Search

Non-typhoidal

Actinobacillus pleuropneumoniae (App) and Mycoplasma hyopneumoniae (Mhp) cause porcine pleuropneumonia and mycoplasmal pneumonia, respectively, and have serious impacts on the swine industry because they retard the growth of pigs. To protect pigs against these diseases, we have developed a multivalent vaccine consisting of App bacterins, APP RTX toxins (Apx toxins), and Mhp bacterin and adhesin protein. This vaccine induced the production of higher levels of antibodies against App and Mhp than the commercial vaccine (Nisseiken Swine APM Inactivated Vaccine). Furthermore, the vaccine efficiently protected pigs against virulent App challenge, showing promise as an efficient vaccine for the prevention of two important respiratory diseases, porcine pleuropneumonia and mycoplasmal pneumonia.

Introduction The pathogenic porcine circovirus type 2 (PCV2) causes significant economic losses in pig production. Emergence of the PCV2d genotype has been linked with PCV2-associated disease (PCVAD) outbreaks. However, no study has been conducted efficacy of an experimental PCV2d-based subunit vaccine in pigs. Therefore, PCV2b- and PCV2d-based capsid (CP) proteins were generated using a baculovirus (Bac) expression system, and we evaluated the protective immune responses in a commercial pig farm where predominant PCV2d is circulating. Methods Eighteen 3-week-old pigs with maternal antibodies were randomly divided into four groups, and were immunized with purified Bac-2dCP, mixed 1:1 ratio with purified Bac-2bCP and Bac-2dCP (Bac-mCP), a commercial PCV2a-based subunit vaccine (VAC) or phosphate-buffered saline (PBS) as controls. Results The Bac-2dCP and Bac-mCP groups had significantly higher PCV2b- or PCV2d- specific IgG and neutralizing antibody without interference by maternal antibody compared to control group in pigs naturally infected with PCV2d. Interestingly, not only serum IL-4 level was significantly increased in the Bac-2dCP group, but also PCV2d viremia level was significantly reduced than the control group. Conclusions The recombinant Bac-2dCP subunit vaccine is a good candidate for the effective reduction against PCV2d infection.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes salmonellosis and mortality worldwide. S. Typhimurium infects macrophages and survives within phagosomes by avoiding the phagosome-lysosome fusion system. Phagosomes sequentially acquire different Rab GTPases during maturation and eventually fuse with acidic lysosomes. Lysophosphatidylcholine (LPC) is a bioactive lipid that is associated with the generation of chemoattractants and reactive oxygen species (ROS). In our previous study, LPC controlled the intracellular growth of Mycobacterium tuberculosis by promoting phagosome maturation. In this study, to verify whether LPC enhances phagosome maturation and regulates the intracellular growth of S. Typhimurium, macrophages were infected with S. Typhimurium. LPC decreased the intracellular bacterial burden, but it did not induce cytotoxicity in S. Typhimuriuminfected cells. In addition, combined administration of LPC and antibiotic significantly reduced the bacterial burden in the spleen and the liver. The ratios of the colocalization of intracellular S. Typhimurium with phagosome maturation markers, such as early endosome antigen 1 (EEA1) and lysosome-associated membrane protein 1 (LAMP-1), were significantly increased in LPC-treated cells. The expression level of cleaved cathepsin D was rapidly increased in LPCtreated cells during S. Typhimurium infection. Treatment with LPC enhanced ROS production, but it did not affect nitric oxide production in S. Typhimurium-infected cells. LPC also rapidly triggered the phosphorylation of IκBα during S. Typhimurium infection. These results suggest that LPC can improve phagosome maturation via ROS-induced activation of NF-κB pathway and thus may be developed as a therapeutic agent to control S. Typhimurium growth.

