Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical mastitis

Serum amyloid A proteins (SAA) are very sensitive acute phase proteins, displaying multiple isoforms in plasma and different body fluids. They are currently under investigation as biomarkers of diseases. The aim of the present study was to compare the concentration and isoform expression of SAA in serum and milk of cows with bacteriologically negative milk (control group) and naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis (subclinical mastitis group). Somatic cell count (SCC) and bacteriological analyses were performed to establish the control and subclinical mastitis group. SAA concentration was evaluated using a commercial ELISA kit, while expression of different isoforms (serum A-SAA and milk M-SAA3 isoforms) was visualized by denaturing isoelectrical focusing and immunoblotting. The SAA concentrations in sera and milk of cows in the subclinical mastitis group were three and 100 times higher than in those from the control group of cows, respectively. Cows in the subclinical mastitis group had more acidic SAA isoforms in serum with the most prominent one at pI 5.5. This isoform was not detected in sera from the control group. Milk samples in the subclinical mastitis group contained abundant highly alkaline M-SAA3 isoforms and most of the serum isoforms, except for that at pI 5.5. In the subclinical mastitis group SAA isoforms with equivalent pI as serum isoforms accounted for 20% of the total SAA concentration in milk. There were significant differences in the concentrations and isoform patterns of SAA in serum and milk between the control and subclinical mastitis groups of cows. Also, we demonstrated that serum SAA isoforms were not transferred to milk proportion to their plasma content.     

Acute phase response in two consecutive experimentally induced E. coli intramammary infections in dairy cows

Background

Acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) have suggested to be suitable inflammatory markers for bovine mastitis. The aim of the study was to investigate acute phase markers along with clinical parameters in two consecutive intramammary challenges with Escherichia coli and to evaluate the possible carry-over effect when same animals are used in an experimental model.

Methods

Mastitis was induced with a dose of 1500 cfu of E. coli in one quarter of six cows and inoculation repeated in another quarter after an interval of 14 days. Concentrations of acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) were determined in serum and milk.

Results

In both challenges all cows became infected and developed clinical mastitis within 12 hours of inoculation. Clinical disease and acute phase response was generally milder in the second challenge. Concentrations of SAA in milk started to increase 12 hours after inoculation and peaked at 60 hours after the first challenge and at 44 hours after the second challenge. Concentrations of SAA in serum increased more slowly and peaked at the same times as in milk; concentrations in serum were about one third of those in milk. Hp started to increase in milk similarly and peaked at 36–44 hours. In serum, the concentration of Hp peaked at 60–68 hours and was twice as high as in milk. LBP concentrations in milk and serum started to increase after 12 hours and peaked at 36 hours, being higher in milk. The concentrations of acute phase proteins in serum and milk in the E. coli infection model were much higher than those recorded in experiments using Gram-positive pathogens, indicating the severe inflammation induced by E. coli.

Conclusion

Acute phase proteins would be useful parameters as mastitis indicators and to assess the severity of mastitis. If repeated experimental intramammary induction of the same animals with E. coli is used in cross-over studies, the interval between challenges should be longer than 2 weeks, due to the carry-over effect from the first infection.     

Haptoglobin and serum amyloid A in milk from dairy cows with chronic sub-clinical mastitis

New tools are needed to detect chronic sub-clinical mastitis, especially in automatic milking systems. Haptoglobin and serum amyloid A (SAA) are the two most sensitive bovine acute phase proteins, and their concentrations increase in milk from cows with clinical mastitis and in milk from cows with experimentally induced chronic sub-clinical Staphylococcus aureus mastitis. The aim of this study was to further evaluate the potential for haptoglobin and SAA in milk as indicators of chronic sub-clinical mastitis. Quarter milk samples were collected from 41 cows with a mean composite milk somatic cell count (CSCC) above 300,000 cells/mL during at least two months prior to sampling. Quarter milk samples were also taken from eleven cows with a mean CSCC below 80,000 cells/mL during at least two previous months. These samples were analysed for haptoglobin, SAA, adenosine triphosphate (ATP) activity and bacterial growth. The samples were grouped according to their ATP, haptoglobin and SAA status. ATP+ samples had ATP > 2 x 10(-10) mol/mL, Hp+ and SAA+ samples had detectable levels of haptoglobin (> or = 0.3 mg/L) and SAA (> or = 0.9 mg/L), respectively. In udder quarter samples from healthy cows, 42 out of 44 samples belonged to the ATP-Hp-SAA- group. Among cows with chronic sub-clinical mastitis, the ATP+Hp+SAA+ group contained 66 out of 164 samples while 44 samples belonged to the ATP+Hp-SAA- group. Detectable levels of haptoglobin and SAA were found in 92 and 80 samples, respectively. Growth of udder pathogens was detected in 28 samples and Staphylococcus aureus was the most common bacteria. In conclusion, haptoglobin and SAA concentrations below the detection limit were considered as good indicators of healthy udder quarters. A substantial variation in haptoglobin and SAA concentrations in milk was observed in udder quarters with chronic sub-clinical mastitis.     

