Involvement of adhesins (EcpD, FdeC, FimH) expressed in mammary pathogenic Escherichia coli on adhesion to bovine mammary epithelial cells.

Mammary pathogenic Escherichia coli (MPEC) causes mastitis, which results in substantial economic losses to the dairy industry. A high percentage of Escherichia coli isolated from cows with clinical mastitis harbor adhesin genes, such as fimH. However, it is unclear whether these adhesins are important in the adhesion of MPEC to bovine mammary epithelial cells (BMECs). Therefore, we investigated the effect of adhesins (EcpD, FdeC, and FimH) in MPEC on adherence to the bovine mammary epithelium using cultured BMECs. For this purpose, we used wild-type MPEC as well as single- and double-mutants of fimH, ecpD, and fdeC, and performed adhesion assays with BMECs. First, BMECs were cultured in the presence of lactogenic hormones to induce milk component production and tight junction formation. The bacterial count of the wild-type strain that adhered to the BMECs increased in a dose-dependent manner. In deletion mutant strains, the ΔfimH strain showed lower adhesion (P < 0.05), whereas the adhesion ratio of the ΔecpD and ΔfdeC strains was not statistically different compared with that of the wild-type strain (P > 0.05). Additionally, the fimH/fdeC double-deletion mutants showed the lowest adhesion to BMECs. In conclusion, FimH is crucial in the adhesion of MPEC to BMECs. Overall, our work identifies FimH or FimH/FdeC as interesting targets for future drugs or vaccines to improve the treatment, prevention or chronicity of mastitis induced by MPEC.

Elevated levels of cyclophilin A secreted in milk during bovine mastitis.

Bovine mastitis is an inflammatory disease that primarily occurs when bacteria invade and proliferate in the mammary gland or such as physical trauma. Mastitis results in a decrease in milk yield and quality, causing huge economic losses. Cyclophilin A (CyPA) is a cytosolic protein known as cyclosporine binding protein. Recent studies have shown that CyPA is secreted from cells and has chemotactic activity, recruiting inflammatory cells and inducing multiple cytokines. In this study, we found that CyPA is detected in milk and is abundantly secreted at the onset of mastitis. A significant correlation was found between somatic cell counts (SCC) and the concentrations of CyPA in milk. To elucidate the relationship between mastitis and CyPA, we gave an intramammary infusion of S. aureus to cattle and investigated the attendant CyPA secretion. In S. aureus infused quarters, we observed an increased expression of CyPA on mammary epithelia and secretion into milk. The temporal profiles of CyPA in milk were synchronous with SCC, and there was a significant correlation between the concentration of CyPA in milk and SCC. These results suggest that CyPA is involved in the migration of immune cells during the onset of mastitis and may be used as a marker for the onset of mastitis.

Staphylococcus aureus-specific IgA antibody in milk suppresses the multiplication of S. aureus in infected bovine udder.

BACKGROUND: Bovine mastitis caused by Staphylococcus aureus (S. aureus) is extremely difficult to control and new methods for its prevention and management are required. Nasal vaccines may prevent initial bovine mastitis infection caused by S. aureus. However, limited information is available regarding induction of mucosal immune response through nasal immunization with antigen and its suppression of S. aureus multiplication during bovine mastitis. This study sought to investigate whether induction of immunoglobulin A (IgA) in milk by nasal immunization could suppress multiplication of S. aureus in the bovine udder. RESULTS: Nasal immunization with formalin-killed S. aureus conjugated with a cationic cholesteryl-group-bearing pullulan-nanogel was performed. Anti-S. aureus-specific IgA antibodies were significantly more abundant in the milk of immunized cows than in non-immunized animals (P < 0.05). S. aureus counts in the quarter were negative in both non-immunized and nasal-immunized cows 1 week after mock infusion. In S. aureus-infused quarters, S. aureus multiplication was significantly suppressed in immunized compared with non-immunized cows (P < 0.05). Furthermore, a significant negative correlation was found between S. aureus-specific IgA antibodies and S. aureus counts in infused quarters of both non-immunized and nasal-immunized cows (r = - 0.811, P < 0.01). CONCLUSION: In conclusion, the present study demonstrates that S. aureus-specific IgA antibodies in milk successfully suppressed the multiplication of S. aureus in infected bovine udders. Although the exact mechanism explaining such suppressive effect remains to be elucidated, nasal vaccines that can induce humoral immunity may help prevent initial infection with S. aureus and the onset of bovine mastitis.

