Influenza A viruses (IAVs) are members of the family Orthomyxoviridae and genus Orthomyxovirus. Avian and mammalian species are the host of IAVs, which includes humans and dogs. Canine influenza virus (CIV) is an emerging pathogen that causes severe and acute respiratory diseases in dogs. This study monitored the antigen and antibody against CIV in dogs in the Republic of Korea (ROK) from 2016 to 2021. One thousand and seventy-two nasal swabs and 1,545 blood samples were collected from animal hospitals and animal shelters. Five nasal swabs in 2017 and seven in 2018 from stray dogs were positive for CIV according to RT-PCR. The prevalence of H3N2 CIV ranged from 9.5% to 24.8%, according to the hemagglutination inhibition (HI) assay. On the other hand, none of the serum samples from 2018 to 2021 showed seropositivity against the avian H5, H7, and H9 viruses. The HI titers for H3N2 ranged from 16 to 512. The distribution of HI titer 16–32 was 57.6% in seropositive samples. The pet dogs were vaccinated against CIV, but the stray and military dogs were unvaccinated. In 2017 and 2018, the seroprevalence of CIV in stray dogs was higher than in the other years, and viral RNA was detected in nasal swabs. It may mean previous exposure of stray dogs to CIV. With the increasing number of pet dogs and the close contact between humans and dogs, canines could serve as an intermediate host for transmitting IAVs to humans. Therefore, continuous surveillance of CIV is needed for public health and the potential emergence of novel zoonotic viruses.
The canine parvovirus (CPV) causes clinical signs, such as severe enteritis, dehydration, diarrhea, vomiting, leukopenia, and hair loss, which may lead to death. Vaccination is still the most important approach, as no specific treatment exists to prevent CPV. Monoclonal antibodies are valuable tools to study the pathogenic mechanisms of CPV and develop effective diagnostic reagents and pharmaceuticals. In this study, two monoclonal antibodies (MAbs) against CPV-2a were obtained through hybridoma technology by fusing myeloma cells and B cells from the spleens of mice immunized with CPV type 2a (CPV-2a). Two MAbs (CPV-330 and CPV-620) were studied on the reactivity of vaccine (CPV-2a) and field strains (CPV-new 2a, -2b, and -2c) by indirect immunofluorescence (IFA), hemagglutination inhibition test (HI), virus neutralization test (VN), and inhibition of virus growth test. Two MAbs showed similar antibody titers for HI and VN. On the other hand, CPV-330 inhibited the viral replication in Crandell-Rees Feline Kidney (CRFK) cells better than CPV-620. These CPV MAbs may provide valuable biological reagents to study the CPV pathogenic mechanisms and work as therapeutic antibodies.
A solid-phase competition enzyme-linked immunosorbent assay (ELISA), recombinant VP2 (rVP2) protein, and monoclonal antibody (mAb) were developed for the specific and sensitive detection of porcine parvovirus (PPV) antibodies in pig sera. A total of 1,544 sera samples were collected from breeding pig farms located in the Gyeongsangbuk-do Province in the Republic of Korea. The optimal operating conditions of SC-ELISA were as follows. The concentration of rVP2 proteins coated on the wells was 4 μg/mL, the swine sera were diluted 1:2, and the HRP-conjugated PPV VP2 mAb (9A8 clone) was used at 500 ng/mL. These results suggest that the SC-rVP-ELISA assay may be a valuable alternative to the current diagnostic tools used to detect PPV-specific monoclonal antibodies and broadly monitor PPV infections in domestic pigs at different breeding stages.
Natural killer (NK) cells have cytotoxic effects on tumor cells and viral pathogens. NK cell-derived exosomes (NK-exosomes) also express typical NK cell markers and cytotoxic molecules, therefore, exert anti-tumor and immune homeostatic activities. In this study, canine NK-exosomes separated from cytotoxic NK cell supernatant carried specific markers such as CD81, Alix, and Perforin 1. We examined the anti-tumor effects of NK-exosomes in an experimental murine model using the canine mammary carcinoma cells, REM134. REM134 cells were xenografted of mammary fat pad of mice. CD133, Bmi-1, MMP-3, IL-6, TNF-α, and PCNA are useful as a molecular marker for tumorigenesis and metastasis. The treatment of canine NK-exosomes inhibited tumor growth and significantly (p<0.01) downregulated the expression of Bmi-1, MMP-3, IL-6, TNF-α, and PCNA in REM134-treated mice. Also, the expression of CD133, potent cancer stem cell marker, was significantly downregulated in the canine NK-exosomes-treated mice compared with that of the tumor group. Collectively, these results suggested that canine NK-exosomes has a potential capacity for regulation of cancer progression and metastasis against canine mammary carcinoma.
