S.L. Perez Martel1, O. Romero2, I. Trevino3,
G. Delgado4, B. Petlacalco4,F. Romero4, H. Hernandez4,
Valencia De Ita Ma.A2  and V. Rodriguez-Hernandez1*

1Colegio de Posgraduados, Campus Puebla-Mexico.
2Centro de Agroecologia-ICUAP; Benemerita Universidad Autonoma de Puebla-Mexico.
3Facultad de Medicina Veterinaria-BUAP.
4CyTA Labs.

ABSTRACT

 Nine dairy cows seven months gestation received via the milk ducts formulation inactivated antigen protein adhesion to fibronectin of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli cells. The analysis of the immunoglobulins G by indirect ELISA against the applied antigens, showed that five of nine cows were stimulated with Escherichia coli, the case for the antigenic stimulus for Streptococcus agalactiae showed that eight of nine cows responded with good indices of significance. Finally, stimulus virulence proteins of Staphylococcus aureus adhesion to fibronectin, revealed response rates all cows (P <0.0002). Intraductal response to antigens promises to be a way for immunization to dairy cows in the drying stage.

Keywords: Acute mastitis, inactivated antigens and analysis of immunoglobulins.

INTRODUCTION

 In order for the mammary gland to become infected with some bacteria, a physical contact between the pathogen and the host is necessary; Where the recognition receptors are exhibited between them, at that time, the innate immune system of the host responds through the interaction of molecules referred to as Pathogen Associated Molecular Patterns (PAMPs)[Ulevitch et al., 2000]. Mastitis cases by Gram negative bacteria are more common, being EscherichiaColi the most predominante; because it contains in its cell wall lipopolysaccharide (LPS) which is common for the whole family (Yoshimura et al., 1999). For the group of Gram positive bacteria, and peptidoglycan (PGN) and lipoteichoic acid (LTA) cell wall representing the repertoire of compounds recognition similar to (LPS) effects, but with other mediators recognition (Schroder et al., 2003).

Staphylococcus aureus is one of the bacteria colonizing the mammary gland tissue destruction effects with high quality and low inflammatory response milk. Its reservoirs scarified skin of the nipple and the stratified epithelium of the nasal mucosa (Kluymants et al., 1997). Current vaccines for Staphylococcus aureus are made with whole bacteria and against the PGN (Baselga et al., 1994; Garcia et al., 1996;Kerro-Dego et al., 2006) and the immune response is limited given the numerous factors virulence that this bacterium exhibits (Anderson et al., 2012). However, facilitates recognition of PAMPs low immune response to infection, also facilitates persistent virus infections immunosuppressants as bovine viral diarrhea BVD, IBR Rhinotraqueitis Infectious Bovine viral Bovine Leukemia BVL (Wilson et al., 1997; Barkema et al., 1998).

The persistence of high risk bact erias in glandular acids such as Staphylococcus aureus, Streptococcus agalactiae,Mycoplasma bovis and some strains of Escherichia coli adherent and invasive, are associated with subclinical mastitis cases high (Dopfer et al., 2002). In turn, prepartum mediated immunosuppression stress and chain hormones are in part the result of dysfunction of phagocytosis of polymorphonuclear neutrophil leukocytes which leads to facilitation of colonization of bacteria in glandular structures generates loss and poor quality of milk production (Burvenich et al., 2003; Fox et al., 2005).

The purpose of this study was to analyze the immune response g enerada application by intra – ductal three bacterial antigens in dairy roof racks to determine the presence of specific immunoglobulins against antigens of the formula.

MATERIAL and METHODS

Bacterial antigens
The application of E coli obtained from cases of acute mastitis was carried out, as well as four serotypes previously characterized as producers of Pilis K-99, 39 and 35 ATCC collection of low and high production of labile thermosensitive and thermosensitive toxins. For Gram + bacteria such as Staphylococcus aureus and Streptococcus agalactiae, they were obtained from clinical cases of acute mastitis and their respective morphological and biochemical characterization until their characterization. The previously obtained recombinant adhesion protein to the fibronectin (Fnb) of Lactococcus lactis previously obtained on the wall of Lactococcus lactis, was also incorporated as a virulence protein that participates for adhesion to cells and host tissue.