An Incremental Sit-to-Stand Exercise for Evaluating Physical Capacity in Older Patients with Type 2 Diabetes.

Exercise is recommended for older patients with type 2 diabetes mellitus (T2DM), and increased physical activity contributes to better management of their condition. The conventional exercise test with treadmill or cycle ergometer (CE) for assessing physical capacity, such as peak oxygen uptake (VO2) and anaerobic threshold (AT), is not always usable for older patients with T2DM. The incremental sit-to-stand (ISTS) exercise is an incremental exercise test using external signals to control the sit-to-stand rate in a given time frame and can be performed in a small space using only a chair. This study aimed to examine the validity of the physical capacity assessment during the ISTS exercise, based on the relationships between the ISTS performance, peak VO2, AT on ISTS exercise and CE test, in older patients with T2DM. Twenty-two patients with T2DM (10 men, 12 women; mean age, 68.0 years; range, 61-77 years) performed ISTS exercise (according to an existing protocol) and CE test in a randomized manner. Peak VO2, AT, and completion time were determined for the ISTS exercise and CE test. Peak VO2 during ISTS exercise was significantly associated with that during the CE test (r = 0.89, p < 0.01). The completion time on the ISTS exercise was significantly associated with peak VO2 (r = 0.80, p < 0.01) and AT on the ISTS exercise (r = 0.78, p < 0.01). The ISTS exercise is a useful tool to determine the physical capacity and estimate peak VO2 and AT in older people with T2DM.

Validity and reproducibility of an incremental sit-to-stand exercise test in healthy middle-aged individuals.

[Purpose] We aimed to evaluate the validity and reproducibility of the incremental sit-to-stand exercise test for aerobic fitness evaluation in healthy middle-aged individuals. [Participants and Methods] Thirteen healthy middle-aged individuals randomly underwent the incremental sit-to-stand exercise and cycle ergometer tests, and the peak oxygen uptake was measured during both tests. Pearson's correlation coefficients were used to assess the strength of the association between the peak oxygen uptake measured during the aforementioned tests. Intraclass correlation coefficients with 95% confidence intervals for peak oxygen uptake obtained during the first, second, and third incremental sit-to-stand exercise tests were used to determine the reproducibility of this test. [Results] The peak oxygen uptake measured during the incremental sit-to-stand exercise test was strongly associated with that measured during the cycle ergometer test (r=0.86). The intraclass correlation coefficients (95% confidence intervals) used to verify the association of the peak oxygen uptake between the first and the second incremental sit-to-stand exercise tests and between the second and third incremental sit-to-stand exercise tests were 0.92 (0.66-0.99) and 0.96 (0.82-0.99), respectively. [Conclusion] The incremental sit-to-stand exercise test is a valid and reproducible tool to evaluate aerobic fitness in healthy middle-aged individuals.

Factors associated with functional rehabilitation outcomes of non-operative treatment for hip fractures: a retrospective study.

[Purpose] Limited data are available regarding the outcomes of non-operative treatment for hip fractures. We investigated the factors associated with functional rehabilitation outcomes in patients undergoing non-operative treatment for hip fractures. [Participants and Methods] We investigated 57 patients with hip fractures who underwent non-operative treatment. We retrospectively analyzed medical or rehabilitation outcomes and functional outcomes (assessed using the Functional Independence Measure tool). We examined the association between functional outcomes and other factors and compared the medical and rehabilitation outcomes between mobile and immobile patients at the time of discharge. [Results] Of the 57 patients investigated, 15 (26.3%) were mobile at discharge. We observed a significant association between the Functional Independence Measure subscores (Motor and Cognitive) and serum albumin levels. Serum albumin levels and the Functional Independence Measure subscores (Motor and Cognitive) were significantly higher in mobile than in immobile patients. [Conclusion] We observed that functional outcomes at discharge in patients undergoing non-operative treatment for hip fractures were associated with serum albumin ratios and the Functional Independence Measure-Cognitive score.

Identification of a novel mechanism of action of bovine IgG antibodies specific for Staphylococcus aureus.

