Dietary-induced negative energy balance has minimal effects on innate immunity during a Streptococcus uberis mastitis challenge in dairy cows during midlactation

Ten multiparous Holstein cows were used to determine the effects of negative energy balance (NEB) on the immune response to a Streptococcus uberis (strain O140J) mastitis challenge during midlactation. Before the study, milk from all quarters of each cow was bacteriologically negative, with a composite somatic cell count of <200,000 cells/mL. Cows were paired based on parity, days in milk, and milk yield. At approximately 77 d in milk, half the cows (n = 5) were feed-restricted to 60% of calculated net energy for lactation requirements to induce NEB. Feed restriction lasted 7 d. Control cows (n = 5) were fed the same diet ad libitum (i.e., positive energy balance; PEB). After 5 d, one rear quarter in all cows was inoculated with 5,000 cfu of Strep. uberis. Jugular blood and aseptic quarter milk samples were collected daily until inoculation and every 6 h postinoculation for 36 h. Blood was analyzed for nonesterified fatty acids, β-hydroxybutyrate, insulin, cortisol, albumin, serum amyloid A (SAA), and haptoglobin (Hp). Periodically throughout the trial period, blood neutrophils were isolated for determination of cell morphology, chemotaxis, and phagocytosis capability in vitro. Quarter milk samples were analyzed for concentrations of SAA, Hp, cytokines (tumor necrosis factor-α, IL-10 and IL-1β), and activity of respiratory burst enzymes (superoxide dismutase and glutathione peroxidase). All cows developed local and systemic signs of mastitis and calculated NEB was similar to that of cows experiencing postpartal NEB. Serum glucose and insulin concentrations increased in both groups after challenge, most likely because of enhanced glycogenolysis and gluconeogenesis; results indicate that immune cell function may be glucose dependent. Serum cortisol concentration was higher in NEB than PEB cows during feed restriction only (before inoculation), and serum albumin concentration was higher in NEB than PEB cows during the infection period. Compared with PEB, cows in NEB had lower SAA concentrations in serum after 5 d of feed restriction but higher SAA concentrations in milk after Strep. uberis challenge. Serum Hp concentration was higher by 36 h postchallenge in NEB than in PEB cows. Phagocytic capability of neutrophils was lower in NEB than in PEB cows at 0 h of infection but decreased in both PEB and NEB cows through 36 h postinfection. Our results indicate that cows subjected to dietary-induced NEB during midlactation had relatively minimal alterations in immune function.     

Between-cow variation in dermal fibroblast response to lipopolysaccharide reflected in resolution of inflammation during Escherichia coli mastitis

Effective response to mammary gland infection depends on efficient early innate immune response. The desired response would be one that is sufficient to clear the infection with a rapid return to the production of high-quality milk and limited tissue damage. In this study, 43 early lactation cows were ranked based on the ability of their fibroblasts to produce IL-8 in response to Escherichia coli lipopolysaccharide. Subsequently, the effect of a low or high response phenotype on the response to E. coli mastitis was determined. Untreated fibroblasts produced no detectable IL-8, whereas the range of IL-8 production in response to LPS (100 ng/mL) was approximately 7-fold between the lowest and highest responding cultures. Similar patterns of between-cow variation were observed in fibroblast production of IL-8 and IL-6 in response to IL-1β and Pam2CSK4 (a synthetic diacylated lipopeptide ligand). Four low and 4 high responder cows were challenged in late lactation with intramammary infusion of E. coli. All cows developed clinical mastitis in the challenged quarters and all cows cleared the infection within 8 d. However, somatic cell count began to decline earlier in the low responder group, and milk BSA concentration (an indicator of tissue damage) was also lower in low responders compared with high responders. Milk production from the challenged quarter was markedly depressed in both groups, but returned toward prechallenge values earlier in low responder cows. Dermal fibroblast cells appear predictive of a cow's response to mastitis. In this study, the low responder phenotype was sufficient to contain an E. coli infection with a more rapid return to the production of high quality milk.     

