The influence of subclinical mastitis on the protein composition and protease activities of raw milk from lactating Thai-crossbred dairy cows

Background and Aim: Mastitis in dairy cattle is associated with a high rate of morbidity and death, which has major implications for milk production and quality. This study aimed to investigate the protein component and the activity of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in raw milk samples with different testing scores determined using the California mastitis test (CMT). Materials and Methods: Thirty cows were employed in the study, and milk from each quarter was tested for subclinical mastitis (SCM). According to the results of CMT, raw milk samples were classified into five categories: Healthy (score 0), trace (score T), weakly positive (score 1), distinctly positive (score 2), and strongly positive (score 3) for somatic cell count (SCC). The total milk protein was analyzed using the Bio-Rad protein assay, and the milk protein composition was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. In addition, gelatin zymography was used to evaluate changes in proteolytic abilities. Results: Milk samples with CMT scores of 1 and 3 had the highest total milk protein levels (32.25 ± 12.60 g/L and 32.50 ± 7.67 g/L, respectively), while the samples from healthy cows (CMT score 0) were only 6.75 ± 1.64 g/L. Globulin and lactoferrin were significantly increased in samples with a CMT score of 3 compared with those with other CMT scores. The bovine serum albumin level in samples with a CMT score of 2 was significantly (p < 0.05) higher than those with other CMT scores. No significant differences in casein abundance were found among samples with different CMT scores. Results from analysis of proteolytic activities demonstrated that the level of MMP-9 in samples with a CMT score of 3 was significantly (p < 0.05) higher than those with other CMT scores. Conclusion: The protein content and gelatinolytic activity of milk were drastically altered by the number of SCC, mainly due to SCM

