Prevention Of Bovine Mastitis Research Articles

Natural sanitizers pre- and post-milking in the prevention of bovine mastitis

Approaches focused on improving animal health are essential strategies to increase the quality and productivity of the livestock sector. Bovine mastitis is a prevalent disease in dairy farming worldwide. There are various ways to prevent infections and mammary inflammation in cows, such as using sanitizers or disinfectants before and after milking to clean the cow's udder surface and reduce contamination. Chlorine-based disinfectants are commonly used, and in some cases, antibiotics are used to prevent mastitis. This review aimed to analyze scientific articles that used plants as a sanitizer for a cow's udder in the last 5 years. There were few records of experiments with plants for disinfecting cows' udders; a total of 10 articles and 12 plant species were mentioned, including twice Piper betle and Acacia nilotica. Among the classes of metabolites indicated as active are terpenoids, flavonoids, and tannins. Studies indicate that plant extracts exhibit antimicrobial properties. Plant-based disinfectants promote sustainable and environmentally friendly sanitation in the livestock production sector, as these products are biodegradable and renewable.

Efecto ecotóxico y preventivo del uso de aceites esenciales en la mastitis bovina

The essential oils of Lippia citriodora(Ort.) and Lippia origanoides(Kunth) have shown antimicrobial activity associated with mastitis. The objective of this study was to evaluate its ecotoxic effect withthe Artemia salinabioassay and the prevention of mastitis through an in vivotest in cattle (n=20) with a product based on these oils using a conventional product as a control. Contact hypersensitivity, the effect on somatic cells, and residuality in milk samples were evaluated. The results of the Artemia salinabioassay were 10.05 and 19.36 (μg/mL) respectively. No negative effects or contact hypersensitivity were observed, and no residual metabolites were found in post-test milk. The somatic cell count showed 75% effectiveness in the prevention of mastitis with essential oils compared to 62.5% with the conventional product. The evaluated formulation could be used in the prevention of bovine mastitis safely, further investigation is required.

Development of a Sanitary Udders Care Regimen to Prevent Bovine Mastitis

The paper provides information on the use of the drug Monclavit-1. To determine the toxicity of the drug on the skin of the udders of cows, we studied the effect of different percentages on the skin of rabbits. As a result, it was found that the drug does not have a toxic effect. At the next stage, the drug was applied in the farms of the livestock complex R-Agro, Imishli district of Azerbaijan. A control examination was carried out to check the teats of the udders of cows of several barns to identify the number of animals predisposed to mastitis. The effectiveness of the drug of various concentrations was determined. In the process of testing, it was determined that the Monclavit-1 solution neutralizes microorganisms on the skin after a certain time after the sanitization of the udders. It has been established that Monklavit-1 has no toxic or allergic effect on the skin of cows’ udders teats. As a result of the research, a solution of the Monclavit-1 preparation was recommended for sanitizing the udders and teats for preventive measures and the prevention of bovine mastitis.

Melatonin Maintains Homeostasis and Potentiates the Anti-inflammatory Response in Staphylococcus aureus-Induced Mastitis through microRNA-16b/YAP1.

Staphylococcus aureus is a highly infectious pathogen and is a considerable threat to food hygiene and safety. Although melatonin is thought to exert an ameliorative effect on bovine mastitis, the regulatory mechanisms are unclear. In this study, we first verified the therapeutic effect of melatonin against S. aureus in vitro and in vivo, a screening of differentially expressed miRNAs and mRNAs among the blank, and S. aureus and melatonin + S. aureus groups by high-throughput sequencing identified miR-16b and YAP1, which exhibited 1.95-fold upregulated and 1.05-fold downregulated expression, respectively. Moreover, epigenetic studies showed that S. aureus inhibited miR-16b expression by methylation (increased DNMT1 expression). Additionally, the DNMT1 expression level was significantly decreased by melatonin treatment, which might indicate that the inhibition of DNMT1 by melatonin reduces the effect of S. aureus on miR-16b. The flow cytometry, scanning and transmission electron microscopy, EdU assay, and cell morphology results indicated that miR-16b in bovine mammary epithelial cells (in vitro) and in mice (in vivo) can modulate the maintenance of homeostasis and potentiate the anti-inflammatory response. In addition, YAP1 was demonstrated to be the target gene of miR-16b through quantitative real-time polymerase chain reaction, western blot, RNA immunoprecipitation, and functional assays. This study indicates that melatonin inhibits S. aureus-induced inflammation via microRNA-16b/YAP1-mediated regulation, and these findings might provide a new strategy for the prevention of bovine mastitis, facilitating further studies good of zoonotic diseases caused by S. aureus infection.

