Acute phase proteins in cattle: discrimination between acute and chronic inflammation.

Acute phase proteins such as serum amyloid A, haptoglobin, and alpha 1-acid glycoprotein have been identified as markers of inflammation in cattle because they are produced by the liver in response to pro-inflammatory cytokines. This study was designed to assess whether they could be used to discriminate between acute and chronic inflammation. Their concentrations were measured in serum samples from 81 cattle in which inflammation was classified by thorough clinical examination, supported by postmortem findings, as being acute in severity in 31 and chronic in 50. The classical haematological markers of inflammation were also determined in blood from the animals. Serum amyloid A had a maximum (100 per cent) clinical sensitivity in discriminating between the acute and chronic cases, and haptoglobin had the highest clinical specificity of 76 per cent; counts of neutrophils and band neutrophils had sensitivities of 71 per cent and 42 per cent and specificities of 30 per cent and 72 per cent, respectively. It was concluded that serum amyloid A and haptoglobin may be used to discriminate between acute and chronic inflammatory conditions.     

Acute phase protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary inflammatory conditions

The concentrations of the two acute phase proteins, serum amyloid A and haptoglobin, in serum and milk were compared in 10 cows with clinical mastitis, 11 cows with extramammary inflammatory conditions and 10 clinically healthy control cows. The concentrations of both acute phase proteins were higher in the serum and milk of the cows with mastitis than in the cows in the other two groups. Four of the cows with extramammary inflammatory conditions had serum amyloid A concentrations in serum above 100 microg/ml, but negligible concentrations in milk, indicating that a pathogen must be present in the mammary gland for serum amyloid A to accumulate in milk. The acute phase protein concentrations in milk increased significantly with increasing somatic cell count, suggesting that they may be indicators of the severity of an infection.     

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.     

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 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.     

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.     

Diagnostic utility of measuring serum amyloid A with a latex agglutination turbidimetric immunoassay in bovine mastitis: Comparison with haptoglobin and alpha 1 acid glycoprotein

This study established the precision and accuracy of a modified latex agglutination turbidimetric immunoassay (LATIA) reagent, and evaluated the ability of the measurement of serum amyloid A (SAA) compared to haptoglobin and α1-acid glycoprotein, which are acute phase proteins (APPs), for diagnosis of clinical mastitis. Concentrations of APPs in cows with mastitis were significantly higher than those in healthy cow. Only the plasma SAA concentration in cows with clinical mastitis (44.90 mg/l; n=15) was significantly higher than that in those with subclinical mastitis (10.70 mg/l; n=16), enabling their diagnosis in contrast to other APPs. Thus, the SAA assay using a LATIA reagent is useful in assessing mastitis severity due to its higher sensitivity and specificity than other APP assays.     

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.     

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).     

An investigation of mastitis due to S agalactiae, S uberis and M smegmatis in a dairy herd

Subclinical mastitis caused by streptococcal infections affected 27 of 83 cows in a commercial dairy herd. Between three and six weeks after intramammary treatment of these cows with cloxacillin, 16 (59 per cent) of the treated cows developed acute clinical mastitis associated with Mycobacterium smegmatis. None of the untreated cows was affected. Infected quarters were moderately hypertrophied and fine clots were present in the milk for three to four weeks. No cows showed systemic signs of illness. Studies carried out over 12 months showed that infected cows shed M smegmatis for three to four months and affected quarters remained hypertrophied in all but one cow after 12 months. The mean milk cell count of affected quarters fell slowly from 4,850,000/ml in the acute stage to 810,000/ml five months later and 620,000/ml 12 months later, suggesting that the organism persisted in the udder. The estimated mean loss in lactation yield for cows with M smegmatis mastitis was 10.8 per cent. Losses were greatest when the hind quarters were involved (mean 28 per cent for cows with both hind quarters affected). Ten of the 16 affected cows were ultimately culled owing to serious reductions in yield.     

Changes in acute-phase proteins and cytokines in serum and milk whey from dairy cows with naturally occurring peracute mastitis caused by Klebsiella pneumoniae and the relationship to clinical outcome

This study was conducted to evaluate the changes in acute-phase proteins and cytokine concentrations in dairy cows with naturally occurring peracute Klebsiella pneumoniae (K. pneumoniae) mastitis and their association with the outcome of the disease. Seventeen Holstein cows with K. pneumoniae mastitis from 8 dairy farms were divided on the basis of outcome after local and systemic therapy into 2 groups comprising 8 euthanized cows and 9 that recovered. Changes in acute-phase proteins and cytokine concentrations in cows with K. pneumoniae mastitis were evaluated at the onset of the disease (day 0) and at days 3, 7 and 14 after therapy and compared with those of 13 healthy dairy cows. The concentrations of haptoglobin (Hp) and interleukin (IL)-6 in serum and α(1)-acid glycoprotein and IL-1β in serum and whey on day 0 were significantly (P<0.05) higher in the euthanized cows than in those that recovered and the healthy cows. A correlation (r=0.90, P<0.01, n=17) was found between IL-6 and Hp concentrations in sera from recovered and euthanized cows at day 0. This indicated that serum concentrations of Hp and IL-6 at the initial examination were prognostic factors for survival, and the cutoff values were 2,020 μg/ml and 32 ng/ml, respectively. These results suggest that IL-6 and Hp concentrations are involved in the manifestation of K. pneumoniae mastitis and may be possible indicators of the prognosis of peracute K. pneumoniae mastitis.     