Acute phase proteins in serum and milk from dairy cows with clinical mastitis

The serum concentrations of haptoglobin, serum amyloid A and alpha1 acid glycoprotein were determined in serum collected from healthy dairy cows and cows with clinical mastitis, graded as mild (clots in milk) or moderate (clots in milk and visible signs of inflammation in the mammary gland/s) to assess their relative diagnostic value in detecting the disease. The concentrations of haptoglobin and serum amyloid A were also measured in milk collected from infected and uninfected quarters. The concentrations of haptoglobin and serum amyloid A were higher in the serum and milk from the cows with mild or moderate mastitis. The diagnostic value of haptoglobin in differentiating between healthy animals and those with mastitis gave sensitivities and specificities of 82 per cent and 94 per cent respectively with serum and 86 per cent and 100 per cent with milk. The diagnostic value of serum amyloid A in differentiating between healthy animals and those with mastitis gave sensitivities and specificities of 83 per cent and 90 per cent with serum and 93 per cent and 100 per cent with milk. The diagnostic value of serum alpha1 acid glycoprotein in differentiating between healthy animals and those with mastitis gave sensitivities and specificities of 62 per cent and 91 per cent.     

Acute phase proteins in milk in naturally acquired bovine mastitis caused by different pathogens

The concentrations of haptoglobin (Hp) and serum amyloid A (SAA) and the activity of N-acetyl-β-D-glucosaminidase (NAGase) in milk from 234 cows with spontaneous mastitis caused by different pathogens were measured to assess whether they corresponded with the clinical signs of mastitis and whether there were any differences between pathogens. Ninety-eight of the cows had clinical mastitis and 136 had subclinical mastitis. There were statistically significant positive correlations between the concentrations of SAA and Hp and the activity of NAGase. Significant differences in the concentrations of acute phase proteins and NAGase activity were found in milk from cows with mastitis caused by different pathogens. The highest concentrations of Hp and NAGase were found in cases of mastitis caused by Escherichia coli and Arcanobacterium pyogenes, and the lowest concentrations were from cases of mastitis caused by coagulase-negative staphylococci. Very low SAA concentrations were found in milk from the cases caused by A pyogenes, in contrast to cases caused by other major mastitis pathogens. The median concentration of SAA was over 10 times higher in cases of mastitis caused by E coli than in mastitis caused by other pathogens. There were significant differences in the mean Hp concentration and NAGase activity between clinical and subclinical mastitis. In approximately one-third of the samples, the Hp concentration was below the detection limit, potentially compromising the use of Hp as a mastitis marker.     

Concentration of serum amyloid A and ceruloplasmin activity in milk from cows with subclinical mastitis caused by different pathogens

The aim of the present study was to determine the concentration of serum amyloid A (SAA) and the activity of ceruloplasmin (Cp) in milk from cows with subclinical mastitis caused by different pathogens. Eighty-four milk samples from cows with subclinical mastitis and fourteen milk samples from healthy cows were examined. SAA concentration was determined using the commercial ELISA kit (Tridelta Development Ltd., Greystones, Wicklow, Ireland). Cp activity was assessed spectrophotometrically, using the Rice method. The results reveal that the concentration of SAA (with exception of CNS) and activity of Cp in cow milk can be regarded as markers of subclinical mastitis, irrespective of the microorganism inducing the disease. In conclusion, measurement of SAA and Cp in milk samples could be a useful method in diagnosing subclinical mastitis in cows, but the method should be adapted for field use.     

