Expression profile of genes associated with mastitis in dairy cattle

In order to characterize the expression of genes associated with immune response mechanisms to mastitis, we quantified the relative expression of the IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF- α genes in milk cells of healthy cows and cows with clinical mastitis. Total RNA was extracted from milk cells of six Black and White Holstein (BW) cows and six Gyr cows, including three animals with and three without mastitis per breed. Gene expression was analyzed by real-time PCR. IL-10 gene expression was higher in the group of BW and Gyr cows with mastitis compared to animals free of infection from both breeds (p < 0.05). It was also higher in BW Holstein animals with clinical mastitis (p < 0.001), but it was not significant when Gyr cows with and without mastitis were compared (0.05 < p < 0.10). Among healthy cows, BW Holstein animals tended to present a higher expression of all genes studied, with a significant difference for the IL-2 and IFN- γ genes (p < 0.001). For animals with mastitis no significant difference in gene expression was observed between the two breeds. These findings suggest that animals with mastitis develop a preferentially cell-mediated immune response. Further studies including larger samples are necessary to better characterize the gene expression profile in cows with mastitis.     

Differential expression of immune response genes associated with subclinical mastitis in dairy buffaloes

Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.     

Effect of clinical mastitis and other diseases on reproductive performance of Holstein cows

The objective of this study was to evaluate the effect of clinical mastitis and (or) other diseases on reproductive performance in lactating Holstein cows. Cows (n=967) from a commercial dairy farm were divided into four groups retrospectively: cows with clinical mastitis and other diseases (MD, n=54), clinical mastitis only (M, n=154), other diseases only (D, n=187), and cows with no record of clinical mastitis or other diseases (H, n=572). Days in milk at first service (DIMFS), services per conception (S/C), days not pregnant (DNP), the rate at which animals became pregnant over time and the proportion of cows that remained non-pregnant during 224 days of lactation were evaluated. Groups MD and M had greater (P<0.05) DNP compared with H (155+/-15 and 140+/-5 vs. 88+/-2, respectively). Moreover, MD and M had greater (P<0.05) S/C compared with H (3.0+/-0.4 and 2.1+/-0.1 vs. 1.6+/-0.1, respectively). The rate at which animals became pregnant over time was less (P<0.05) for MD and M and tended (P=0.1) to be less for D when compared with H. In addition, proportion of cows that remained non-pregnant by 224 days of lactation was greater (P<0.05) in MD, M, and D compared with H. Cows with mastitis were also divided into three groups according to the day of occurrence of the first case of clinical mastitis: (1) clinical mastitis occurred before 56 days postpartum (MP1); (2) clinical mastitis occurred between 56 and 105 days after parturition (MP2); and (3) clinical mastitis occurred after 105 days postpartum (MP3) Regardless of the time of occurrence, DNP was greater (P<0.05) for cows with mastitis compared with H. Time of mastitis occurrence affected S/C in that cows in MP2 and MP3 had a greater S/C compared with H cows (P<0.05). Reproductive efficiency was decreased by the presence of clinical mastitis alone because a greater proportion of cows with mastitis remained non-pregnant over time. Moreover, a greater proportion of cows with mastitis or diseases remained non-pregnant by 224 postpartum. Furthermore, the negative effects on reproduction were exacerbated when cows experienced both clinical mastitis and other diseases.     

Transition Diseases in Grazing Dairy Cows Are Related to Serum Cholesterol and Other Analytes