Actinobacillus pleuropneumoniae (APP) is a causative agent of porcine pleuropneumonia. Therefore, the development of an effective vaccine for APP is necessary. Here, we optimized the culture medium and conditions to enhance the production yields of Apx toxins in APP serotype 1, 2, and 5 cultures. The use of Mycoplasma Broth Base (PPLO) medium improved both the quantity and quality of the harvested Apx toxins compared with Columbia Broth medium. Calcium chloride (CaCl2) was first demonstrated as a stimulation factor for the production of Apx toxins in APP serotype 2 cultures. Cultivation of APP serotype 2 in PPLO medium supplemented with 10 μg/ml of nicotinamide adenine dinucleotide (NAD) and 20 mM CaCl2 yielded the highest levels of Apx toxins. These findings suggest that the optimization of the culture medium and conditions increases the concentration of Apx toxins in the supernatants of APP serotype 1, 2, and 5 cultures and may be applied for the development of vaccines against APP infection.

Non-typhoidal Salmonella (NTS) is one of the most frequent causes of bacterial foodborne illnesses. Considering that the main reservoir of NTS is the intestinal tract of livestock, foods of animal origin are regarded as the main vehicles of Salmonella infection. In particular, poultry colonized with Salmonella Typhimurium (S. Typhimurium), a dominant serotype responsible for human infections, do not exhibit overt signs and symptoms, thereby posing a potential health risk to humans. In this study, comparative genomics approaches were applied to two S. Typhimurium strains, ST1539 and ST1120, isolated from a duck slaughterhouse and a pig farm, respectively, to characterize their virulence and antimicrobial resistance-associated genomic determinants. ST1539 containing a chromosome (4,905,039 bp; 4,403 CDSs) and a plasmid (93,876 bp; 96 CDSs) was phylogenetically distinct from other S. Typhimurium strains such as ST1120 and LT2. Compared to the ST1120 genome (previously deposited in GenBank; CP021909.1 and CP021910.1), ST1539 possesses more virulence determinants, including ST64B prophage, plasmid spv operon encoding virulence factors, genes encoding SseJ effector, Rck invasin, and biofilm-forming factors (bcf operon and pefAB). In accordance with the in silico prediction, ST1539 exhibited higher cytotoxicity against epithelial cells, better survival inside macrophage cells, and faster mice-killing activity than ST1120. However, ST1539 showed less resistance against antibiotics than ST1120, which may be attributed to the multiple resistanceassociated genes in the ST1120 chromosome. The accumulation of comparative genomics data on S. Typhimurium isolates from livestock would enrich our understanding of strategies Salmonella employs to adapt to diverse host animals.

Salmonella enterica serovar Typhimurium, with a broad-host range, is a predominant cause of non-typhoidal Salmonella infection in humans, and the infectious source is highly associated with food animals, especially poultry. Considering the horizontal transmission of S. Typhimurium from farm animals to humans, vaccination has been strongly recommended in industrial animals. In an effort to eradicate S. Typhimurium in poultry farms, a live candidate vaccine strain lacking the phoBR genes, which encode the PhoB/PhoR two-component regulatory system responsible for cellular phosphate signaling, was evaluated in mice and chickens. Lack of the phoBR genes promoted overgrowth of intracellular Salmonella. However, notably, in BALB/c mouse models, the ΔphoBR mutant showed attenuated virulence and instead, provided protection against infection with virulent Salmonella, thereby clearing out Salmonella in the spleen and liver. Accordingly, immunization with the ΔphoBR mutant increased immunoglobulin (Ig)G and IgM antibody responses and also tended to increase the IgG2a/IgG1 ratio, which is indicative of T helper (Th)1-mediated cellular immunity. In chicken challenge models, immunization with the ΔphoBR mutant significantly boosted the production of IgG and IgM antibodies after the second vaccination. The vaccinated chickens ceased fecal shedding of challenged Salmonella earlier than the non-vaccinated ones and showed no Salmonella in their caecum and ileum. These results demonstrate the potential of the S. Typhimurium ΔphoBR mutant as a vaccine in chickens.

