2. Materials and methods

The diagnosis of the situation of subclinical mastitis in small dairy herds' family production was made under the causal model based on the clinical diagnosis of bovine mastitis and its association with the main infectious agents present in the herds and the S. aureus level infection and virulence factors in the different regions of the State of Mexico located in the Mexico central region. The determination of the S. aureus infection was associated to subclinical mastitis in dairy herds during the period of 2015–2017.

### 2.1 Determination of Staphylococcus aureus and frequency of subclinical mastitis

Milk samples obtained from 2749 cows of 182 dairy herds of family production in different municipal regions of the State of Mexico (Almoloya de Juarez, Zinacantepec, Chapultepec, Temoaya, Toluca, Tenango del Valle, and Lerma y Atlacomulco) were studied, to detect the mastitis frequency, the S. aureus herd level infection distribution, and dairy cow density. The isolation and identification of S. aureus in the milk samples was carried out using the protocol established by the National Mastitis Council [23]. The municipal regions of the entity were grouped taking into account the livestock inventory and the territorial extension of the municipalities studied to determine the population density expressed as the average number of cows per km<sup>2</sup> . The population density of the region was classified as: low (BA), mean (ME), and high (AA). The results were evaluated using the proportion estimation test corresponding to the design (p < 0.05).

The level of association between the reaction of the Wisconsin Test and the isolation frequency of S. aureus in dairy herds was determined. A total of 243 milk pooled samples obtained from the four glandular quarters of the cows from different ages and lactation stages were obtained at random from small dairy herds of the Toluca Valley, Mexico. The inclusion criterion of cows in the study was a positive reaction to the mastitis Wisconsin Test [23]. The milk samples, were inoculated 0.01 mL, on blood agar plates, MacConkey and Vogel Jhonson agar plates (0.001 g/L potassium tellurite), blood agar plates CAMP-esculine, the plates were incubated at 37°C for 18–24 hours [24]. The identification of the isolates was carried out by routine bacteriological procedures Gram staining, coagulase test tube, catalase, Voges-Proskauer, and the standardized commercial systems API Staph and API 20E. The estimated number of somatic cells in milk was determined from tubes of the reaction level of the mastitis Wisconsin Test. The results were evaluated using the proportions estimation test corresponding to the design (p < 0.05).

### 2.2 Staphylococcus aureus isolation and phenotypic characterization

The isolation of S. aureus and its phenotypic characterization was carried out in a transversal longitudinal study during the fall-spring period of 2016, by randomly sampling 87 dairy family herds in the dairy system with an average herd size of 14.3 cows from different municipalities in the Valley of Toluca, State of Mexico. We obtained 1256 composed milk samples from the four glandular quarters to carry out the bacteriological study. The small-scale production system was characterized in the type of small dairy herd family production of rustic rural types. The racial phenotypes in dairy cow herds were Holstein, Creole and hybrid Holstein, European Swiss and Bos indicus crosses. A traditional productive management, hand dairy milking predominantly, feeding with diurnal grazing in native pastures and use of agricultural corn husks (Zea mays), a minimum supplementation diet with feedstuffs. Isolation of S. aureus was carried out by routine microbiological protocols [24]. About 0.01 mL was inoculated on Vogel Jhonson agar plates (0.001 g potassium tellurite/L). The agar plates were incubated at 37°C for 24 hours; colony forming units were identified by Gram stain, catalase, coagulase test tubes and aerobic fermentation of maltose, trehalose, and anaerobic mannitol tests. The final identification of the S. aureus was confirmed by API Staph system (Biomerieux Vitek, Durham, NC, USA).

The biotypes of S. aureus were identified from 90n isolates, those were grown on crystal violet media (brain heart infusion agar plates, added with 1:10000 violet crystal). The agar plates were incubated at 37°C for 24 hours, biotypes were identify when biotypes were identify when observing the colony forming units, associated with the biotypes A, B, C and D. The positive crystal violet reaction was considered with violet coloration and a slightly yellow halo formation. Biotype A was characterized as positive violet crystal, biotype B showed a whitish coloration, biotype C showed a yellowish color, and the absence of growth on the medium was related to biotype D.

