3. Results

The S aureus subclinical mastitis frequency determination in dairy cow population density expressed as cows number/km<sup>2</sup> shows the municipalities that were identified as the regions: Low (1.12) Toluca and Metepec; and Median (2.7): Atlacomulco, Chapultepec, Lerma, Tenango del Valle, Temoaya, and Zinacantepec. The high municipal livestock density (5.6) was observed only at the municipality of Almoloya de Juárez (Table 1).The overall S. aureus rate of the isolates was 21%. The S. aureus infection level in the herds was higher in the municipal region of Almoloya de Juárez. The S. aureus infection rate showed a higher tendency when the density/km<sup>2</sup> of cows increased.

The bacterial isolation rate of reaction level in the Wisconsin Test obtained from 243 milk samples was 46%, the main agent isolated was S. aureus with an overall rate of 22.4% in the studied milk samples, and the coagulase-negative Staphylococcus frequency was 12.9% (Table 2). Low frequencies of other environmental pathogens and minor pathogens were identified in the bacterial isolates.

When evaluating the level of somatic cells in milk by the Wisconsin Test, a significant proportion of 39% of isolates were observed in the range 1700–2500 ° 103 cells/mL of the Wisconsin Test reaction distribution and the proportion of the bacterial isolates in the population sample studied (Table 3).

The isolation frequency of S. aureus in the dairy cow herds studied was 22.8%, compared to 12.29% of coagulase negative Staphylococcus (SCN), observed in the study (p < 0.001). The identification of the types of hemolysin and their relationship with the biotypes is observed (Table 4). The expression of hemolysins in isolates of S. aureus alpha-toxin was higher than other types of the identified hemolysins.

The relationship among the S. aureus biotypes and hemolysin type was observed; the predominant hemolysin type was α-toxin mostly related to the biotypes C and A, and β hemolysin was observed with biotype A mainly and to a less proportion


### Table 1.

Dairy cows density and frequency of staphylococcus aureus isolates in municipalities of the State of Mexico.


### Table 2.

Bacterial isolation frequency in dairy cows with subclinical mastitis.


### Table 3.

Distribution of mastitis Wisconsin Test reactions and bacterial isolates.

with C. The biotype C often expressed all hemolysin types: α-toxin, β, γ, and δ. The simultaneous expression among other hemolysin types such as α β and αβδ was observed in the biotype C.

The S. aureus antibiotypes resistant was frequent in β-lactam antibiotics with the highest observed proportion of antibiotics to the β-lactamases resistant was observed in the dicloxacillin (Table 5).

The distribution of in vitro sensitivity to antibiotics of the S. aureus isolates in the resistance pattern observed was: 65.7% and 90.3% for penicillin and ampicillin, 8.2% for dicloxacillin, 5.5% for cefotaxime, 6.0% for erythromycin, and 25.4% for lincomycin (p < 0.05). The evidence of the antibiotic resistance suggests a potential risk to health by antimicrobial resistance mainly to antibiotics β-lactam antibiotics and the possibility of identifying resistant strains ORSA/MRSA S. aureus methicillin and oxacillin resistant in the cow in the small family dairy herds studied [34].

The in vitro sensitivity to β-lactam antibiotics and the β-lactamase production was observed in a high percentage of the isolates evaluated (Table 6).

In vitro Evaluation of the Phagocytosis Activity of Neutrophils… DOI: http://dx.doi.org/10.5772/intechopen.83834


### Table 4.

Hemolysin types associated with biotypes of Staphylococcus aureus in dairy herds of family production in Toluca Valley.


### Table 5.

In vitro sensitivity to Staphylococcus aureus antimicrobials in dairy herds of the Toluca Valley.

The β-lactamase production was observed in relationship with β-lactam antibiotic in S. aureus resistant isolates mostly observed with penicillin and ampicillin. The oxacillin resistant isolates produce β-lactamase in a proportion of 20%.