The objectives of this study were to describe the incidence of postpartum disease and to evaluate the association with serum cholesterol concentrations during the first 3 weeks after calving in grazing dairy cows. The association between non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), calcium and postpartum diseases was also evaluated. A total of 307 Holstein dairy cows from 6 commercial grazing herds in Osorno, Chile, were monitored from calving until 21 days in milk. Cases of retained placenta, clinical hypocalcemia and clinical mastitis were recorded by the farmer using established definitions. Twice weekly, cows were evaluated for metritis by the same veterinarian based on vaginal discharge and body temperature. Postpartum blood samples were collected weekly and analyzed for serum concentrations of cholesterol, NEFA, BHBA and calcium. Cows were considered as having subclinical ketosis if BHBA >1.2 mmol/L, and subclinical hypocalcemia if calcium <2.0 mmol/L in any of the 3 weekly samples. Overall, 56% of the cows studied developed at least one clinical or subclinical disease after calving. Incidence of individual diseases was 8.8% for retained placenta, 4.2% for clinical hypocalcemia, 11.7% for clinical mastitis, 41.1% for metritis, 19.9% for subclinical hypocalcemia and 16.6% for subclinical ketosis. Lower postpartum cholesterol in cows was associated with developing severe metritis or having more than one clinical disease after calving. For every 0.4 mmol/L decrease in serum cholesterol cows were nearly twice as likely to be diagnosed with multiple clinical diseases after calving. Higher BHBA concentrations and lower calcium concentrations during week 1 were associated with severe cases of metritis. Low serum calcium concentration during week 1 was also associated with developing more than one clinical disorder after calving. In conclusion, the incidence of postpartum diseases can be high even in grazing herds and lower serum cholesterol concentrations were associated with occurrence of clinical postpatum disorders.     

Effect of timing of first clinical mastitis occurrence on lactational and reproductive performance of Holstein dairy cows

Objectives of this study were to determine the influence of timing of first clinical mastitis case occurrence on lactational and reproductive performance in high producing lactating dairy cows during the first 320 days in milk (DIM). Holstein cows, 1001, from two commercial dairy farms in California were retrospectively divided into four treatment groups according to timing of first clinical mastitis case caused by environmental pathogens: control with no recorded clinical cases of mastitis (C; n=501); first clinical mastitis prior to first postpartum AI (MG1; n=250); first clinical mastitis between first postpartum AI and pregnancy diagnosis (MG2; n=147); and first clinical mastitis after diagnosed pregnant (MG3; n=103). Clinical cases of mastitis were identified at every milking by the herd personnel based on abnormal milk or swelling of the mammary gland. A fore sample of milk was aseptically collected from every clinical case for microbiological culture. Mastitis decreased yields of milk, 3.5% fat-corrected milk, and milk components, but the effect was only observed for MG1 and MG2. Cows in the control group had lower linear somatic cell count (SCC) score throughout the lactation. Culling was increased by mastitis, and cows in the mastitis groups left the study earlier than controls. Conception rate at first postpartum AI and pregnancy rate at the end of the study were both decreased by mastitis prior to or after first AI, and MG1 and MG2 cows had extended days open. Furthermore, cows experiencing mastitis during lactation had a higher incidence of abortions. The negative effects of mastitis on reproduction were observed regardless of clinical case being caused by either Gram positive or negative bacteria. Mastitis either prior to or after first postpartum AI impairs lactation performance, increases culling, and decreases reproductive efficiency in high producing Holstein dairy cows.     

Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis

During clinical mastitis in dairy cows, the quantity of milk produced decreases and the composition of the milk is altered. As the severity of inflammation associated with the disease increases, the chemical composition of milk approaches that of blood as a consequence of increased permeability of the blood mammary barrier, or de novo intramammary synthesis, as has been suggested for mammary associated serum amyloid A3. A better understanding of these events may provide new approaches for the diagnosis and treatment of mastitis. The objective of this study was to document the changes in the protein composition of milk during clinical mastitis using a proteomic approach, with the objective of identifying new diagnostic markers of mastitis. Whey from dairy cows with clinical mastitis was compared to whey from healthy animals by two-dimensional gel electrophoresis (2-DE) with colloidal Coomassie staining and matrix-assisted desorption/ionization mass spectrometry. Increases in the concentrations of proteins of blood serum origin, including serotransferrin and albumin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins alpha-lactalbumin and beta-lactoglobulin were reduced in mastitic whey. Mass spectrometry subsequently confirmed the location of albumin, alpha-lactalbumin and beta-lactoglobulin on the 2-DE gels at M(r)/pI of 69 294/5.8, 14 200/4.5 and 19 883/4.9 respectively.     