Staphylococcus aureus is a major pathogen that causes subclinical mastitis associated with huge economic losses to the dairy industry. A few vaccines for bovine mastitis are available, and they are expected to induce the production of S. aureus-specific antibodies that prevent bacterial adherence to host cells or promote opsonization by phagocytes. However, the efficacy of such vaccines are still under debate; therefore, further research focusing on improving the current vaccines by seeking additional mechanisms of action is required to reduce economic losses due to mastitis in the dairy industry. Here, we generated S. aureus-specific bovine IgG antibodies (anti-S. aureus) that directly inhibited bacterial growth in vitro. Inhibition depended on specificity for anti-S. aureus, not the interaction between Protein A and the fragment crystallizable region of the IgG antibodies or bacterial agglutination. An in vitro culture study using S. aureus strain JE2 and its deletion mutant JE2ΔSrtA, which lacks the gene encoding sortase A, revealed that the effect of anti-S. aureus was sortase-A-independent. Sortase A is involved in the synthesis of cell-wall-associated proteins. Thus, other surface molecules, such as membrane proteins, cell surface polysaccharides, or both, may trigger the inhibition of bacterial growth by anti-S. aureus. Together, our findings contribute insights into developing new strategies to further improve the available mastitis vaccine by designing a novel antigen on the surface of S. aureus to induce inhibitory signals that prevent bacterial growth.

Development of an Incremental Sit-to-Stand Exercise for Aerobic Fitness Evaluation.

Incremental sit-to-stand exercise (ISTS) is an incremental exercise test using external signals to control the sit-to-stand rate in a given time frame. This study aimed to investigate the concurrent validity and reproducibility of the ISTS in aerobic fitness evaluation among healthy elderly women. Sixteen elderly women performed the ISTS and cycle ergometer test at 3-day to 2-week intervals, and six of the participants performed the ISTS twice. Peak oxygen uptake (VO2), peak heart rate and completion time on the ISTS and cycle ergometer test were determined. Measured peak VO2 on the cycle ergometer test was significantly related to the peak VO2 (r=0.80, P<0.05) and the completion time (r=0.65, P<0.05) on the ISTS. The intraclass correlation coefficients were 0.96 for peak VO2 values and 0.91 for the completion time values during the two ISTSs. In conclusion, the ISTS is a valid, reproducible and safe test for aerobic fitness evaluation in healthy elderly women.

Cyclophilin A is a new M cell marker of bovine intestinal epithelium.

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches contribute to the mucosal immune response by the transcytosis of microorganisms. The mechanism by which M cells take up microorganisms, and the functional proteins by which they do this, are not clear. In order to explore one such protein, we developed a 2H5-F3 monoclonal antibody (2H5-F3 mAb) through its binding to bovine M cells, and identified the antibody reactive molecule as cyclophilin A (Cyp-A). The localization patterns of Cyp-A were very similar to the localization pattern of cytokeratin (CK) 18-positive M cells. Cyp-A was identified at the luminal surface of CK18-positive M cells in bovine jejunal and ileal FAE. The membranous localization of Cyp-A in the bovine intestinal cell line (BIE cells) increased as cells differentiated toward M cells, as determined by flow cytometry analysis. Additionally, BIE cells released Cyp-A to the extracellular space and the differentiation of BIE cells to M cells increased the secretion of Cyp-A, as determined by western blotting. Accordingly, Cyp-A may be localized in M cells in the small intestinal epithelium of cattle. The rise of the membranous localization and secretion of Cyp-A by differentiation toward M cells indicates that Cyp-A has an important role in the function of M cells. While Cyp-A of the M cell membrane may contribute to the uptake of viruses with peptidyl-prolyl cis-trans isomerase activity, in the extracellular space Cyp-A may work as a chemokine and contribute to the distribution of immuno-competent cells.

Effect of Different Seat Heights during an Incremental Sit-To-Stand Exercise Test on Peak Oxygen Uptake in Young, Healthy Women.

