Brian J. Nonnecke

Bovine milk exosome proteome.

Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology.

Bovine milk proteome: Quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis

UNLABELLED Milk protein expression in healthy cows and cows with mastitis will provide information important for the dairy food industry and immune function in the mammary gland. To facilitate protein discovery, milk was fractioned into whey, milk fat globule membranes (MFGM) and exosomes from healthy and Staphylococcus aureus infected cows. Amine-reactive isobaric tags (iTRAQ) were used to quantify protein changes between milk fractions isolated from healthy and S. aureus infected cows. 2971 milk proteins were identified with a false discovery rate of 0.1%. Greater than 300 milk proteins associated with host defense were identified and 94 were significantly differentially regulated in S. aureus infected milk compared to their uninfected controls. These differentially regulated host defense proteins were selectively segregated in the 3 milk compartments examined. An example of this segregation of host defense proteins was the partitioning and high concentration of proteins indicative of neutrophil extracellular traps (NETs) formation in the MFGM preparations from S. aureus infected milk as compared to exosomes or whey. Protein composition changes found in milk exosomes, MFGM and whey during an infection provides new and comprehensive information on milk protein composition in general as well as changes occurring during an infection. BIOLOGICAL SIGNIFICANCE The significance of this study is the identification and quantification of the individual components of the neutrophil extracellular traps (NET) functional proteome in an apparent stable complex with MFGM and/or milk fat globules during an intra-mammary infection. NETs could be functionally relevant in intra-mammary infection, as it is known that during an infection neutrophils ingest large amounts of milk fat that down regulates many of their traditional immune functions. Thus the presence of NETs in milk fat provides new insights to mammary immune function and suggests a role for NETs in clinical mastitis. These in vivo NETs can now be tested to determine if they retain functional antimicrobial activity when primarily associated with milk fat. Then we can estimate their real world functional relevance during an intra-mammary infection, which is one key to understanding clinical mastitis in dairy cows.

Use of Recombinant ESAT-6:CFP-10 Fusion Protein for Differentiation of Infections of Cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis

ABSTRACT Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-γ)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-α), IFN-γ, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-γ and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-γ responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.

Efficacy and immunogenicity of Mycobacterium bovis ΔRD1 against aerosol M. bovis infection in neonatal calves

An attenuated Mycobacterium bovisRD1 deletion (DeltaRD1) mutant of the Ravenel strain was constructed, characterized, and sequenced. This M. bovis DeltaRD1 vaccine strain administered to calves at 2 weeks of age provided similar efficacy as M. bovis bacillus Calmette Guerin (BCG) against low dose, aerosol challenge with virulent M. bovis at 3.5 months of age. Approximately 4.5 months after challenge, both DeltaRD1- and BCG-vaccinates had reduced tuberculosis (TB)-associated pathology in lungs and lung-associated lymph nodes and M. bovis colonization of tracheobronchial lymph nodes as compared to non-vaccinates. Mean central memory responses elicited by either DeltaRD1 or BCG prior to challenge correlated with reduced pathology and bacterial colonization. Neither DeltaRD1 or BCG elicited IFN-gamma responses to rESAT-6:CFP-10 prior to challenge, an emerging tool for modern TB surveillance programs. The DeltaRD1 strain may prove useful for bovine TB vaccine programs, particularly if additional mutations are included to improve safety and immunogenicity.

Effects of stress on leukocyte trafficking and immune responses: implications for vaccination.

Increased susceptibility of animals to infectious disease during the periparturient period results in suffering and economic losses. Stress appears to delay inflammation by reducing efficiency of CD62L-mediated immune surveillance by phagocytes. It is important to note that the effects of stress are not limited to alteration of leukocyte trafficking patterns since various stressors (e.g., transport, parturition, and castration) also decrease IFN-gamma secretion by lymphocytes, and may decrease antigen presentation efficiency by down-regulating class II molecule expression on antigen presenting cells, and delay or impair immune responses to vaccination. Documented immunosuppression in periparturient animals, particularly the bias toward Th2 immune responses, and also changes in general leukocyte trafficking patterns suggest that vaccination intending to elicit cell-mediated immunity may not be efficacious at this point of the production cycle. Based on findings of numerous periparturient studies on immunosuppression in cattle, waiting at least 30 days after parturition before administering routine vaccinations is recommended.