The S. aureus capsular characterization results showed that total isolates of S. aureus expressed capsular exopolysaccharide phenotypes, expressed diffuse capsule, with the absence of the compact type in the milk serum soft agar in tube 63.33% (57/90) of the capsular strains were positive for serotyp. 5, 22.22% (20/90) for serotyp. 8 and 14.44% (13/90) were nontypable (NT) (p < 0.05). The municipal distribution of capsular serotypes 5 and 8 was similar (p > 0.05). In the Almoloya de Juárez region, a higher prevalence of capsular serotypes 5 and 8, 31.11 and 4.4%, respectively, was observed. In the municipality of Toluca, 1.75% S. aureus of the capsular serotyp. 5 was observed, indicating the absence of serotyp. 8; the results of the isolates were confirmed in the polymerase chain reaction (PCR) test (Figure 1).

The S. aureus capsular genes cap 5 and cap 8 were PCR confirmed, corresponding to the genes amplicons observed in the isolates obtained from dairy cows in small dairy family farms corresponding to the serotypes 5 and 8. In other hand, the serotypes non typiables NT, was not corresponded with the PCR evaluated amplicons.


### Table 6.

β-lactam In vitro sensitivity of Staphylococcus aureus of cows with subclinical mastitis in small dairy family herds.

The in vitro apoptosis of bovine neutrophils was evaluated by light field optical microscopy having a positive and negative control. The apoptosis values were 95.47 ˜ 3.07, compared with control groups. It was appreciated that S. aureus of capsular typ. 5 induced a greater proportion of neutrophils with apoptosis in vitro. The neutrophils apoptosis was confirmed in the May-Grunwald-Giemsa stained smears showing neutrophils with chromatin condensation and fragmentation.

On the other hand, the in vitro induction of apoptosis by S. aureus in bovine neutrophils was evaluated using light field and epifluorescence microscopy (Figure 2). The mean and standard deviation of the treatments are observed by the techniques of light field microscopy (CM) and fluorescence microscopy (MF); CC (+) positive control of neutrophilsincubated with cyclophosphamide (400 μG/100 μL); negative control CC (°) only neutrophils; CC compact S. aureus strain; and CP capsular S. aureus strain serotyp. 5. The resultsshowed differences between treatments (p < 0.05). The increased apoptosisinduced byCP due to capsularserotyp. 5 was compared with the control groups. CC strain showed lessin vitro neutrophil apoptosis induction.

The S. aureus mec A and nuc genes identified by PCR, results obtained from the characterization of the S. aureus phenotype isolates evaluated detecting the nuc gene (Figure 3), when confirming the MRSA strains mec A are shown with the PCR reaction products (Figure 4), that confirm presence of the amplicons in the S.

### Figure 1.

Agarose gel showing the 173 bp amplicons obtained by PCR related to the cap 8 gene. Lanes 1: Molecular weight marker, 2: positive control Staphylococcus aureus gene Nuc, 3 and 4: positive controls Staphylococcus aureus Cap8, 5: Positive control Staphylococcus aureus Cap5, 6 and 8: Positive samples of Staphylococcus aureus Cap8, 10–12: Positive samples for Staphylococcus aureus Cap5, 7, 9, 13, 14: Negative samples to Staphylococcus aureus.

In vitro Evaluation of the Phagocytosis Activity of Neutrophils… DOI: http://dx.doi.org/10.5772/intechopen.83834

### Figure 2.

Evaluation of the induction of apoptosis in vitro in bovine neutrophils under light microscopy and epifluorescence.

aureus isolates strains evaluated identified as S. aureus phenotypically as ORSA/ MRSA, the nuc and mec A genes appreciated in the strains were identified is a such as 305, 123, 18, 25, 38, 44 and A53 previously identified as MRSA.

