, it may be used for clinical cases as well as the determination of herd health.

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 ß-hydroxybutyrate (BHBA) before and during the response to an intramammary lipopolysaccharide (LPS) challenge. Thirteen dairy cows received intravenously either a Na-DL-ß-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-ß-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.

Development of an indirect ELISA for detection of E. Coli antibodies in cow serum using a recombinant OmpT as antigen.

The aim of this article was to develop an indirect enzyme-linked immunosorbent assay (ELISA) for efficient detection of the infection of E. coli in cattle. OmpT, a highly conserved protease in all E. coli strains, was successfully expressed in E. coli XL-1-Blue strain with PET32a vector. Molecular weight of recombinant protein was identified by analyzing SDS-PAGE and the immunogenicity of OmpT was confirmed by Western Blotting. The recombinant OmpT was then employed as capture antigen in the ELISA. The antigen concentration and serum dilution were determined using a checkerboard titration. Results showed that the optimal concentration of coated antigen was 1 µg/ml at a serum dilution of 1:640 and the cut-off value of the assay was 0.335. In addition, the cross-reactivity assay showed that the OmpT was E. coli specific and the reproducibility experiments displayed good repeatability of the assay. Three hundred and forty cattle serum samples were tested by rOmpT-ELISA and sera coagulation tests. The ELISA has showed relative sensitivity of 100% and specificity of 96.47%. Results of these experiments indicated that the rOmpT-ELISA is a simple, rapid, and convenient method for detection the infection of E. coli with different serotype strains.

Short communication: Oxidative status and incidence of mastitis relative to blood α-tocopherol concentrations in the postpartum period in dairy cows.

Vitamin E supplementation, when combined with high blood α-tocopherol (>6.25 µg/mL) at dry off, has been reported to unexpectedly increased the risk for clinical mastitis in dairy cows. Furthermore, higher levels of oxidative stress in the postpartum period were related to higher risk of mastitis. The objective of the present study was to determine the relationship between various serum biomarkers of oxidative status, incidence of mastitis, and blood α-tocopherol concentrations at dry off and at calving. A total of 146 dairy cows from a commercial farm were used in an observational field study. All cows were supplemented with 3,000 and 50 IU/cow per day of all-rac-α-tocopherol during the dry period and lactation, respectively. Blood samples were collected at dry off and at calving. Serum was analyzed for α-tocopherol, levels of reactive oxygen metabolites (ROM), thiol groups (SH), and ferric-reducing ability. Three α-tocopherol groups at calving were created: high (>3 µg/mL), medium (2-3 µg/mL), and low (<2 µg/mL). Three α-tocopherol groups at dry off were created: high (>6.25 µg/mL), medium (4.25-6.25 µg/mL), and low (<4.25 µg/mL). All cases of clinical mastitis that occurred during the dry period and the entire subsequent lactation were verified by a veterinarian. No differences were observed in the incidence of mastitis between the 3 α-tocopherol groups based on the serum levels at dry off. Incidence of mastitis was 4 times lower in the high and medium groups when compared with the corresponding value for the low-α-tocopherol group based on the serum levels at calving. Lower levels of ROM and SH at dry off and at calving were found in the group of cows with the highest α-tocopherol values at dry off when compared with the corresponding values in the low-α-tocopherol group. The ROM values at dry off but not at calving were lower in the group of cows with the highest α-tocopherol values at calving when compared with the corresponding values in the low-α-tocopherol group. No differences were observed in ferric-reducing ability values between the 3 α-tocopherol groups at dry off or calving. No differences were observed in all biomarkers of oxidative status between healthy cows and those with mastitis. Thus, blood α-tocopherol is inversely related to certain biomarkers of oxidative stress in the postpartum period and incidence of mastitis. However, reduction in the incidence of mastitis is not mediated through a reduction in the levels of various biomarkers of oxidative stress.