Milk small extracellular vesicles (sEV) contain proteins that provide potential information of host physiology and immunology. Bovine leukemia virus (BLV) is an oncogenic virus that causes ...progressive B-cell lymphosarcoma in cattle. In this study, we aimed to explore the proteomic profile of milk sEV from BLV-infected cattle compared with those from uninfected cattle. Milk sEV were isolated from three BLV-infected and three uninfected cattle. Proteomic analysis was performed by using a comprehensive nanoLC-MS/MS method. Furthermore, gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to evaluate the candidates for uniquely or differentially expressed proteins in milk sEV from BLV-infected cattle. Proteomic analysis revealed a total of 1330 common proteins in milk sEV among BLV-infected cattle, whereas 118 proteins were uniquely expressed compared with those from uninfected cattle. Twenty-six proteins in milk sEV were differentially expressed proteins more than two-fold significant difference (p < 0.05) in BLV-infected cattle. GO and KEGG analyses indicated that the candidates for uniquely or differentially expressed proteins in milk sEV had been involved in diverse biological activities including metabolic processes, cellular processes, respond to stimulus, binding, catalytic activities, cancer pathways, focal adhesion, and so on. Taken together, the present findings provided a novel insight into the proteomes of milk sEV from BLV-infected cattle.
Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) ultracentrifugation with ExoQuick precipitation, (3) ultracentrifugation with density gradient ...centrifugation, and (4) human milk exosome isolation. Methods 1 and 4 failed due to differences between bovine and human milk. Exosomes were efficiently isolated by ultracentrifugation with either ExoQuick precipitation (method 2) or density gradient centrifugation (method 3). The highest yield of exosomes was achieved using ultracentrifugation with ExoQuick precipitation, whereas higher quality exosome isolation with intact morphological structures was achieved by ultracentrifugation with density gradient centrifugation.
Northern fur seals (Callorhinus ursinus) have a distinct life history pattern comprising annual terrestrial breeding and oceanic migration, and the physiological changes associated with these ...patterns are of particular interest for understanding their environmental adaptations. However, owing to their oceanic distribution, limited information is available on the reproductive physiology of wild individuals during the immature stage and the winter migration period. This study aimed to determine the relationships among the seasonal hormone profiles, body growth, age, and pregnancy using monthly serum samples collected over 3–5 years from two male and two female captive individuals during pubescence and sexual maturation. Small increases in the serum testosterone signaled puberty in males aged 3 and 4 years. Thereafter, males showed considerable increases in testosterone during breeding seasons, indicating sexual maturity. Immature female serum progesterone was maintained at low levels, but after pubescence, females showed an increase in serum progesterone in August, the month next to the peak of delivery, followed by a decrease. In non-pregnant females, progesterone did not increase significantly until the next breeding season, but in pregnant females, they increased again from February to March and then gradually decreased. Immature males increased body mass constantly and reached puberty when their body mass exceeded 20 kg, and they showed seasonal weight fluctuations after puberty. These results provide fundamental information for determining sexual maturity and pregnancy in this species based on sex steroid hormones and body mass measurements.
Bovine milk extracellular vesicles (EVs) attract research interest as carriers of biologically active cargo including miRNA from donor to recipient cells to facilitate intercellular communication. ...Since toxicity of edible milk seems to be negligible, milk EVs are applicable to use for therapeutics in human medicine. Casein separation is an important step in obtaining pure EVs from milk, and recent studies reported that adding hydrochloric acid (HCl) and acetic acid (AA) to milk accelerates casein aggregation and precipitation to facilitate EV isolation and purification; however, the effects of acidification on EVs remain unclear. In this study, we evaluated the acidification effects on milk-derived EVs with that by standard ultracentrifugation (UC). We separated casein from milk by either UC method or treatment with HCl or AA, followed by evaluation of EVs in milk serum (whey) by transmission electron microcopy (TEM), spectrophotometry, and tunable resistive pulse sensing analysis to determine EVs morphology, protein concentration, and EVs size and concentration, respectively. Moreover, we used anti-CD9, -CD63, -CD81, -MFG-E8, -HSP70, and -Alix antibodies for the detection of EVs surface and internal marker proteins by western blot (WB). Morphological features of EVs were spherical shape and similar structure was observed in isolated EVs by TEM. However, some of the EVs isolated by HCl and AA had shown rough surface. Although protein concentration was higher in whey obtained by UC, EV concentration was significantly higher in whey following acid treatment. Moreover, although all of the targeted EVs-marker-proteins were detected by WB, HCl- or AA-treatments partially degraded CD9 and CD81. These findings indicated that acid treatment successfully separated casein from milk to allow efficient EV isolation and purification but resulted in partial degradation of EV-surface proteins. Our results suggest that following acid treatment, appropriate EV-surface-marker antibodies should be used for accurate assess the obtained EVs for downstream applications. This study describes the acidification effects on EVs isolated from bovine milk for the first time.