Acute Phase Response in Dairy Cows with Experimentally Induced Escherichia coli Mastitis

Six Finnish Ayrshire cows were challenged intramammarily with 1500 CFU of Escherichia coli (E. coli) into single udder quarters, and the challenge was repeated into contralateral quarters 3 weeks later. All cows received flunixine meglumine once, and 3 of them were also treated with enrofloxacin. At the 2nd challenge, treatments were changed vice versa. The development of mastitis was followed by monitoring of systemic and local clinical signs, and with serial milk and serum samples. Intramarnmary challenge with E. coli produced clinical mastitis in all cows, the severity of the disease varying greatly between the animals. No significant changes between the 2 treatment regimens or sequent challenges were found for any of the clinical parameters. The response of each cow followed the same pattern after both challenges; three of the cows became mildly and the other 3 either moderately or severely affected. Two severely affected cows had to be euthanized because of severe mastitis. Serum haptoglobin and amyloid-A concentrations peaked 2–3 days after bacterial challenge. Serum haptoglobin did not correlate with the severity of the disease. Serum amyloid-A rose gradually in the severely affected cows, and significant differences were found between severely versus moderately or mildly affected cows at day 4. Serum tumor necrosis factor alpha concentrations increased only in the severely affected cows. Serum Cortisol response was prolonged in the severely diseased animals, and was significantly lower after the second challenge. Serum nitrite/nitrate concentration increased in the severely affected cows. This indicated excess nitric oxide production during acute E. coli mastitis. Strongly decreased milk production, and high bacterial growth in the infected quarters were best predictors for the outcome from acute E coli mastitis.     

Genetically enhanced cows resist intramammary Staphylococcus aureus infection

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.     

Short communication: Variance and autocorrelation of deviations in daily milk yield are related with clinical mastitis in dairy cows

Daily milk production, and fluctuations therein, can provide information on health and resilience of dairy cows. We studied variance and autocorrelation of deviations in daily milk yield in relation to the occurrence of clinical mastitis (no, early or later in lactation). Individual lactation curves were fitted to 305-d lactations of 414 dairy cows using quantile regression. Log-transformed variance (lnVar) and autocorrelation of the quantile residuals of daily milk yield (predicted – observed) were evaluated for intervals until 30 and until 305 days in milk (DIM). Cows were classified as having no mastitis (n = 249), early mastitis that first occurred before 30 DIM (n = 29); or later mastitis (n = 136). Subsequently, linear models were used to assess effects of mastitis and parity class (primiparous or multiparous) on lnVar and autocorrelations; and logistic regression analyses were performed to predict mastitis from lnVar or autocorrelation and parity. From 10 to 30 DIM, lnVar was greater for cows with early mastitis than for cows with no or late mastitis, and autocorrelation tended to be lower for cows with early mastitis than for cows with no mastitis. The lnVar and autocorrelation from 10 to 30 DIM were not predictive of late mastitis. From 10 to 305 DIM, lnVar was greater and autocorrelation was lower for both cows with early and late mastitis than for cows with no mastitis; and both were predictive of having mastitis in the 305-d lactation. Primiparous cows had lower lnVar than multiparous cows. In cows without mastitis, autocorrelation values were positively correlated with lnVar. Results confirm that increased lnVar is associated with clinical mastitis.     

Engineering Disease Resistant Cattle

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk’s nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

The relationship between bovine major histocompatibility complex class II polymorphism and disease studied by use of bull breeding values

The predictive value of class II DQ and DYA polymorphisms of the bovine major histocompatibility (MHC) complex (BoLA) for the incidence of disease in dairy cattle was estimated in a sample of 196 progeny-tested AI bulls of the Swedish Red and White breed. The BoLA DQ and DYA types of the bulls were determined by analysing restriction fragment length polymorphisms (RFLPs). Breeding values of bulls for clinical mastitis, all diseases including clinical mastitis and diseases other than clinical mastitis were used as measures of disease resistance or susceptibility. The relationship between MHC polymorphism and bull breeding values for disease resistance was evaluated statistically by linear regression analysis. A significant association between the haplotype DQ1A and susceptibility to clinical mastitis was revealed. No other DQ haplotype nor the DYA locus has a significant effect on any of the disease traits studied.     