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.     

Prostaglandins and glutathione peroxidase in bovine mastitis

Prostaglandin (PG) levels in milk and plasma samples from mastitic cows were determined by radioimmunoassay and compared with erythrocyte glutathione peroxidase (GSH-Px) and other relevant parameters in milk and blood. The overall levels of PGE2, PGF2 alpha and thromboxane B2 (TXB2) in milk were two to four times higher than in blood plasma both in healthy and diseased animals (P less than 0.01). In mastitic milk the PG levels were 24 to 55 per cent and in blood plasma 41 to 95 per cent higher than in healthy animals. The changes were significant and largest for the PGF2 alpha values. In milk, the PG concentrations correlated with the markedly elevated cell count (r = 0.49 to 0.57), and TXB2 values also correlated with milk yield. In blood, PGE2 and PGF2 alpha correlated positively with serum albumin, and PGE2 also correlated with glutathione (GSH). PGE2 and PGF2 alpha correlated negatively with GSH-Px and gamma-glutamyl transferase. The substantial decline in GSH-Px in mastitic animals (P less than 0.01) may be related to changes in lipid peroxidation and PG formation. The possible implications of these findings in the treatment of mastitis are discussed.     

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.     

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.     

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.     

Diagnostic significance of lactose level of milk. 2. Lactose level in first quarter milk samples in various udder disorders]

The behaviour of lactose levels in first quarter milk was studied in the context of a large dairy herd in the presence of various udder health defects. The results were related to other criteria generally used in mastitis diagnosis. The lactose level in first quarter milk was found to be a diagnostic criterion which was properly applicable to detecting and identifying clinical and subclinical mastitis. A limit value of 4.6 per cent has been commonly assumed in literature and proved to be a sound basis for udder health assessment, provided that the given phase of lactation is taken into due consideration (exclusion of cows in the first month post partum as well as of aged milking cows from the seventh month of lactation). Also reported in this paper are results regarding milk lactose and its correlations with other criteria for mastitis diagnosis. A close correlation was found to exist between lactose level and electric conductivity of first quarter milk.     

Acute phase proteins in cattle after exposure to complex stress

Stressors such as weaning, mixing and transportation have been shown to lead to increased blood concentrations of acute phase proteins (APP), including serum amyloid A (SAA) and haptoglobin, in calves. This study was therefore undertaken to assess whether SAA and haptoglobin levels in blood mirror stress in adult cattle. Six clinically healthy Holstein cows and two Holstein heifers were transported for four to six hours to a research facility, where each animal was housed in solitary tie stalls. Blood samples for evaluation of leukocyte counts and serum SAA and haptoglobin concentrations were obtained before (0-sample) and at 8, 24 and 48 hours after the start of transportation. Upon arrival the animals gave the impression of being anxious, and they appeared to have difficulty coping with isolation and with being tied on the slippery floors of the research stable. Serum concentrations of SAA and haptoglobin increased significantly in response to the stressors (P < 0.01 and 0.05 at 48 hours, respectively). Additionally, the animals had transient neutrophilia at 8 and 24 hours (P < 0.05). In conclusion, the results of the study suggest that SAA and haptoglobin may serve as markers of stress in adult cattle.     

Disposal and disease rates in 340 British dairy herds

Data derived from 340 dairy herds, mainly in southern England, between April 1998 and March 1999, showed that the average total culling rate was 22.1 per cent, with 5.6 per cent for infertility, 3.6 per cent for mastitis, 1.7 per cent for lameness, 2.0 per cent for poor milk yield, 3.7 per cent for age and 5.5 per cent for miscellaneous reasons which included death. The average annual rate of assisted calvings was 8.7 per cent, of injury 0.9 per cent, digestive disease 1.3 per cent ketosis 0.4 per cent, hypomagnesaemia 0.7 per cent, hypocalcaemia 5.3 per cent, mastitis 36.6 per cent, and lameness 23.7 per cent. There was a significant association (P<0.001) between higher rates of mastitis in cows housed in straw yards as opposed to cubicles and also between higher rates of lameness in cows housed in cubicles as opposed to yards (P<0.015). However, there were farms with low rates of mastitis in cows kept in straw yards and low rates of lameness in cows kept in cubicles. Larger herds tended to have more problems with lameness and higher bulk milk somatic cell counts (BMSCC). There was a positive association between BMSCC and mastitis rate.     

Prevalence of subclinical mastitis and associated risk factors in smallholder dairy cows in Tanzania

A cross-sectional study was carried out on 200 randomly selected farms in each of the Iringa and Tanga regions of Tanzania to estimate the prevalence and risk factors for subclinical mastitis in dairy cows kept by smallholders. Subclinical mastitis was assessed using the California mastitis test (cmt), and by the bacteriological culture of 1500 milk samples collected from 434 clinically normal cows. The percentages of the cows (and quarters) with subclinical mastitis were 75.9 per cent (46.2 per cent) when assessed by the cmt and 43.8 per cent (24.3 per cent) when assessed by culture. Factors significantly associated with an increased risk of a cmt-positive quarter were Boran breed (odds radio [or]=3.51), a brought-in cow (rather than homebred) (or=2.39), peak milk yield, and age. The stripping method of hand milking was associated with a significantly lower prevalence of cmt-positive quarters (or=0.51). The cmt-positive cows were more likely to be culture positive (or=4.51), as were brought-in (or=2.10) and older cows.