Mastitis biomarkers and tests have been proposed

Mastitis is one of the most important of  infectious diseases which is associated with
oxidative stress(Lykkesfeldt and
Svendsen 2007). In
both clinical and subclinical mastitis bacteria and leukocytes in the infected
quarters release products, which are chemoattractants for leukocytes.
Polymorphonuclear cells (PMN) move rapidly from the blood into the parenchyma
causing an increase in SCC in milk. Subclinical mastitis leading
to substantial economic losses in whole of
dairy industry (Halasa et al. 2007; Radostitis et al. 2007) which is reported to
be even larger than that caused by clinical mastitis(Huijps et al. 2008).

biomarkers and tests have been proposed to detect subclinical mastitis
including milk bacterial culture, SCC, California Mastitis Test (CMT),
electrical conductivity, milk enzymes such as N-acetyl-?-d-glucosaminidase
(NaGase), lactoperoxidase, xanthine
oxidase, catalase  and l-lactate dehydrogenase, acute phase proteins such as
milk haptoglobulin and milk amyloid A and antioxidative and oxidative
parameters(Lindmark-Månsson and Åkesson 2000; Viguier et al. 2009).

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culture is the standard method for identifying subclinical mastitis. However
time consuming and financial considerations in sampling of all quarters have
prevented widespread adoption of this technique in the dairy industry.

The SCC has been widely implemented as a screening test
to identify subclinical mastitis in
lactating cows. A threshold of 200,000 cells/ml has been shown to have a high
sensitivity and specificity for detecting IMI(Dohoo and Leslie
1991) However, thresholds for SCC during the lactation period
or at dry-off did not provide accurate identification of inflammation status. SCC
in early of lactation can be used for screening with major pathogens but not
suitable for minor pathogens(Sargeant et al. 2001). It has proven that between 1 to 3 weeks after calving
oxidative burst activity of neutrophils as main part of SCC decreased(Dosogne et al. 1999) so in post calving period the SCC does not necessarily
indicate neutrophil functionality.   

The CMT is fairly accurate cow side test indirectly measuring somatic
cells in milk which is simple, and little equipment is needed. Scoring the test
may be subjective and false positive reactions occur frequently on cows that
have 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 the
absence of optimal amounts of antioxidants are leading to oxidative stress which leading to necrosis and apoptosis(Lykkesfeldt
and Svendsen 2007; Su et al. 2002). Oxidative stress
is usually defined as imbalance between the concentrations of reactive oxygen
species(ROS) and reactive nitrogen species(RNS) and the antioxidative defense
mechanisms(Nathan and Shiloh 2000). Oxidative stress and
subsequently oxidative damage leading to modification of cellular components
and cell death by apoptosis or necrosis(Lykkesfeldt and Svendsen 2007).

Relationships between severity of bovine mastitis and
oxidative production in infected udders have been reported. In a way that increase
of SCC have been associated with increase of malondialdehyde concentrations in milk which is an element
for lipid peroxidation (Suriyasathaporn
et al. 2006). It has been showed that during lactation the total oxidant capacity
levels were significantly higher in milk samples from mammary glands with
subclinical mastitis compared to normal mammary glands(Atakisi et al. 2010).

Nitric oxide
(NO) is produced by nitric oxide
synthases during inflammatory processes(Knowles and Moncada
1994) and, nitrogen dioxide(NO2) is a major
oxidation product derived from NO, and increased NO2 levels can
often 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 enhancement
of 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 that
affected with subclinical mastitis(Silanikove et al. 2014).

total antioxidant capacity levels were
numerically lower in milk samples from mammary glands with subclinical mastitis
compared to those from mammary glands without subclinical mastitis(Atakisi et al. 2010) on the other hand antioxidant
parameters 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 antioxidant
power in solutions like plasma and milk(Chen et al. 2003) which assess the ability of sample to reduce the ferric ion to
ferrous ion(Benzie and Strain
1996). Small molecules such as ascorbic acid, uric acid, and ?-tocopherol
were found to have ferric-reducing ability. Silanikove et al have been reported
that FRAP levels were 26.2% higher in uninfected mammary glands than infected mammary
glands of goat (Silanikove et al. 2014) and it was
higher 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 of
subclinical mastitis at early lactation is not satisfying, the aim of the
present study is to evaluate the diagnostic accuracy of milk oxidative (DTNB)
and anitioxidative (FRAP) parameters for detecting subclinical mastitis at
early lactation based on culture results as gold standard. Moreover cut-offs
definition, sensitivity and specificity for the mentioned assays have been


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