Non-typhoidal Salmonella (NTS) causes gastrointestinal infection, which is commonly self-limiting in healthy humans but may lead to invasive infection at extraintestinal sites, leading to bacteremia and focal systemic infections in the immunocompromised. However, a prophylactic vaccine against invasive NTS has not yet been developed. In this work, we explored the potential of a ΔyjeK mutant strain as a live attenuated vaccine against invasive NTS infection. YjeK in combination with YjeA is required for the post-translational modification of elongation factor P (EF-P), which is critical for bacterial protein synthesis. Therefore, malfunction of YjeK and YjeA-mediated EF-P activation might extensively influence protein expression during Salmonella infection. Salmonella lacking YjeK showed substantial alterations in bacterial motility, antibiotics resistance, and virulence. Interestingly, deletion of the yjeK gene increased the expression levels of Salmonella pathogenicity island (SPI)-1 genes but decreased the transcription levels of SPI-2 genes, thereby influencing bacterial invasion and survival abilities in contact with host cells. In a mouse model, the ΔyjeK mutant strain alleviated the levels of splenomegaly and bacterial burdens in the spleen and liver in comparison with the wild-type strain. However, mice immunized with the ΔyjeK mutant displayed increased Th1- and Th2-mediated immune responses at 28 days post-infection, promoting cytokines and antibodies production. Notably, the Th2-associated antibody response was highly induced by administration of the ΔyjeK mutant strain. Consequently, vaccination with the ΔyjeK mutant strain protected 100% of the mice against challenge with lethal invasive Salmonella and significantly relieved bacterial burdens in the organs. Collectively, these results suggest that the ΔyjeK mutant strain can be exploited as a promising live attenuated NTS vaccine.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes salmonellosis and mortality worldwide. S. Typhimurium infects macrophages and survives within phagosomes by avoiding the phagosome-lysosome fusion system. Phagosomes sequentially acquire different Rab GTPases during maturation and eventually fuse with acidic lysosomes. Lysophosphatidylcholine (LPC) is a bioactive lipid that is associated with the generation of chemoattractants and reactive oxygen species (ROS). In our previous study, LPC controlled the intracellular growth of Mycobacterium tuberculosis by promoting phagosome maturation. In this study, to verify whether LPC enhances phagosome maturation and regulates the intracellular growth of S. Typhimurium, macrophages were infected with S. Typhimurium. LPC decreased the intracellular bacterial burden, but it did not induce cytotoxicity in S. Typhimurium-infected cells. In addition, combined administration of LPC and antibiotic significantly reduced the bacterial burden in the spleen and the liver. The ratios of the colocalization of intracellular S. Typhimurium with phagosome maturation markers, such as early endosome antigen 1 (EEA1) and lysosome-associated membrane protein 1 (LAMP-1), were significantly increased in LPC-treated cells. The expression level of cleaved cathepsin D was rapidly increased in LPC-treated cells during S. Typhimurium infection. Treatment with LPC enhanced ROS production, but it did not affect nitric oxide production in S. Typhimurium-infected cells. LPC also rapidly triggered the phosphorylation of IκBα during S. Typhimurium infection. These results suggest that LPC can improve phagosome maturation via ROS-induced activation of NF-κB pathway and thus may be developed as a therapeutic agent to control S. Typhimurium growth.

Porcine epidemic diarrhea virus (PEDV) targets the intestinal mucosa in pigs. To protect against PEDV invasion, a mucosal vaccine is utilized effectively. In this study, we generated a recombinant adenovirus vaccine encoding the heat-labile enterotoxin B (LTB) and the core neutralizing epitope (COE) of PEDV (rAd-LTB-COE). The fusion protein LTB-COE was successfully expressed by the recombinant adenovirus in HEK293 cells, and the immunogenicity of the vaccine candidate was assessed in BALB/c mice and piglets. Three intramuscular or oral vaccinations with rAd-LTB-COE at two-week intervals induced robust humoral and mucosal immune responses. Moreover, a cell-mediated immune response was promoted in immunized mice, and the neutralizing antibody inhibited both the vaccine strain and the emerging PEDV isolate. Immunization experiments in piglets revealed that rAd-LTB-COE was immunogenic and induced good immune responses in piglets. Further studies are required to evaluate the efficacy of rAd-LTB-COE against a highly virulent PEDV challenge.