The identification of the different types of hemolysins α, β, γ, and δ of S. aureus was made from the observation of hemolysis in blood agar plates supplemented with 7% erythrocytes obtained from: bovine, rabbit, equine, and human type O+ [25]. The agar plates were incubated at 37°C for 24 hours under aerobic conditions and under a reduced atmosphere of CO2, the type of hemolysis was observed in the different blood agar plates; they were compared with the control strains and the type was determined.

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

The S. aureus antibiotypes were characterized by in vitro sensitivity test performed by the agar diffusion method of National Committee for Clinical Laboratory Standards [26]. On the Mueller Hinton agar plates inoculated with the isolates of S. aureus, antibiotic discs were placed on the agar: penicillin 10 IU, 10 μg ampicillin, 1 μg dicloxacillin, 10 μg streptomycin, 30 μg cefotaxime, 30 μg cephalosporin, 2 μg lincomycin, 15 μg erythromycin, 30 μg novobiocin, and 100 μg spiramycin. Agar plates were incubated at 37°C for 18–24 hours. The bacterial growth inhibition halos on the agar plates were expressed in mm, compared to the bacterial growth inhibition halos of the control strain of S. aureus ATCC25923. The results were evaluated using the proportion estimation test corresponding to the design (p < 0.05).

### 2.2.1 Staphylococcus aureus capsular polysaccharides characterization

The capsular exopolysaccharide phenotypes were characterized from 90n S. aureus isolates obtained previously from small dairy family herds. They were studied for the expression of the capsule that was performed on Columbia agar added with 2% NaCl incubated at 37°C for 24 hours; the capsular expression was confirmed by capsule staining. The capsular type was observed in 4% whey soft agar tubes (brain and heart agar added with 4% v/v milk whey). The capsular serotype was determined with a rabbit polyclonal antiserum against the capsular serotypes 5 and 8 of S. aureus. The results obtained were evaluated by the Chi-square test (p < 0.05), based on the observed frequencies of bacterial isolation and the level of infection. The comparison between the growth inhibition halos was carried out by means of the hypothesis test of two proportions of the same group with mutually exclusive characteristics.

### 2.2.2 Staphylococcus aureus capsular genes

The identification of the cap5 and cap8 genes related to S. aureus capsular types was performed from isolates of S. aureus, using the polymerase chain reaction (PCR) test, were performed using the oligonucleotides Cap 5 k1 (5-GTCAAAGATT ATGTGATGCTACTGAG-3) and Cap 5 k2 (5-ACTTCGAATATAAACTTG AATCAATGTTATACAG-3) for the detection of the capsular typ. 5 and for the capsular typ. 8 the following primers 8 k1 (5GCCTTATGTTAGGTGATAAACC-3) 8 k2 (5-GGAAAAACACTATCATAGCAGG-3) were used, obtaining the PCR products amplified of 361 bp for cap 5 and 173 bp cap 8 [27].

### 2.3 In vitro induction of apoptosis in bovine neutrophils

The effect of the capsular serotyp. 5 of S. aureus on the induction of in vitro apoptosis of neutrophils from dairy cattle was evaluated by means of light field microscopy and smear staining method, May-Grünwald-Giemsa stain. The in vitro induction of apoptosis was used as a phagocytosis substrate of heat-inactivated S. aureus (120°C for <sup>20</sup> minutes) suspension (2 ˜ <sup>10</sup><sup>8</sup> CFU/mL) at 4°C. The in vitro assay was performed in 1:10 neutrophil: bacteria ratio incubated during 1 hour, and then smears using May-Grünwald-Giemsa stain were prepared [28]. The microscopic observation of neutrophil apoptosis was confirmed under the epifluorescence microscope preparing ethidium bromide solution (100 μg/mL) and acridine orange (100 μg/mL). A number of apoptotic neutrophils were determined from the reddish coloration of the chromatin and nuclear condensation or fragmentation against viable neutrophils that showed a green coloration of the chromatin [29].