The MRSA strains were confirmed from ORSA/MRSA phenotypes detected previously; in the polymerase chain reaction (PCR) reactions, the S. aureus isolates were characterized by nuc gene. The MRSA isolates were identified as S. aureus methicillin resistant strains MRSA, identifying the mec A gene by PCR. In the ORSA/MRSA, isolates were considered to show phenotypical resistance in the different concentrations of oxacillin related to the production of β-lactamase. Those that showed resistant to 4 and 6 μg of oxacicline concentrations were confirmed by showing in vitro differences in the bacterial inhibition halos at 37 and 42°C, and they were considered presumptive MRSA strains. The results confirm that 90 S. aureus isolates were previously evaluated to detect β-lactam antibiotic-resistant phenotypes in the small dairy family farms studied, 93% the isolates produce β-lactamases and 20% of the isolates were considered ORSA/MRSA antibiotypes in which mec A gen of the MRSA strains confirmed by PCR were found.

### Figure 3.

PCR agarose gel electrophoresis, amplification products of the nuc gene of S. aureus chromosomal DNA control strains ATCC 25293, ATCC 29213 S. aureus strains as positive controls and ATCC 12228. S. epidermidis strain as negative control. Lanes: M, DNA manufacturer of molecular mass (100-bp ladder). Field strains; 1-8, 9-10, 305 and 113. PC1 (S. aureus ATCC 25293), NC (ATCC 12228 S. epidermidis), PC2 (S. aureus ATCC 29213). All have an identical 270-Pb band pattern, corresponding to the S. aureus nuc gene.

### Figure 4.

Agarose gel electrophoresis shows the amplification products of the chromosomal DNA of the working strains of S. aureus, the positive and negative controls line: M, molecular weight marker, field strains lines 1–6, the last lines correspond to SA 305, CN1 ATCC 12228 S. epidermidis CN2 ATCC 25923 are negative controls, CP ATCC 43300 S. aureus. SA 305 and ATCC 43300 showed a 310-bp amplicon corresponding to the mec A gene of S. aureus.

### 4. Discussion

The wide municipal distribution of S. aureus mastitis and the dairy herd infection level by S. aureus in the studied cows were considered high as 22.4%, and S. aureus was identified as the main agent in the subclinical mastitis dairy cows family herds of Toluca Valley in the municipalities of central region of Mexico [35]. The herd infection level represents an important risk to the health herd and public health due to the contamination of the milk and fresh unpasteurized dairy products in the small dairy family farms in the municipal regions studied. The findings indicate the importance of S. aureus in the development and persistence of intramammary infection in subclinical mastitis in dairy cows [36, 37]. In it considered that dairy cows herds in family production in Mexico are widely distributed throughout the national territory, with an important contribution to regional socioeconomic development [38, 39]. In Toluca Valley, small-scale family-type production units are predominant with traditional production model, employment of family labor [40]. The danger of S. aureus mammary gland infection in the dairy herds occurred by poor hygiene at milking time, by increasing the bacterial contamination of the nipple and the challenge mammary gland resistance mechanisms [6, 41, 42]. In other cases, the possibility that the season of the year and the stress conditions that the cow undergoes are also indirectly affected mammary gland resistance mechanisms [43, 44]. Animal hygiene and udder health contributes to milk quality and safety food for the consumers, in opposite to subclinical mastitis, in which inflammatory reaction affects quality and milk safety [45].

Actually the importance of support of the sustainability of small cattle herds has an effect that can moderate the methane production and the adverse effects in the phenomenon of global climate change [46] because it will have a greater impact in agricultural production in geographic regions with less socioeconomic development affecting quality of life by increasing demand for food, deterioration of natural resources, water sources, and biodiversity [47]. One of the main expected effects of climate change is associated with changes in temperature and extreme weather disturbances that seriously affect the ecosystem, biodiversity, and agro-food production [48, 49]; there is a direct ecological and socioeconomic impact on human activities, the health of the human and animal population [50], and animal acclimation response in their adaptation processes [51]. Adaptive process in dairy cattle develops metabolic and behavioral physiological compensatory mechanisms to