Actinobacillus pleuropneumoniae (APP) causes a form of porcine pleuropneumonia that leads to significant economic losses in the swine industry worldwide. The apxIBD gene is responsible for the secretion of the ApxI and ApxII toxins and the pnp gene is responsible for the adaptation of bacteria to cold temperature and a virulence factor. The apxIBD and pnp genes were deleted successfully from APP serotype 1 and 5 by transconjugation and sucrose counter-selection. The APP1ΔapxIBDΔpnp and APP5ΔapxIBDΔpnp mutants lost hemolytic activity and could not secrete ApxI and ApxII toxins outside the bacteria because both mutants lost the ApxI- and ApxII-secreting proteins by deletion of the apxIBD gene. Besides, the growth of these mutants was defective at low temperatures resulting from the deletion of pnp. The APP1ΔapxIBDΔpnp and APP5ΔapxIBDΔpnp mutants were significantly attenuated compared with wild-type ones. However, mice vaccinated intraperitoneally with APP5ΔapxIBDΔpnp did not provide any protection when challenged with a 10-times 50% lethal dose of virulent homologous (APP5) and heterologous (APP1) bacterial strains, while mice vaccinated with APP1ΔapxIBDΔpnp offered 75% protection against a homologous challenge. The ΔapxIBDΔpnp mutants were significantly attenuated and gave different protection rate against homologous virulent wild-type APP challenging.

A cold-adapted porcine reproductive and respiratory syndrome virus (CA-VR2332) was generated from the modified live virus strain VR2332. CA-VR2332 showed impaired growth when cultured at 37°C with numerous mutations (S731F, E819D, G975E, and D1014N) in the hypervariable region of the NSP2, in which the mutation S731F might play a vital role in viral replication at 30°C. Conserved amino acid sequences of the GP5 protein suggests that CA-VR2332 is a promising candidate for producing an effective vaccine against PRRSV infection. Further studies on replication and immunogenicity in vivo are required to evaluate the properties of CA-VR2332.

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease, which is characterized by systemic wasting syndrome, including respiratory problems worldwide. Most commercial PCV2 vaccines are derived from recombinant capsid protein or inactivated whole-virus. We compared average daily weight gain, interferon-γ, and neutralizing antibody levels of a recombinant protein vaccine and an inactivated whole-virus vaccine in a pilot study. Both PCV2 vaccines showed similar effect on immunity against PCV2 and a better average daily weight gain was found in both vaccinated groups compared to non-vaccinated animals.

Porcine epidemic diarrhea (PED) is characterized by acute enteritis, watery diarrhea, weight loss, dehydration, and death with high mortality in neonatal piglets. In this study, 3 virus isolates collected in Vietnam between 2016 and 2017 were successfully propagated in Vero cells at high virus titers. Sequence analysis of the full-length spike (S) gene revealed that all 3 isolates belong to genogroup 2a, which is closely related to other prevalent Asian strains. Amino acid sequence comparisons revealed 98.19% to 99.13% homology with the Vietnam isolates circulating during 2013–2015, suggesting that field PED viruses (PEDVs) evolve continuously. Experiments in animals demonstrated that antisera from guinea pigs immunized with the vaccine strain resulted in higher levels (5 log2) of neutralizing antibody against the homologous strain, and showed a relatively lower level of neutralizing antibody against the field isolates. This finding would be helpful in choosing a PEDV strain for vaccine development.