Udder Health Monitoring for Prevention of Bovine Mastitis and Improvement of Milk Quality.

To maximize milk production, efficiency, and profits, modern dairy cows are genetically selected and bred to produce more and more milk and are fed copious quantities of high-energy feed to support ever-increasing milk volumes. As demands for increased milk yield and milking efficiency continue to rise to provide for the growing world population, more significant stress is placed on the dairy cow's productive capacity. In this climate, which is becoming increasingly hotter, millions of people depend on the capacity of cattle to respond to new environments and to cope with temperature shocks as well as additional stress factors such as solar radiation, animal crowding, insect pests, and poor ventilation, which are often associated with an increased risk of mastitis, resulting in lower milk quality and reduced production. This article reviews the impact of heat stress on milk production and quality and emphasizes the importance of udder health monitoring, with a focus on the use of emergent methods for monitoring udder health, such as infrared thermography, biosensors, and lab-on-chip devices, which may promote animal health and welfare, as well as the quality and safety of dairy products, without hindering the technological flow, while providing significant benefits to farmers, manufacturers, and consumers.

Ethanol Extract of Artemisia Annua Prevents LPS-Induced Inflammation and Blood-Milk Barrier Disruption in Bovine Mammary Epithelial Cells.

This experiment evaluated the pre-protective effect of AAE on inflammatory injury and tight junction disturbance in bMECs induced by LPS. The bMECs were treated with AAE (3, 6, 12 μg/mL) for 3 h and then incubated with 10 μg/mL lipopolysaccharide (LPS) for 12 h. Our results showed that LPS significantly increased the mRNA and protein expression of CD36, induced the phosphorylation of IκBα and p65 and elevated the levels of TNF-α, IL-1β and IL-6 mRNA, which further resulted in ultrastructural damage, disrupted the expression of tight junction proteins (occludin, zonula occludens (ZO-1) and claudin-1) and decreased the viability of bMECs (p < 0.05). More importantly, AAE pretreatment attenuated the expression of CD36, suppressed the activity of the NF-κB signaling pathway and down-regulated the levels of inflammatory factors in LPS-stimulated bMECs (p < 0.05). Therefore, AAE can effectively protect bMECs against inflammatory injury and tight junction dysfunction, which has important research value for the prevention of bovine mastitis.

Antimicrobial resistance profile of Staphylococcus spp. isolates in cattle herds from Western Amazon

Staphylococcus is one of the most frequent etiological agents of bovine mastitis, causing economic losses to milk farming. This study aimed to describe the milk production and management practices in 15 herds located in three microregions of the State of Rondônia and to identify the antimicrobial resistance profile of 97 isolates of the genus Staphylococcus from these herds. The isolates were subjected to antimicrobial susceptibility tests using the agar diffusion method. Twenty-nine S. aureus isolates were selected to determine their minimum inhibitory concentration (MIC). In the farms studied, the breeding system was semi-intensive, with the Girolando breed being predominant. Milking was performed predominantly using a mechanical milking system (86.7%), twice a day (66.7%), in the presence of the calf (53.8%). The average number of lactating cows in the farms was 24, with an average milk production of 204.9 L d-1 and a milk productivity of 10.2 L animal-1 d-1. The use of antimicrobials for the treatment and prevention of bovine mastitis was reported for all properties, and therapy for dry cows was adopted in 80% of the herds. The percent susceptibility to antimicrobials ranged from 85.5% to 100% for S. aureus, 22.2% to 88.9% for coagulase-positive staphylococci (CPS), and 69.2% to 100% for coagulase-negative staphylococci (CNS), with the highest resistance frequencies for penicillin, ampicillin, and tetracycline. Among the S. aureus isolates, 100% susceptibility to oxacillin, cefalexin, and gentamicin was observed, and among the CNS isolates, 100% susceptibility to gentamicin was observed. None of the antimicrobials tested showed 100% in vitro effectiveness for CPS. CPS and CNS presented lower percentages of susceptibility to penicillin, ampicillin, and tetracycline than the S. aureus isolates. Twelve resistance patterns were detected, six of which were multiresistance patterns. The most prevalent resistance patterns were penicillin and ampicillin (PEN-AMP) and penicillin, ampicillin, and tetracycline (PEN-AMP-TET). Results of the MIC assay revealed that all 29 S. aureus isolates were susceptible to cephalothin, cefoxitin, tetracycline, and erythromycin, whereas 25 (86.2%) were susceptible to penicillin.

