In vitro Evaluation of the Phagocytosis Activity of Neutrophils and Characterization of Staphylococcus aureus Mastitis in Dairy Cows of Small Family Farms

Abstract

A total of 269 cows in small family herds in the central region of México from different municipalities of México State were studied. Composed milk samples were obtained to detect subclinical mastitis and S. aureus infection and for characterization of phenotypes as follows: biotypes, capsular exopolysaccharide 5 and 8, ORSA/MRSA and MRSA strains; and in vitro phagocytosis neutrophil activity and apoptosis by S. aureus serotype 5. Results were evaluated by estimating proportions and chi-square test (p < 0.05). The microbial isolation rate was 46%; S. aureus isolation rate was 23.4–21.0% among cow herds; 39% of microbial isolates were in 1500–2500 cells/mL, with Wisconsin test. The phenotypes of S. aureus were: biotypes A and C are identified frequently that produce alpha and beta-hemolysin toxins, and a smaller proportion other hemolysins types. S. aureus isolates capsular serotypes 5 and 8 show differences in the in vitro neutrophil phagocytosis activity and apoptosis. The ORSA/MRSA isolates show that MRSA strains? mec A gene was confirmed by PCR. The S. aureus infection level in the dairy cow herds shows a wide municipal distribution, identifying different S. aureus pathotypes enclosed to virulence factors and MRSA to establish a potential health risk in small dairy cow herds in México.

1. Introduction

Bovine mastitis is a limiting disease of the production in dairy herd, commonly caused by Staphylococcus aureus. The inflammation of the glandular tissue constitutes a mechanism of natural resistance of the mammary gland infection in dairy cattle [1]. The appearance of inflammation in the mammary gland to increase the number of somatic cells (CCS), with an increased proportion of the leukocytes in milk is related to the agents present in the infection and the severity of the inflammatory response in the mammary gland [2, 3]. In addition, the inflammation provokes physical and chemical changes in the milk affecting the quality and its nutritional composition [4]. The significant increase in CCS in milk may be influenced by S. aureus herd infection level which stands out as one of the main contagious pathogens that are capable of producing a persistent infection and chronic inflammation in the mammary gland [5, 6]. The pathogenicity of S. aureus strains are related to the different virulence factors of the agent, considered primary in the development of mammary gland infection. The production of the alpha toxin shows a cytotoxic and cytolytic activity responsible for cases of gangrenous mastitis due to S. aureus [7]; the capsular exopolysaccharide is responsible for interfering with phagocytosis and complement activation [8]. In addition, colonization of udder skin in cows before parturition increases the risk of developing an intramammary postpartum infection, when the S. aureus infection level in the herd is high. The environment and body sites of animals are a frequent source of infection from cows with persistent S. aureus infection [9]. The potential risk for an epidemic colonization by S. aureus in the dairy herd occurs from the skin contamination of the nipple, and the udder lesions present favor the development of intraglandular infection. The first phase of lactation in cows is considered the most risky to new infections by S. aureus in cattle herds [10, 11]. Other environmental pathogens such as Escherichia coli and Streptococcus agalactiae increase the infection risk in fresh cows during postparturition period. The design of the milking parlors has a determining influence on the infection rate in the dairy herd and the elevated milk pipe lines producing large vacuum fluctuations that increase intramammary infection risk in dairy cows [12]. In comparison, the low pipe lines in the milking parlors reduce the proportion of new infections in the mammary glands. The risk of a cross infection increases at the milking time, when milking techniques are inappropriate or the milking equipment malfunctions. These factors increase the bacterial contamination of the nipple affecting the resistance mechanisms of the mammary gland. The reduction of the risk of bacterial colonization in the skin is achieved with the application of the nipple disinfectants, decreasing the skin contamination by S. aureus and other pathogens, without appreciably affecting the physical characteristics of the nipple skin. Keratin in the nipple duct is a natural barrier that reduces bacterial colonization and limits the development of the mammary gland infection [13, 14]. In the presence of mastitis, the chemical composition of keratin changes, and there is an increase in the proportion of polyunsaturated fatty acids and a decrease in the content of short-chain fatty acids [15, 16]. On the other hand, when the keratin is removed experimentally from the teat canal, the rate of infection with Str. agalactiae increases; consequently, there is a high excretion of bacteria in milk and an elevated somatic cell count in milk. This shows the importance of keratin as a natural barrier that contributes to reduce mammary glandular infection [17]. On the other hand, in infection of S. aureus in the mammary gland, significant changes of glandular tissue structure occur in which hyperplasia, stratification, and keratinization of the milk cistern and reduction of the glandular lumen are observed [18]. These changes are caused by the inflammatory reaction and local leukocyte infiltration, with the formation of plaques of cellular debris and keratin present in the amorphous material. S. aureus experimental infection in dairy cows is affected by the lactation stage and the number of milk somatic cells [6]. The S. aureus strain type and the infective dose influence the development and the infection persistence in the mammary gland. In dairy cows with less than 4 months of lactation, when the number of somatic cells is less than 500,000 cells/mL, resistance to infection occurs. In turn, other studies indicate that the pathogenicity of S. aureus strains contributes in a decisive way to the development of mammary gland infection and its potential spread in the herd [19]. The persistence of infection by S. aureus in the udder of the cows increases the levels of antibodies present in the milk; this correlates positively with the number of lactations and the number of somatic cells in the cows [20]. The diagnosis and timely treatment decrease the evolution of clinical signs and the severity of damage to the mammary gland caused by S. aureus, Str. agalactiae, and Str. uberis, resulting in a reduction in the incidence of bovine mastitis in the dairy herd [21]. The diagnostic tests allow early identification of physical-chemical milk changes caused by mastitis. The detection of agents, antigens, and antibodies favors the identification of an intramammary infection and its clinical evolution in the herd [22]. The diagnosis of the situation of subclinical mastitis in the herd, under a causal model based on clinical diagnosis, establishes an important association between the environmental factors and the causal conditions in the transmission of infectious agents in the population. In this case, the S. aureus infection is able to affect seriously the udder health and milk production. To evaluate herd infection level and to characterize Staphylococcus aureus phenotypes, dairy cows from small family farms in the Central Mexico region were studied.

