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Equine hyperimmune serum protects mice against Clostridium difficile spore challenge.

Weiwei YAN ; Kang Soon SHIN ; Shih Jon WANG ; Hua XIANG ; Thomas DIVERS ; Sean MCDONOUGH ; James BOWMAN ; Anne ROWLANDS ; Bruce AKEY ; Hussni MOHAMED ; Yung Fu CHANG

Journal of Veterinary Science.2014;15(2):249-258. doi:10.4142/jvs.2014.15.2.249

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.
Animals ; Antibodies, Bacterial/blood/*immunology/therapeutic use ; Bacterial Proteins/immunology/therapeutic use ; Bacterial Toxins/immunology/therapeutic use ; Clostridium Infections/microbiology/prevention & control/*veterinary ; Clostridium difficile/*immunology ; Enterotoxins/immunology/therapeutic use ; Female ; Horse Diseases/microbiology/*prevention & control ; Horses ; Immune Sera/*immunology ; Immunization, Passive/*veterinary ; Mice ; Mice, Inbred C57BL ; Spores, Bacterial/immunology

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Isolation, in vitro propagation, genetic analysis, and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil.

Rosiane Nascimento ALVES ; Susana Elisa RIECK ; Carlos UEIRA-VIEIRA ; Marcelo Bahia LABRUNA ; Marcelo Emilio BELETTI

Journal of Veterinary Science.2014;15(2):241-248. doi:10.4142/jvs.2014.15.2.241

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlandia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlandia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlandia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlandia and Sao Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.
Animals ; Antigens, Bacterial/blood/*diagnostic use ; Bacterial Outer Membrane Proteins/genetics/metabolism ; Bacterial Proteins/*genetics/metabolism ; Base Sequence ; Brazil ; Dog Diseases/diagnosis/*microbiology ; Dogs ; Ehrlichia canis/*genetics/*immunology/isolation & purification ; Ehrlichiosis/diagnosis/microbiology/*veterinary ; Fluorescent Antibody Technique, Indirect/veterinary ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; RNA, Ribosomal, 16S/genetics/metabolism ; Sequence Alignment/veterinary

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A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis.

Miguel SALGADO ; Cristobal VERDUGO ; Cord HEUER ; Pedro CASTILLO ; Patricia ZAMORANO

Journal of Veterinary Science.2014;15(2):233-239. doi:10.4142/jvs.2014.15.2.233

PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.
Animals ; Bacteriological Techniques/economics/*veterinary ; Cattle ; Cattle Diseases/diagnosis/*microbiology ; DNA, Bacterial/chemistry/genetics ; Female ; Mycobacterium avium subsp. paratuberculosis/*genetics ; Paratuberculosis/diagnosis/*microbiology ; Real-Time Polymerase Chain Reaction/veterinary ; Reproducibility of Results

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Identification of abnormal gene expression in bovine transgenic somatic cell nuclear transfer embryos.

Jongki CHO ; Sungkeun KANG ; Byeong Chun LEE

Journal of Veterinary Science.2014;15(2):225-231. doi:10.4142/jvs.2014.15.2.225

This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogenetic embryos, and in vitro fertilization (IVF) embryos. Transgenic SCNT embryos showed significantly lower rates of development to the blastocyst stage than non-transgenic ones. To investigate normal gene expression, RNA was extracted from ten blastocysts derived from parthenogenesis, IVF, non-transgenic, and transgenic SCNT embryos and reverse-transcribed to synthesize cDNA. The cDNA was then subjected to PCR amplification and semi-quantified. More DNMT1 mRNA was detected in the transgenic SCNT group than the other three groups. Hsp 70.1 mRNA was detected in the IVF embryos, while lower levels were found in SCNT and parthenogenetic embryos. Mash2 mRNA was present at the highest levels in transgenic SCNT embryos. In conclusion, the higher levels of methylation and lower protein synthesis after heat shock in the transgenic SCNT embryos expected based on our results may cause lower embryonic development.
Animals ; Animals, Genetically Modified/genetics ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism ; Cattle/embryology/*genetics ; DNA (Cytosine-5-)-Methyltransferase/*genetics/metabolism ; Embryo, Mammalian/embryology/metabolism ; Female ; Fertilization in Vitro ; *Gene Expression Regulation, Developmental ; HSP70 Heat-Shock Proteins/*genetics/metabolism ; Nuclear Transfer Techniques/veterinary ; Parthenogenesis ; Pregnancy ; RNA, Messenger/genetics/metabolism ; Transcription, Genetic

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Oxidative response of neutrophils to platelet-activating factor is altered during acute ruminal acidosis induced by oligofructose in heifers.