泌乳後期荷蘭乳牛對乳房內益生性腸球菌SF68之耐受性

過去慣用抗生素有使整個牛場之牛隻系統化乾乳，為防止抗生素濫用，歐洲已經禁止使用抗生素作為促進生長之使用。為防止病原體感染乳腺並降低抗生素使用，應用益生菌經由乳頭灌注提供了一個新的替代方法。應用益生菌經由乳頭灌注，迄今為止只有局部免疫反應經過驗證。屎腸球菌菌株SF68（SF68）最初是從人體腸道內分離出來，其經由飼糧中添加途徑在豬的耐受性已被驗證，而牛隻則被建議使用於乾乳。本篇實驗的目的在於評估利用SF68來部份取代商業用之抗生素，探討荷蘭牛泌乳後期之乳房對SF68的耐受性。本實驗共有四隻荷蘭牛用來乳房之灌流研究，於最後榨乳的一天(d1)，其中一側之前部和後部的分房分別注入劑量為5 × 108 CFU ( SF68 每一分房)，對側分房則使用商業乾乳之抗生素（控制四分之一）同時處理。 試驗結果顯示:在d3可暫時減少血液中白血球總數，然而在d7乳房分泌物中體細胞數（MSCC）仍有持續增加，特別是來自以SF68處理之乳房。MMP-2在血漿中的表現量亦相似，至d7仍上升，但血漿中的MMP-9則過低而無法測出。MMP-2的活性在乳房分泌物中至d7仍持續增加，但MMP-9之增加更急劇，尤其是在SF68灌流之乳房中。血液中白血球的活性氧（ROS）產生，在d3短暫增加，而乳細胞數在d7快速上升，特別是在SF68灌流的乳房中。在第二個試驗中，灌注一個月後母牛其血漿助孕素(progesterone)、雌二醇(estradiol)和皮質醇(cortisol)在血漿濃度與灌流第一天相比沒有顯著差異。血液中總白血球數在產後和灌流後第一天相比無顯著之差異。血漿中助孕素濃度在產後與灌流後幾天相比有一個明顯的下降(p<0.05)。血漿中的基質金屬蛋白酶（明膠酶A）的表現量在d30、產後與灌流後幾天相比無顯著差異。新生犢牛的血液生理指標均在正常生理範圍。Laemmli SDS-PAGE分析顯示在第一週的乳分泌物中乳鐵蛋白和血清白蛋白含量豐富，初乳中γ-球蛋白與控制組的分房中γ-球蛋白和白蛋白相比無太大變化，但SF68灌流的乳房有較高傾向。利用酵素結合免疫吸附分析法（ELISA）測定乳腺乾乳分泌物和初乳中乳鐵蛋白和免疫球蛋白G（IgG）濃度，結果顯示以SF68灌流的乳房相較於控制組乳房具有較高的濃度。 本研究之結果顯示:SF68在短期效應上有助於乾乳母牛乳腺體液免疫，而在長期上有利於新生仔牛之被動免疫，SF68可以作為牛隻乾乳時取代抗生素之有效替代方案。Systematic drying-off with antibiotics contributes a major part to the total use of antibiotics in dairy herds. As European has banned the use of antibiotics as a production promoter, the novel route of probiotic application via teat canal provides an alternative way to protect mammary gland against pathogen infection for dairy animal although so far only local immune responses have been validated. Enterococcus faecium strain SF68 (SF68) was originally isolated from human gut, and its tolerance through dietary supplementation in porcine has been validated and is recommended to intramammary application in dry-off cows. The purpose of the current study was to evaluate the intramammary tolerance of late lactation Holstein cows towards SF68 using SF68 as a substitute of commercial antibiotics in dry cow formula. Totally 4 Holstein cows were used in the intramammary infusion study, in which one of their front and rear quarters was respectively infused with total SF68 dosage of 5 × 108 CFU (SF68 quarter) at the day of last milking (d1) and the contralateral quarters was treated with commercial antibiotic dry cow formula (antibiotics quarter) meanwhile. Results showed a temporarily minor decrease in blood total leukocyte count at d3 and microscopic somatic cell count (MSCC) increased continuously to d7 especially in SF68 quarters. The level of gelatinase A in plasma remained similar up to d7 but plasma gelatinase B was not detectable throughout the study. The level of gelatinase A in quarter secretion increased up to d7 but gelatinase B increased even more drastically, especially in SF68 quarters. Reactive oxygen species (ROS) production of blood leukocytes temporarily increased at d3, while that of milk cells increased up to d7 especially in SF68 quarters. In the second trial, during the month post-infusion, plasma concentrations of progesterone, estradiol, and cortisol were not significantly changed as compared to the infusion day. Blood total leukocyte count during the month post-infusion and at partum were not significantly different with those of the infusion day, and were ranged within a normal physiological range. A typical drop (P < 0.05) in plasma progesterone concentration at partum as compared to the infusion day was observed. Plasma matrix metalloproteinase (Gelatinase A) level at d 30 and at parturition was not significantly changed as compared to the infusion day. Blood physiological parameters of the newborn calve were within a normal physiological range. Native Laemmli SDS-PAGE analysis suggested that the abundance of lactoferrin and bovine serum albumin in the first-week dry secretion, as well as γ-globulin in the initial colostrum, tended to be higher in SF68 quarter as compared to control quarter and plasma γ-globulin and albumin were maintained relatively constant. Lactoferrin and immunoglobulin G (IgG) concentrations estimated by an enzyme-linked immunosorbent assay (ELISA) in the mammary secretion and initial colostrum were greater in SF68-infused quarters as compared to control quarters. Results suggested that SF68 treatment in dry-cows exerts a short-term beneficial effect on the humoral defense of drying-off mammary gland and has a long-term beneficial effect on the transfer of passive immunity to the neonates. SF68 can serve as an alternative to antibiotics in dry-cow formula.Page ACKNOWLEDGMENTS i ABSTRACT iii TABLE OF CONTENTS vii LIST OF TABLES x LIST OF FIGURES xi CHAPTER 1: Introduction……………………………………………………………………..… 1 1. Rationale and background………………………………………………... 2 2. Aim of this study………………………..…………...…………………….. 3 CHAPTER 2: Literature review……………………………………………………………….…. 4 1. Bovine mammary gland anatomy ……………………………………….. 5 2. Mammary development………………………………………………….. 10 3. Development of epithelial cells....…………………………………………. 14 4. Mammary remodeling.………………………..…………………………... 15 5. Dry and periparturient periods susceptibility.…………………….……….. 15 6. General aspects of the bovine immune system…………………………… 16 7. Anatomical defenses ……………………………………………………... 17 8. Innate immunity…………………………………………………………... 18 9. Adaptive immunity ………………………………………………………. 23 10. Mammary gland immune response ………………………………………. 24 11. Milk components ………………………………………………………… 28 12. Lactoferrin ………………………………………………………………... 28 13. Immunoglobulins ……………………………………………………….... 30 14. Immune colostrum ……………………………………………………….. 32 15. Matrix metalloproteinases (MMPs)………………………………………. 32 16. Probiotics in man and animals ……………………………........................34 17. Probiotic modulation of the immune system …………………………….. 36 CHAPTER 3: Materials and Methods……………………………………………………………. 41 CHAPTER 4: Systemic and local bactericidal reactions in late lactation Holstein cows following a combined antibiotics and Enterococcus faecium SF68 dry-cow treatment………………………………………………………………………….. 49 1. Abstract…………………………………………………………………… 50 2. Introduction……………………………………………………………….. 51 3. Results…………………………………………………………………….. 52 4. Discussion………………………………………………………………… 54 CHAPTER 5: Probiotic dry cow treatment in late gestation did not clinically affect the following pregnancy, parturition of Holstein cows, and their neonatal calves health ……………………………………………………………………………... 65 1. Abstract…………………………………………………………………… 66 2. Introduction……………………………………………………………….. 67 3. Results…………………………………………………………………….. 68 4. Discussion………………………………………………………………… 68 CHAPTER 6: Electrophoretic profiles of the proteinous components of plasma and mammary secretion of Holstein cows having received Enterococcus faecium SF68 dry-cow treatment………………………………………………………………………….. 78 1. Abstract…………………………………………………………………… 79 2. Introduction……………………………………………………………….. 80 3. Results…………………………………………………………………….. 81 4. Discussion………………………………………………………………… 83 CHAPTER 7: Determination of Lactoferrin and Immunoglobulin G of plasma and mammary secretion of Holstein cows received Enterococcus faecium SF68 dry-cow treatment………………………………………………………………………….. 89 1. Abstract …………………………………………………………………... 90 2. Introduction……………………………………………………………….. 91 3. Results…………………………………………………………………….. 92 4. Discussion………………………………………………………………… 94 CONCLUSIONS...………………………………………………………………... 100 REFERENCES…………………………………………………………………… 102 APPENDICES……………………………………………………………………. 14