### In vitro Evaluation of the Phagocytosis Activity of Neutrophils… DOI: http://dx.doi.org/10.5772/intechopen.83834

reduce the adverse effects of climate related to the region's racial genotype [52, 53]. The risk of suffering thermal stress is increased in the animal population in certain regions with negative effects on livestock production and animal welfare [54, 55]. In extreme weather, events with a high ambient temperature, solar radiation, relative humidity and air velocity increase. Under these climatic conditions, cattle are susceptible to developing heat stress [56–59]. Thermotolerant animals expressed certain genes related to cellular stress induced by a high environmental temperature, increasing the secretion of growth hormone (b-GH), milk proteins β-casein (CSN3), and lactalbumin (LAA) [60–64].

Neutrophils phagocytosis activity in bovine mammary gland is the first line of cellular defense; its phagocytosis activity is reduced affecting its microbicidal capacity, by the presence of fat, casein [65, 66]. The bovine neutrophils are different in their capacity of phagocytosis on S. aureus in the mammary gland, the nipple canal and its permeability [67]. Leukocytic infiltration of the teat canal and glandular tissue occurs in response to S. aureus, infection at the time of mastitis [68]. Phagocytosis of leukocytes in the mammary gland shows differences in phagocytosis in vitro; bactericidal activity shown by neutrophils in the presence of milk whey stimulated is higher. The opsonization and intracellular killing of the S. aureus is affected by alpha toxin. It is possible that this effect increases intracellular survival causing failures in antibiotic therapy and mammary gland persistence infection, and the severity of the infection and the evolution of mastitis seriously affect the activity of phagocytosis with increased apoptosis and necrosis of neutrophils.

Other physiological factors of the cow modify the activity of phagocytosis in the mammary gland; during the first week of the dry period, the activity of phagocytosis increases, decreasing at the end of the dry period [69]. In other studies, one reveals a difference in the phagocytosis activity of neutrophils obtained from the glandular secretion of nulliparous and multiparous cows, when evaluating chemilumincence and peroxidase activity [70]. In lactating cows and heifers, peroxidase activation was associated with fat globules, casein, similar to that shown by zymosan phagosomes in the control group explained by the low activity of leukocyte xanthine oxidase. Other studies show differences in alkaline phosphatase of neutrophils from cows with mastitis and healthy cows. In the same way, another condition that can influence the resistance of the mammary gland is the ontogeny of the myeloid cells and their differentiation, by identifying the absence of the transferrin receptor and the expression of the antigens [43, 67, 68], BOCD11 A and BOWC5 [71]. The different surface receptors in the cell membrane of neutrophils are involved in chemotaxis, phagocytosis, and the activation of the respiratory explosion in neutrophils, evidencing the polymorphism of the functional sites of phagocytes and their modulation in phagocytosis [72]. Low neutrophils functional activity is shown at parturition, assuming an increase in susceptibility to infection at the beginning of lactation [73]. When evaluating the parameters of phagocytosis activity and milk production, a negative correlation was obtained. However, at present, there is a tendency to genetic selection of dairy cows to look for natural resistance to bovine mastitis, when choosing the progeny for the estimated data of the somatic cell count and the heritability index, evaluating the neutrophils phagocytosis activity and capacity [74].

During the S. aureus mammary gland infection, the somatic cells in milk are increased; these are composed of a cellular proportion of the mammary gland epithelium and another cellular portion of the leukocytes [75]. The leukocytes proportion present represent the severity inflammatory reaction affected by bovine mastitis, which causes physical-chemical and cellular alterations in milk that compromise the quality and safety of milk to increase the somatic cell count [76]. The changes that occur in milk are detected in the field and laboratory by diagnostic

techniques for detection of subclinical mastitis [77]. Other diagnostic methods for mastitis include determination of ions, leukocyte enzymes, proteins of the acute phase of inflammation, serum amyloid, and haptogloblin whose set of tests may determine the clinical course of the disease [78].