'Sit-to-stand' exercise uses the repetitive motion of standing up and sitting down in a chair, a common activity of daily living. A new assessment using an incremental sit-to-stand exercise test employs an external sound to control the speed of standing-up and allows increases in work rate. The aims of the study were to examine the effect of different seat heights on peak oxygen uptake (peak VO2) during an incremental sit-to-stand exercise and to assess any difference between peak VO2 values during incremental sit-to-stand exercise compared with a cycle ergometer test. Thirteen healthy young women (age: 23.1 ± 2.6 years, height: 1.61 ± 0.06 m, body mass: 51.9 ± 7.4 kg·m-2) participated in four incremental sit-to-stand tests with different seat heights and cycle tests in random order. The seat heights were adjusted to 100%, 80%, 120%, and 140% of knee height distance (100%, 80%, 120%, and 140% incremental sit-to-stand exercise, respectively). The peak VO2 and completion time were measured during incremental sit-to-stand and cycle ergometer tests, and repeated-measures analysis of variance and Student's paired t-test with Holm's method were used to evaluate differences between these variables. The peak VO2 values increased by about 10-12 mL·min-1·kg-1 as the seat height on the ISTS decreased over a 60% range of lower leg lengths. The peak VO2 values on the 80%, 100%, 120%, and 140% incremental sit-to-stand tests were about 11%, 25%, 40%, and 50% lower than that on the cycle ergometer test, respectively. The peak VO2 on the incremental sit-to-stand test increased as seat height decreased. These findings are useful to determine which seat height on the incremental sit-to-stand tests test is suitable for different populations.

Validity and Reproducibility of an Incremental Sit-To-Stand Exercise Test for Evaluating Anaerobic Threshold in Young, Healthy Individuals.

Sit-to-stand exercise (STS) is a common activity of daily living. The objectives of the present study were: 1) to assess the validity of aerobic fitness measurements based on anaerobic thresholds (ATs), during incremental sit-to-stand exercise (ISTS) with and without arm support compared with an incremental cycle-ergometer (CE) test; and 2) to examine the reproducibility of the AT measured during the ISTSs. Twenty-six healthy individuals randomly performed the ISTS and CE test. Oxygen uptakes at the AT (AT-VO2) and heart rate at the AT (AT-HR) were determined during the ISTSs and CE test, and repeated-measures analyses of variance and Tukey's post-hoc test were used to evaluate the differences between these variables. Pearson correlation coefficients were used to assess the strength of the relationship between AT-VO2 and AT-HR during the ISTSs and CE test. Data analysis yielded the following correlations: AT-VO2 during the ISTS with arm support and the CE test, r = 0.77 (p < 0.05); AT-VO2 during the ISTS without arm support and the CE test, r = 0.70 (p < 0.05); AT-HR during the ISTS with arm support and the CE test, r = 0.80 (p < 0.05); and AT-HR during the ISTS without arm support and the CE test, r = 0.66 (p < 0.05). The AT-VO2 values during the ISTS with arm support (18.5 ± 1.9 mL·min(-1)·kg(-1)) and the CE test (18.4 ± 1.8 mL·min(-1)·kg(-1)) were significantly higher than those during the ISTS without arm support (16.6 ± 1.8 mL·min(-1)·kg(-1); p < 0.05). The AT-HR values during the ISTS with arm support (126 ± 10 bpm) and the CE test (126 ± 13 bpm) were significantly higher than those during the ISTS without arm support (119 ± 9 bpm; p < 0.05). The ISTS with arm support may provide a cardiopulmonary function load equivalent to the CE test; therefore, it is a potentially valid test for evaluating AT-VO2 and AT-HR in healthy, young adults. Key pointsThe ISTS is a simple test that varies only according to the frequency of standing up, and requires only a small space and a chair.The ISTS with arm support is valid and reproducible, and is a safe test for evaluating AT in healthy young adults.For evaluating the AT, the ISTS may serve as a valid alternative to conventional CPX, using either a cycle ergometer or treadmill, in cases where the latter methods are difficult to implement.

Bovine neutrophils stimulated with Streptococcus uberis induce neutrophil extracellular traps, and cause cytotoxicity and transcriptional upregulation of inflammatory cytokine genes in bovine mammary epithelial cells.

This study aimed to understand the response of neutrophils stimulated by Streptococcus uberis, a major cause of mastitis. It was found that the production of neutrophil extracellular traps (NETs) was induced in milk clots from mastitic milk produced by S. uberis-infected bovine udders. The release of NETs from neutrophils stimulated by S. uberis was investigated. Bovine neutrophils cocultured with S. uberis in vitro released the components of NETs, which contained extracellular DNA and elastase. Bovine mammary epithelial cells (BMECs) incubated in coculture supernatants containing components of NETs, caused cytotoxicity and transcriptional upregulation of inflammatory cytokines, including of interleukin (IL) -1ß, tumor necrosis factor (TNF)-α, IL-6, and IL-8, in BMECs. These findings suggest that bovine neutrophils stimulated by S. uberis induce responses that cause exacerbated inflammation, such as NET formation, cytotoxicity against BMECs, and increased production of inflammatory cytokines. Bovine neutrophil responses stimulated by S. uberis could be involved in the progression of S. uberis-induced mastitis.