Prevention of Bovine Mastitis through Vaccination.

Cow mastitis is one of the main factors of economic damage in modern animal husbandry. It is registered to affect almost everywhere, taking into account the subclinical form from 30% to 50% of the livestock per year. Economic losses of farms from the diseases of cows with mastitis are due to a decrease in milk productivity, precocious cow disposal, a decrease in the grade and sale price of market milk, and treatment costs. This study aimed to use a safer method in the prevention and control of mastitis in cows, and vaccinations could be a solution to this problem. Vaccination promotes acquired immunity to a specific pathogen and also has few side effects. A bacteriological study of clinical and subclinical forms of mastitis was conducted to study the etiology of mastitis in cows in various farms of Kazakhstan. A total of 1,068 milk samples from 767 cows were examined. The studies were carried out according to "Guidelines for bacteriological studies of milk and udder secretions of cows". The primary selection of cultures was carried out based on growth characteristics on media and microscopy of preparations from individual colonies. Morphological, cultural, and biochemical properties of the isolated cultures were studied according to generally accepted schemes. Identification of the selected cultures was carried out using Bergey's determinant. The strain numbers of Staphylococci, Streptococci, Escherichia, Klebsiella, Diplococci, and Protea were 590 (55.2%), 240 (22.4%), 151 (14.1%), 50 (4.7%), 24 (2.3%), and 13 (1.3%), respectively. The greatest strain numbers of Staphylococci (n=351) and Streptococci (n=129) were isolated from cow's milk with subclinical mastitis. The effectiveness of the polyvalent vaccine used was determined by the manifestation of clinical and subclinical forms of mastitis. Out of 600 immunized cows, 9 (1.5%) and 13 (2.3%) animals developed subclinical and clinical mastitis, respectively. Furthermore, out of 150 cows taken into control, 12 (8%) and 10 (6.6%) animals developed subclinical and clinical mastitis, respectively. Furthermore, out of 12 cows with clinical mastitis, 5 cows previously had a subclinical form. Vaccination takes a significant place in the control of infectious diseases. The success of vaccine prevention depends on the quality of vaccines and timely vaccination coverage of threatened populations. Modern immunology and vaccine prevention have summed up the theoretical basis and outlined ways to improve vaccines in the direction of creating new harmless effective vaccines.

Identification of a New Antimicrobial Agent against Bovine Mastitis-Causing Staphylococcus aureus

Staphylococcus aureus RF122 is a major pathogen that causes bovine mastitis, which is the most prevalent and costly disease in the milk and dairy industry. S. aureus expresses various virulence factors that are especially highly associated with iron metabolism, and the bacterial ferrous iron transport system Feo is important for bacterial growth or virulence in mammalian hosts. In this study, we evaluated a new antimicrobial agent, PHT-427, targeting the S. aureus RF122 Feo system for the prevention of bovine mastitis. Various analyses on in vitro enzymatic assays, growth inhibition, virulence expressions, and toxicity of animal model systems were conducted to characterize the inhibition properties of PHT-427. This small molecule efficiently inhibited enzyme activity of FeoB and bacterial growth. PHT-427 attenuated various virulence factors related to milk quality, including staphyloxanthin production, biofilm formation, and coagulation. Considering the high frequency of antibiotic-resistant S. aureus in bovine mastitis isolates, PHT-427 synergistically enhanced bacterial antibiotic susceptibility and further inhibited global Gram-positive bacterial growth. Unlike its effects on bacteria, the inhibitor did not show any toxicity on animal model systems. These results indicate that the S. aureus Feo system represents a good target for antimicrobial strategies, and this new antimicrobial agent may represent a promising biotechnological application for preventing S. aureus-induced bovine mastitis in the milk and dairy industry.

