*4.3.2.2. Lactoferrin*

Ceruloplasmin has been evaluated as a marker of animal health and welfare [187]. Several studies in cattle indicate its diagnostic use with applications in many disease conditions, including uterine bacterial contamination, as well as clinical and subclinical mastitis [177, 188]. Hussein et al. [189] evaluated ceruloplasmin activity in dairy cows in different lactation stages, showing higher values in fresh-lactation stage. López-Alonso et al. [190] measured ceruloplasmin as a potential marker of hepatic copper accumulation in cattle. Studies in young animals have shown that the concentrations of ceruloplasmin in the serum increases during induced pneumonic pasteurellosis, with the highest concentration observed 2 and

The β-globulins belong to group of globular proteins that migrate faster than γ-globulins in electrically charged solutions, but more slowly than α-globulins. The main components of the β-globulin fraction are transferrin and complement, which may correspond to the 2 subfrac-

and angiostatin. Furthermore, in response to the stimulation by different antigens, some IgM immunoglobulins may migrate in the β region, while the IgA and IgE immunoglobulins in

Transferrin (Tf), the iron-binding protein of serum has been described as a negative acutephase protein. It is a strong chelator that is able to bind iron tightly but reversibly. The transferrin molecule has high affinity to bind two atoms of ferric iron (Fe3+), being higher in the extracellular pH of 7.4 and decreases in the acidified endosomes, allowing the dissociation of Fe3+ [192]. The primary role of transferrin is to transport iron safely around the body to supply growing cells [193]. Essentially, all iron circulating in the blood normally is bound to transferrin. It renders iron soluble under physiologic conditions, prevents iron-mediated free radical toxicity, and facilitates transport into cells [194]. Similar to lactoferrin, transferrin inhibits multiplication and growth of certain viral, bacterial, and fungal organisms by iron inhibition. Moser et al. [195] evaluated the concentrations of transferrin in cattle in various physiological states, in energy-deficient (ketotic) cows, in cases of several acute and chronic infections, as well as after the administration of endotoxins. The values of transferrin in healthy animals ranged from 2.0 to 6.6 g/l. While in animals with acute infections and ketosis, the values were in the range of 1.5 and 8.5 g/l, chronic infectious diseases (such as paratuberculosis) were associated with relatively low values (below 2 g/l). The evaluation of the effect of age on transferrin concentrations showed its lower values in adult animals compared to young animals [195]. Tóthová et al. [122] presented a marked increase of transferrin concentrations from day 7 of life, reflecting acceptable rate of protein synthesis and good nutritional status. Furthermore, the concentrations of transferrin increased in veal calves with iron deficiency above 8 g/l,

resulting in negative correlation between hemoglobin and transferrin [195].

) identified in some animal species [81]. Other important proteins belonging


subfraction, identified in some animal

4 hours after the inoculation [191].

118 Ruminants - The Husbandry, Economic and Health Aspects

the β-γ interzone, which may also correspond to the β<sup>2</sup>

*4.3.2. The β-globulins*

and β<sup>2</sup>

to this fraction are: β<sup>2</sup>

tions (β<sup>1</sup>

species [5].

*4.3.2.1. Transferrin*

Lactoferrin (Lf), also known as lactotransferrin, is a multifunctional protein of the transferrin proteins capable of binding and transferring Fe3\* ions. Lactoferrin is a globular glycoprotein with a molecular weight of about 80 kDa, which shows high affinity for iron [196]. Although the overall structure of lactoferrin is very similar to that of transferrin, they differ in their relative affinities for Fe and the propensity for release of Fe [197]. The capability of lactoferrin to bind iron is two times higher than that of transferrin [198]. This bound is very strong and can resist pH values of as low as 4 [199]. The ability to keep iron bound even at low pH is important, especially at sites of infection and inflammation where, due to metabolic activity of bacteria, the pH may fall under 4.5 [200]. The most of bacterial pathogens necessitate Fe for metabolic activities, growth, and proliferation. Since lactoferrin has Fe-binding capacity, it reduces the growth of Fe-requiring pathogenic bacteria including enteropathogenic *E. coli* [201]. Lactoferrin is a major component of the innate immune system of mammalians and represents one of the first defense systems against microbial agents, which invaded the organism mostly by mucosal tissues [202]. It affects the growth and proliferation of many infectious agents including both Gram-positive and Gram-negative bacteria, viruses, protozoa, and fungi [203].

Lactoferrin is expressed in most biological fluids, including milk, saliva, and nasal secretions. It is present in blood, plasma, or serum in relatively low concentrations, but its concentrations increase during infection, inflammation, excessive intake of iron, or tumor growth [204]. Higher concentrations of lactoferrin were observed in bovine and human milk, or colostrum. The lactoferrin values in milk of healthy cows are quite variable and may range from 1.15 to 485.63 μg/ml. On the other hand, sub-clinical and clinical mastitis may lead to rapid increase of its concentrations positively correlating with SCC, stage of lactation, and milk yield [205, 206]. The concentrations of lactoferrin are higher in colostrum (varying between 1 and 5 mg/ml), during drying-off and early mammary involution period than during lactation [207, 208].