Performance evaluation of a rapid immunochromatographic test kit in detecting bovine mastitis-causing streptococci.

Mastitis causes significant economic losses to the dairy industry due to decreased milk production in infected cows. Identification of mastitis-causing pathogens, such as streptococci, is necessary for selecting an effective antibiotic for treating mastitis. Although bacterial cultivation is widely used for pathogen identification, it requires more than 24 hr to complete. Contrarily, Lateral flow assays are simple, rapid, and inexpensive testing procedures. In this study, the effectiveness of an immunochromatographic test kit for detecting streptococci in milk samples from cows with clinical mastitis was evaluated as an alternative to bacterial cultivation. The performance of the immunochromatographic test kit for detecting mastitis-causing pathogens was compared with that of bacterial cultivation and real-time quantitative polymerase chain reaction (qPCR). The sensitivity and specificity of the immunochromatographic test kit were 0.800 and 0.875, respectively, compared with bacterial cultivation. Additionally, the κ statistic values of the immunochromatographic test kit was 0.667, indicating substantial agreement with the results of bacterial cultivation. Statistically, sensitivity and specificity of the immunochromatographic kit and real-time qPCR did not differ significantly; thus, the immunochromatographic test kit detected mastitis-causing streptococci as effectively as real-time qPCR. Therefore, the immunochromatographic kit is a rapid, inexpensive, and simple method for detecting streptococci and contributes to the timely selection of appropriate antibiotics for treatment and promotes early recovery from mastitis.

A novel vaccine strategy using quick and easy conversion of bacterial pathogens to unnatural amino acid-auxotrophic suicide derivatives.

We propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after the removal of the uAA. The survival time after uAA removal could be controlled by the choice of the uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105 fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates. IMPORTANCE: Live vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of uAAs to control gene expression and to conditionally kill host bacteria as a biological containment system. This report proposes a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system. The balance between safety and immunogenicity can be modulated by the selection of the genetic devices used. Moreover, the uAA-auxotrophy can prevent the vaccine from infecting other individuals or establishing the environment.

Effect of myostatin on chemokine expression in regenerating skeletal muscle cells.

Transforming growth factor-ß (TGF-ß) is implicated in the regulatory expression of chemokines that control multiple steps in myogenesis. However, it remains to be established whether myostatin, a member of the TGF-ß superfamily, affects chemokine expression in skeletal muscle. We investigated the effects of myostatin on the expression of mRNAs and proteins for 4 chemokines (CXCL1, CXCL2, CXCL6, CCL2) in intact and regenerating musculus longissimus thoracis from normal-muscled (NM) and double-muscled (DM) cattle. These chemokines were expressed in regenerating muscle, and their expression was always lower in DM than in NM cattle. Immunohistochemistry revealed that CXCL1 and CXCL6 were detected in the regenerating areas of myoblasts and myotubes in both NM and DM cattle. In cultures of myoblasts isolated from the regenerating muscles, significantly less CXCL1, CXCL2 and CCL2 mRNA was expressed in DM myoblasts than in NM myoblasts during the proliferating stage (P-stage). The expression of CXCL1, CXCL2 and CCL2 mRNAs in NM myoblasts and CXCL1, CXCL2 and CXCL6 mRNAs in DM myoblasts decreased upon switching from P-stage to fusion stage (F-stage). Also, the expression of CXCL1, CXCL2 and CXCL6 mRNAs was significantly lower in DM than in NM myoblasts during the F-stage. The addition of 100 ng/ml myostatin during the F-stage attenuated the expression of CXCL1 and CXCL2 mRNAs and augmented that of CCL2. These results show for the first time that myostatin regulates the differential expression of chemokines in skeletal muscle cells.

Estimation of silent phenotypes of calf antibiotic dysbiosis.

