Staphylococcus aureus is one of the most detected bacteria in mastitis cases (Tiwari et al., Reference Tiwari, Babra, Tiwari, Williams, Wet, Gibson, Paxman, Morgan, Costantino, Sunagar, Isloor and Mukkur2013). S. aureus is able to invade mammary glands deeply, and a considerable number of infected cattle have weak responses to treatment, need prolonged antibiotic therapy or should be culled (Ruegg, Reference Ruegg2018). It has been reported that the difference between spontaneous and antibiotic-associated cure rates was not significant for subclinical mastitis caused by S. aureus (43% vs. 49%, respectively) (Wilson et al., Reference Wilson, Gonzalez, Case, Garrison and Grohn1999). Therefore, not all cattle with subclinical mastitis need or benefit from antibiotic treatment, and spontaneous cure rates for untreated cases are considerable. Efficient protocols for identifying the chances of spontaneous cure would enable more judicious use of antibiotics (Ruegg, Reference Ruegg2018).
Mastitis triggers an acute phase response, which results in increased acute-phase proteins such as haptoglobin and serum amyloid A (SAA) in milk, even before visible changes in the milk or the onset of clinical signs (Safi et al., Reference Safi, Khoshvaghti, Jafarzadeh, Bolourchi and Nowrouzian2009). As at least some of these proteins are synthesized in the mammary gland (eg mammary-associated SAA3, Eckersall et al., Reference Eckersall, Young, Nolan, Knight, McComb, Waterston, Hogarth, Scott and Fitzpatrick2006), their concentration increases significantly in both clinical and subclinical mastitis and there is also a correlation between their concentration and severity of mastitis. Increased SAA in milk has been used to detect subclinical mastitis caused by S. aureus (Gronlund et al., Reference Gronlund, Hulten, Eckersall, Hogarth and Waller2003) and increased haptoglobin concentrations were associated with higher SCC at first test-milking (Nyman et al., Reference Nyman, Emanuelson, Holtenius, Ingvartsen, Larsen and Persson Waller2008). The concentrations of milk proteins, including albumin, α-lactalbumin, β-lactoglobulin and immunoglobulins, also change following subclinical mastitis and their measurement has been used diagnostically (Shirazi-Beheshtiha et al., Reference Shirazi-Beheshtiha, Safi, Rabbani, Bolourchi, Ameri and Khansari2012). Numerous enzymes, such as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP), have also been used for the detection of bovine mastitis, and they have shown high correlation with acute-phase proteins and SCC as indicators of mastitis (Chagunda et al., Reference Chagunda, Larsen, Bjerring and Ingvartsen2006; Larsen et al., Reference Larsen, Røntved, Ingvartsen, Vels and Bjerring2010). Oxidative stress, which occurs owing to metabolic changes and microorganism challenges, exacerbates the immune system impairment that occurs in the periparturient period, so that it may predispose dairy cattle to mastitis. Neutrophils, as the predominant cell type in the mammary gland during mastitis, display their bactericidal activities via a respiratory burst generating free radicals. Oxidative stress markers, such as malondialdehyde (MDA) and total antioxidant capacity, have recently been used to detect subclinical mastitis (Sadek et al., Reference Sadek, Saleh and Ayoub2017; Amiri et al., Reference Amiri, Fallah Rad, Heidarpour, Azizzadeh and Khoramian2020).
Although acute phase proteins, enzymes, metabolic parameters and oxidative indices have been applied for diagnosis of subclinical mastitis, no study has compared these factors before and after spontaneous cure, and their effects on the outcome of S. aureus subclinical mastitis have not been evaluated. The present study aimed to investigate the usefulness of some milk metabolites, proteins and enzymes as indicators of spontaneous cure in subclinical mastitis caused by S. aureus.
Materials and methods
Animals
The current research was approved by the Animal Welfare Committee of the Ferdowsi University of Mashhad with the code of 3/46067 under the institutional, national and international guidelines. The study was performed from July to September 2019 in a large commercial dairy herd (Mashhad, Northeast Iran) consisting of 1000 lactating cows. Cows were kept in an open shed and received a well-balanced total mixed ration and were milked three times a day. The average daily milk production was 39–41 kg/cowand the voluntary waiting period was 60 d. The first examination and milk sampling took place between 20 and 30 d in milk. If the cows had any apparent disease, such as retained fetal membranes, milk fever, metritis, lameness, ketosis, displaced abomasum and clinical mastitis they were excluded from the study.