Lactoferrin plays a key role in the defense mechanisms of the mammary gland, contributing to the prevention of microbiological infection diseases [209]. Therefore, the concentrations of lactoferrin in milk are markedly influenced by the health status of the cows. Harmon et al. [210] induced *E. coli* infection in bovine mammary gland. In these cows, they found a 30-fold increase of lactoferrin values in the mammary secretion 90 h after the inoculation. Furthermore, they concluded that acute mastitis is associated with 30-fold increase of the concentrations of lactoferrin in the milk with the greatest production in the infected quarter.

#### *4.3.2.3. C-reactive protein*

C-reactive protein (CRP) was the first identified acute-phase protein, which was named according to its ability to bind to C-polysaccharide of Gram-positive bacteria [211]. It is a non-glycosylated protein from the group of pentraxins, and is composed of 5 subunits that firmly bind to C-polysaccharides [212]. Following bacterial infection, CRP binds to pathogen and activates the classical complement pathway leading to the opsonization of the bacteria [213]. It also plays a role in the destruction of the infectious agent through the interaction with specific receptors on phagocytes, which may help in the reduction of tissue damage, and contribute to the tissue repair and regeneration [162].

The immunoglobulins are glycoproteins composed of two heavy (H) and two light (L) chains linked by disulfide bridges [223]. According to the structure of the H chain, immunoglobulins are classified into the following classes: IgG, IgM, IgA, IgE, and IgD. The L chain consists of either kappa (κ) or lambda (λ) chain, which indicates the type of immunoglobulins. Based on the structural variations in the variable regions of H or L chains, immunoglobulins can be further divided into subtypes and subclasses. For example, two subclasses of IgG have been

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Most viral, bacterial, and toxin antibodies are of the IgG type and are present in all animals. It is the predominant type of immunoglobulins found in the body and has the longest serum half-life. IgE is involved in allergic and anaphylactic reactions, whereas IgA can be found in the secretions of the respiratory, genitourinary, and gastrointestinal tracts [7]. IgM functions be opsonizing antigens for destruction and fixing complement, and usually are associated with the first line of defense [224]. IgD is found in very low concentrations in the serum and

Several factors, including non-pathological and pathological conditions, may influence the concentrations of proteins in the serum, thus the entire profile of serum proteins [226]. Many disease processes are associated with abnormal serum protein profiles. Changes in the protein profile commonly occur as secondary symptoms in numerous diseases, but may be also the primary symptom of some specific disease conditions [227]. Thus, the results of the electrophoretic analyses of serum or plasma proteins may provide a basis for the establishment of further specific diagnostic procedures and may be helpful by the differential diagnosis of several disease processes. However, abnormalities in the serum protein profile must be interpreted with regards to many influences that are not associated with pathological processes.

Variations in the serum protein profile and shifts in albumin and globulin concentrations may occur not only under pathological, but also under physiological conditions [102]. Animal age is one of these important factors that may affect the concentrations of the different serum protein fractions or their electrophoretic pattern [103]. It has been shown in young and adult cattle [228], where the most important age-related differences were observed in the α- and

had higher γ-globulin concentrations. In particular, it has been stated that the most important changes occur in the first month of the life of calves, and are associated with the changes in nutrition and adaptation processes during the neonatal period [229]. The total serum proteins and γ-globulin concentrations increase rapidly 1 day after the intake of colostrum, and then decrease gradually till the end of the first month of age. According to Hammon et al. [230], the concentrations of total proteins in the serum are very low at birth, due to the minimal quantities of immunoglobulins but, it increases during the first 24 hours of life as a result


has a short half-life. The functions of circulating IgD are not well understood [225].

**5. Changes in the serum protein electrophoretic pattern**

**5.1. Serum protein pattern variations related to non-pathological conditions**

identified in cattle (IgG1 and IgG2) [7].

γ-globulin fractions. While the values of α<sup>1</sup>

There are considerable species differences in the magnitude and duration of changes in CRP concentrations during health disorders. In humans, dogs, and pigs, CRP is the major acutephase protein with approximately 1000-fold increase in serum concentrations during acute inflammatory states [214]. In cattle, CRP has been reported to be a constitutive protein with only a minor increase during disease processes [7]. Despite this disadvantage, Schrodl et al. [215] evaluated the CRP concentrations in cows with mastitis, and found approximately 10-fold higher values in these cows (1083 ± 93 ng/ml) compared with healthy ones (82 ± 66 ng/ml). The data recorded by Lee et al. [216] showed a correlation between serum CRP concentrations and the health condition of dairy cattle.