Reducing antibiotic usage among livestock animals to prevent antimicrobial resistance has become an urgent issue worldwide. This study evaluated the effects of administering chlortetracycline (CTC), a versatile antibacterial agent, on the performance, blood components, fecal microbiota, and organic acid concentrations of calves. Japanese Black calves were fed with milk replacers containing CTC at 10 g/kg (CON group) or 0 g/kg (EXP group). Growth performance was not affected by CTC administration. However, CTC administration altered the correlation between fecal organic acids and bacterial genera. Machine learning (ML) methods such as association analysis, linear discriminant analysis, and energy landscape analysis revealed that CTC administration affected populations of various types of fecal bacteria. Interestingly, the abundance of several methane-producing bacteria at 60 days of age was high in the CON group, and the abundance of Lachnospiraceae, a butyrate-producing bacterium, was high in the EXP group. Furthermore, statistical causal inference based on ML data estimated that CTC treatment affected the entire intestinal environment, potentially suppressing butyrate production, which may be attributed to methanogens in feces. Thus, these observations highlight the multiple harmful impacts of antibiotics on the intestinal health of calves and the potential production of greenhouse gases by calves.

Cytokeratin 18 is a specific marker of bovine intestinal M cell.

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.

Intramammary infection caused by Staphylococcus aureus increases IgA antibodies to iron-regulated surface determinant-A, -B, and -H in bovine milk.

The aim of this study was to identify virulence factors that have high immunogenicity. An in vivo-expressed Staphylococcus aureus antigen was identified by probing bacteriophage expression libraries of S. aureus with antibodies in bovine mastitis milk. Eighteen clones were isolated, and their proteins were identified as 5 characterised proteins (IsdA, Protein A, IsdB, autolysin, and imidazole glycerol phosphate dehydratase) and 13 hypothetical proteins. We focused on IsdA, IsdB, and IsdH as virulence factors that have a high immunogenicity and are capable of inducing a specific humoral immune response in S. aureus-infected quarters. The optical density (OD) values of IsdA and IsdB IgA and IgG antibodies in milk affected by naturally occurring mastitis caused by S. aureus increased significantly compared to those in healthy milk. In the experimental infection study, the OD values of IsdA- and B-specific IgA and IgG antibodies were significantly increased from 2 to 4 weeks after S. aureus infection compared to day 0 (P < 0.05). On the other hand, we demonstrated that milk from natural and experimental intramammary infections caused by S. aureus are associated with significantly higher IgA levels against IsdH (P < 0.05), but no significant change in IgG levels. Our findings facilitated our understanding of the pathogenicity of S. aureus in bovine mastitis, as well as the mechanisms by which specific humoral immune responses to S. aureus infection are induced. In addition, the results obtained could provide insight into how bovine mastitis can be controlled, for example, through vaccination.

Evaluation of a rapid coliform detection kit from clinical mastitis milk using colloidal gold nanoparticle-based immunochromatographic strips.

The accurate identification of mastitis-causing bacteria assists in effective management by both dairy farmers and veterinarians and can be used to implement the selective use of antimicrobials for treatment. The purpose of this study was to evaluate the ability of our developed anti-ribosomal protein-L7/L12 antibody-coated immunochromatographic strip (ICS) test to detect coliforms in milk by comparing the results with the bacteriological culture method. We investigated the performance of the ICS test as compared with the bacteriological culture method using 308 milk samples from clinical bovine mastitis. First, to determine the optimal ICS test cutoff point for detecting coliform mastitis, we developed a receiver-operating characteristic curve. The result showed that the cutoff point was at 0.5 of our index. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of the ICS test were 81.3%, 84.8%, 69.2%, and 91.54%, respectively. As the clinical signs increased in severity, the F-measure, a weighted harmonic mean of the sensitivity and overall PPV performance, increased. Because it is especially important to treat clinical mastitis appropriately in the early stages of detection, the ICS test, which can be used by both dairy farmers and veterinarians on dairy farms, is considered to be a useful tool for detecting coliform mastitis, which often presents with severe signs.

The bacterial load in milk is associated with clinical severity in cases of bovine coliform mastitis.

We evaluated the relationship between the severity of coliform mastitis and bacterial load in 106 quarter milk samples. We found no significant relationship between somatic cell count and coliform bacterial load in milk in bovine clinical coliform mastitis. Results of the Cochran-Armitage test for trend in milk bacterial load proportions indicated a significant decreasing low group (P<0.001), increasing medium group (P<0.002) and increasing high group (P<0.02) with increasing clinical grade. The present study indicates that the coliform bacterial load in milk is significantly associated with clinical severity states in cases of bovine coliform mastitis, and can be a useful indicator for optimal management of this disease.