The presence of S. aureus subclinical mastitis was determined by bacterial culture on quarter milk samples collected aseptically from each cow according to the procedure described by the National Mastitis Council (NMC) and by SCC measurement with Fossomatic device 5000 (Hillerød, Denmark). Cattle with SCC count ≥ 200 000 cell/ml were considered as subclinical mastitis and they were included in the study if no bacteria except S. aureus was isolated in bacterial culture. Cattle with SCC count less than 200 000 cell/ml and negative bacterial culture were considered as healthy control animals. The second and third examinations and sampling were made on days 7 and 14 after initial sampling and diagnosis.
Based on the results of three milk samples (at the time of diagnosis and on days 7 and 14 after diagnosis), three groups of animals were selected: cows with spontaneous cure (SCC ≥200 000 cell/ml and positive S. aureus culture at first sample, but SCC <200 000 cell/ml and negative S. aureus culture at second and third samples; n = 23), cows without spontaneous cure (SCC ≥200 000 cell/ml and positive S. aureus culture at all three samples; n = 29) and healthy cattle (SCC <200 000 cell/ml and negative S. aureus culture at all three samples; n = 23).
Biochemical analysis
For measurement of total antioxidant capacity and MDA, the milk samples were defatted through centrifugation at 4000 g at 4°C for 20 min. Total antioxidant capacity and MDA concentration were assessed by ferric reducing antioxidant power (FRAP) (Chen et al., Reference Chen, Lindmark-Mansson, Gorton and Akesson2003) and trichloroacetic acid assays (Suriyasathaporn et al., Reference Suriyasathaporn, Chewonarin and Vinitketkumnuen2012), respectively.
The concentration of MUN and the activities of ALP and LDH were measured in defatted milk samples through commercial kits (Pars Azmoon, Iran) using an autoanalyzer (Mindray, BS 200, China). The concentrations of Cl and K were determined with Ion Selective Electrode technology (Starlyte Electrolyte Analyzer, Alfa Wassermann, USA).
For separation and measurement of different protein fractions, whole milk was centrifuged at 1000 g for 20 min at 4°C and the skim milk recovered. The whey fraction was obtained by precipitating casein at pH 4.6 with 1 N hydrochloric acid and then centrifugation at 2000 g for 20 min at room temperature (Ishikawa et al., Reference Ishikawa, Shimizu, Hirano, Saito and Nakano1982). The supernatant was assayed for the measurement of total protein by the biuret method and albumin, α-lactalbumin, β-lactoglobulin, and immunoglobulin concentrations by acetate cellulose electrophoresis (Bell and Stone, Reference Bell and Stone1978).
For measurement of acute-phase proteins, whole milk samples were diluted with diluent buffer for SAA and haptoglobin assays, as recommended by the manufacturer. The concentrations of haptoglobin and SAA were measured using ELISA assay (Bioassay Technology Laboratory, Shanghai, China). The ELISA automatic washer (BioTek, ELX-50, Winooski, USA) and reader (BioTek, ELx-800, Winooski, USA) were utilized.
Statistical analysis
The biochemical parameters measured at the time of diagnosis and on day 14 after treatment were used for statistical analysis. One way ANOVA was used to compare measured parameters between the trial groups at each sampling time. Paired samples T-test was used to analyze the effect of time of sampling in each trial group. All data are reported as means ± se. A P value less than 0.05 was considered as statistically significant. Data analysis was performed by SPSS software (version 16, USA).
Results
The comparison of measured parameters at the time of diagnosis revealed that healthy cows and subclinical mastitis cows with spontaneous cure had lower ALP and haptoglobin levels than the subclinical mastitis group without subsequenbt spontaneous cure (P < 0.05, Table 1).
Table 1. The results (mean ± se) of milk acute phase proteins, ALP, and oxidative stress markers in S. aureus subclinical mastitis cows with or without spontaneous cure at different times
In each row, parameters with different lowercase superscripts a, b, c are significantly different at the time of diagnosis (P < 0.05).