Bovine mastitis prevention and control in the post-antibiotic era.

Mastitis is the most important disease in the dairy industry. Antibiotics are considered to be the first choice in the treatment of the disease. However, the problem of antibiotic residue and antimicrobial resistance, in addition to the impact of antibiotic abuse on public health, leads to many restrictions on uncontrolled antibiotic therapy in the dairy sector worldwide. Researchers have investigated novel therapeutic approaches to replace the use of antibiotics in mastitis control. These efforts, supported by the revolutionary development of nanotechnology, stem cell assays, molecular biological tools, and genomics, enabled the development of new approaches for mastitis-treatment and control. The present review discusses recent concepts to control mastitis such as breeding of mastitis-resistant dairy cows, the development of novel diagnostic and therapeutic tools, the application of communication technology as an educational and epidemiological tool, application of modern mastitis vaccines, cow drying protocols, teat disinfection, housing, and nutrition. These include the application of nanotechnology, stem cell technology, photodynamic and laser therapy or the use of traditional herbal medical plants, nutraceuticals, antibacterial peptides, bacteriocins, antibodies therapy, bacteriophages, phage lysins, and probiotics as alternatives to antibiotics.

Characterization of Staphylococcus aureus Isolates From Cases of Clinical Bovine Mastitis on Large-Scale Chinese Dairy Farms.

Bovine mastitis is a prevalent disease that causes serious economic problems globally in the dairy industry. Staphylococcus aureus is an important pathogen of bovine mastitis. This study was conducted to characterize S. aureus isolates from clinical bovine mastitis cases in large-scale dairy herds in China. S. aureus was isolated from 624 clinical mastitis cases and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 62 S. aureus isolates were obtained. Cluster analysis, genetic diversity, quantification of biofilm formation, antimicrobial resistance, and detection of virulence genes were performed on these isolates of S. aureus. Eight isolates harbored the mecA gene and were sensitive to oxacillin. MALDI-TOF MS cluster analysis revealed that the 62 isolates were divided into three major clusters (I, II, III) and eight main groups (A–H) at the distance level of 700. The agr II was the most prevalent (56.5%). The 62 S. aureus isolates were assigned to seven spa types. The most common spa type was t529(58.1%), followed by t2196 (14.5%), t518 (14.5%), t571(6.5%), t034 (3.2%), t2734 (1.6%), and t730 (1.6%). Five STs were identified from seven representative isolates as follows: ST630/CC8, ST97/CC97, ST50, ST398, and ST705. All isolates had the ability to form biofilm. Antimicrobial resistance was most frequently observed to ciprofloxacin (29%), followed by penicillin (24.2%), and streptomycin (9.6%). All isolates harbored the fnbA, clfB (100%), icaA, and icaD genes. This study provides the basis for the development of bovine mastitis prevention program on large-scale dairy farms.

Intramammary inoculation with lactic acid bacteria at dry-off triggers an immunomodulatory response in dairy cows.

