Protective efficacy of attenuated Salmonella Typhimurium strain expressing BLS, Omp19, PrpA, or SOD of Brucella abortus in goats

Background@#Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. @*Objectives@#The study aimed to determine the protective efficacy of attenuated Salmonellastrain expressing highly conserved Brucella immunogens in goats. @*Methods@#Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 10 9 CFU/mL and challenge of all groups was done at 6 weeks after vaccination. @*Results@#Among these vaccines inoculated at 5 × 10 9 CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Realtime polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the nonvaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. @*Conclusions@#These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

Protective efficacy of attenuated Salmonella Typhimurium strain expressing BLS, Omp19, PrpA, or SOD of Brucella abortus in goats

Background@#Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. @*Objectives@#The study aimed to determine the protective efficacy of attenuated Salmonellastrain expressing highly conserved Brucella immunogens in goats. @*Methods@#Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 10 9 CFU/mL and challenge of all groups was done at 6 weeks after vaccination. @*Results@#Among these vaccines inoculated at 5 × 10 9 CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Realtime polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the nonvaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. @*Conclusions@#These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

Attraction and Repellent Behaviors of Culicoides Biting Midges toward Cow Dung, Carbon Dioxide, and Essential Oils

Culicoides biting midges (Diptera: Ceratopogonidae) are hematophagous arthropod vectors that transmit epizootic arthropod-borne viruses (arboviruses). Arboviruses are recognized as causes of pregnancy loss, encephalomyelitis, and congenital malformations in ruminants. Therefore, continuous monitoring and control of Culicoides, which causes significant damage to industrial animals are necessary. We performed attraction and repellent tests in Culicoides using various essential oils, cow dung, and carbon dioxide (CO2). Culicoides tended to move more to cow dung (60.8%, P<0.0001) and CO2 (63.8%, P<0.01). To the essential oils as repellents, 26.1% (P<0.0001), 18.7% (P<0.001), and 25.5% (P<0.01) of the Culicoides moved to the lavender, lemongrass, and eucalyptus chamber, respectively. The Culicoides that moved to the 3 essential oils chambers showed markedly low activity. Collectively, it was showed that Culicoides tended to be attractive to cow dung and CO2, and repellent from the 3 essential oils.

Pathogenicity of severe fever with thrombocytopenia syndrome virus in mice regulated in type I interferon signaling Severe fever with thrombocytopenia and type I interferon / 한국실험동물학회지

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease, which causes high fever, thrombocytopenia, and death in humans and animals in East Asian countries. The pathogenicity of SFTS virus (SFTSV) remains unclear. We intraperitoneally infected three groups of mice: wild-type (WT), mice treated with blocking anti-type I interferon (IFN)-α receptor antibody (IFNAR Ab), and IFNAR knockout (IFNAR−/−) mice, with four doses of SFTSV (KH1, 5 × 105 to 5 × 102 FAID50). The WT mice survived all SFTSV infective doses. The IFNAR Ab mice died within 7 days post-infection (dpi) with all doses of SFTSV except that the mice were infected with 5 × 102 FAID50 SFTSV. The IFNAR−/− mice died after infection with all doses of SFTSV within four dpi. No SFTSV infection caused hyperthermia in any mice, whereas all the dead mice showed hypothermia and weight loss. In the WT mice, SFTSV RNA was detected in the eyes, oral swabs, urine, and feces at 5 dpi. Similar patterns were observed in the IFNAR Ab and IFNAR−/− mice after 3 dpi, but not in feces. The IFNAR Ab mice showed viral shedding until 7 dpi. The SFTSV RNA loads were higher in organs of the IFNAR−/− mice compared to the other groups. Histopathologically,coagulation necrosis and mononuclear inflammatory cell infiltration in the liver and white pulp atrophy in the spleen were seen as the main lesions in the IFN signaling lacking mice. Immunohistochemically, SFTSV antigens were mainly detected in the marginal zone of the white pulp of the spleen in all groups of mice, but more viral antigens were observed in the spleen of the IFNAR−/− mice. Collectively, the IFN signaling-deficient mice were highly susceptible to SFTSV and more viral burden could be demonstrated in various excreta and organs of the mice when IFN signaling was inhibited.