Acute phase proteins in the diagnosis of bovine subclinical mastitis

Background: The California mastitis test (CMT) and somatic cell count (SCC) are commonly used for diagnosis of subclinical mastitis in cattle. Acute phase proteins (APPs), as alternative biomarkers of mastitis, may increase in concentration in the absence of macroscopic changes in the milk, or may precede the onset of clinical signs. Objective: The aim of this study was to compare the accuracy of APPs measured in milk and in serum with bacterial culture for the diagnosis of bovine subclinical mastitis. Methods: One hundred and seventy‐five Holstein cows were randomly selected from 7 dairy farms. Quarter milk and serum samples were taken from all cows. Milk samples were analyzed using a CMT and SCC, and for haptoglobin (MHp) and amyloid A (MAA) concentrations, and were also submitted for bacterial culture. Serum samples obtained concurrently were analyzed for haptoglobin (SHp) and amyloid A (SAA). Two‐sample Wilcoxon (Mann–Whitney) test was used to compare SCC, MAA, MHp, SAA, and SHp concentrations between culture‐positive and culture‐negative animals. Receiver‐operating characteristic analysis was used to assess the performance of each test using bacterial culture as the reference method. Results: MAA concentration was the most accurate of the 5 tests, with a sensitivity of 90.6% and specificity of 98.3% at concentrations >16.4 mg/L. MAA and MHp had significantly larger areas under the curve than the respective serum proteins, SAA and SHp. Conclusions: The results suggest that measuring haptoglobin and amyloid A in milk is more accurate than serum analysis for the diagnosis of subclinical mastitis in Holstein cows.     

Arachidonic acid metabolites in milk of cows during acute coliform mastitis

Concentrations of prostaglandin F2 alpha (PGF2 alpha) and thromboxane B2 (TXB2) were evaluated in the milk of cows with naturally occurring (n = 3) and experimentally induced (n = 5) acute coliform mastitis. These arachidonic acid metabolites were measured by radioimmunoassay in unextracted milk. Experimental infections were induced by inoculating 600 to 1,200 colony-forming units of Escherichia coli into 1 mammary quarter per experimental cow. In the experimental cows, milk was collected before inoculation and at 12, 24, 36, 48, 60, and 72 hours after inoculation. Somatic cell concentrations, bovine serum albumin, and concentrations of PGF2 alpha and TXB2 were determined in milk collected at each sampling. Mild-to-moderate increases in milk PGF2 alpha and TXB2 concentrations were observed in cows with naturally occurring mastitis. the increases corresponded to the clinical severity and course of mastitis. In the experimental cows, increases in milk PGF2 alpha and TXB2 concentrations were observed, but the increases were not significant, using a statistical model that included factors of treatment, cows, hours after inoculation, cows-by-treatment and hours-by-treatment interactions, and random error (residual). Results of the present study indicated a large biological variability in milk arachidonic acid metabolite concentrations in cows with acute coliform mastitis, and that arachidonic acid metabolites may be important in the pathophysiologic process of acute coliform mastitis.     

Milk amyloid A: correlation with cellular indices of mammary inflammation in cows with normal and raised serum amyloid A

This study compared concentrations of amyloid A in bovine milk with the cell-based indicators of intramammary inflammation, somatic cell count and California Mastitis Test. Mammary quarter data pertaining to 180 cows were categorised according to concentrations of serum amyloid A in the cow of origin. Ranked correlation, ranked regression and receiver operator characteristics all demonstrated acceptable agreement between milk amyloid A concentrations and cell-based indices. There were some indications of reduction in this agreement, in cows with raised concentrations of serum amyloid A. However, there were also indications that serum amyloid A did not significantly influence milk amyloid A. The results of the current study indicate that milk amyloid A exhibits good correlation with established cell-based indicators of intramammary inflammation.     