The use of antibiotics to prevent bovine mastitis is responsible for the emergence and selection of resistant strains. Lactic acid bacteria (LAB) could be introduced into animal feed as an alternative prevention method that would bypass the risk of resistance development. In previous research, we demonstrated that two probiotic LAB strains isolated from bovine milk were capable of stimulating the production of antibodies and the host's immune cellular response in the udder. The present study aimed to elucidate whether the antibodies of animals inoculated with these strains were able to increase phagocytosis by neutrophils and inhibit the growth of different mastitis-causing pathogens. Moreover, the effect of LAB on the expression of pro-inflammatory cytokines was assessed. Ten animals were inoculated intramammarily with 106 cells of the two strains at dry-off. The blood serum was tested for its ability to opsonize bovine mastitis pathogens, the in vitro bactericidal activity of bovine blood and milk against these pathogens was determined, and cytokine mRNA expression was quantified in milk somatic cells. The inoculated animals did not show abnormal signs of sensitivity to the LAB. Their blood serum significantly enhanced the phagocytosis of Staphylococcus spp. and the LAB. Escherichia coli and Streptococcus uberis were inhibited by the milk serum but not the blood serum, whereas Staphylococcus aureus and Staphylococcus haemolyticus were inhibited by both. In regard to cytokine expression, interleukin (IL)-1β increased markedly for up to 4 h post-inoculation, and an increase in IL-8 was observed 4, 12 and 24 h after inoculation. Tumour necrosis factor-α mRNA increased 1 and 2 h after inoculation and a significant difference was registered at 6 h for interferon-γ. This rapid immunomodulatory response shows that inoculating animals with LAB at dry-off, when they are especially susceptible, could be a useful strategy for the prevention of bovine mastitis.

Microtiter Screening Reveals Oxygen-Dependent Antimicrobial Activity of Natural Products Against Mastitis-Causing Bacteria.

In this study we investigated the influence of oxygen availability on a phenotypic microtiter screen to identify new, natural product inhibitors of growth for the bovine mastitis-causing microorganisms; Streptococcus uberis, Staphylococcus aureus, and Escherichia coli. Mastitis is a common disease in dairy cattle worldwide and is a major cause of reduced milk yield and antibiotic usage in dairy herds. Prevention of bovine mastitis commonly relies on the application of teat disinfectants that contain either iodine or chlorhexidine. These compounds are used extensively in human clinical settings and increased tolerance to chlorhexidine has been reported in both Gram-positive and Gram-negative microorganisms. As such new, non-human use alternatives are required for the agricultural industry. Our screening was conducted under normoxic (20% oxygen) and hypoxic (<1% oxygen) conditions to mimic the conditions on teat skin and within the mammary gland respectively, against two natural compound libraries. No compounds inhibited E. coli under either oxygen condition. Against the Gram-positive microorganisms, 12 inhibitory compounds were identified under normoxic conditions, and 10 under hypoxic conditions. Data revealed a clear oxygen-dependency amongst compounds inhibiting growth, with only partial overlap between oxygen conditions. The oxygen-dependent inhibitory activity of a naturally occurring quinone, β-lapachone, against S. uberis was subsequently investigated and we demonstrated that this compound is only active under normoxic conditions with a minimum inhibitory concentration and minimum bactericidal concentration of 32 μM and kills via a reactive oxygen species-dependent mechanism as has been demonstrated in other microorganisms. These results demonstrate the importance of considering oxygen-availability in high-throughput inhibitor discovery.

Development and applications of safranine-loaded Pluronic® F127 and P123 photoactive nanocarriers for prevention of bovine mastitis: In vitro and in vivo studies

Photodynamic therapy (PDT) is a therapeutic modality that exploits the formation of singlet oxygen through a photosensitizing compound. This compound is capable of eradicating sick cells or several strains of microorganisms. Its main advantage is associated with high selectivity and ability to avoid the self-resistance of pathogens, as often expected after the prolonged use of conventional antibiotics. These characteristics made it possible to the explore the performance of Safranine-O (Sf) photosensitizer incorporated in F127 and P123 Pluronic® 4% (w/v) as nanocarriers solubilizer in the Photodynamic Inactivation of Microorganisms (PDIM) for post-dipping procedures in the prevention of bovine mastitis. Spectroscopic studies have shown the effective monomerization of Sf after being loaded into nanostructured micellar systems. The Sf-F127 and Sf-P123 showed bond molar absorptivity coefficients (ɛ) in the order of 103 L mol−1 and preferential relative locations in the hydrophilic hydrated micelle region (shell), as verified through the high Stern-Volmer constants (11.4 for Sf-F127 and 7.3 for Sf-P123) and proximity between apparent pKa (pKaap) values (Sf-F127 ∼ Sf-P123) with a pKa of Sf in water. Sf singlet oxygen quantum yield in water was 0.24. This was determined by the indirect method of photodegradation of the chemical probe tryptophan. In vitro studies of the Sf-F127 and Sf-P123 systems proved to be efficient in inactivating the bacteria that cause bovine mastitis. In vivo evaluations in Dutch dairy cows demonstrated the viability of the system as a form of treatment or prevention of bovine mastitis in post-dipping procedures.