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 km². 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.

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 20 minutes) suspension (2 × 10⁸ 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].

2.4 Staphylococcus aureus methicillin-resistant strains identification

With the identified resistant oxacillin and methicillin antibiotypes (ORSA/MRSA), the in vitro susceptibility test on antimicrobials with 90n S. aureus isolates was carried out by the agar diffusion method of National Committee for Clinical Laboratory Standards [30], S. aureus isolates were incubated in Mueller Hilton broth for 4 hours at 37°C, compared to McFarland standard 0.5; Mueller Hinton agar plates were inoculated by applying on the plates the antibiotic unidisks (BBL, Lawrence, KS, USA): 10 U penicillin, 30 μg ampicillin, 1 μg oxacillin, and 10 μg cephalothin. The S. aureus strains of ATCC25923 and ATCC 29213 and the S. epidermidis strain ATCC 12228 were used as controls. The strain of S. aureus ATCC 43300 was used as a control of methicillin resistance. To confirm the MRSA strains, Mueller Hinton agar plates containing 4% NaCl were inoculated and incubated at 35 and 42°C for 24 hours. The bacterial inhibition halos of the bacterial growth on the agar plates were expressed in mm and compared to the established values for the test with a difference >4 mm in diameter between the halos of inhibition containing unidisks with 1, 2, 4, and 6 μg of oxacicline [31]. The in vitro production of β-lactamase was determined by the modified iodometric method [32].

2.4.1 Identification of the mec A gene in Staphylococcus aureus by polymerase chain reaction (PCR) test

Isolation of chromosomal DNA was obtained from S. aureus 90n isolates, from the study groups of work strains 1, 56, 305, and 123, with the control strains of S. epidermidis ATCC 12228 and S. aureus ATCC 25923 as negative controls, and S. aureus ATCC 43300 as a positive control. The washed bacterial pellet was suspended in a buffered phosphate solution of pH 7.2 to proceed to bacterial lysis. The bacterial DNA solution was standardized at 0.5 ng per 1 μL and stored at −20°C. The PCR reactions were carried out with a commercial PCR kit (BioTecnologías Universitarias, México), using a thermal cycler (Genius Techne, Duckford, UK). The PCR for the identity of the S. aureus isolates was carried out by the amplification of nuc gene, using the oligonucleotides (Gibco BRL, Rockville, MD), Sa1 (5′GCGATTGATGGTGATACGGTT-3′), and Sa2 (5′AGCCAAGCCTTGACGAACTAAAGC-3′) [33]; the amplification product of PCR obtained was 270 bp related to the nuc gene. The amplification of the mec A gene was performed to identify the MRSA strains of S. aureus, using the primers (Gibco BRL, Rockville, MD), mec1 (5′-TGGCTATCGTGTCACAATCG3′), and mec2 (5′-CTGGAACTTGTTGAGCAGAG-3′) to obtain an amplification product of 310 bp; initial denaturation was performed at 92°C for 3 minutes and 30 cycles under the amplification conditions: 92°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute, and a final extension of 2 minutes at 72°C. The amplified product was kept at 4°C, before being visualized on an agarose gel. The amplification conditions of the gene were similar with an alignment temperature of 56°C. The nuclear DNA amplification products were separated by electrophoresis in a horizontal chamber (Horizon 5B, Gibco BRL, Goithersburg, MD, USA), with a power supply of 70 Volts for 90 minutes applied to 2% agarose gel (Gibco BRL, Rockville, MD. USA), in TBE in a run with a 100 bp molecular weight marker (Gene Ruler, Fermentas, Burlington, Ontario, CA). The strains of S. aureus ATCC 29213, ATCC 43300, and S. epidermidis ATCC 12228 were used as a control. The gels were stained with ethidium bromide. The gels were photographed in a UV transluminator (UVP, Upland, CA. USA), visualized with a DC 120 digital camera (Kodak Eastman, Rochester, NY, USA) and image analysis program (ID image analysis software Windows; and 3.0, Kodak digital science, Rochester, NY, USA). The analysis of results was made from the frequency of isolation and the expression of virulence factors of S. aureus related to the biotype, by estimating the absolute and relative frequency; The statistical evaluation was performed with the Pearson’s Chi-square test (χ2), with an α level of 0.05 and a 95% confidence interval using Epi-Info 6.0 software (CDS, Atlanta, GA, USA). The in vitro sensitivity was estimated from the mean ± standard deviation of the inhibition halos (in mm) of the antibiotics.