In this study, the near-complete genome sequence of the novel reassortant H1N2 influenza A virus strain A/swine/Korea/KS60/2016 is reported. Sequences of the hemagglutinin (HA), neuraminidase (NA), and polymerase basic 2 (PB2) genes were analyzed, revealing that the isolates contain segments from previous Korean swine H1N2 strains. Additionally, the remaining genes of this strain originated from human H1N1 strains in 2009.
Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and thus hold therapeutic implications in field of regenerative medicine and reproductive biotechnology. In the present study, we compared the stem cell properties of bovine ear skin tissue (ESK)- and nasal mucosa (NM)-derived MSCs. Bovine ESK-MSCs and NM-MSCs were successfully isolated by collagenase digestion and maintained proliferative capacity during the 20 consecutive passages. Both ESK-MSCs and NM-MSCs showed similar morphology and expressed common cell surface markers (CD29, CD44, CD90, and CD105). Also, we compared differentiation potentials of bovine ESK-MSCs and NM-MSCs into osteogenic, adipogenic, and chondrogenic lineages through specific staining and quantitative real-time RT-PCR. As results, bovine ESK-MSCs and NM-MSCs could differentiate into mesodermal cell lineages. However, bovine ESK-MSCs and NM-MSCs exhibited difference in expression of differentiation-related specific markers. Specifically, NM-MSCs exhibited increased expression levels of osteocalcin, peroxisome proliferator-activated receptor gamma, and aggrecan compared to ESK-MSCs. Also, ESK-MSCs exhibited increased expression levels of collagen type I, II, and lipoprotein lipase compared to NM-MSCs. We suggest that the nasal mucosa of bovine could be used as a source of bovine MSCs.
Canine mammary tumors account for ~30% of all tumors in the female dogs and approximately 50% of the tumors are malignant. Exosomes have been the focus of great interest, as they appear to be involved in numerous important cellular processes. In this study, we examined the anti-tumor effects of canine mesenchymal stem cells-derived exosomes (MSC-exosomes) in an experimental murine mammary tumor model using canine mammary carcinoma cells, REM134. The MSC-exosomes were injected tumor site and tail vein of REM134 xenografted mice. We found that tumor size of the MSC-exosomes-treated group decreased compared to those of the only tumor group in REM134-driven tumorigenic mouse model. In addition, the MSC-exosomes-treated tumor group showed meaningfully reduced expression levels of the MMP-3, IL-1β, IL-6, and TNF-α compared to those in the tumor group. Specifically, we confirmed that the expression level of the CD133, potent cancer stem cell (CSC) markers, decreased in the MSC-exosomes-treated tumor group compared to the tumor group. This study suggests that the MSC-exosomes exhibited anti-tumor effects through downregulating CSC-related markers in the canine mammary tumor murine model. Further study is needed in the future, and we are conducting research on the detailed anti-tumor mechanism of the MSC-exosomes.
Canine parvovirus (CPV) remains a leading infectious cause of death in canines, especially in young puppies. Though vaccination is being carried out regularly, immunization failures occur, and puppies may be exposed to infection. Virus-like particles (VLPs) act like a subunit vaccine, mimicking the structure of authentic viruses. Therefore, VLPs have the potential to be used as vaccine candidates. Since Viral Protein 2 (VP2), a major structural protein of CPV, is the crucial antigen for CPV, the purpose of this study was to produce a recombinant VP2 of new canine parvovirus-2a using the baculovirus expression system in SF9 insect cells. The results revealed that recombinant VP2 assembles to form VLPs with antigenic properties similar to those of natural CPV, the recombinant VLP can produce a hemagglutination assay (HA) titer (1:210) in SF9 cells. Expression of the recombinant 6-His-tagged VP2 in SF9 cells was confirmed by western blotting. These findings suggest that the recombinant VP2 expressed in this study could be used as an efficient subunit vaccine against CPV infection.
Porcine parvovirus 2 (PPV2) was recently detected in the Republic of Korea. This paper reports two near-complete genome sequences of PPV2 identified for the first time in the lung tissue of aborted pig fetuses.
Influenza A virus (IAV) causes respiratory disease in birds and mammals, including pigs and humans. Infection by IAV in pigs increases not only economic losses in the swine industry but also the emergence of novel IAV variants via gene reassortment, which is important due to the susceptibility of both birds and humans to IAV. This study provides serological data obtained during a study to detect IAV infections in pigs in the Republic of Korea during 2018 and 2019. A total of 1,559 samples were collected from 74 domestic pig farms. Hemagglutination inhibition (HI) assays were performed using the A/Swine/Korea/25-13(H1N1), A/Swine/Korea/E102 (H1N2), and A/Swine/Korea/Cy10/2007 (H3N2) viruses as antigens. The HI assay results showed that 266 of the 1,559 samples were seropositive (17.0%). Among these, H1N1, H1N2, and H3N2 comprised 7.3% (114), 6.0% (93), and 8.8% (137) of the 1,559 samples, respectively. Co-infections of H1N1/H1N2, H1N1/H3N2, H1N2/H3N2 and H1N1/H1N2/H3N2 were observed in 2.1% (31), 1.5% (23), 1.5% (24), and 0.8% (13) of the 1,559 samples, respectively. Interestingly, IAV infections were detected in all nine provinces of the country.