Moderate inflammatory reaction during experimental Escherichia coli mastitis in primiparous cows

Nineteen primiparous cows were experimentally infected in 2 quarters with 1 x 10(4) (group A) or 1 x 10(6) (group B) cfu of Escherichia coli P4:O32 per quarter within 2 to 4 wk after parturition. Blood and milk samples were collected from all primiparous cows at regular time intervals from d -4 to d +3 relative to inoculation. Milk production rapidly decreased in both groups during E. coli mastitis, but recovery appeared to be faster in group B at d + 1 postinfusion (p.i.). The milk production losses in the noninfected quarters were substantial on the day of inoculation, which is probably due to pronounced systemic effects. However, on d + 2 p.i. milk production in the noninfected quarters nearly reached preinfection levels, indicating a moderate clinical severity following intramammary inoculation. None of the other severity criteria evolved towards a severe response pattern. Reticulorumen motility was inhibited in both groups during E. coli mastitis. The clinical episode was short lasting in both groups. Rectal temperature, heart rate, blood leukocyte count, number of colony-forming units, milk somatic cell count and several indicators for the disintegration of the blood-milk barrier returned to normal values within 24 to 72 h p.i. Primiparous cows reacted with a moderate inflammatory response following intramammary infusion with a relatively high dose of E. coli. Despite the use of a high inoculum dose, primiparous cows in both groups showed pronounced resistance against severe intramammary E. coli infection. A possible effect of the inoculum dose could be present, however, further research into the effect of the inoculum dose on the inflammatory response should be performed.     

Local and systemic response to intramammary lipopolysaccharide challenge during long-term manipulated plasma glucose and insulin concentrations in dairy cows

The metabolic load during periods of high milk production in dairy cows causes a variety of changes of metabolite blood concentrations including dramatically decreased glucose levels. These changes supposedly impair the immune system. The goal of this study was, therefore, to evaluate adaptations of the cow's immune system in response to an intramammary lipopolysaccharide (LPS) stimulation during a 3-d modification of plasma glucose and insulin induced by different clamp infusions. Seventeen midlactating dairy cows received a hypoglycemic hyperinsulinemic clamp induced by insulin infusion (HypoG; n=5), a euglycemic hyperinsulinemic clamp induced by insulin and glucose infusion (EuG; n=6), or infusion of saline solution (NaCl; n=6) for 56 h. At 48 h of infusion, 2 udder quarters were challenged with 200 μg of Escherichia coli LPS. At 48 h of infusion (immediately before LPS challenge), tumor necrosis factor α, lactoferrin, and serum amyloid A (SAA) mRNA abundance was increased in HypoG and Il-1β mRNA abundance was decreased in EuG. After LPS challenge, plasma glucose concentration did not decrease, although plasma insulin increased simultaneously in all groups either due to enhanced endogenous release (NaCl) or due to increased insulin infusion rate (HypoG; EuG). Plasma cortisol, rectal temperatures, and milk somatic cell count of challenged quarters increased, whereas plasma nonesterified fatty acid concentrations were similarly decreased across treatments. In mammary biopsies, increased mRNA expression of tumor necrosis factor α, IL-1β, IL-8, and IL-10, and SAA were observed in LPS-treated quarters of all groups, with a more pronounced increase in IL-1β, IL-10, and SAA expression in EuG. Nuclear factor-κB mRNA expression was upregulated in NaCl and EuG but not in HypoG in response to LPS. Lactoferrin, toll-like receptor 4, and cyclooxygenase-2 mRNA expression was increased in LPS-treated quarters of EuG only, and 5-lipoxygenase mRNA expression was decreased in LPS-treated quarters only in treatments HypoG and NaCl. In conclusion, intramammary LPS induces local and systemic inflammatory responses, as well as systemic insulin resistance. The observed treatment differences of the mammary mRNA expression of several immune parameters both before and after LPS challenge indicate a direct influence of changed glucose and insulin concentrations during the course of lactation on the immune defense against mastitis pathogens.     