Salmonella enterica subsp. enterica serovar Typhimurium, one of the most common foodborne pathogens, is transmitted mainly through contaminated food derived from infected animals. In this study, S. Typhimurium ST1120, an isolate from pig feces in Korea, was subjected to whole-genome analysis to understand its genomic features associated with virulence. The genome of ST1120 was found to have a circular chromosome of 4,855,001 bp (GC content 52.2%) and a plasmid of 6,863 bp (GC content 46.0%). This chromosome was predicted to have 4,558 open reading frames (ORFs), 17 pseudogenes, 22 rRNA genes, and 86 tRNA genes. Its plasmid was predicted to have three ORFs. Comparative genome analysis revealed that ST1120 was phylogenetically close to S. Typhimurium U288, a critical isolate in piggery farms and food chains in Europe. In silico functional analysis predicted that the ST1120 genome harbored multiple genes associated with virulence and stress resistance, including Salmonella pathogenicity islands (SPIs containing SPI-1 to SPI-5, SPI-13, and SPI-14), C63PI locus, ST104 prophage locus, and various antibiotic resistance genes. In accordance with these analysis results, ST1120 showed competence in invasion and survival abilities when it was added to host cells. It also exhibited robust resistance against antibiotics in comparison with other S. Typhimurium strains. This is the first report of the complete genome sequence of S. Typhimurium isolated from swine in Korea. Comparative genome analysis between ST1120 and other Salmonella strains would provide fruitful information toward understanding Salmonella host specificity and developing control measures against S. Typhimurium infection.

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host’s immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

With the increase in international human and material exchanges, contagious and infectious epidemics are occurring. One of the effective methods of epidemic inhibition is the rapid development and supply of vaccines. Considering the safety of the brain during vaccine development is very important. However, manuals for brain safety assays for new vaccines are not uniform or effective globally. Therefore, the aim of this study is to establish a positive-control protocol for an effective brain safety test to enhance rapid vaccine development. The blood-brain barrier's tight junctions provide selective defense of the brain; however, it is possible to destroy these important microstructures by administering lipopolysaccharides (LPSs), thereby artificially increasing the permeability of brain parenchyma. In this study, test conditions are established so that the degree of brain penetration or brain destruction of newly developed vaccines can be quantitatively identified. The most effective conditions were suggested by measuring time-dependent expressions of tight junction biomarkers (zonula occludens-1 [ZO-1] and occludin) in two types of mice (C57BL/6 and ICR) following exposure to two types of LPS (Salmonella and Escherichia). In the future, we hope that use of the developed positive-control protocol will help speed up the determination of brain safety of novel vaccines.