In this study, antibody responses after vaccination against equine influenza were investigated among 1,591 horses in the Republic of Korea using the hemagglutination inhibition (HI) test. Equine influenza has not occurred since 2011 and a commercial vaccine against H3N8 has been used. The equine influenza virus, A/equine/South Africa/4/03 (H3N8), was used as the antigen in the HI assay. The mean seropositive rate was 90.5% in 2019. Except for stallion whose seropositive rate was 78.5%, all seropositive rates of other horse types were over 90%. Regionally, except for Gangwon-do and Jeju-do whose seropositive rates were 89.0% and 87.1%, all seropositive rates in other provinces were over 90%. In the future, more through vaccination against equine influenza needs to be done based on this investigation result.
Viral protein 2 (VP2), which is the structural protein of parvovirus, can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. VP2 of canine parvovirus (CPV) is responsible for neutralizing antibodies in immunized animals. In this study, VP2 protein of canine parvovirus-2c was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells. The results show that VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and a high hemagglutination (HA) titer (1:27). The recombinant 6-His-tagged VP2 protein with a molecular mass of about 65 kDa was detected by anti-His antibody and anti-PPV serum. This study provides a foundation for the application of VP2 protein in the clinical diagnosis of CPV and in the vaccination against CPV.
The culture of the intestinal epithelium into three dimensional (3D) structures typically termed organoid culture. Organoid culture is based on the ability of intestinal stem cells (ISCs), at the base of the crypt, perpetually to divide and produce a fully differentiated, polarized epithelium. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) positive ISCs isolated from the intestine can form organoids in long-term culture. Thus, when cultured under the appropriate 3D conditions, single Lgr5+ ISCs undergo cycles of self-renewal, differentiation and morphogenesis, and self-organize into crypt-villus domains that house cycling ISCs and differentiated intestinal epithelial cells (IECs). In this study, we performed isolation, characterization and consecutive subculture of small intestinal crypts from BALB/c-nude mouse. Briefly, isolated mouse crypts were embedded in matrigel, cast into 40 μL droplets at the bottom of well in a 48-well plate. Following polymerization, the gels were overlaid with ISCs expansion medium containing B27, N2, N-acetylcysteine, epidermal growth factor, noggin, and R-spondin 1. As a result, mouse crypt-derived ISCs had enteroids and spheroid morphologies. We also confirmed by quantitative real-time RT-PCR that expression of ISCs-related specific genes (Lgr5, sox9) and IECs-related specific genes (chromogranin A, defensin-5, mucin-1, mucin-2, and villin) was maintained at eight passages or more. Thus, we observed that expression of specific markers and consecutive self-renewing in the mouse small intestinal crypt-derived organoids.
Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. Regulation of cytotoxicity in NK cells is mediated by inhibitory receptors that bind major histocompatibility complex class I (MHC-I) molecules on target cells. Human myelogenous leukemia K562 cells are readily attacked by NK cells, because K562 cells expressed very low levels of MHC-I molecules for inhibitory NK cell receptors. In this study, we compared the ability of γ-irradiated- or mitomycin C (MMC)-treated K562 feeder cells to support expansion and activation of canine NK cells. We isolated CD5 negative cells from canine peripheral blood mononuclear cells by immunomagnetic separation and co-cultured with γ-irradiated (100 Gy)- or MMC (20 μg/mL)-treated K562 cells in the presence of interleukin (IL)-2, IL-15 and IL-21 for 21 days. As a result, number of CD5 negative cells, co-cultured with γ-irradiated- or MMC-treated K562 cells (56.72 ± 13.77 fold or 32.99 ± 10.83 fold), was increased than those of CD5 negetive cells (2.99 ± 1.42 fold). Also, we found that gene expression markers of activated NK cells such as NKp30, NKp44, NKp46, Ly49, NKG2D, CD244, perforin, and granzyme B and production of interferon gamma were similarly upregulated in NK cells co-cultured with γ-irradiated- or MMC-treated K562 cells, respectively. Next, we observed that cytotoxicity of NK cells co-cultured with γ-irradiated K562 cells was more sensitively reacted to canine mammary carcinoma cells than those of MMC-treated K562 cells. These results revealed that γ-irradiated K562 cells are more efficient feeder cells than MMC-treated K562 cells for enhancing NK cells expansion and activation.