Effect of carprofen treatment following experimentally induced Escherichia coli mastitis in primiparous cows

Acute Escherichia coli mastitis is one of the major sources of economic loss in the dairy industry due to reduced milk production, treatment costs, discarded milk, and occasional fatal disease. Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used as adjunctive therapy to antibiotics. The objective of the current study was to evaluate the effect of carprofen treatment following infusion of Escherichia coli into the mammary glands of primiparous cows during the periparturient period. Severity of mastitis was scored based on the average milk production in the uninfected quarters on d +2 postinoculation and a clinical severity score. Carprofen was administered intravenously at 9 h postchallenge, when clinical signs of mastitis appeared. In previous work, efficacy of NSAIDs was mainly evaluated using clinical symptoms. In the present study, the effect of carprofen on innate immune response was also assessed by quantification of inflammatory mediators. All primiparous cows reacted as moderate responders throughout the experimental period. Primiparous cows were intramammarily inoculated with 1 × 10⁴ cfu of E. coli P4:O32 in 2 left quarters. Analysis of blood and milk parameters, including IL-8, complement component C5a, lipopolysaccharide-binding protein (LBP), soluble CD14, prostaglandin E₂, and thromboxane B₂ was performed from d 0 to d +6 relative to intramammary inoculation. Rectal temperature in carprofen-treated animals was lower than in control animals at 3 and 6 h posttreatment. Treatment also restored the decreased reticulorumen motility that occurs during E. coli mastitis to preinfection levels faster than in control animals. Carprofen treatment resulted in an earlier normalization of the clinical severity score. Eicosanoid (prostaglandin E₂ and thromboxane B₂) production in milk tended to be inhibited by carprofen. No significant differences in the kinetic patterns of somatic cell count, IL-8, complement component C5a, LBP, and soluble CD14 were observed. In conclusion, carprofen treatment improved general clinical condition by effective antipyrexia and restoration of reticulorumen motility but did not significantly inhibit eicosanoid production. Carprofen treatment did not result in a significant decrease of chemotactic inflammatory mediators, IL-8 and C5a, and early innate immune molecules, sCD14 and LBP. Therefore, major modulatory effects from NSAID administration were not observed in this mastitis model, although a larger study might confirm some apparent trends obtained in the present results.     

Factors associated with concentrations of select cytokine and acute phase proteins in dairy cows with naturally occurring clinical mastitis

The objective of the current observational study was to determine the potential associations between cow factors, clinical mastitis (CM) etiology, and concentrations of select acute phase proteins and cytokines in milk from affected quarters of cows with CM. Cows with CM (n = 197) were grouped based on systemic disease severity, milk culture result, parity, days in milk (DIM), previous CM occurrence, and season of the year when CM occurred. Concentrations of lipopolysaccharide-binding protein (LBP), haptoglobin (Hp), BSA, IFN-γ, tumor necrosis factor-α (TNF-α), IL-1β, IL-8, IL-10, IL-12, transforming growth factor (TGF)-α, and TGF-β and activity of lactate dehydrogenase (LDH) were evaluated. Differences in the least squares means log₁₀ transformed concentrations of these proteins were compared using multiple linear regression mixed models. The milk concentrations of LBP, Hp, IL-1β, IL-10, and IL-12, and activity of LDH in milk were higher in cows with moderate to severe versus mild systemic disease. The concentrations of Hp, BSA, IL-1β, and IL-10 in milk were higher in cows with a gram-negative versus gram-positive milk culture result. Season of the year when CM occurred was associated with the concentration of all proteins evaluated except for IL-1β and IL-12. Concentrations were higher in the winter versus summer except for Hp and TGF-β, for which the opposite was true. Concentrations of LBP, IL-10, and IL-12, and LDH activity in milk were associated with DIM group. Except for LBP, these proteins were lower in cows with CM during the first 60 DIM versus those in mid or later lactation. Interferon-γ, TNF-α, and IL-8 were undetectable in 67, 31, and 20% of samples, respectively. Detection of IFN-γ and IL-8 was associated with season, and detection of TNF-α and IL-8 was associated with systemic disease severity. The current study provides the most comprehensive report of milk concentrations of innate immune response proteins in cows with naturally occurring CM and identifies factors that potentially influence those concentrations. Further investigation into the seasonal variation of cytokine production and its potential effect on the outcome of CM is warranted. Furthermore, the results of this study provide useful data for planning future studies examining the role of the innate immune response in CM.     