1,25-Dihydroxyvitamin D3 inhibits secretion of interferon-γ by mitogen- and antigen-stimulated bovine mononuclear leukocytes☆

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active metabolite of vitamin D, and delta 22-26-F3-1,25-dihydroxyvitamin D3 (delta 22-26-F3-1,25(OH)2D3), a synthetic analog with a high affinity for the vitamin D receptor, significantly inhibited interferon-gamma (IFN-gamma) secretion in 24- and 48-h cultures of pokeweed mitogen (PWM) and ovalbumin (OVA) stimulated peripheral blood mononuclear leukocyte (MNL) from adult, OVA-sensitized dairy cattle. Vitamin D-induced inhibition of IFN-gamma production was most pronounced in MNL cultures supplemented with 1,25(OH)2D3 at 1.0 nM or more, a concentration equal to or exceeding that in plasma of cows with clinical hypocalcemia. Secreted IFN-gamma was undetectable in all resting MNL cultures. Ultra-low concentrations (0.0001, 0.001, and 0.01 nM) of 1,25(OH)2D3 had no effect on IFN-gamma secretion by PWM-stimulated bovine MNL, unlike a previous study in other species demonstrating enhancement of IFN-gamma secretion at these concentrations. Preincubation of MNL with.

Modulation of Mycobacterium bovis-Specific Responses of Bovine Peripheral Blood Mononuclear Cells by 1,25-Dihydroxyvitamin D3

ABSTRACT Historically, administration of vitamin D has been considered beneficial in the treatment of tuberculosis. The interaction of this vitamin {i.e., 1,25-dihdroxyvitamin D3[1,25(OH)2D3]} with the antitubercular immune response, however, is not clear. In the present study, in vitro recall responses of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis were used to study the immune-modulatory effects of 1,25(OH)2D3 on M. bovis-specific responses in vitro. Addition of 1 or 10 nM 1,25(OH)2D3 inhibited M. bovis-specific proliferative responses of PBMC from M. bovis-infected cattle, affecting predominately the CD4+ cell subset. In addition, 1,25(OH)2D3 inhibited M. bovis-specific gamma interferon (IFN-γ) production yet enhanced M. bovis-specific nitric oxide (NO) production. Lymphocyte apoptosis, measured by flow cytometry using annexin-V staining, was diminished by addition of 1,25(OH)2D3 to PBMC cultures. These findings support the current hypothesis that 1,25(OH)2D3enhances mycobacterial killing by increasing NO production, a potent antimicrobial mechanism of activated macrophages, and suggest that 1,25(OH)2D3 limits host damage by decreasingM. bovis-induced IFN-γ production.

Differential Expression of Cytokines in Response to Respiratory Syncytial Virus Infection of Calves with High or Low Circulating 25-Hydroxyvitamin D3