Systemic and local bactericidal potentiality in late lactation Holstein-Friesian cows following a combined antibiotics and Enterococcus faecium SF68 dry-cow treatment

Antibiotic dry-cow treatment contributes a major part to the total use of antibiotics in dairy herds. Enterococcus faecium strain SF68 (SF68) was of human origin but has been authorized in EU as probiotic feed additive. In the present study, one of the front and rear quarters of twelve late lactation Holstein-Friesian cows were infused once with a commercial antibiotic dry-cow formula (antibiotics quarter) on the first milk-stasis day (d 1), when the contrallateral quarters were infused with 5 × 108-CFU SF68 plus half-dose antibiotic dry-cow formula (SF68/antibiotics quarter) meanwhile. Gelatinase level and cellular reactive oxygen species (ROS) production capacity were measured for blood and quarter secretion. The results showed that the count of blood total leukocytes minorly decreased on d 3 only but the microscopic somatic cell count (MSCC) continuously increased up to d 7, especially in SF68/antibiotics quarters. Plasma level of gelatinase A remained similar up to d 7 but gelatinase B was not detectable in plasma throughout the study. The level of gelatinase A in quarter secretion increased up to d 7 but gelatinase B increased even more drastically, especially in SF68/antibiotics quarters. The ROS production capacity of blood leukocytes increased temporarily only on d 3, but that of milk cells continuously increased up to d 7, especially in SF68/antitiotics quarters. Overall, late lactation Holstein-Friesian cows were systemically adaptable to the combined antibiotics and SF68 dry-cow treatment, while the local bactericidal potentiality in mammary gland was actively responsive to additional SF68 intramammary treatment

Involvement of TNF- and MAPK pathway in the intramammary MMP-9 release via degranulation of cow neutrophils during acute mammary gland involution

Neutrophil-derived MMP-9 activity is regulated more promptly and efficiently at the level of degranulation than at other levels of regulation. In human neutrophils, degranulation is one of the earliest responses to TNF- stimulation, which involves protein kinase C and mitogen-activating protein kinase (MAPK) pathways. The level of MMP-9 in mammary secretion of cows increases drastically following milk-stasis, which is partially explained by increases of both neutrophil infiltration and neutrophil degranulation per se. Since MMP- 9 represents one of the major remodeling capacities in the mammary gland of cows during early dry period, the current study attempted to explore the involved intracellular mechanisms in the up-regulated MMP-9 secretion. We repeatedly measured on the somatic cells of mammary secretion along the early dry period of cows the expression of TNF- protein and the phosphorylation of p38 MAPK, ERK, and JNK. Also, cultures of bovine peripheral neutrophils were conducted to examine the mode of short-term MMP-9 secretion in response to TNF- stimulation and the blocking effects of TNF- antibody and inhibitors of MAPK pathways. Ex vivo measurements show that conventional cow milk has fully transformed into a neutrophil-abundant, lactoferrin-rich, and high-MMP-9 mammary secretion by d 7 in milk-stasis. No significant (P > 0.05) change, however, was found in the expression of TNF- or the phosphorylation extent of MAPK pathway intermediates on the somatic cells of mammary secretion during the first 3 weeks in milk-stasis. In vitro studies indicate linear increase of short-term MMP-9 release in response to TNF- stimulation in dosages between 0.1 and 10 ng/ml. In the presence of preparations of d 7-dry secretion of cows, the short-term release of MMP-9 from bovine peripheral neutrophils was significantly (P < 0.05) blocked by inhibitor of p38 MAPK but was significantly (P < 0.05) promoted by ERK inhibitor while TNF- antibody or JNK inhibitor exerted no effect. In conclusion, the current ex vivo measurements suggest no apparent association of TNF- and MAPK pathway with long term intramammary accumulation of MMP-9 during the early dry period of cows, whereas cultures of bovine peripheral neutrophils under a simulated acute involution intramammary environment of cows suggest a role played by TNF- and MAPK pathways in the short-term MMP-9 release via degranulation