// Jeong Hyun Cho 1, * , Hyo-Ji Lee 1, * , Hyun-Jeong Ko 2 , Byung-Il Yoon 3 , Jongseon Choe 4 , Keun-Cheol Kim 1 , Tae-Wook Hahn 3 , Jeong A. Han 5 , Sun Shim Choi 6 , Young Mee Jung 7 , Kee-Ho Lee 8 , Yun-Sil Lee 9 , Yu-Jin Jung 1 1 Department of Biological Sciences, Kangwon National University, Chuncheon, Republic of Korea 2 College of Pharmacy, Kangwon National University, Chuncheon, Republic of Korea 3 Department of Veterinary Medicine, Kangwon National University, Chuncheon, Republic of Korea 4 Department of Microbiology, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea 5 Department of Biochemistry and Molecular Biology, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea 6 Department of Medical Biotechnology, College of Biomedical Science, Kangwon National University, Chuncheon, Republic of Korea 7 Department of Chemistry, Kangwon National University, Chuncheon, Republic of Korea 8 Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Nowon-gu, Seoul, Republic of Korea 9 Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seodaemun-gu, Seoul, Republic of Korea * These authors have contributed equally to this work Correspondence to: Yu-Jin Jung, email: yjjung@kangwon.ac.kr Keywords: melanoma, TLR7, imiquimod (IMQ), autophagy, radiotherapy Received: February 17, 2016 Accepted: January 23, 2017 Published: February 15, 2017 ABSTRACT Toll-like receptor (TLR) ligands are strongly considered immune-adjuvants for cancer immunotherapy and have been shown to exert direct anti-cancer effects. This study was performed to evaluate the synergistic anti-cancer and anti-metastatic effects of the TLR7 agonist imiquimod (IMQ) during radiotherapy for melanoma. The pretreatment of B16F10 or B16F1 cells with IMQ combined with γ-ionizing radiation (IR) led to enhanced cell death via autophagy, as demonstrated by increased expression levels of autophagy-related genes, and an increased number of autophagosomes in both cell lines. The results also confirmed that the autophagy process was accelerated via the reactive oxygen species (ROS)-mediated MAPK and NF-κB signaling pathway in the cells pretreated with IMQ combined with IR. Mice subcutaneously injected with melanoma cells showed a reduced tumor growth rate after treatment with IMQ and IR. Treatment with 3-methyladenine (3-MA), ameliorated the anti-cancer effect of IMQ combined with IR. Additionally, the combination therapy enhanced anti-cancer immunity, as demonstrated by an increased number of CD8 + T cells and decreased numbers of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) in the tumor lesions. Moreover, the combination therapy decreased the number of metastatic nodules in the lungs of mice that were injected with B16F10 cells via the tail vein. In addition, the combination therapy enhanced systemic anti-cancer immunity by increasing the abundances of T cell populations expressing IFN-γ and TNF-α. Therefore, these findings suggest that IMQ could serve as a radiosensitizer and immune booster during radiotherapy for melanoma patients.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses to the global swine industry. The present study aimed to evaluate the efficacy of a commercial PRRSV modified live virus (MLV) vaccine in conventionally reared growing/finishing pigs. Four barns were designated for groups A, B, C and D in the growing-to-finishing site. All pigs of the A barn were vaccinated with a commercial PRRSV MLV vaccine, whereas pigs of the B, C or D barn as control groups were unvaccinated. Twenty pigs randomly selected and tagged from each barn were serially bled at 0, 20, 40 and 60 day-post-vaccination, and tested for serological response with a commercial enzyme-linked immunosorbent assay kit. Body weights were measured to calculate the average-daily-weight gain (ADG). Serological assays indicated that the seropositivity of the PRRSV-vaccinated group was higher than that of the unvaccinated groups at 40 day-post-vaccination. ADG of group A was significantly higher than that of groups B and C, and the mean weights of groups A, B, C and D were 0.82 ± 0.017, 0.76 ± 0.016, 0.74 ± 0.019 and 0.81 ± 0.018 kg, respectively. In conclusion, the present study reports the serological responses and growth performance parameters by the PRRSV MLV vaccine in growing/finishing pigs under field conditions.

Phagocytosis is an essential mechanism in innate immune defense, and in maintaining homeostasis to eliminate apoptotic cells or microbes, such as Mycobacterium tuberculosis, Salmonella enterica, Streptococcus pyogenes and Legionella pneumophila. After internalizing microbial pathogens via phagocytosis, phagosomes undergo a series of ‘maturation’ steps, to form an increasingly acidified compartment and subsequently fuse with the lysosome to develop into phagolysosomes and effectively eliminate the invading pathogens. Through this mechanism, phagocytes, including macrophages, neutrophils and dendritic cells, are involved in the processing of microbial pathogens and antigen presentation to T cells to initiate adaptive immune responses. Therefore, phagocytosis plays a role in the bridge between innate and adaptive immunity. However, intracellular bacteria have evolved diverse strategies to survive and replicate within hosts. In this review, we describe the sequential stages in the phagocytosis process. We also discuss the immune evasion strategies used by pathogens to regulate phagosome maturation during intracellular bacterial infection, and indicate that these might be used for the development of potential therapeutic strategies for infectious diseases.