Gentisaldehyde and Its Derivative 2,3-Dihydroxybenzaldehyde Show Antimicrobial Activities Against Bovine Mastitis Staphylococcus aureus.

Bovine mastitis is a worldwide disease of dairy cattle associated with significant economic losses for the dairy industry. One of the most common pathogens responsible for mastitis is Staphylococcus (S.) aureus. Due to the development and spreading of antibiotic resistance, the search for novel antimicrobial substances against S. aureus is of great importance. The aim of this study was to evaluate two dihydroxybenzaldehydes for the prevention of bovine mastitis. Therefore we determined the minimal inhibitory concentration (MICs) of gentisaldehyde (2,5-dihydroxybenzaldehyde) and 2,3-dihydroxybenzaldehyde of a diverse set of 172 bovine mastitis S. aureus isolates using an automated robot-based microdilution method. To characterize the bovine isolates we determined the genotype by spa-typing, the antimicrobial resistance to eight antibiotic classes using the disk diffusion method and the MICs of three commonly used antiseptics (benzalkonium chloride, chlorhexidine, and iodine). Further we investigated the cytotoxicity of gentisaldehyde and 2,3-dihydroxybenzaldehyde in bovine mammary epithelial MAC-T cells using the XTT assay. The S. aureus strains showed a high genetic diversity with 52 different spa-types, including five novel types. Antibiotic susceptibility testing revealed that 24% of isolates were resistant to one antimicrobial agent and 3% of isolates were multi-resistant. The occurrence of antibiotic resistance strongly correlated with the spa-type. Both dihydroxybenzaldehydes showed antimicrobial activities with a MIC50 of 500 mg/L. The MIC of gentisaldehyde significantly correlated with that of 2,3-dihydroxybenzaldehyde, whereas no correlation was observed with the MIC of the three antiseptics. Cytotoxicity testing using bovine mammary epithelial MAC-T cells revealed that gentisaldehyde and 2,3-dihydroxybenzaldehyde show low toxicity at MIC50 and MIC90 concentrations. In conclusion, gentisaldehyde and 2,3-dihydroxybenzaldehyde exhibited antimicrobial activities against a diverse range of bovine mastitis S. aureus strains at low-cytotoxic concentrations. Therefore, both compounds are potential candidates as antiseptics to prevent bovine mastitis and to reduce the use of antibiotics in dairy cows.

In Vitro Characterization of Lactic Acid Bacteria Isolated from Bovine Milk as Potential Probiotic Strains to Prevent Bovine Mastitis.

Bovine mastitis causes economic losses on dairy farms worldwide. Lactic acid bacteria (LAB) in animal health are an alternative tool to avoid antibiotic therapy on the prevention of bovine mastitis. In previous studies, 12 LAB isolated from bovine milk were selected taking into account some of the following characteristics: hydrophobicity, auto aggregative capability, inhibition of indicator pathogens, hydrogen peroxide, and capsular polysaccharide production. These LAB were considered because of their beneficial properties. In this work, we also analyzed the antimicrobial activity and the co-aggregation against mastitis causing bacteria, auto-inhibition, adhesion to bovine teat canal epithelial cells (BTCEC), and growth kinetic curves for the 12 LAB. Two of them, Lactococcus lactis subsp. lactis CRL 1655 and Lactobacillus perolens CRL 1724, were selected because they had an interesting pattern of adhesion to BTEC, the inhibition of pathogens and the co-aggregation with the 100% of the assayed pathogens. They showed a predictable difference in the PFGE genomic pattern bands. The kinetic growth of these two strains was similar between them and with the rest of the assayed LAB. The strains selected in the present study showed indispensable characteristics for their inclusion in a probiotic formulation to be used at dry-off period for the prevention of bovine mastitis.