Selection criteria for pre-calving and intramammary cows
In the region of Atlixco, Puebla-Mexico nine next to dry gestation diagnosed by palpation of about 7 months and low milk production in lactating cows were selected earlier. The herd of 200 breeding receives annual immunization against the virus of the respiratory and reproductive cattle complex, however, n or applied biological control of mastitis, this depends on control of hygiene and milking management on a daily basis.

Analysis of total protein and Immunoglobulins
Of each cow s blood samples were given Vacutainer tubes in obtaining d serum before and after 30 days of immunization. The serum is centrifuged do for 10 minutes at 1,000 rpm, was later deposited in Ependorff tubes and frozen at minus 20 oC. Each sample of 2 cc was evaluated relative to the total protein concentration using a refractometer Brix ATAGO Master-M. For the determination of total protein busied 0.5 mL of serum, diluted 1:2 in buffer solution of phosphates (PBS), serum was transferred to a beaker on ice with magnetic stirring co ntinua, adding 1.5 mL of ammonium sulfate at pH 7.8 up to saturation, subsequently conducted the analysis of the total protein concentration by the Bradford method which is based on the binding of blue dye Coomassie (G-250) to proteins. The acid solution dye is bound to form a protein-dye complex with a higher extinction coefficient than the free dye. This method is sensitive 1-15 mg.

For precipitac ion total immunoglobulin was used 0.5 mL of serum from each sample of nine dairy cows, ammonium sulfate was incorporated in slowly and under constant stirring for 30 minutes at 4°C for precipitation of globulins. The precipitate was re – suspended by gentle agitation for five minutes and centrifuged at 10,000 G for 15 minutes at 4 °C. E l supernatant was discarded and the button is re Cupero in 0.5 mL of PBS with a final saturation of 40% salt. Subsequently the fraction of globulins γ was transferred to a dialysis bag against saline 0.85% making 6 changes using barium chloride in a 1:1 with water for 24 to 36 hours at 4 °C until the ammonium sulfate it was no longer detectable. Globulins were pr eservadas frozen at 20 oC.

Quantification of immunoglobulins in animals immunized by the ELISA method
The ELISA its acronym EnglishLinked Immuno Sorbent-Enzyme Assay: is a specific technique to detect Bound antibodies an enzymecapable of generating a product (Lequin, 2005). Indirect ELISA test was developed for the quantitative determination of antibodies in serum in each of dairy cows for the different antigens. 100 μL of a 108 suspension of each bacterium to be tested was added in triplicate on 96-well ELISA plates (Costar). It was left at 4 °C for 12 hours. subsequently they were washed with PBS and applied 100 μL of purified immunoglobulins as the first antibody, incubated at room temperature with moderate stirring for one hour. Do that time passes washed three times with PBS solution and applied 100 μL bovine IgG peroxidase – labeled goat as second antibody. It was read in spectrophotometer for ELISA, Diagnostic Pasteur model LP 400.

RESULTS AND DISCUSSION

Immune System immune ma participates significantly in the resolution of infections of viral or bacterial origin. Some germs are producers of compounds that inhibit the immune system, so the chances of the germ (s) in colonizing and invading the host (Nakawa et al., 1993). In the particular case of bacterial infection in the mammary gland in the immediate postpartum period, polymorphonuclear neutrophils are inhibited in its function of phagocytosis, probably mediated by the stress of labor (Lee et al., 2003). Udders of dairy cows receiving immunization in the drying period, did not have any physical alteration, besides the electrical conductivity of the milk on the fifth day after postpartum, it turned with measurements above the cutpoint (300 +/- 50), which means that the milk had no alterations of bacterial origin.

E.coli is one of the common pathogens found in milk immediate postpartum especially in cows second and third lactation where injury to the sphincter through the nipple are more frequent and loss of function predisposes to colonization of environmental bacteria (Russol et al., 2007). In this research c inco nine cows stimulated Eantigens.coli showed higher antibody concentration in statistically significant (Fig.1). The use of antibiotic products as a curative and / or preventive treatment in the drying stage is a common practice among milk producers and there is little control over their use (Russol et al., 2007). Also the control of antimicrobial resistance of bacteria that infect the mammary gland of cattle is nil or limited in Mexico.