Molecular subtyping and antimicrobial susceptibility of Streptococcus dysgalactiae subspecies equisimilis isolates from clinically diseased pigs

Background@#Streptococcus dysgalactiae subspecies equisimilis (SDSE) acts as an etiological agent for lameness, neurological signs, and high mortality in pigs. Despite its importance in pig industries and zoonotic potential, little is known about the effects of this pathogen. @*Objectives@#This study aimed to determine the molecular characteristics and antimicrobial resistance of SDSE strains isolated from diseased pigs. @*Methods@#A total 11 SDSE isolates were obtained from diseased pigs. Bacterial identification, PCR for virulence genes, emm typing, and antimicrobial resistance genes, multilocus sequence typing, and antimicrobial susceptibility test were performed. @*Results@#Nine isolates were from piglets, and 8 showed lameness, sudden death, or neurological signs. The isolates were PCR-positive for sla (100%), sagA (100%), and scpA (45.5%), and only 1 isolate amplified the emm gene (stL2764). Eight different sequence types were detected, categorized into 2 clonal complexes and 4 singletons. All the isolates in this study were included in a small cluster, which also contained other strains derived from humans and horses. The minimum inhibitory concentrations for the tested beta-lactams were low, while those for macrolides, tetracyclines, and fluoroquinolones were relatively high. PCR analysis of the macrolide and tetracycline resistance genes demonstrated that the isolates carried erm(B) (18.2%, n = 2), mef(A/E) (9.1%, n = 1), tet(M) (18.2%, n = 2), and tet(O) (90.2%, n = 10). Two isolates presented a mutation in parC, which is associated with fluoroquinolone resistance. @*Conclusion@#This study provided insight into swine-derived SDSE, as it is related to veterinary medicine, and elucidated its zoonotic potential, in the context of molecular epidemiology and antimicrobial resistance in public health.

Pathogenicity of severe fever with thrombocytopenia syndrome virus in mice regulated in type I interferon signaling Severe fever with thrombocytopenia and type I interferon / 한국실험동물학회지

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease, which causes high fever, thrombocytopenia, and death in humans and animals in East Asian countries. The pathogenicity of SFTS virus (SFTSV) remains unclear. We intraperitoneally infected three groups of mice: wild-type (WT), mice treated with blocking anti-type I interferon (IFN)-α receptor antibody (IFNAR Ab), and IFNAR knockout (IFNAR−/−) mice, with four doses of SFTSV (KH1, 5 × 105 to 5 × 102 FAID50). The WT mice survived all SFTSV infective doses. The IFNAR Ab mice died within 7 days post-infection (dpi) with all doses of SFTSV except that the mice were infected with 5 × 102 FAID50 SFTSV. The IFNAR−/− mice died after infection with all doses of SFTSV within four dpi. No SFTSV infection caused hyperthermia in any mice, whereas all the dead mice showed hypothermia and weight loss. In the WT mice, SFTSV RNA was detected in the eyes, oral swabs, urine, and feces at 5 dpi. Similar patterns were observed in the IFNAR Ab and IFNAR−/− mice after 3 dpi, but not in feces. The IFNAR Ab mice showed viral shedding until 7 dpi. The SFTSV RNA loads were higher in organs of the IFNAR−/− mice compared to the other groups. Histopathologically,coagulation necrosis and mononuclear inflammatory cell infiltration in the liver and white pulp atrophy in the spleen were seen as the main lesions in the IFN signaling lacking mice. Immunohistochemically, SFTSV antigens were mainly detected in the marginal zone of the white pulp of the spleen in all groups of mice, but more viral antigens were observed in the spleen of the IFNAR−/− mice. Collectively, the IFN signaling-deficient mice were highly susceptible to SFTSV and more viral burden could be demonstrated in various excreta and organs of the mice when IFN signaling was inhibited.

Pathogenicity of clade 2.3.4.4 H5N6 highly pathogenic avian influenza virus in three chicken breeds from South Korea in 2016/2017

In 2016, novel H5N6 highly pathogenic avian influenza virus emerged in Korea. During the outbreak, the virus caused the largest culling, especially in brown chicken lines. We determined the pathogenicity and transmissibility of the virus in 2 white chicken lines of the specific pathogen-free chickens, broilers and brown chicken line of Korean native chicken (KNC). A KNC had a longer virus shedding period and longer mean death time than others. Our study showed that this characteristic in the KNC might have contributed to a farm-to-farm transmission of the brown chicken farms.

