Mastitis biomarkers and tests have been proposed

Mastitis is one of the most important of  infectious diseases which is associated withoxidative stress(Lykkesfeldt andSvendsen 2007).

Inboth clinical and subclinical mastitis bacteria and leukocytes in the infectedquarters release products, which are chemoattractants for leukocytes.Polymorphonuclear cells (PMN) move rapidly from the blood into the parenchymacausing an increase in SCC in milk. Subclinical mastitis leadingto substantial economic losses in whole ofdairy industry (Halasa et al.

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2007; Radostitis et al. 2007) which is reported tobe even larger than that caused by clinical mastitis(Huijps et al. 2008).Numerousbiomarkers and tests have been proposed to detect subclinical mastitisincluding milk bacterial culture, SCC, California Mastitis Test (CMT),electrical conductivity, milk enzymes such as N-acetyl-?-d-glucosaminidase(NaGase), lactoperoxidase, xanthineoxidase, catalase  and l-lactate dehydrogenase, acute phase proteins such asmilk haptoglobulin and milk amyloid A and antioxidative and oxidativeparameters(Lindmark-Månsson and Åkesson 2000; Viguier et al.

2009).Bacteriologicalculture is the standard method for identifying subclinical mastitis. Howevertime consuming and financial considerations in sampling of all quarters haveprevented widespread adoption of this technique in the dairy industry.The SCC has been widely implemented as a screening testto identify subclinical mastitis inlactating cows. A threshold of 200,000 cells/ml has been shown to have a highsensitivity and specificity for detecting IMI(Dohoo and Leslie1991) However, thresholds for SCC during the lactation periodor at dry-off did not provide accurate identification of inflammation status. SCCin early of lactation can be used for screening with major pathogens but notsuitable for minor pathogens(Sargeant et al.

2001). It has proven that between 1 to 3 weeks after calvingoxidative burst activity of neutrophils as main part of SCC decreased(Dosogne et al. 1999) so in post calving period the SCC does not necessarilyindicate neutrophil functionality.   The CMT is fairly accurate cow side test indirectly measuring somaticcells in milk which is simple, and little equipment is needed. Scoring the testmay be subjective and false positive reactions occur frequently on cows thathave been fresh less than ten days, or on cows that are nearly dry(Rice 1997).

The PMN produces reactive oxygen species(ROS) for killing microorganisms(Babior 1999) and an excess of ROS and theabsence of optimal amounts of antioxidants are leading to oxidative stress which leading to necrosis and apoptosis(Lykkesfeldtand Svendsen 2007; Su et al. 2002). Oxidative stressis usually defined as imbalance between the concentrations of reactive oxygenspecies(ROS) and reactive nitrogen species(RNS) and the antioxidative defensemechanisms(Nathan and Shiloh 2000). Oxidative stress andsubsequently oxidative damage leading to modification of cellular componentsand cell death by apoptosis or necrosis(Lykkesfeldt and Svendsen 2007).Relationships between severity of bovine mastitis andoxidative production in infected udders have been reported. In a way that increaseof SCC have been associated with increase of malondialdehyde concentrations in milk which is an elementfor lipid peroxidation (Suriyasathapornet al.

2006). It has been showed that during lactation the total oxidant capacitylevels were significantly higher in milk samples from mammary glands withsubclinical mastitis compared to normal mammary glands(Atakisi et al. 2010).Nitric oxide(NO) is produced by nitric oxidesynthases during inflammatory processes(Knowles and Moncada1994) and, nitrogen dioxide(NO2) is a majoroxidation product derived from NO, and increased NO2 levels canoften be detected in situations where NO production is elevated(Pryor and Squadrito 1995). After addition of TNB to milk, continuous formation of NO2,causing its conversion to DTNB(5,5′-dithiobis-2-nitrobenzoate) so enhancementof free radicals can be assessed by conversion of TNB to DTNB(Van Der Vliet et al. 1997).

Significant enhancement of  TNB to DTNB conversion  has been reported in milk of goats thataffected with subclinical mastitis(Silanikove et al. 2014).total antioxidant capacity levels werenumerically lower in milk samples from mammary glands with subclinical mastitiscompared to those from mammary glands without subclinical mastitis(Atakisi et al.

2010) on the other hand antioxidantparameters measured in cows with clinical and subclinical mastitis were not significantly different compared with healthy cows(Ghasemian Karyak et al. 2011; Politis et al. 2012). FRAP assay has been presented as a method for assessing antioxidantpower in solutions like plasma and milk(Chen et al. 2003) which assess the ability of sample to reduce the ferric ion toferrous ion(Benzie and Strain1996).

Small molecules such as ascorbic acid, uric acid, and ?-tocopherolwere found to have ferric-reducing ability. Silanikove et al have been reportedthat FRAP levels were 26.2% higher in uninfected mammary glands than infected mammaryglands of goat (Silanikove et al. 2014) and it washigher in milk of healthy glands compare to mastitic glands of cow as well(Atakisi et al.

2010).In spite of the fact that accuracy of SCC for diagnosis ofsubclinical mastitis at early lactation is not satisfying, the aim of thepresent study is to evaluate the diagnostic accuracy of milk oxidative (DTNB)and anitioxidative (FRAP) parameters for detecting subclinical mastitis atearly lactation based on culture results as gold standard. Moreover cut-offsdefinition, sensitivity and specificity for the mentioned assays have beendetermined.


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