Norfloxacin pharmacokinetics in lactating cows with sub-clinical and clinical mastitis

Single-dose pharmacokinetics of norfloxacin after intravenous administration of norfloxacin nicotinate at 10 mg norfloxacin/kg body weight was investigated in cows with healthy udders and in cows with chronic subclinical and postacute clinical mastitis. An HPLC method was used to determine the norfloxacin concentrations in serum and milk. Significant differences were observed in norfloxacin pharmacokinetics when administered to cows with infected udder quarters. The clearance (Cl) values were 10.4+/-2.5, 13.2+/-1.9 and 14.2+/-2.1 mL/min/kg (mean +/-SD) in the control (healthy udder) cows and in cows with subclinical and postacute clinical mastitis, respectively. There appeared to be a trend of increasing clearance according to severity of the disease. The volume of distribution at steady state (Vss) in the respective groups was 3.1+/-0.7, 2.2+/-0.6 and 1.3+/-0.2 L/kg. The volume of distribution was significantly decreased in the cows with postacute clinical mastitis. The half-lives (t1/2) and mean residence times (MRT) of norfloxacin were 353, 206 and 115 min (harmonic means) and 306+/-76, 168+/-39 and 95+/-9 min in control cows or in cows with subclinical and postacute clinical mastitis, respectively. The half-lives in the clinical mastitis group were significantly shorter than in the control group and the mean residence times were significantly shorter in the two mastitis groups when compared to the control group. Norfloxacin concentrations in milk were extremely high when compared to the respective serum concentrations. The area under the concentration vs. time curve (AUC) of norfloxacin in milk was 23899+/-6206 mg/L x min in the control cow group. The AUC in milk was significantly lower in the infected udder quarters of the mastitis groups (5075+/-1887 mg/L x min and 7484+/-4645 mg/L x min in the subclinical and the clinical group). The AUC values were significantly lower in milk from the infected udder quarters of the cows with chronic subclinical and postacute clinical mastitis when compared to the values in milk from the healthy quarters of the same udder. Norfloxacin was marginally bound to serum protein. The binding was concentration dependent and was 19, 13 and 6% at 0.2, 1.0 and 8.4 mg/L, respectively. Binding to milk protein was 46-51% and concentration independent. An in vitro dialysis model was used to simulate drug transport between serum and milk as a function of protein binding. The results showed that the rate of norfloxacin disposition from milk to serum was slower than from serum to milk, which was in agreement with the findings obtained in the pharmacokinetic study. Norfloxacin was poorly soluble in organic solvents and our results suggest that changes in the degree of ionization of the drug in different body fluids considerably affect its disposition.     

Conglutinin, mannan-binding protein, and serum amyloid P component concentrations in sera from cows: changes associated with mastitis.

To study the roles of conglutinin (Kg), mannan-binding protein (MBP), and serum amyloid P component (SAP) in the protection of cattle against infections, the concentrations of these proteins in the sera from cows with mastitis were determined by sandwich enzyme-linked immunosorbent assays. The Kg and MBP concentrations in the sera from cows with mastitis were much lower than those from uninfected (or clinically healthy) ones, and increased after recovery. No significant difference in the SAP concentrations was found between healthy and infected cows. With the sera from cows experimentally infected with bovine leukemia virus, the Kg concentrations were also lower than those from uninfected ones, whereas the MBP concentrations were not. From these findings, the Kg concentration is suggested to be serologically a possible indicator for clinical diagnosis of treatment for mastitis although both Kg and MBP concentrations were found to decrease in cows with mastitis and to increase after recovery.     

Indicators of inflammation in the diagnosis of mastitis

Mastitis affects the quality of milk and is a potential health risk for the other cows. In a well managed dairy herd, in addition to clinical mastitis, subclinical mastitis should be efficiently detected. Bacteriological sampling is not feasible as a routine test to identify subclinical mastitis, and indirect tests of mastitis are more suitable for selecting cows with intramammary infections for subsequent bacteriological sampling. Mastitis affects the composition of milk, and the degree of changes depends on the infecting agent and the inflammatory response. Indicators of inflammation in the milk which can be determined using rapid, reliable and easy routine techniques, can be used for the early detection of mastitis. The measuring of the somatic cell count in milk is the standard method, but the analysis technique is problematic for routine use in herds. The most promising parameters for monitoring subclinical mastitis are milk N-acetyl-beta-D-glucosaminidase activity, lactose, and electrical conductivity along with some other indicators such as optical and milk flow measurements, preferably with an inter-quarter evaluation included in the test. Acute phase proteins, haptoglobin and serum amyloid A, are also potential candidates for mastitis monitoring. New mastitis detection systems which can be adapted into on-line use are urgently needed, since dairy units are growing bigger and automatic milking systems are being taken into use.     