TLR-4 在慢性阻塞性肺病患者肺组织表达的研究

目的:观察COPD患者肺组织中TLR-4,IL-8,MUC5AC的表达,并探讨其在气道炎症、气道高分泌中的作用。方法:非COPD、COPD组男性肺癌病人各20例,取其肺叶切除后的外周肺组织,对肺组织标本行HE及AB-PAS染色,用免疫组织化学方法检测肺组织中TLR-4,IL-8,MUC5AC的表达并分析其相关性。结果:①COPD患者肺组织中TLR-4,IL-8,MUC5AC表达较对照组增高(P<0.05)。TLR-4主要在气道上皮细胞、肺巨噬细胞及血管内皮细胞表达,IL-8在气道壁、肺泡间隔、血管壁及肺组织内浸润的单核细胞、巨噬细胞、多形核白细胞均有表达,MUC5AC主要在气道上皮杯状细胞中表达。②TLR-4、IL-8表达与气道炎细胞评分成正相关(P<0.05)。TLR-4与IL-8、MUC5AC表达成正相关(P<0.05)。结论:COPD患者肺组织中TLR-4高表达可能参与了COPD的气道炎症及气道高分泌,这可能是通过增加IL-8与MUC5AC的表达来实现的。     

Endometrial expression of selected transcripts involved in prostaglandin synthesis in cows with endometritis

Several cytokines and prostaglandins play an important role in preparing the endometrium for implantation and mediating pro-inflammatory events. The aim of the present study was to examine mRNA expression of interleukin 1α (IL-1α), interleukin receptor antagonist (IL-1-RN), cytosolic prostaglandin E synthase (cPGES), microsomal PGES (mPGES-1 and mPGES-2) and lipocalin-type PGDS (L-PGDS) in the bovine endometrium. Endometrial epithelium samples were collected ex vivo from cows with different status of health at day 21-27 postpartum on a dairy farm. Three groups (n = 9 animals each) were defined: (1) healthy cows with no signs of endometritis (control group), (2) cows with subclinical endometritis, and (3) cows with signs of clinical endometritis. Oestrous cycle-dependent mRNA expression pattern was investigated using bovine endometrial epithelial cells from healthy uteri collected at the abattoir. These uteri were classified into post-ovulatory, early-to-mid luteal, late luteal or pre-ovulatory phase (n = 8 animals for each cycle phase). After collecting endometrial epithelium using the cytobrush-method, mRNA analysis was performed by real-time RT-PCR. L-PGDS, IL-1α and IL-1-RN mRNA were expressed significantly higher (P < 0.05) in the endometrium of cows with subclinical or clinical endometritis compared with healthy cows. A twofold lower cPGES mRNA expression (P < 0.05) was detected in cows with subclinical endometritis compared to healthy cows. L-PGDS and IL-1-RN mRNA expression was increased (P < 0.05) after ovulation compared with the pre-ovulatory or luteal phase, respectively. These results support the hypothesis that a dys-regulated cytokine and/or prostaglandin profile in the uterus could be induced by subclinical endometritis or clinical endometritis.     

Ghrelin and orotic acid increased in subclinical mastitis

Hormone ghrelin and orotic acid accelerate wound healing as well as controlling inflammation and immunity. We have, therefore, investigated the serum and milk levels of ghrelin and orotic acid in dairy cows with (n = 21) or without (n = 21) subclinical mastitis. Acylated and des-acylated ghrelin as well as orotic acid concentration were detected by using high performance liquid chromatography (HPLC). The results revealed that ghrelin level in milk and serum was significantly higher in dairy cows with subclinical mastitis than that of dairy cows without subclinical mastitis. This was also the case when the orotic acid concentrations in dairy cows with subclinical mastitis were compared with those dairy cows without subclinical mastitis. In conclusion, ghrelin and orotic acid occur in particularly high concentrations in subclinical mastitis, and might, therefore, be required in greater amounts for tissue repair and may be also used as a indicator for subclinical mastitis.     