Randomized clinical trial to evaluate the efficacy of a 5-day ceftiofur hydrochloride intramammary treatment on nonsevere gram-negative clinical mastitis

The objective of this study was to evaluate the efficacy of intramammary treatment with ceftiofur hydrochloride of nonsevere, clinical coliform mastitis. One hundred four cases on 5 farms met the enrollment criteria for the study. Escherichia coli was the most common coliform species identified in milk samples from cows with mild to moderate clinical mastitis, followed by Klebsiella spp. and Enterobacter spp. At enrollment, a milk sample from the affected quarter was taken and used for on-farm culture or submitted to the laboratory. For cows in the treatment group, treatment was initiated with ceftiofur hydrochloride via intramammary infusion at 24-h intervals for 5 d according to label standards. Cows in the control group did not receive treatment. Culture results were available on the day after enrollment and only cows with coliform mastitis continued in the treatment and untreated control groups. Bacteriological cure was defined based on 2 posttreatment milk samples. Molecular typing was used for final definition of bacteriological cure. Treatment of nonsevere clinical gram-negative mastitis with ceftiofur hydrochloride resulted in a significant increase in bacteriological cure compared with nontreated controls in animals infected with E. coli or Klebsiella spp. Treated animals clinically improved significantly more compared with control cows. No significant differences were observed between treated and control animals in milk production or linear score before or after clinical mastitis. Treated animals left the study less frequently compared with control animals.     

Determination of milk and blood concentrations of lipopolysaccharide-binding protein in cows with naturally acquired subclinical and clinical mastitis

Blood and milk concentrations of the acute phase protein lipopolysaccharide-binding protein (LBP) were evaluated in cows with naturally occurring mastitis. Blood and milk samples were collected from 101 clinically healthy dairy cows and 17 dairy cows diagnosed with clinical mastitis, and the LBP concentrations of the samples were measured by an ELISA. Concentrations of LBP were greater in the blood and milk of cows with clinical mastitis than in those with healthy quarters. Concentrations of LBP also differed between uninfected and subclinically infected quarters with low somatic cell count. Blood concentrations of LBP in cows with subclinical intramammary infections could not be differentiated from those of cows with all healthy quarters. Together, these data demonstrate that increased blood and milk concentrations of LBP can be detected in dairy cows with naturally acquired intramammary infections that cause clinical mastitis.     

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 μg of LPS or 20 μg of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12 h relative to the challenge to measure mRNA expression of tumor necrosis factor-α (TNFα), IL-1β, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFα. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFα in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFα, IL-1β, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.     

Short communication: Pheromonicin-SA affects mRNA expression of toll-like receptors, cytokines, and lactoferrin by Staphylococcus aureus-infected bovine mammary epithelial cells

Pheromonicin-SA (Ph-SA) is a newly developed, engineered multidomain peptide that has a bactericidal effect against Staphylococcus aureus. The objective of this study was to characterize innate immune responses by Staph. aureus-stimulated bovine mammary epithelial cells (BMEC) following treatment with Ph-SA. Primary BMEC from one lactating Holstein cow were isolated and exposed to Staph. aureus for 2 h, and then treated with rifampicin or Ph-SA. Total RNA was isolated from BMEC at 0, 2, 6, 12, and 24 h postinfection, and the mRNA expression of selected genes, including toll-like receptor (TLR)2 and TLR4, IL-1β, IL-6, IL-8, tumor necrosis factor α (TNF-α), and lactoferrin, was quantified by real-time PCR. In the rifampicin group, increases in the expression of mRNA for TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection and in the expression of mRNA for TLR2 but not for TLR4 at 12 h postinfection. In the Ph-SA group, increases in the mRNA expression of TLR2, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection, and an increase in TLR4 mRNA expression was observed at 24 h postinfection. At 24 h postinfection, the mRNA expression of TLR4, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin was higher in the Ph-SA group than in the rifampicin group. In conclusion, Ph-SA might promote the expression of mRNA for TLR2, TLR4, the pro-in?ammatory cytokines IL-1, IL-6, and TNF-α, the chemotactic factor IL-8, and lactoferrin in Staph. aureus-infected BMEC. Moreover, Ph-SA may be of value as an antibiotic in promoting innate immune responses by Staph. aureus-infected bovine mammary epithelial cells.     