Vaccination of goats with a combination Salmonella vector expressing four Brucella antigens (BLS, PrpA, Omp19, and SOD) confers protection against Brucella abortus infection

Salmonella is an intracellular pathogen with a cellular infection mechanism similar to that of Brucella, making it a suitable choice for use in an anti-Brucella immune boost system. This study explores the efficacy of a Salmonella Typhimurium delivery-based combination vaccine for four heterologous Brucella antigens (Brucella lumazine synthase, proline racemase subunit A, outer-membrane protein 19, and Cu/Zn superoxide dismutase) targeting brucellosis in goats. We inoculated the attenuated Salmonella delivery-based vaccine combination subcutaneously at two different inoculation levels; 5 × 10⁹ colony-forming unit (CFU)/mL (Group B) and 5 × 10¹⁰ CFU/mL (Group C) and challenged the inoculations with virulent Brucella abortus at 6 weeks post-immunization. Serum immunoglobulin G titers against individual antigens in Salmonella immunized goats (Group C) were significantly higher than those of the non-immunized goats (Group A) at 3 and 6 weeks after vaccination. Upon antigenic stimulation, interferon-γ from peripheral blood mononuclear cells was significantly elevated in Groups B and C compared to that in Group A. The immunized goats had a significantly higher level of protection as demonstrated by the low bacterial loads in most tissues from the goats challenged with B. abortus. Relative real-time polymerase chain reaction results revealed that the expression of Brucella antigens was lower in spleen, kidney, and lung of immunized goats than of non-immunized animals. Also, treatment with our combination vaccine ameliorated histopathological lesions induced by the Brucella infection. Overall, the Salmonella Typhimurium delivery-based combination vaccine was effective in delivering immunogenic Brucella proteins, making it potentially useful in protecting livestock from brucellosis.

Morphological and Molecular Characterization of Toxocara tanuki (Nematoda: Ascaridae) from Korean Raccoon Dog, Nyctereutes procyonoides koreensis

Present study was performed to describe the morphological and molecular characterization of Toxocara tanuki (Nematoda: Ascaridae) from Korean raccoon dog, Nyctereutes procyonoides koreensis, naturally infected in the Republic of Korea (Korea). Juvenile and adult worms of T. tanuki were recovered in 5 out of 10 raccoon dogs examined and the larval worms were detected in 15 out of 20 muscle samples (75%). Small lateral alae were observed on the cranial end of the body in male and female adults and 2 long spicules (3.0–3.5 mm) were characteristically observed in the posterior end of males. In SEM observation, 18 pairs of proximal precloacal, a precloacal median, a postcloacal median and 5 pairs of postcloacal papillae were uniquely revealed in the posterior portion of males, but the proximal papillae were not shown in the lateral ends of females. Molecular analysis on the 18S rRNA partial DNA sequences was revealed the same finding in both samples, adult worms and muscle larvae, which are closely related to T. tanuki. In conclusion, it was confirmed for the first time that T. tanuki is indigenously distributed, the Korean raccoon dog is acted as the natural definitive host of this nematode in Korea and the morphological characteristics of T. tanuki were shown in specific structure for single postcloacal median papilla in male.

Analysis of Five Arboviruses and Culicoides Distribution on Cattle Farms in Jeollabuk-do, Korea

Arthropod-borne viruses (Arboviruses) are transmitted by arthropods such as Culicoides biting midges and cause abortion, stillbirth, and congenital malformation in ruminants, apparently leading to economic losses to farmers. To monitor the distribution of Culicoides and to determine their relationship with different environmental conditions (temperature, humidity, wind speed, and altitude of the farms) on 5 cattle farms, Culicoides were collected during summer season (May-September) in 2016 and 2017, and analyzed for identification of species and detection of arboviruses. About 35% of the Culicoides were collected in July and the collection rate increased with increase in temperature and humidity. The higher altitude where the farms were located, the more Culicoides were collected on inside than outside. In antigen test of Culicoides against 5 arboviruses, only Chuzan virus (CHUV) (2.63%) was detected in 2016. The Akabane virus (AKAV), CHUV, Ibaraki virus and Bovine ephemeral fever virus (BEFV) had a positive rate of less than 1.8% in 2017. In antigen test of bovine whole blood, AKAV (12.96%) and BEFV (0.96%) were positive in only one of the farms. As a result of serum neutralization test, antibodies against AKAV were generally measured in all the farms. These results suggest that vaccination before the season in which the Culicoides are active is probably best to prevent arbovirus infections.

Salmonella enterica subsp. enterica infections in eastern great egrets (Ardea alba modesta)

Five eastern great egrets with a history of ataxia, wry neck, and wet feathers were submitted to the Veterinary Diagnostic Center for pathologic examination. Slightly enlarged livers with diffuse white-grayish nodules were observed. Microscopically, the hepatic and lung parenchyma contained granulomatous lesions consisting of central necrosis. Some hearts showed myofiber necrosis with infiltration of histiocytes and heterophils. Partial 16SrRNA and gyrB gene sequences of all isolates showed high similarities (99-100%) to those of Salmonella (S.) enterica subsp. enterica. Based on pathological and molecular biological results, S. enterica subsp. enterica systemic infections were diagnosed in eastern great egrets of Korea.