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

Thirty-one Campylobacter jejuni isolates (22 from various local sources, 9 from imported chicken meats) weresubtyped with PFGE and flaA typing to investigate their genetic relatedness. Based on a 90% similarity criterion, 23 and 21genotypic patterns were formed by PFGE and flaA typing, respectively. The discriminatory indices for PFGE, flaA typing, anda composite analysis of PFGE and flaA typing were 0.9785, 0.9527, and 0.9871, respectively. Similar patterns in compositeanalysis were observed between sources (cattle and chicken, and cattle and human), indicating that reservoir animals mayhave been the source of human campylobacteriosis. Therefore, strict hygiene measures from farm to table should beimplemented to prevent diseases due to C. jejuni in humans.

Ginseng has been widely used in Korea as a natural medicine due to its saponin contents. Although the total amount of ginseng stem and leaf saponins (GSLS) is 4~5 times higher than that of saponin in the root, the root is mainly used. This is due to two reasons: nervous system-stimulant activity of GSLS and pesticide residues in GSLS. In this study, residual agricultural pesticides were removed from GSLS using two types of bacterial treatments. Two GSLS treatment groups of chickens (GSLS-1 and GSLS-2) were established. The chickens were fed 0.4% GSLS- 1 or GSLS-2 mixed with crop. We then evaluated the effects of GSLS on bodyweight and several immune parameters. At the end of the experiments, chickens fed GSLS-1 and red ginseng saponin had significantly higher growth rates (16.6% and 8.0%, respectively) compared to the vaccine control group treated with Noblis Salenvac-T. The group fed GSLS-1 also had the highest IgG titer that was significantly different at the end of experiments compared to the other groups. These findings imply that GSLS-1 is a good candidate feed additive for the chicken industry.

In the present study, transposon mutagenesis was used to further attenuate Brucella abortus RB51 vaccine strain. Two purD and purF mutants were constructed, characterized and evaluated for attenuation via intracellular survival in murine macrophage-like RAW264.7 and HeLa cells, and by clearance in BALB/c mice. The purD and purF mutants showed significantly decreased intracellular survival, and complementation of these mutants with intact copies of purD or purF genes of RB51 strain was able to restore these defects. In addition, the pur mutants presented significantly lowered persistence in mice. Immunization with purD and purF mutants protected mice against a challenge with the virulent B. abortus strain 544 at a level similar to that of the parent RB51. These data suggest that genes encoding the early stages of purine biosynthesis (purD and purF) are required for intracellular survival and virulence of B. abortus.

(Received: July 4, 2014; Revised: October 15, 2014; Accepted: October 21, 2014)Abstract : Uterine sterilization is important for improving fertility in cattle. This study compared bacterial flora in theuterus between healthy and repeat breeder cows (RBCs). The uterine flushing of six heifers, 13 healthy HanWoo cowsand eight RBCs (HanWoo) were sampled, and 15 frozen semen samples were selected. Overall, 35 bacteria wereidentified from in HanWoo uterine flushing and semen. The bacterial genera identified from HanWoo uterine flushingwere Alloiococcus, Bacillus, Enterobacter, Enterococcus, Erysipelothrix, Gardnerella, Granulicatella, Kocuria, Pantoea,Pasteurella , Rothia, Serratia, Sphingomonas, Staphylococcus, Stenotrophomonas and Streptococcus. The bacterial generaidentified from HanWoo semen were Bacillus, Escherichia, Kocuria, Oligella, Pseudomonas, Serratia, Sphingomonas,Staphylococcus, Stenotrophomonas and Streptococcus. The prevalence and presence of the identified bacteria betweenhealthy cows and RBCs differed significantly. Further studies are needed to determine the role of these bacteria inthe uterus of HanWoo cattle with reproductive disorder.Keywords : bacterial flora, HanWoo, repeat breeder, semen, uterus

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx1, stx2c, stx2e, or stx1 + stx2b) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.