Increased milk levels of transforming growth factor-alpha, beta1, and beta2 during Escherichia coli-induced mastitis

Among the gram-negative bacteria that cause mastitis, Escherichia coli are the most prevalent. The innate immune system provides initial protection against E. coli infection by detecting the presence of the foreign pathogens and by mounting an inflammatory response, the latter of which is mediated by cytokines such as IL-1β, IL-8, and tumor necrosis factor (TNF)-α. Although changes in these cytokines during mastitis have been well-described, it is believed that other mediators moderate mammary gland inflammatory responses as well. The growth factors/cytokines transforming growth factor (TGF)-α, TGF-β1, and TGF-β2 are all expressed in the mammary gland and have been implicated in regulating mammary gland development. In other tissues, these growth factors/cytokines have been shown to moderate inflammation. The objective of the current study was to determine whether TGF-α, TGF-β1, and TGF-β2 milk concentrations were altered during the course of E. coli-induced mastitis. The contralateral quarters of 11 midlactating Holstein cows were challenged with either saline or 72 cfu of E. coli, and milk samples were collected. Basal milk levels of TGF-α, TGF-β1, and TGF-β2 were 98.81 ± 22.69 pg/mL, 3.35 ± 0.49 ng/mL, and 22.36 ± 3.78 ng/mL, respectively. Analysis of whey samples derived from E. coli-infected quarters revealed an increase in milk levels of TGF-α within 16 h of challenge, and these increases persisted for an additional 56 h. Elevated TGF-β1 and TGF-β2 milk concentrations were detected in E. coli-infected quarters 32 h after challenge, and these elevations were sustained throughout the study. Because TGF-α, TGF-β1, and TGF-β2 have been implicated in mediating inflammatory processes, their induction during mastitis is consistent with a role for these molecules in mediating mammary gland host innate immune responses to infection.     

Evaluation of an alternative dosing regimen of a J-5 mastitis vaccine against intramammary Escherichia coli challenge in nonlactating late-gestation dairy cows

The objective of the study was to evaluate the efficacy of an alternative vaccination regimen of a J-5 bacterin against intramammary Escherichia coli challenge in nonlactating late-gestation dairy cows. The parameters analyzed to assess the effect of vaccination were milk yield, milk conductivity, somatic cell count, J-5-specific serum IgG titers, and clinical signs. Twenty multiparous Holstein cows from the Cornell teaching and research dairy herd were paired by days in milk and were randomly selected to receive either the alternative off-label regimen of commercial J-5 bacterin or act as nonvaccinated controls. Cows received a first dose of bacterin 15 d before dry off, a second dose with the same product at the day of dry off, and the third dose 2 wk after dry off. The cows in both groups were challenged 10 d before the expected calving date. Serum IgG (total, IgG1 and IgG2) levels were higher in vaccinates compared with control cows. Eighty-five percent of challenged quarters became infected between both groups of animals. Eight of the 10 vaccinated and 9 of the 10 control cows showed signs of clinical mastitis postfreshening. A non-severe clinical mastitis was observed 24 to 48 h postparturition, characterized by flakes or clots in milk and mild swelling or pain. Off-label vaccination did reduce the clinical severity of clinical mastitis in the vaccinated group of cows as evidenced by reduced California mastitis test score, fewer flakes and lower overall clinical mastitis score. No significant differences between vaccinated and control groups were detected for rectal temperature. In conclusion, the alternative off-label vaccination scheme used in our study and evaluated in a novel E. coli challenge model did not prevent new intramammary infections but reduced clinical severity of experimentally induced E. coli mastitis.     