Effects of continuous low dose infusion of lipopolysaccharide on inflammatory responses,milk production and milk quality in dairy cows

The objective of this study was to evaluate the effects of continuous low dose infusion of lipopolysaccharide (LPS) on inflammatory responses and milk production and quality in lactating dairy cows. Eight Holstein cows were assigned to two treatments in a cross‐over experimental design. Cows were infused intravenously either with saline solution or with saline solution containing LPS from Escherichia coli O111:B4 at a dose of 0.01 μg LPS/kg body weight for approximately 6 hr each day during a seven‐day trial. The clinical symptoms and milk production performance were observed. Milk samples were analysed for conventional components, fatty acids and amino acids. And jugular vein and mammary vein plasma samples were analysed for concentrations of cytokines and acute phase proteins. LPS infusion decreased feed intake and milk yield. An increase in body temperature was observed after LPS infusion. LPS infusion also increased plasma concentrations of interleukin‐1β, serum amyloid A, LPS‐binding protein, C‐reactive protein and haptoglobin. LPS infusion decreased the contents of some fatty acids, such as C17:1, C18:0, C18:1n9 (trans) and C18:2n6 (trans), and most amino acids except for methionine, threonine, histidine, cysteine, tyrosine and proline in the milk. The results indicated that a continued low dose infusion of LPS can induce an inflammatory response, decrease milk production and reduce milk quality.     

Relationship between haptoglobin, serum amyloid A, and clinical status in a survey of dairy herds during a 6-month period

BACKGROUND: Haptoglobin and serum amyloid A are major acute phase proteins in cattle. Dairy cattle often develop pathologic conditions in the peripartum period; acute phase proteins may be useful in their diagnosis. OBJECTIVES: The purpose of this study was to compare the accuracy of serum haptoglobin (Hp) and serum amyloid A (SAA) concentrations with clinical health status for diagnosing disease during the peripartum period in dairy cattle. METHODS: Dairy cows from 4 herds were evaluated every 15 days over a 6-month period. Health status was determined by thorough clinical examination. Haptoglobin and SAA concentrations were measured in serum using validated methods and the results were classified as positive or negative based on defined cutoff points. Disease prevalence, sensitivity, and specificity were compared using clinical examination as the gold standard. RESULTS: A total of 1896 samples from 158 cows were analyzed. Significant increases in mean Hp and SAA concentrations were observed in the week following parturition in both primiparous and multiparous cows, although high interindividual variability was observed. Both Hp and SAA had low sensitivity but higher specificity in determining disease status compared with clinical examination. Increased concentrations of Hp and SAA were found in <10% of samples from clinically healthy cows, except in the week after parturition. CONCLUSIONS: Haptoglobin and serum amyloid A should be used with caution as markers of inflammation in the week after calving. Poor sensitivity in other postpartum periods could be related to the higher incidence of chronic (vs acute) inflammation. Haptoglobin may be appropriate for routine screening, but further work needs to be done to assess its value as an indicator of herd health.     

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. Intramammary 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.     

Concentrations of IL-6 in Serum and Whey from Healthy and Mastitic Cows

Inflammatory cytokines, such as interleukin (IL)-6, have been shown to reflect clinical signs in certain conditions in diseased animals. In this study, we quantified the IL-6 concentrations in the serum and milk whey from 94 dairy cows with acute clinical mastitis and 55 healthy lactating cows. The IL-6 concentrations in serum from mastitic cows were significantly higher on the first day of illness compared to those of normal cows. Higher concentrations of IL-6 were also detected in the whey from mastitic cows, whereas low concentrations of IL-6 were detected in both serum and whey samples from normal cows. IL-6 concentrations in the serum taken at the onset of illness from cows that later required euthanasia were significantly higher than those in samples from cows that later recovered. These results suggest that serum IL-6 concentrations may be of prognostic value in identifying cows with severe mastitis.     