In each row, parameters with different uppercase superscript letters A, B, are significantly different on day 14 after diagnosis (P < 0.05).
M-SAA, milk-serum amyloid A; ALP, alkaline phosphatase; MDA, malondialdehyde; FRAP, ferric reducing antioxidant power; MUN, milk urea nitrogen.
Other parameters measured in the study are presented as online Supplementary Table S1.
On day 14 after diagnosis, lower ALP, haptoglobin and MDA levels were noted in the healthy cows and subclinical mastitis cows with spontaneous cure in comparison with the subclinical mastitis cows without spontaneous cure (P < 0.05, Table 1). FRAP concentration at this time was also lower in the subclinical mastitis cows with spontaneous cure in comparison with the subclinical mastitis cows without spontaneous cure (P < 0.05, Table 1).
In cows with spontaneous cure ALP and haptoglobin showed a decreasing trend and their levels decreased with time (P < 0.05, data not shown), whereas no significant change was observed for these parameters in healthy cows and subclinical mastitis cows without spontaneous cure. MDA also showed a decreasing trend in healthy cows and subclinical mastitis cows with spontaneous cure and its level decreased with time (P < 0.05; day 0 and day 14 data in Table 1). On the other hand, FRAP and MDA concentrations in subclinical mastitis cows without spontaneous cure increased significantly with time and their concentrations on day 14 after diagnosis were significantly higher than those at the time of diagnosis (P < 0.05; Table 1). Other measured parameters that did not show differences between groups or across time are reported in online Supplementary Table S1.
Discussion
According to the results of the present study, there is an association between the spontaneous cure of S. aureus subclinical mastitis and the values of acute-phase protein, ALP and oxidative stress indices maeasured at initial diagnosis. The hypothesis, that these parameters might be indicative of the outcome of S. aureus subclinical mastitis in dairy cows, is supported by the findings of the present study.
It has been shown that SAA and haptoglobin are synthesized in infected mammary glands. Consequently, they are considered as valuable biomarkers of subclinical mastitis, when there are no visible alterations in milk and mammary glands, and can also provide a quantitative estimate of the severity of subclinical mastitis (Gronlund et al., Reference Gronlund, Hulten, Eckersall, Hogarth and Waller2003; Eckersall et al., Reference Eckersall, Young, Nolan, Knight, McComb, Waterston, Hogarth, Scott and Fitzpatrick2006). The measurement of haptoglobin and SAA in milk samples is more accurate than blood serum samples for the diagnosis of bovine subclinical mastitis (Safi et al., Reference Safi, Khoshvaghti, Jafarzadeh, Bolourchi and Nowrouzian2009). These proteins have been used in many studies to assess subclinical mastitis and a relationship between them and SCC has been demonstrated (Lindmark-Mansson et al., Reference Lindmark-Mansson, Branning, Alden and Paulsson2006; Akerstedt et al., Reference Akerstedt, Waller and Sternesjo2007; Gerardi et al., Reference Gerardi, Bernardini, Elia, Ferrari, Iob and Segato2009; Simojoki et al., Reference Simojoki, Orro, Taponen and Pyorala2009). The present study is the first one that has measured acute phase protein concentration before and after spontaneous cure in cows with S. aureus subclinical mastitis. In the present study, cows with spontaneous cure had a lower haptoglobin level at the time of diagnosis compared with the subclinical mastitis group without spontaneous cure. Furthermore, after spontaneous cure, mean milk haptoglobin concentration dropped significantly in cows so that their milk haptoglobin level at the second sampling time (after spontaneous cure) was comparable to those of healthy cows. However, haptoglobin concentration in cows without spontaneous cure was still high at the second sampling time. Therefore, it seems that haptoglobin might be useful for prognostic purposes, and S. aureus subclinical mastitis cows with low haptoglobin concentration are more likely to show spontaneous cure. Similarly, the level of milk haptoglobin was higher in euthanized dairy cows with Klebsiella pneumonia mastitis than in recovered cows, suggesting that it may be involved in the manifestation of K. pneumoniae mastitis and can be a possible biomarker of the prognosis of this infection (Hisaeda et al., Reference Hisaeda, Arima, Sonobe, Nasu, Hagiwara, Kirisawa, Takahashi, Kikuchi and Nagahata2011). Acute-phase proteins are induced by pro-inflammatory cytokines such as IL-6. There is a correlation between acute-phase proteins and inflammatory cytokines concentrations and the severity of inflammation (Hisaeda et al., Reference Hisaeda, Arima, Sonobe, Nasu, Hagiwara, Kirisawa, Takahashi, Kikuchi and Nagahata2011).