Physicochemical properties of potato chip treated with superheated steam (SHS) at various temperatures (120, 150, 180, and 220oC) and durations (2, 5, 8, and 10 min) were measured to assess the potential application of SHS in the production of unfried chips. A faster drying rate was obtained at a higher SHS temperature due to a lower degree of water condensation on the surface of the potato chip. A higher temperature of SHS resulted in higher volume shrinkage, indicating the dependency of shrinkage on the volume of water loss. SHS treatment did not cause any significant defects in the appearance of potato chips although pillowing and burnt spots were observed on the surface of the sample processed at 220oC for 10 min. Damaged starch content and Rapid Visco Analyzer profiles showed that partial gelatinization occurred during SHS treatment. Potato chips treated with SHS showed the shrinkage of parenchyma cells, resulting to compressed cell layers at the surface and inside. As treatment proceeded, air cells were formed internally. These results suggested that SHS combined with post drying process would be appropriate in the production of unfried potato chips by reducing drying time without causing any deterioration in quality.
Effects of superheated steam (SHS) temperature (120, 150, 180, 220°C) and time (2, 5, 8, 10 min) on product quality attributes of snack-type ginseng chips were studied. Ginseng chips dried at faster rate under a higher SHS temperature. Higher SHS temperature resulted in ginseng chips with lower moisture content and water activity. After SHS treatment, lightness (L values) and redness (a values) of ginseng chips decreased significantly whereas yellowness (b values) of those increased. Although crude saponin content in ginseng chips reduced slightly after SHS treatment, chips treated at 150°C for 5 min had the highest saponin retention among samples. Scanning electron microscope analysis revealed that SHS treatment cause the shrinkage at the surface first, forming compressed cell layers. As treatment proceeded, pores and cavities were formed internally, resulting porous structure. These results suggested that SHS treatment shows great promise in production of snack-type ginseng chips by reducing drying time without any quality deterioratio
Effects of superheated steam (SHS) temperature (120, 150, 180, 220°C) and time (2, 5, 8, 10 min) on physicochemical properties of garlic chips were examined. Higher the SHS temperature, a faster drying rate was observed due to the lower extent of water condensation on the garlic chip’s surface at the initial stage of treatment. Garlic chips treated at 220°C for 10 min showed the lowest water content and water activity. However appearance of these was inappropriate due to burnt spots on the surface. Scanning electron microscope analysis showed that garlic chips treated at higher SHS temperature showed compressed smooth cell layers at the surface with more compact internal structure due to higher degree of dehydration when compared to chips treated at lower temperature. These results confirmed that SHS could be utilized as an effective drying method for the production of garlic chips. Moreover, it is recommended that SHS treatment combined with other conventional post drying process would result in garlic chips with better quality in terms of color.
The sensitivities of PrP Sc detection methods, western blotting (WB), immunohistochemistry (IHC) and protein mis-folding cyclic amplification (PMCA) techniques were compared from brains, spleens and blood of mice challenged with PrP Sc of murine-adapted BSE strain 301C. PrP Sc was detected in the spleen from 30 dpi by IHC and at 60 dpi by WB. At 30 dpi, disease-specific signals of PrP Sc was observed in only two follicles of a single spleen. PrP Sc was detected in spleen at 10 dpi with PMCA after 5 rounds of amplification. Clinical signs were obviously shown from 240 dpi, and coincided with first detection of PrP Sc in brains by WB, IHC and PMCA after one round amplification. In addition, PrP Sc was also detected in blood at 60, 180 and 240 dpi with PMCA after 5 rounds of amplification. The FDC-M1 epitope, which appears in immature FDCs, and PrP Sc were detected in follicles first at 30 dpi, whilst the FDC-M2 epitope of mature FDCs was detected at 60 dpi. More FDC-M2 epitope and PrP Sc were detected in follicles as disease progressed. The CD21/35 epitope is expressed on both FDCs and germinal center B cells. The pattern of CD21/35 expressing cells was similar to but less dominant than that of FDCs.
Abnormal prions are infectious agents involved in a neuro-degenerative disease, which occurs naturally such as Chronic Wasting Disease (CWD) in deer and elk, Bovine Spongiform Encephalopathy (BSE) in cattle, Scrapie in sheep and goats and Creutzfeldt-Jakob Disease (CJD) in humans. The cellular prion protein of the elk consists of 233 amino acids (residues 25-257), which represents an autonomous folding unit with three α-helices and two-stranded anti-parallel β-sheets. Here, we demonstrated elk-recPrP (Elk recombinant prion protein) which can be obtained as follows; (1) Cloning of elk PrP gene, (2) Expression of a histidine-tagged full-length elk PrP by induction with IPTG in E. coli and (3) Purification by affinity chromatography using Ni-NTA agarose resin. In Western blot and ELISA analysis, elk-recPrP showed specific activity against anti-PrP monoclonal antibody. Thus, our elk-recPrP would be a useful tool for the understanding of basic structure and mechanism studies of PrPSC formation.