Factors affecting conception rate after artificial insemination and pregnancy loss in lactating dairy cows

Objectives were to determine factors associated with conception rate (CR) and pregnancy loss (PL) in high producing lactating Holstein cows. In Study 1, CR was evaluated in 7633 artificial inseminations (AI) of 3161 dairy cows in two dairy farms. Pregnancy diagnosis was performed by palpation per rectum 39+/-3 days after AI. Environmental temperature was recorded at different intervals prior to and after AI. In Study 2, 1465 pregnancies from 1393 cows diagnosed at 31+/-3 days after AI by ultrasonography on three dairy farms were re-examined 14 days later to determine PL. Temperature > or =29 degrees C was considered to be heat stress (HS). Exposure to HS was defined as following: NH, no heat stress; HS1, exposure to at least 1 day of maximum temperature > or =29 degrees C and average daily maximum temperature (ADMT) <29 degrees C; and HS2, exposure to ADMT > or =29 degrees C. In Study 1, exposure of cows to HS1 and HS2 from 50 to 20 prior to AI was associated with reduced CR compared to cows not exposed to HS (28.8, 23.0, and 31.3%, respectively). Post-AI HS was not associated with CR. Cows inseminated following estrus detection or timed AI had similar CR. As the number of AI increased, CR decreased. Multiparous cows had lower CR than primiparous cows, and occurrence of milk fever and retained placenta was associated with decreased CR. In Study 2, PL was not associated with exposure to HS either prior to or after AI. Cows diagnosed with clinical mastitis experienced increased PL, but parity, number of AI, AI protocol, milk production, and days postpartum at AI were not associated with PL. In conclusion, CR was affected by HS prior to AI, parity, number of AI, and postparturient diseases, whereas PL was affected by clinical mastitis.     

Preliminary field evidence for the association of clinical mastitis with altered interestrus intervals in dairy cattle

Clinical mastitis and reproductive records from two southern California dairy herds were used in a cross-sectional study to determine the risk of an altered interestrus interval following clinical mastitis. An altered interestrus interval was defined as cycles occurring at either less than 18 day or more than 24-day intervals. The data were stratified by herd to assess herd differences and by lactation number to assess confounding by cow parity. The predominant pathogen isolated from mastitis cases in Herd 1 was Staphylococcus aureus , whereas the predominant pathogens in Herd 2 were gram-negative isolates. Cows in Herd 1 which had coliforms cultured from mastitic milk were excluded in the analysis for comparison with Herd 2. In Herd 1, cows with clinical mastitis were less likely to have an altered interestrus interval (Relative Risk [RR]=0.9; 95% Confidence Interval [CI]=0.6,1.6) than herdmates without clinical mastitis. However, cows in Herd 2 were almost two times more likely to have an altered interestrus interval following an episode of clinical mastitis compared to herdmates without clinical mastitis (RR=1.6; 95% CI=1.3,2.0). Because herd management practices differ and could cause differences in mastitis and reproductive outcomes at the dairies, this preliminary field evidence should be followed by prospective studies of luteal function after clinical coliform mastitis.     

Lipopolysaccharide pretreatment of the udder protects against experimental Escherichia coli mastitis

Exposure to pathogen-associated molecular patterns such as LPS can cause an immune refractory state in mammals known as endotoxin tolerance (ET), resulting in a decreased inflammatory response after pathogen contact. This ET concept was used to reduce the severity of an experimentally-induced clinical mastitis. Cows were pretreated with 1?μg LPS per udder quarter and challenged 72?h (group L72EC) or 240?h (group L240EC) later with 500 CFU Escherichia coli. Pretreated animals showed no leukopenia after challenge, no (L72EC), or only slightly (L240EC), elevated body temperature and significantly reduced systemic and local clinical scores compared with cows that were not pretreated. Whereas an increase of milk somatic cell count after the E. coli challenge was abrogated in L72EC animals, it was significantly delayed in the L240EC group. In both pretreated groups the bacterial load in milk was markedly reduced. Based on the expression of inflammation-related genes in lobulo-alveolar mammary tissue, the tolerizing effect of LPS pretreatment is based on the inhibited up-regulation of inflammatory (TNF-α, IL-6, CXCL8, CCL20) and anti-inflammatory genes (IL-10, IRAK-M). These findings indicate that the concept of ET may be usefully applied as mastitis prophylaxis facilitating a rapid response to microbial infection and avoiding dysregulated inflammation.