The measurement of milk oxidative stress markers can be applied as a diagnostic indicator for early detection of subclinical mastitis (Ellah, Reference Ellah2013). Alterations in milk oxidant capacity and antioxidant power have been reported in dairy animals with subclinical mastitis (Suriyasathaporn et al., Reference Suriyasathaporn, Vinitketkumnuen, Chewonarin, Boonyayatra, Kreausukon and Schukken2006; Atakisi et al., Reference Atakisi, Oral, Atakisi, Merhan, Pancarci, Ozcan, Marasli, Polat, Colak and Kaya2010, Silanikove et al., Reference Silanikove, Merin, Shapiro and Leitner2014). In the present study, oxidative stress markers (MDA and FRAP) showed an increasing trend in the subclinical mastitis group without spontaneous cure, so that their values on the day 14 d after diagnosis were higher than those at the time of diagnosis. This was not the case, however, in the healthy cows and cows with spontaneous cure. Immune cells are especially sensitive to oxidative stress due to higher levels of polyunsaturated fatty acids in their membrane and generation of large amounts of reactive oxygen species (ROS) by stimulated cells (Spears, and Weiss, Reference Spears and Weiss2008). Oxidative stress can diminish the functional capabilities of immune cells and may prevent cows from mounting an efficient immune response in mammary glands (Sordillo and Aitken, Reference Sordillo and Aitken2009). Furthermore, excess ROS generation in infected mammary glands and resulting oxidative stress can enhance tissue damage and disturb mammary function (Zhao and Lacasse, Reference Zhao and Lacasse2008). ROS not only react with macromolecules, such as DNA, proteins and polyunsaturated fatty acids, but also inactive protease inhibitors. As a result, over-activation of enzymes like gelatinase, collagenase and elastase would degrade interstitial matrix as well as epithelial cells (Brambilla et al., Reference Brambilla, Mancuso, Scuderi, Bosco, Cantarella, Lempereur, Benedetto, Pezzino and Bernardini2008). It seems that improving oxidative stress by antioxidant supplementation, might be beneficial to increase the cure rate in dairy cows with S. aureus subclinical mastitis.
Enzymes like ALP are considered as indigenous constituents of milk (Andrews, Reference Andrews and Fox1991). Tissue damage due to inflammation in the mammary gland epithelium can result in increased enzyme activity in milk. However, their increase in mastitic milk also originates from other sources. It has been suggested that inflammatory mediators, which are released during mastitis, enhance the expression of ALP in leukocytes and consequently increase the milk ALP activity (Heyneman and Burvenich, Reference Heyneman and Burvenich1992). Milk enzymes have also been used as indices of the degree of mammary gland inflammation and for discrimination between major and minor pathogens (Chagunda et al., Reference Chagunda, Larsen, Bjerring and Ingvartsen2006). In the present study, the comparison of ALP activity at all sampling times (before and after cure) indicated that its activity in healthy cows and subclinical mastitis cows with spontaneous cure was lower than in the subclinical mastitis group without spontaneous cure. These findings show an association between milk ALP activity and the outcome of S. aureus subclinical mastitis, and higher ALP activity can be a manifestation of more severe inflammation in cows without spontaneous cure.
In conclusion, the findings of the present study suggested that increased milk haptoglobin concentration, enhanced oxidative stress and higher ALP activity could be indicative for the persistence of S. aureus subclinical mastitis in dairy cows. S. aureus subclinical mastitis cows with lower milk haptoglobin, ALP and oxidative stress are more likely to cure spontaneously and cows showing higher values of these parameters may need and benefit more from antibiotic treatment.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029921000613.
Acknowledgements
The present study was supported by Ferdowsi University of Mashhad (grant number 3/46067).