The mammary gland infection and their relationship between the average somatic cell in some cases to reflect observing persistent somatic cell counts in milk >1000,000 cells an infection with minor pathogens. The risk of intramammary infection increases when the somatic cell count of milk is >1500,000 cells/mL. When evaluating the dairy herds with high somatic cell counts in milk, the generated information is a useful collection by evolution to prevent the herd infection level [79]. Infection with Escherichia coli produces a pronounced increase in the somatic cell count in cows suffering from acute mastitis, in order to rapidly decline the somatic cell count after infection occurs [80, 81]. Unlike infection caused by contagious pathogens, cows have high somatic cell counts with a significantly high proportion of neutrophils [82]. The individual somatic cell count of milk and in the milk collection tank is a basic indicator of the level of mastitis in the dairy herd and health of the mammary gland of the cow [83].

The phagocytosis activity intervenes in cellular resistance and the modulation of glandular inflammation limiting the development of intraglandular mammary infection in the different stages of production of dairy cows [84]. S. aureus mammary gland infection in dairy cattle is important in the development of mastitis and epidemiological health risk to the population in animal and human. The phenotypic variation and genetic variability of strains of S. aureus allows the expression of a greater pathogenicity potential of bacteria for the host, depending on the conditions of resistance and immunity of the gland mammary [85]. The infection by S. aureus is determined by the conditions of herd management and hygiene, due to the absence of measures of prevention and control of glandular disease. Intramammary infection by S. aureus causes a drastic reduction in production and deterioration of milk quality [86].

The infection by S. aureus in dairy herds develops from the contamination of the udder skin and the subsequent bacterial colonization of the nipple, which are determinants in the development of intramammary infection when proliferating the S. aureus in the glandular alveolus [87]. The colonization of the udder skin in cows before calving increases the risk of postpartum intramammary infection, when the level of infection in the herd by S. aureus is high. The production environment and the body sites of the animals are a source of infection of the agent from chronically infected cows.

The occurrence of S. aureus mastitis in cows increases the possibility of infection to the human population [88] through milk and unpasteurized dairy products. The pathogenicity of strains of S. aureus related to virulence factors are considered primary in the development of mammary infection. The production of the toxins shows a cytotoxic activity responsible for the cases of S. aureus mastitis. The biotypes A and C in the strains evaluated of S. aureus indicate their possible human and bovine origin, in which it suggests the possibility of cross infection of cow-man, extending the range of interspecies infection [89]. In isolates of S. aureus, the association of α and β toxins increases the cytotoxic and leukocidal capacity on neutrophils of the mammary gland, favoring the development and persistence of glandular infection [90]. The association of α hemolysin in the biotypes A and C manifests a potential risk to human health due to exotic strains of S. aureus of bovine origin. The capsular exopolysaccharide of S. aureus is responsible for interfering with phagocytosis and complement activation [67]. The capsular types of S. aureus predominant in dairy herds were of capsular serotypes 5 and 8. They show substantial differences in surface proteins and their ability to bind lactoferrin,

In vitro Evaluation of the Phagocytosis Activity of Neutrophils… DOI: http://dx.doi.org/10.5772/intechopen.83834