Fig. 1. ELISA absorbance values to E. coli

The production of specific immunoglobulins against epitopes of pili and fimbriae of E. coli mediated immunization are factors internal support for the facilitation of opsonization, control and resolution to challenges of E. coliin cattle milk production (EcoStaph PM + 3 2015) [González et al., 1989]. (EcoStaph PM + 3 2015) [González et al., 1989]. Although E. coli serotypes among animals livestock production species maintain their niches regarding colonization, it is necessary to mention that artisanal producers of fresh bovine milk cheese curdling made without pasteurization what it defines as potential zoonosis to those infections.

Streptococcus agalactiae is a bacterium streptococcus group B and other family that prevails in the intestinal tract of various mammals, including humans. In bovines it induces mastitis considered within the pathogens of importance by the reduction of milk production from which its surname derives. It is a germ with high sensitivity to antimicrobial beta lactams, so its treatment both lactating and the drying period eliminates infection (Rossitto et al., 2002), however, the antimicrobial resistance what little Is known in Mexico, is a factor that drives the improvement of hygiene and the application of vaccines.

Yokomizo and Norcross (1978), described the benefits in immunizing vaccine Streptococcus by way intra- parenteral and intro-mamaria, other experimental groups developed significant increase in IgG, 1 and 2, IgM and IgA. Furthermore, Lindahl et al. (2005) has developed vaccines for Streptococcus agalactiae neonatal infections linked to the men, where bovine strains involved are linked to virulence and capsule formation.

In this paper we find that the antigenic stimulus for S. agalactiae by way intra tanker, was favorable in eight of nine cows, which responded with good titers of antibodies to this antigen with gnificancia of p <0.0002 (Fig. 2) which confirms previous work by (Yokomizo and Norcross, 1978).MastitisforS. agalactiae is common in ruminants such as; Cows, sheep, goats and monogastricos as; Bristles, bitches, cats, rabbits, etc. significantly reducing the quality of milk (Bergonier, 2014).

Fig. 2. ELISA absorbance values to Streptococcus agalactiae

The  absorbance values in the ELISA test for Staphylococcus aureus were concentration values for inmunoglobulinas with some statistical significance for 2 of 9 cows (Fig. 3). These results are in agreement with previous studies where the low response.

Fig. 3. ELISA absorbance values Staphylococcus aureus

However, stimulation with the virulence protein adhesion to fibronectin S.aureus, revealed response in all cows with signi cance rates between p <0.0002 (Fig.4).

Fig. 4. ELISA absorbance values at (Fnb), S. aureus protein Recombinant fibronectin binding

On the other hand, the immuno stimulation via the milk duct, resulted in production of immuno globulins specific G with significance values in; E coli,Streptococcus agalactiaeand Protein FNB of the S.aureus. Antigens applied and its response indicates that the tank via intra can be used for the induction of immune response in the drying step. Uncapsulated lymphoid tissue located in the Fürstenberg rosette and permeability of the epithelium of that region suggest sites of antigen presentation to immune cells. It remains to be studied the production of IgA by this route where the mucosal response does with synthesis of this immunoglobulin. Staphylococcus aureus bacteria from the group of Gram positive found more frequently in cases of bovine mastitis (Barkema et al., 1998). The infections whose origin was S.aureus infections occur with sub clinical or chronic medical that usually persist for the life of the infected animal.

The results described in table 1, show that the sera prepartum of 5 cows were lower compared to sera obtained after delivery. The study by refractometer is often used to assess concentrations of immunoglobulins in the colostrum milk, however, their sensitivity limits are not as accurate as others.

Table 1. Protein measurement by refractometer

Sample number Brix Brix
  Pre-delivery whey Postpartum Sera
10 8 10
19 9 10
23 10 11
65 7 9
307 9 9
410 10 10
484 7 12
798 11 11
990 10 10

 

CONCLUSIONS

 The age of the cow, nutritional status, its environment and vaccination schedules will determine the quality and quantity of serum immunoglobulins present in the bloodstream. During the pre-natal period, the cow disseminate their blood capillaries, into the light immunoglobulins of glandular acinos of the mammary gland, forming the protein rich secretion, hormonal factors, lipids, known as colostrum. In cattle, there are particular conditions of immune suppression “stress” pre and post delivery, which favors the invasion of opportunistic pathogens to different organs such as the uterus, vagina and mammary gland (Lioyd, 1993).

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