Dependence between acute phase response, oxidative status and mastitis of cows

There are many studies exploring the topic of acute phase response and oxidative status in inflammation of the mammary gland of cows. However, many phenomena are relatively not well known. Mastitis is associated with significantly higher concentrations of inflammatory and oxidative mediators in the cells and blood. Results of experiments have shown that there are evident changes in serum interleukins (IL), acidglycoprotein (alpha1AG), tumor necrosis factor (TNF), and haptoglobin (Hp). Thus, local as well as systemic inflammation might play important roles in increased mammary oxidative stress. Reactive oxygen species (ROS) have been implicated in the pathogenesis of a variety of diseases, including mastitis and in transgenic technology leading to production of new bacterial proteins, very important in prevention of mastitis. We can also observe an interaction between inflammatory and oxidative mediators. These results suggest an important role played by acute phase response and oxidative status in inflammation of the mammary gland.     

Serum amyloid A as an marker of cow֨ s mastitis caused by Streptococcus sp.

The aim of the study was to evaluate the concentrations of amyloid A in serum (SAA) and in milk (MAA) of cows with mastitis caused by Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis and healthy cows. The blood and milk samples were obtained from Holstein-Friesian cows with clinical signs of mastitis from two tie-stall housing systems herds in the Lublin region in Poland. A total of 80 milk and serum samples from 30 cows with mastitis and 10 healthy cows were selected for study. In the quarter milk samples from cows with mastitis Streptococcus strains were isolated: Strep. agalactiae (7 cows), Strep. dysgalactiae (9 cows) and Strep. uberis (14 cows). The present study indicates that amyloid A concentration was significantly higher in milk of cows with mastitis compared to control cows (1134.25 ng/mL and 324.50 ng/mL, P < 0.001). The highest concentration of amyloid A was found in milk of cows with mastitis caused by Strep. agalactiae and Strep. uberis whereas lowest in the milk of cows with mastitis caused by Strep. dysgalactiae (3882.50 ng/mL, 2587.75 ng/mL and 812.00 ng/mL, respectively). No statistically significant difference in amyloid A concentration in serum was revealed between all unhealthy cows and control group (2140.00 ng/mL and 2510.00 ng/mL, P > 0.05). There was also no statistically significant difference between the level of amyloid A in serum and in milk of cows with mastitis caused by Strep. agalactiae and Strep. uberis. Whereas, in the case of Strep. dysgalactiae, like in the group of healthy cows, the level of amyloid A was significantly higher in serum compared to this in milk (2100 ng/mL and 812.00 ng/mL, P < 0.001; 2510.00 ng/mL and 324.50 ng/mL, P < 0.001; respectively).     

Altered leukocyte responsiveness in dairy cows with naturally occurring chronic Staphylococcus aureus mastitis

Changes in inflammatory parameters, leukocyte surface markers, functional responses and cytokine mRNA expression of leukocytes of dairy cows with naturally occurring chronic Staphylococcus aureus (S. aureus) mastitis and healthy cows were determined to elucidate the leukocyte responses to S. aureus infection of the mammary gland. Increased values in inflammatory parameters and matrix metalloproteinase activities in milk revealed the characteristics of cows with chronic mastitis. Expression of L-selectin and CD18 molecules on neutrophils and proportion of CD8 cells in milk from cows with S. aureus mastitis were significantly (P<0.05) increased compared with those found in healthy cows. The FcR-stimulated CL response of blood neutrophils was significantly (P<0.05) decreased in cows with S. aureus mastitis. Significantly (P<0.05) decreased mitogenic responses of lymphocytes were found in cows with S. aureus mastitis; however, the values were not restored to those of healthy cows when stimulated with both mitogens and the cytokine IL-1β. The mRNA expression of TNF-α, IL-1β and IL-8 on milk leukocytes from cows with S. aureus was found to be increased compared with that of healthy cows. The changes of immune responses found in cows with S. aureus mastitis appear to be influenced by the severity and duration of inflammation in infected quarters. The down-regulation of the leukocyte functions found in cows with S. aureus mastitis appears to be associated with the progress of the chronic stage of S. aureus mastitis.     

Host response in bovine mastitis experimentally induced with Staphylococcus chromogenes

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein-Friesian cows were challenged with S. chromogenes 4 weeks after calving. One udder quarter of each cow was inoculated with 2.1 x 10(6)cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-beta-D-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on-off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.