fibrinogen, fibronectin, and IgG in isolates of S. aureus. Different studies confirm the importance of serotypes 5 and 8 of S. aureus in the epidemiology of mastitis in dairy cattle [91]. The increasing occurrence of S. aureus strains carrying the R<sup>+</sup> factor to antibiotics modifies the response to antibiotic therapy. Antibiotic resistance and multidrug resistance frequently occur in isolates of S. aureus in livestock farms when antibiotics are used indiscriminately in drug therapy. Isolates of S. aureus showed resistance to β-lactam antibiotics associated with the production of βlactamase and, to a lesser extent, related to ORSA/MRSA antibiotypes [92]. Since there is intraglandular infection due to S. aureus, dissemination may occur in cows and human. The mec A gene in the ORSA/MRSA phenotypes confirms a low proportion of MRSA strains in dairy cattle in family production herds. The results are similar with the low frequency reported in studies conducted in dairy herds from other countries. It is possible that human infection can occur through the consumption of dairy products contaminated with strains of animal origin and by the management of animals carrying ORSA/MRSA strains. The phenotypic expression of the virulence factors of S. aureus establishes additional risk conditions in the population for MRSA strains of an epidemic nature, related to their geographical distribution and genetic variability.

The MRSA strains identified in the study may be of the epidemic type (EMRSA), when related to the production of the α-toxin, which is considered a predictive marker of the virulence of S. aureus in EMRSA strains. The PCR amplification products demonstrated the nuc gene of S. aureus-specific thermonuclease in the strains evaluated. The detection of the genetic determinant of the mec A gene in the ORSA/MRSA phenotype allowed the identification of MRSA strains. The study confirms that the ORSA/MRSA phenotype carries the mec A gene that characterizes MRSA strains. The ORSA/MRSA phenotype also included the ORSA strains, which are considered sensitive methicillin (MSSA), which may include strains of βlactamase producers called Border line that suggest a heterogeneous resistance. The use of the PCR allowed distinguishing the ORSA/MRSA isolates, the presence of MRSA strains. The procedure can be useful for the diagnosing and monitoring MRSA infection in dairy cattle [35, 93].

The case of bovine mastitis is a multifactorial disease in which several predisposing factors are identified, such as the stage of lactation, the number of births, the time of year, milking hygiene, the size, and technological level of the herd [94]. In the production environment, the presentation of subclinical mastitis is accentuated in the larger dairy herds compared with those of smaller size [95–97]. The monitory of the somatic cells in milk is an indicator of the inflammatory response of the mammary gland, and under stress, it suggests a condition of immunosuppression in cows [98, 99]. In the presence of mastitis, milk production and quality are affected by the disease presenting physicochemical and cellular alterations. When the somatic cells of total bacteria in milk increases and at the same time as it deteriorates the sanitary quality of the milk [100, 101], the poor milk quality contributes to the deterioration of the dairy products in the industrialization and increases the risk to the health of the consumers [102]. The physical-chemical alterations of milk are associated with the inflammatory reaction of the mammary gland, due to the increase in the number of leukocytes and the presence of enzymes and bacterial inhibitors that are incorporated into milk, as well as some components of blood plasma [103, 104]. The components of blood plasma contain proteases and lipases, which accelerate the decomposition of milk fat and proteins [105–107]. The increase in the number of somatic cells is related to the increase of proteins and nitrogenous substances in milk [108, 109]. The concentration of α-lactalbumin and β-lactoglobulin in milk serum decreases substantially [110]. The concentration of lactose decreases to maintain the ionic balance and the osmotic pressure of the milk, thereby producing a variation of the mineral profile of the milk [111]. When this changes occurred, the thermotolerance of milk is reduced [112]. The glandular inflammation decreases the synthesis of casein, consequently decreasing the content of Zn, Ca, and P bound to the casein; the presence of blood serum in milk provokes an increase of the Cu, Fe, and Mn being united to serum albumin and ceruloplasmin, lactoferrin and transferrin [113, 114].

The milk pH increases from 6.6 to 7.0, due to the presence of bicarbonate, without affecting the titratable acidity and its buffer capacity and electrical conductivity, and increasing the freezing point of milk [110]. When the riboflavin and ascorbic acid concentration decreases in milk, it affects the fermentation and acidification capacity in dairy production [115]. There are many intrinsic and extrinsic factors that affect the quality of milk in the presence of mastitis, which is why a health problem is currently considered.