3. Results

The S aureus subclinical mastitis frequency determination in dairy cow population density expressed as cows number/km² 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/km² of cows increased.

Density (cows/km^2)	Municipalities	Number of cows	Isolations; Staphylococcus aureus
				%
High [5.6]	Almoloya de Juárez	1021	268	46.2
Median [2.7]	Atlacomulco, Chapultepec, Lerma, Tenango del Valle, Temoaya, and y Zinacantepec	1237	211	36.3
Low [1.12]	Toluca, Metepec	491	101	17.4
Total		2749	580	21.0

Table 1.

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

p < 0.01.

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.

Agents	Isolation number	%
Staphylococcus aureus	82a	22.4
Staphylococcus coagulase negative	45a	12.29
Streptococcus agalactiae	14	3.82
Escherichia coli	6	1.63
Bacillus spp.	3	0.81
Micrococcus spp.	5	1.36
Klebsiella spp.	4	1.09
Enterobacter spp.	1	0.2
Streptococcus dysgalactiae	4	1.09
Streptococcus uberis	2	0.54
Negative isolations	197	53.82
Total	366	100

Table 2.

Bacterial isolation frequency in dairy cows with subclinical mastitis.

^a

Significant differences (p < 0.05).

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).

Wisconsin test estimated somatic cells × 10^3 mL	Bacterial isolates (%)
<100	10.0
100–500	8.0
500–1000	20.0
1000–1700	14.0
1700–2500	39.0
>2500	8.5
Total	100

Table 3.

Distribution of mastitis Wisconsin Test reactions and bacterial isolates.

^a

Significant differences (p < 0.05).

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.

				Hemolysin types
Biotype	Total	α	β	γ	δ	α β	α δ	γ	α	α β δ
A	48	9	11	0	2	19	2	0	3	1
C	36	23	4	1	1	5	3	1	2	0
B	6	0	0	3	0	0	0	0	0	0
Total	90	32	15	4	3	24	5	1	5	1
%	100	35.5	17.0	4.4	3.3	26.6	5.5	1.1	5.5	1.1

Table 4.

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

p < 0.01; total isolates of S. aureus evaluated (90 n)

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 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 α β δ 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).

Antimicrobial	Isolations of Staphylococcus aureus
	Sensitive (%)	Intermediate (%)	Resistant (%)
Penicillin	8.2	26.1	65.7
Ampicillin	8.2	1.5	90.3
Novobiocin	69.4; 91.8	5.2; 0.0	25.4; 8.2
Dicloxacillin	90.3	1.5	8.2
Cephalosporin	8.2	40.3	5.5
Cefotaxime	91.8	2.2	6.0
Erythromycin	94.8	1.5	3.7
Spiramycin	69.4	5.2	25.4
Lincomycin	65.0	8.9	26.1

Table 5.

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

p < 0.05.

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). 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%.

Antimicrobial		Halo inhibition (mm); Halo growth inhibition			Strains resistant (%)	Production strains, β-lactamase (%)
	Maximum	Minimum	Average	SD±
Penicillin	30	8	18.4	6.7	93.0	93.0
Ampicillin	29	10	17.3	5.7	93.0	93.0
Cefotaxime	30	0	19.1	10.2	7.0	59.0
Oxacicline	16	0	9.7	6.9	21.0	20.0

Table 6.

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

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).

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.

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.

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 neutrophils incubated 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 results showed differences between treatments (p < 0.05). The increased apoptosis induced by CP due to capsular serotyp. 5 was compared with the control groups. CC strain showed less in vitro neutrophil apoptosis induction.

Figure 2.

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

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. 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.

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.

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.

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 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, 57, 58, 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, 61, 62, 63, 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, 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⁺ 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 β-lactamase 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 β-lactamase 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, 96, 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, 106, 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.

5. Conclusion

The S. aureus infection is prevalent in dairies of family production and associated to the expression of virulence factors that characterize the predominant S. aureus pathotypes in dairy cows in the Toluca Valley in México central region. Phenotypes and genotypes of S. aureus associated with the production of toxins, capsular exopolysaccharide, and MRSA strains, to establish a potential risk to health, are highlighted. The S. aureus infection level in the cow herds can be compromised with food safety and establishing animal and human health risk.

Acknowledgments

To the members collaborating in the project, “Red de Cuerpos Académicos en Salud Animal e Inocuidad Alimentaria: México-Uruguay”.