Haptoglobin concentrations in blood and milk after endotoxin challenge and quantification of mammary Hp mRNA expression

Haptoglobin (Hp), an acute phase protein mostly secreted by the liver, is an inflammatory marker. To use the full diagnostic potential of Hp measurements for mastitis, we developed and validated an ELISA sensitive to quantify even basal and subclinical concentrations in both blood and milk. Bovine Hp was purified from serum and was used as a standard and to generate polyclonal antiserum. The limit of detection was 0.07 microg of Hp/mL. From 6 cows challenged by intracisternal injection of lipopolysaccharide (LPS) into one quarter, blood samples were collected 0, 3, 6, 9, and 12 h after LPS administration. Milk samples from the treated and from the contralateral quarters were collected 0, 3, 6, 9, 12, 24, 36, 48, and 60 h after LPS administration. Haptoglobin concentrations in blood were increased above basal at 9 h, whereas milk Hp concentration increased 3 h after LPS administration. We therefore evaluated Hp mRNA synthesis within the mammary gland and specifically demonstrated Hp mRNA expression in parenchymal tissue, in tissue around the cisternal milk ducts and also in teat tissue by RT-PCR. Haptoglobin mRNA expression was then quantitatively evaluated by real-time RT-PCR in mammary biopsies collected from the treated and the control quarter before, and 3, 6, 9, and 12 h after LPS challenge from 6 other cows. Haptoglobin mRNA expression in the treated vs. the control quarters was different. The relation between mammary Hp expression and milk Hp concentrations needs further investigation, but the results suggest good diagnostic potential of this parameter for mastitis.     

Efficacy of nisin in treatment of clinical mastitis in lactating dairy cows

Nisin is an antimicrobial polypeptide produced by Lactococcus lactis and is believed nontoxic to humans. The objective of this study was to evaluate a nisin-based formulation for the treatment of bovine clinical mastitis in lactating dairy cattle. A total of 92 cows with 107 clinically mastitic quarters were randomly assigned to nisin- (48 cows with 51 quarters) and gentamicin (GM)-treated (44 cows with 56 quarters) groups. In the nisin-treated group, cows received an intramammary infusion of nisin at a dose of 2,500,000 IU; in the GM-treated group, intramammary infusion of GM was administered at a dose of 0.8 g. Results indicated that nisin offered a clinical cure rate similar to GM (90.2 vs. 91.1%) and no difference in bacteriological cure rate than GM-treated group (60.8 vs. 44.6%, respectively). Proportion of the quarters with milk somatic cell counts <500,000 cells/mL was not different in the nisin-treated group (50.0 and 47.8%) compared with the GM-treated group (33.3 and 37.3%) 1 and 2 wk after treatment. Of 17 Staphylococcus aureus isolates, 82.5% were resistant to penicillin, and 35.3% to GM, but none of them to nisin. Nisin therapy eliminated 54.5% (6 of 11) of S. aureus IMI, whereas GM eliminated 33.3% (2 of 6). Nisin in milk (4.5 ± 0.8 IU/mL) was detected only at 12 h following intramammary infusion, which was much lower than the upper limit (500 mg/mL) allowed as preservative in milk by the China authority. Because of its efficacy in the treatment of bovine clinical mastitis, especially resistant Staph. aureus-caused IMI, as well as its safety in humans, nisin deserves further study to clarify its effects on mastitis caused by different mastitis pathogens on a larger scale.     

Short communication: Changes in micromineral, magnesium, cytokine, and cortisol concentrations in blood of dairy goats following intramammary inoculation with Staphylococcus aureus