Expression analysis of ciliary rootlet coiled coil protein mRNA during Xenopus development / 대한수의학회지

Ciliary rootlet coiled coil protein (CROCC), the structural component that originates from the basal body at the proximal end of the ciliary rootlet, plays a crucial role in maintaining the cellular integrity of ciliated cells. In the current study, we cloned Xenopus CROCC and performed the expression analysis. The amino acid sequence of Xenopus laevis was related to those of Drosophila, cow, goat, horse, chicken, mouse and human. Reverse transcription polymerase chain reaction analysis revealed that CROCC mRNA encoding a coiled coil protein was present maternally, as well as throughout early development. In situ hybridization indicated that CROCC mRNA occurred in the animal pole of embryo during gastrulation and subsequently in the presumptive neuroectoderm at the end of gastrulation. At tailbud stages, CROCC mRNA expression was localized in the anterior roof plate of the developing brain, pharyngeal epithelium connected to gills, esophagus, olfactory placode, intestine and nephrostomes of the pronephric kidney. Our study suggests that CROCC may be responsible for control of the development of various ciliated organs.

Synthetic Prion Peptide 106-126 Resulted in an Increase Matrix Metalloproteinases and Inflammatory Cytokines from Rat Astrocytes and Microglial Cells

It has been shown that the accumulation of prion in the cytoplasm can result in neurodegenerative disorders. Synthetic prion peptide 106-126 (PrP) is a glycoprotein that is expressed predominantly by neurons and other cells, including glial cells. Prion-induced chronic neurodegeneration has a substantial inflammatory component, and an increase in the levels of matrix metalloproteinases (MMPs) may play an important role in neurodegenerative development and progression. However, the expression of MMPs in PrP induced rat astrocytes and microglia has not yet been compared. Thus, in this study, we examined the fluorescence intensity of CD11b positive microglia and Glial Fibrillary Acidic Protein (GFAP) positive astrocytes and found that the fluorescent intensity was increased following incubation with PrP at 24 hours in a dose-dependent manner. We also observed an increase in interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) protein expression, which are initial inflammatory cytokines, in both PrP induced astrocytes and microglia. Furthermore, an increase MMP-1, 3 and 11 expressions in PrP induced astrocytes and microglia was observed by real time PCR. Our results demonstrated PrP induced activation of astrocytes and microglia respectively, which resulted in an increase in inflammatory cytokines and MMPs expression. These results provide the insight into the different sensitivities of glial cells to PrP.

Generation of CD2+CD8+ NK Cells from c-kit+ Bone Marrow Cells in Porcine

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-kit+ bone marrow cells (c-kit+ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-kit+ BM cells that give rise to CD2+CD8+ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-kit+ BM cells than those of controls. And, we compared the ability of the cytotoxicity of CD2+CD8+ NK cells differentiated by cytokines from c-kit+ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100:1 for 4 h once a week. In results, CD2+CD8+ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-kit+ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-kit+ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.

Multiple congenital anomalies in a Korean native calf (Bos taurus coreanae) / 대한수의학회지

Supernumerary ectopic limb(s) (SEL) is a congenital anomaly defined as the presence of accessory limb(s) attached to various body regions. This paper describes a case of SEL with ectopic lung and ectopia cordis in a newborn calf, based on macroscopic, microscopic and radiographic findings. External features of multiple congenital anomalies included an ectopic lung growing over the middle of the backbone and covered with normal haired skin. Ectopia cordis was found in the abdominal cavity and attached to the liver. Two extra abnormal limbs originated separately from within the ectopic lung. Most of the abdominal organs were exposed to the outside through the opened abdominal cavity. Microscopically the ectopic lung tissue had edema in the connective tissue around the bronchus and artery. Changes in other organs included congestion of the renal medulla, infiltration of inflammatory cells (lymphocytes and eosinophils) around the hepatic portal tract, and edema surrounding blood vessels and neurons in the brain. The rudimentary humerus of the forelimb was attached to the thoracic spine, as viewed radiographically. The hindlimb was consisted of an irregularly shaped femur, short tibia and fibula, two tarsal bones, one metatarsal bone, and three phalanges. This is the first description of congenital anomalies involving the SEL, ectopic lung and ectopia cordis in a calf.