Bovine mastitis prevention: humoral and cellular response of dairy cows inoculated with lactic acid bacteria at the dry-off period.

The use of lactic acid bacteria (LAB) in animal feed, constitute an alternative tool for bovine mastitis prevention. Previously, two LAB strains were isolated from bovine milk and selected for their probiotics properties. So far, immune response of inoculating LAB in bovine udders at dry-off period has not been investigated. The immunoglobulin isotype levels and memory cell proliferation in blood and milk of animals inoculated with Lactobacillus lactis subsp. lactis CRL1655 and Lactobacillus perolens CRL1724 at dry-off period was studied. Ten animals were inoculated intramammarily with 106 cells of each LAB (IG) and 2 animals used as control (NIG). Milk and blood samples were taken before inoculation and 1, 2, 4, 6, 12 and 24 h and 7 and 14 days after inoculation. Somatic cell count (SCC) in milk, the presence of bovine mastitis pathogens, the levels of antibodies and lymphocyte proliferation were determined. In the IG, the SCC was <250,000 cells/ml up to 4 h after intramammary inoculation. Six and 12 h after inoculation, the SCC increased up to 600,000 and 2,000,000 cells/ml, respectively. In the NIG, the SCC reached the maximum value 7 days after inoculation. Microbiological analysis showed that all samples were negative for major bovine mastitis pathogens after 24-48 h of incubation. In general, LAB inoculation increased the amount of IgG isotypes in blood and milk, and these antibodies were able to recognise Staphylococcus aureus epitopes. Lymphocytes proliferation was significantly higher in the IG at all time points assayed, following LAB or S. aureus stimulation. The lymphocytes of animals inoculated with LAB do not react in vitro to the presence of S. aureus antigen.. The results showed that probiotic microorganisms could be a natural and effective alternative in the prevention of bovine mastitis at dry-off period and act as immunomodulatory stimulating local and systemic defence lines.

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.

Development of a Teat Bio-sealant and Evaluation of its Technological and Functional Properties.

A teat bio-sealant was developed using Weissella cibaria, and the bio-sealant's technological and functional properties were assessed. The development included four experimental phases that were analyzed using independent experimental designs. Initially, sterilized or pasteurized Aloe vera gels were used, and the effect of heat treatment was investigated. In the second phase, the effects of time, storage temperature, and addition of cryopreservatives on the viability of the probiotic were observed. The third phase consisted of evaluating the synergistic effects of the cryopreservatives. The fourth phase involved selecting a material that would provide viscosity to the teat sealant. Technological and functional properties were measured in terms of viability of W. cibaria, and antimicrobial activity against Staphylococcus aureus and Streptococcus agalactiae was also analyzed. A mixture of milk powder and glycerol preserved this antimicrobial activity. Pullulan provided greater viscosity and maintained the technological and functional properties of the bio-sealant for 29days. This teat bio-sealant can be used as an alternative for the prevention of bovine mastitis.

Expression of the lingual antimicrobial peptide (LAP) in the bovine mammary gland with Staphylococcus aureus

Mastitis is one of the most important infectious diseases in dairy cows around the world that causes great economic loses. To prevent and treat mastitis is important to understand the immunology of the mammary gland. Mammalian β -defensins are peptides of the local innate host defense with a potential as therapeutic agents. With the aim to better understand the role of these local antimicrobial peptides in the prevention of bovine mastitis, in the present study the mRNA expression of the lingual antimicrobial peptide ( LAP )was determinedin 5 localizations of the bovine mammary gland with mastitis applying a quantitative real time PCR. The results showed that LAP was mainly expressed in the cisternal tissue with an average of 1.36 7 x 10 copies of mRNA, followed by 9.7 x 10 6 copies in parenchyma, and the lowest expression was obtained streak canal. Therefore, LAP could be involved in the local defense of the mammary gland since is overexpressed in these regions where S. aureus mainly invades and multiplies.