Two cases of coinfection with bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) in dairy calves in Tochigi Prefecture, Japan, are reported. Sequences of BPSV and PCPV were ...simultaneously detected in the same polymerase chain reaction (PCR) amplicons, which were obtained from the DNA of two dairy calves using a pan-parapoxvirus primer set. PCR amplification using BPSV- and PCPV-specific primer sets were able to distinguish between the two viruses in coinfected clinical samples. Based on these data, further studies on the occurrence BPSV/PCPV coinfections in cattle in Japan are warranted.
Parapoxvirus (PPV) causes papular stomatitis and contagious pustular dermatitis in ruminants worldwide. The virus is generally transmitted through close contact with skin lesions containing PPV in ...infected animals and indirectly through PPV-contaminated materials. PPV-infected animals frequently do not show clinical signs and the route of PPV transmission is sometimes unclear. In this study, the possibility of mechanical transmission of PPV by houseflies (Musca domestica) was investigated using polymerase chain reaction (PCR) gene surveillance. Samples were collected from cattle, sheep, barn environments, direct wash solution of the body surface of houseflies, and indirect wash solution of the body surface and feces of the flies. Bovine papular stomatitis virus, pseudocowpox virus, and orf virus were detected in the oral cavity and body surface of cattle and sheep without clinical signs of PPV infection or barn environments; PPV was considered to have been retained on the farm. PPVs were also detected in the direct wash solution of the body surface of houseflies, and the indirect wash solution of the body surface and feces of the flies. The viral sequence determined from the indirect wash solution of the body surface and feces of the flies was identical to that determined from the body surface of cattle and barns. These results suggested that houseflies may mechanically transmit PPV to both cattle and sheep.
Objective: This study aimed to establish a rapid and simple method for isolating exosomes from raw bovine milk and to compare the quality of the isolated exosomes with those isolated by a standard ...method involving ultracentrifugation (UC).
Methods: To remove caseins, which are major milk proteins consisting more than 80% of milk protein (35% in human breast milk) and hamper isolation and purification of exosomes, hydrochloride (HCl) was added to milk for isoelectric precipitation (IP). The effects of acidification on morphological features, particle size distribution, surface charge, and exosome surface proteins were analyzed by electron microscopy, tunable resistive pulse sensing (TRPS), and Western blot (WB) analysis, respectively.
Results: Electron microscopy showed that some of the exosomes isolated using IP had rough surfaces; most exosomes were successfully isolated without breakage, and their morphological features were similar to those of exosomes isolated by UC. TRPS showed that their surface charge and peaks (mode) for particle size distribution did not significantly differ between both methods. WB analysis using antibodies against the exosome surface marker proteins - milk fat globule-epidermal growth factor 8 (MFG-E8) and CD63 - revealed that the structures of exosome surface proteins were not affected by adding HCl.
Conclusions: IP can be used to remove caseins to reduce operation time. This method will be useful for efficient isolation and purification of bovine milk exosomes and contribute to progression of research on health management of dairy cattle and drug delivery systems in human medicine, which require large amounts of milk exosomes.
Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by bovine leukemia virus (BLV) infection. In Japan, cattle diagnosed with EBL are not permitted for human consumption by the law, ...thereby causing serious economic losses to farmers. The prevalence of BLV is high in Japan (40.9% in dairy cattle and 28.7% in beef cattle, respectively), which makes it difficult to perform the test-and-slaughter of BLV-infected cattle. This necessitates preventing the spread of BLV infection in cattle by early detection, segregation, and the removal of BLV-infected cattle with high proviral load, which are considered high risk for BLV transmission. We aimed to identify cattle that were at high risk for BLV transmission by comparing microRNA (miRNA) profiles in milk small extracellular vesicles (sEV). At first, miRNA profiles in sEV were compared among 4 uninfected cattle and 4 BLV-infected cattle with high proviral load by using a microarray containing mixed probes for miRNA of cattle and humans. Significantly lower amounts of hsa-miR-557 and hsa-miR-19b-1-5p, and insignificantly but higher amounts of hsa-miR-424-5p were observed in milk sEV from BLV-infected cattle than those from uninfected cattle. Next, to evaluate the utility of the aforementioned miRNAs for the identification of cattle that were at high risk for BLV transmission, we performed quantitative real-time PCR using milk sEV newly collected from 5 uninfected cattle and 17 BLV-infected cattle with high proviral load. The cycle threshold value of hsa-miR-424-5p was significantly lower in milk sEV from BLV-infected cattle. The PCR detection was unavailable or a significant difference was not observed for hsa-miR-557 and hsa-miR-19b-1-5p, respectively. These results suggest that the amount of hsa-miR-424-5p was higher in milk sEV from BLV-infected cattle and increasing the hsa-miR-424-5p in milk sEV could be one of the characteristic trends in cattle that are high risk for BLV transmission. Moreover, assessing characteristic miRNA amounts in milk sEV, which can be recovered twice a day by milking, could be useful for the routine monitoring of cattle in dairy herds instead of blood collection.
In recent years, there has been an increase in infectious diseases in marine mammals, including brucellosis, infections of morbillivirus, herpesvirus, and poxvirus. Several serological diagnostic ...methods, including enzyme-linked immunosorbent assays, immunofluorescence assays (ELISA), and western blotting, have been used to detect antibodies against pathogens in marine mammals. However, options for commercial secondary antibodies used to detect antibodies in marine mammals are limited; therefore, the use of proteins A, G, or chimeric protein AG may provide a suitable alternative. This study aimed to assess the use of proteins A, G, and chimeric protein AG to detect marine mammal immunoglobulins. Currently, there are no comparative studies on the use of proteins A, G, and chimeric protein AG for the detection of immunoglobulins in marine mammals. In this study, we used ten pinnipeds’ species (Baikal seal, California sea lion, harbor seal, northern fur seal, ringed seal, South American fur seal, South American sea lion, spotted seal, Steller sea lion, and walrus) and five cetacean species (beluga whale, bottlenose dolphin, harbor porpoise, killer whale, and Pacific white-sided dolphin) and compare binding ability to proteins A, G, or chimeric protein AG by ELISA. The results revealed that the immunoglobulins from pinniped and cetacean species reacted more strongly to protein A than protein G. In addition, the immunoglobulins of pinnipeds and cetaceans showed a strong binding ability to chimeric protein AG. These results suggest that proteins A, G, and chimeric protein AG would be used to help further develop serological assays.
Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). Most BLV-infected cattle show no clinical signs and only some develop EBL. The pathogenesis of EBL ...remains unclear and there are no methods for predicting EBL before its onset. Previously, it was reported that miRNA profiles in milk small extracellular vesicles (sEVs) were affected in cattle in the late stage of BLV infection. It raised a possibility that miRNA profile in milk sEVs from EBL cattle could be also affected. To characterize the difference in milk of EBL cattle and healthy cattle, we examined the miRNA profiles in milk sEVs from four EBL and BLV-uninfected cattle each using microarray analysis. Among the detected miRNAs, three miRNAs—bta-miR-1246, hsa-miR-1290, and hsa-miR-424-5p—which were detectable using quantitative real-time PCR (qPCR) and are associated with cancers in humans—were selected as biomarker candidates for EBL. To evaluate the utility of these miRNAs as biomarkers for EBL, their levels were measured using milk that was freshly collected from 13 EBL and seven BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p, but not hsa-miR-1290, were detected using qPCR and their levels in milk sEVs from EBL cattle were significantly higher than those in BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p in sEVs may promote metastasis by targeting tumor suppressor genes, resulting in increased amounts in milk sEVs in EBL cattle. These results suggest that bta-miR-1246 and hsa-miR-424-5p levels in milk sEVs could serve as biomarkers for EBL.
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