Deficiency of serum levels of 25-hydroxyvitamin D3 has been related to increased risk of lower respiratory tract infections in children. Respiratory syncytial virus (RSV) is a leading cause of low respiratory tract infections in infants and young children. The neonatal calf model of RSV infection shares many features in common with RSV infection in infants and children. In the present study, we hypothesized that calves with low circulating levels of 25-hydroxyvitamin D3 (25(OH)D3) would be more susceptible to RSV infection than calves with high circulating levels of 25(OH)D3. Calves were fed milk replacer diets with different levels of vitamin D for a 10 wk period to establish two treatment groups, one with high (177 ng/ml) and one with low (32.5 ng/ml) circulating 25(OH)D3. Animals were experimentally infected via aerosol challenge with RSV. Data on circulating 25(OH)D3 levels showed that high and low concentrations of 25(OH)D3 were maintained during infection. At necropsy, lung lesions due to RSV were similar in the two vitamin D treatment groups. We show for the first time that RSV infection activates the vitamin D intracrine pathway in the inflamed lung. Importantly, however, we observed that cytokines frequently inhibited by this pathway in vitro are, in fact, either significantly upregulated (IL-12p40) or unaffected (IFN-γ) in the lungs of RSV-infected calves with high circulating levels of 25(OH)D3. Our data indicate that while vitamin D does have an immunomodulatory role during RSV infection, there was no significant impact on pathogenesis during the early phases of RSV infection. Further examination of the potential effects of vitamin D status on RSV disease resolution will require longer-term studies with immunologically sufficient and deficient vitamin D levels.

In vitro modulation of proliferation and phenotype of resting and mitogen-stimulated bovine mononuclear leukocytes by 1,25-dihydroxyvitamin D3

Effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the proliferation and phenotype of normal bovine peripheral blood mononuclear leukocytes (MNL) were studied in vitro. Resting and pokeweed mitogen (PWM)-stimulated MNL cultures were supplemented with 1.0 nM of 1,25(OH)2D3 at the beginning of the culture period. Leukocytes were removed from 2-, 4-, 6-, 8-, 10-, 12-, and 14-day unsupplemented and 1.25(OH)2D3-supplemented cultures, and were counted and phenotyped using monoclonal antibodies to bovine leukocyte surface antigens. Cell numbers in resting MNL cultures decreased with time and were unaffected by 1,25(OH)2D3 supplementation. The progressive increase in cell numbers in PWM-stimulated MNL cultures was suppressed, but not abolished by 1,25(OH)2D3. Suppression was greatest in PWM-stimulated 6- to 12-day cultures. Cellular composition of resting MNL cultures was unaffected by 1,25(OH)2D3. Pokeweed mitogen-stimulated cultures supplemented with 1,25(OH)2D3 had fewer total T-cells and CD4+ T-cells at 6-14 days. In contrast, numbers of CD8+ T-cells were significantly higher in 1,25(OH)2D3-supplemented cultures at 6, 10, and 14 days. Effects of 1,25(OH)2D3 on CD4+ and CD8+ T-cell populations were manifested by a significant reduction in the CD4+:CD8+ T-cell ratios in 6- to 14-day cultures. Proliferation of IL-2 receptor+ and MHC class II antigen+ cells was also reduced in supplemented 6- to 14-day cultures, indicating events associated with PWM-induced MNL activation were suppressed by 1,25(OH)2D3. These findings indicate that 1,25(OH)2D3 modulates the proliferation and differentiation of bovine MNL in vitro. Our results also suggest that changes in plasma or tissue 1,25(OH)2D3 concentrations that occur during the peripartum period and in clinical cases of milk fever may regulate the bovine immune system in vivo.

Regulation of mitogenic responses by bovine milk leukocytes.

Bovine milk lymphocytes are less responsive to in vitro mitogen stimulation than peripheral blood lymphocytes (PBL). In this study, milk leukocytes (ML) or their soluble products, were co-cultured with mitogen stimulated PBL to determine if suppression could be transferred to normally responsive cells. Addition of either ML (treated with mitomycin C to prevent cell division), or supernatant from ML cultures to cultures of autologous PBL resulted in a reduction of mitogenesis by the PBL, but no suppression was seen with addition of treated PBL or PBL supernatant. Suppression was greater when the ML were from animals with chronic staphylococcal infection. Suppression by ML supernatant was not due to toxicity to the responders, since addition at the latter stages of culture had no effect on the response. These results indicate that reduced mitogenesis by milk lymphocytes may be due to the presence of suppressor cells or molecules.