The Potential of Metalloproteinase-9 Administration to Accelerate Mammary Involution and Boost the Immune System at Dry-Off.

The dry period is decisive for the milking performance of dairy cows. The promptness of mammary gland involution at dry-off affects not only the productivity in the next lactation, but also the risk of new intra-mammary infections since it is closely related with the activity of the immune system. Matrix metalloproteinase-9 (MMP-9) is an enzyme present in the mammary gland and has an active role during involution by disrupting the extracellular matrix, mediating cell survival and the recruitment of immune cells. The objective of this study was to determine the potential of exogenous administration of a soluble and recombinant version of a truncated MMP-9 (rtMMP-9) to accelerate mammary involution and boost the immune system at dry-off, avoiding the use of antibiotics. Twelve Holstein cows were dried abruptly, and two quarters of each cow received an intra-mammary infusion of either soluble rtMMP-9 or a positive control based on immunostimulant inclusion bodies (IBs). The contralateral quarters were infused with saline solution as negative control. Samples of mammary secretion were collected during the week following dry-off to determine SCC, metalloproteinase activity, bovine serum albumin, lactoferrin, sodium, and potassium concentrations. The soluble form of rtMMP-9 increased endogenous metalloproteinase activity in the mammary gland compared with saline quarters but did not accelerate either the immune response or involution in comparison with control quarters. The results demonstrated that the strategy to increase the mammary gland immunocompetence by recombinant infusion of rtMMP-9 was unsuccessful.

Short communication: Recombinant mammary serum amyloid A3 as a potential strategy for preventing intramammary infections in dairy cows at dryoff.

Mammary serum amyloid A3 (M-SAA3) has shown potential in stimulating innate immunity during intramammary infections, at calving and at dryoff. In this study, we produced recombinant caprine M-SAA3 to test its ability to reduce intramammary infections with Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli, which are all common mastitis-producing pathogens. Recombinant production of M-SAA3 (followed by lipopolysaccharide removal to avoid lipopolysaccharide-nonspecific stimulation of the immune system) was successfully achieved. Mammary serum amyloid A3 stimulated the expression of IL-8 in a dose-dependent manner in primary mammary cultures. Although a direct killing effect on Staph. aureus by M-SAA3 was not detected, this acute phase protein was able to reduce Staph. aureus, Strep. uberis, and Strep. dysgalactiae infections by up to 50% and induced a reduction in E. coli counts of 67%. In general, the best concentration of caprine M-SAA3 for inhibiting infections was the lowest concentration tested (10 µg/mL), although higher concentrations (up to 160 µg/mL) increased its antimicrobial potential against some pathogens.

Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology.

UNLABELLED: Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest. STATEMENT OF SIGNIFICANCE: The development of both natural and synthetic biomaterials for biomedical applications is a field in constant development. In this context, E. coli is a bacteria that has been widely studied for its ability to naturally produce functional biomaterials with broad biomedical uses. Despite being effective, products derived from this species contain membrane residues able to trigger a non-desired immunogenic responses. Accordingly, exploring alternative bacteria able to synthesize such biomaterials in a safe molecular environment is becoming a challenge. Thus, the present study describes a new type of functional protein-based nanomaterial free of toxic contaminants with a wide range of applications in both human and veterinary medicine.

Detection of porcine anelloviruses in pork meat and human faeces.

Torque teno viruses (TTV) are icosahedral, single-stranded circular DNA viruses infecting several vertebrate species. Currently, these viruses are considered non-pathogenic although they are suggested to be co-factors in several diseases. Recently single-stranded circular DNA viruses have been found in human faeces. Considering the consumption of pork meat products and the ubiquitous nature of swine TTV (Torque tenosus virus, TTSuV), the human population is frequently exposed to these viruses. To determine if TTSuVs could be delivered through food, human faecal samples were analysed for their presence. Indeed, the results of this study show that up to 25% of faecal samples were positive for known TTSuVs by PCR and sequencing. Additionally, all commercially available pork products purchased in Spanish supermarkets contained DNA of TTSuV.