The aim of this study was to investigate mineral metabolism and immune response in dairy goats following intramammary inoculation with varying doses of Staphylococcus aureus. Blood samples were collected at 0, 2, 4, 8, 12, 24, 48, and 72 h after intramammary inoculation. Lowered plasma Fe concentrations were observed from 12 to 24 h postinoculation in groups SAA (Staph. aureus at 10⁴ cfu, n = 5) and SAB (Staph. aureus at 10⁸ cfu, n = 5). Plasma Cu concentrations increased in group SAB 2 h after inoculation and maintained greater concentrations until the end of the experiment compared with the control group (phosphate-buffered saline, n = 5). Increased plasma Zn concentrations in group SAB were observed 48 h after inoculation, and the concentration was still greater 72 h after inoculation compared with the control group. Greater plasma Mg concentrations were detected in groups SAA and SAB compared with the control group at all timepoints after inoculation. Plasma Mg concentrations were generally greater in group SAA than in group SAB through 72 h (except at 2 h). Plasma tumor necrosis factor-alpha concentrations were unchanged following intramammary inoculation with Staph. aureus throughout the study. Plasma IL-6 concentrations in groups SAA and SAB increased gradually compared with the control group and peaked at 48 h after inoculation. In group SAB, serum cortisol concentrations started to increase from 8 h postinoculation and peaked at 12 h postinoculation. In conclusion, increasing the inoculum dose does not induce more rapid proinflammatory cytokine responses, whereas the data indicate that mineral metabolic alterations occur during the course of Staph. aureus mastitis in the goat.     

Characterization of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae

Gram-negative bacteria are responsible for almost one-half of the clinical cases of mastitis that occur annually. Of those gram-negative bacteria that induce mastitis, Klebsiella pneumoniae remains one of the most prevalent. Detection of infectious pathogens and the induction of a proinflammatory response are critical components of host innate immunity. The objective of the current study was to characterize several elements of the bovine innate immune response to intramammary infection with Klebsiella pneumoniae. The inflammatory cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), 2 proteins that contribute to host recognition of gram-negative bacteria, were studied. The contralateral quarters of 7 late-lactating Holstein cows were challenged with either saline or K. pneumoniae, and milk and blood samples were collected. Initial increases in the chemoattractants C5a and IL-8, as well as TNF-alpha, were evident in infected quarters within 16 h of challenge and were temporally coincident with increases in milk somatic cells. Augmented levels of TNF-alpha and IL-8 were observed in infected quarters until >48 h postchallenge, respectively. Elevated levels of IL-12, IFN-gamma, and the antiinflammatory cytokine, IL-10, which were first detected between 12 and 20 h postinfection, persisted in infected quarters throughout the study (>96 h). Initial increases in milk LBP and sCD14 were detected 16 and 20 h, respectively, after challenge. Together, these data demonstrate that intramammary infection with K. pneumoniae elicits a host response characterized by the induction of proinflammatory cytokines and elevation of accessory molecules involved in LPS recognition.     

Haptoglobin and serum amyloid A in milk and serum during acute and chronic experimentally induced Staphylococcus aureus mastitis

Local and systemic changes in the acute phase proteins, haptoglobin and serum amyloid A (SAA), were studied in six dairy cows during the acute and chronic phases of experimentally induced Staphylococcus aureus mastitis. Haptoglobin and SAA were measured in serum, and in milk from infected and healthy control udder quarters within each cow. Concentrations of haptoglobin and SAA increased rapidly in both serum and milk during the acute phase of mastitis and followed a similar pattern. Significantly raised milk concentrations of SAA were also found during chronic subclinical mastitis. Serum concentrations of SAA also tended to be higher during the chronic phase than pre-infection. Increases in milk haptoglobin and SAA were specific for the infected udder quarters. In conclusion, measurement of SAA in milk samples could be a useful tool in diagnosing mastitis.     

Efficacy of 5-day parenteral versus intramammary benzylpenicillin for treatment of clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro

The efficacy of parenteral (intramuscular) or intramammary (IMM) benzylpenicillin treatment for clinical mastitis caused by gram-positive bacteria susceptible to penicillin in vitro was investigated. Cows with clinical mastitis in 1 udder quarter were randomly placed into 2 treatment groups. The preliminary bacteriological diagnosis of intramammary infection (IMI) was based on on-farm culturing, and the bacteriological diagnoses were later confirmed by a quantitative PCR assay. Clinical mastitis caused by gram-positive bacteria susceptible to benzylpenicillin was treated with penicillin via either the parenteral route (20 mg/kg) or IMM route (600 mg) once per day for 5 d. The outcome of the treatment was evaluated 3 to 4 wk after the onset of the treatment. The affected quarter was examined to assess the clinical cure, and milk samples were collected from the affected quarter to determine the bacteriological cure and milk N-acetyl-β-d-glucosaminidase activity. The survival and the composite milk somatic cell counts of the treated cows were followed up for 6 and 3 mo after treatment, respectively. A total of 140 cows with clinical mastitis were included in the study, 61 being treated with benzylpenicillin parenterally and 79 via the IMM route. From all quarters treated, 108 of 140 (77.1%) were cured clinically and 77 of 140 (55.0%) were cured bacteriologically. The route of treatment did not significantly affect the outcome of the treatment; 80.3% of the quarters with parenteral treatment and 74.7% of the quarters with IMM treatment showed a clinical cure, and 54.1 and 55.7% a bacteriological cure, respectively. The milk N-acetyl-β-d-glucosaminidase activity was significantly lower in the quarters with a clinical or bacteriological cure than in the quarters with no cure. The 6-mo survival and the proportion of cows with composite milk somatic cell counts <200,000/mL among the treated cows during the 3-mo follow-up period did not significantly differ between the treatment groups. In conclusion, the outcome of either parenteral or IMM benzylpenicillin treatment of clinical mastitis caused by penicillin-susceptible bacteria was similar.     

Behavioral and patho-physiological response as possible signs of pain in dairy cows during Escherichia coli mastitis: A pilot study

Bovine mastitis is one of the most common diseases in the dairy industry and it is a major welfare problem. Pain during mastitis is generally assessed through behavior but a combination of indicators would increase the chances of detecting pain and assessing its intensity. The aim of this study was to assess behavioral and patho-physiological responses as possible signs of pain experienced by cows after experimental intramammary challenge (mastitis) with Escherichia coli. Six Holstein-Friesian cows received an inoculation of E. coli P4 in one healthy quarter. Evolution of the disease was assessed using bacteriological growth and somatic cell counts (SCC). Cows' response to the challenge was monitored by direct behavioral and clinical observations, data loggers, rumen temperature sensors, and indicators of inflammation, stress, and oxidative status. From all data recorded, the variables that contributed most to the discrimination of mastitis phases were obtained by factorial discriminant analysis. Baseline levels of all indicators corresponded to values before challenge. Specifically, we weighted data relating to lying behavior by the observations at the same hour of the day before challenge to eliminate the circadian rhythm effect. We identified 3 phases that were discriminated by factorial discriminant analysis with good performance. Nine indicators varied according to the phase of the disease: cows' attitude toward their surroundings, tail position, clinical signs, ear position, variation of postural changes, concentrations of haptoglobin and serum amyloid A (SAA), cortisol blood levels, and rumen temperature (as a surrogate for body temperature). In phase 1 (4 to 8 h postinoculation), E. coli proliferated exponentially in milk but inflammation indicators remained at baseline levels. Cows were less attentive toward their surroundings (median score, 0.63), and postural changes (lying/standing) were less frequent (0.75 times from baseline). In phase 2 (12 to 24 h postinoculation), bacterial concentrations peaked around 12 h and then began to decrease concomitantly with a sharp SCC increase. Cows were less attentive toward their surroundings (score, 0.54), had high plasma cortisol (31.3 ng/mL) and SAA (100.3 µg/mL) concentrations, and rumen temperature was increased (40.3°C). In phase 3 (32 to 80 h postinoculation), bacterial concentrations decreased concomitantly with high SCC levels. Cows had high levels of haptoglobin (0.57 mg/mL) and SAA (269 µg/mL) but showed no behavioral changes. Dairy cows displayed changes of behavioral, inflammatory, and stress parameters after E. coli mammary inoculation. Our results suggest that cows may have experienced discomfort in the preclinical phase (phase 1) and pain in the acute phase (phase 2) but neither discomfort nor pain in the remission phase (phase 3). Although larger controlled studies are needed to confirm our findings, this knowledge could be useful for early detection of E. coli mastitis and for decision-making regarding the initiation of pain-relief treatment during mastitis in dairy cows. This would improve animal welfare and potentially faster disease remission.