Claudia CONCHA ; Maria Daniella CARRETTA ; Pablo ALARCON ; Ivan CONEJEROS ; Diego GALLARDO ; Alejandra Isabel HIDALGO ; Nestor TADICH ; Dante Daniel CACERES ; Maria Angelica HIDALGO ; Rafael Agustin BURGOS

Journal of Veterinary Science.2014;15(2):217-224. doi:10.4142/jvs.2014.15.2.217

Reactive oxygen species (ROS) production is one of the main mechanisms used to kill microbes during innate immune response. D-lactic acid, which is augmented during acute ruminal acidosis, reduces platelet activating factor (PAF)-induced ROS production and L-selectin shedding in bovine neutrophils in vitro. This study was conducted to investigate whether acute ruminal acidosis induced by acute oligofructose overload in heifers interferes with ROS production and L-selectin shedding in blood neutrophils. Blood neutrophils and plasma were obtained by jugular venipuncture, while ruminal samples were collected using rumenocentesis. Lactic acid from plasma and ruminal samples was measured by HPLC. PAF-induced ROS production and L-selectin shedding were measured in vitro in bovine neutrophils by a luminol chemiluminescence assay and flow cytometry, respectively. A significant increase in ruminal and plasma lactic acid was recorded in these animals. Specifically, a decrease in PAF-induced ROS production was observed 8 h after oligofructose overload, and this was sustained until 48 h post oligofructose overload. A reduction in PAF-induced L-selectin shedding was observed at 16 h and 32 h post oligofructose overload. Overall, the results indicated that neutrophil PAF responses were altered in heifers with ruminal acidosis, suggesting a potential dysfunction of the innate immune response.
Acidosis/chemically induced/immunology/*veterinary ; Animals ; Blood ; Cattle ; Cattle Diseases/chemically induced/*immunology ; Female ; Flow Cytometry/veterinary ; *Immunity, Innate ; L-Selectin/metabolism ; Neutrophils/*drug effects ; Oligosaccharides/*pharmacology/toxicity ; Platelet Activating Factor/*pharmacology ; Reactive Oxygen Species/metabolism ; Rumen

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Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus.

Lishan LV ; Xiaoming LI ; Genmei LIU ; Ran LI ; Qiliang LIU ; Huifang SHEN ; Wei WANG ; Chunyi XUE ; Yongchang CAO

Journal of Veterinary Science.2014;15(2):209-216. doi:10.4142/jvs.2014.15.2.209

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.
Animals ; Antibodies, Viral/blood ; *Chickens ; Chimera/genetics/immunology ; Coronavirus Infections/prevention & control/*veterinary/virology ; Female ; *Immunity, Innate ; Infectious bronchitis virus/genetics/*immunology ; Influenza A Virus, H5N1 Subtype/genetics/immunology ; Injections, Intramuscular/veterinary ; Mice ; Mice, Inbred BALB C ; Neuraminidase/genetics ; Poultry Diseases/*prevention & control/virology ; Recombinant Fusion Proteins/genetics/immunology ; Spike Glycoprotein, Coronavirus/genetics/*immunology ; Vaccines, Synthetic/administration & dosage/genetics/immunology ; Vaccines, Virus-Like Particle/administration & dosage/genetics/*immunology ; Viral Proteins/genetics

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Recombinant Kluyveromyces lactis expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP5 elicits mucosal and cell-mediated immune responses in mice.

Haiyan ZHAO ; Yalan WANG ; Zhitao MA ; Yongqiang WANG ; Wen Hai FENG

Journal of Veterinary Science.2014;15(2):199-208. doi:10.4142/jvs.2014.15.2.199

Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-gamma in spleen CD4+ and CD8+ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-gamma secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future.
Administration, Oral ; Animals ; Antibodies, Viral/*immunology ; B-Lymphocytes/immunology/virology ; Enzyme-Linked Immunosorbent Assay ; *Immunity, Cellular ; *Immunity, Mucosal ; Injections, Subcutaneous ; Kluyveromyces/genetics ; Mice ; Mice, Inbred BALB C ; Porcine respiratory and reproductive syndrome virus/*immunology ; Recombinant Proteins/genetics/immunology ; T-Lymphocytes/immunology/virology ; Viral Envelope Proteins/*genetics/*immunology ; Viral Vaccines/administration & dosage/*pharmacology

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Accuracy of sonographic diagnosis of pneumoperitoneum using the enhanced peritoneal stripe sign in beagle dogs.

Song Yeon KIM ; Ki Tae PARK ; Seong Chan YEON ; Hee Chun LEE

Journal of Veterinary Science.2014;15(2):195-198. doi:10.4142/jvs.2014.15.2.195

The objective of this study was to evaluate the feasibility and accuracy of estimating the smallest amount of abdominal free gas detectible in a large population of beagles by ultrasonography. Healthy dogs were randomly divided into three groups: group A that received 0.1 mL of air injected into the peritoneal cavity, group B that received 0.2 mL of air injected into the peritoneal cavity, and group C that received 0.5 mL of intraperitoneal air. Randomly, some dogs in each group did not receive air injection for the negative control. All ultrasonographic procedures were performed by individuals blinded to group assignments and the presence of intraperitoneal air. The minimum volume of consistently detectable air with good accuracy and reliability was 0.2 mL. Results of the study demonstrated that the enhanced peritoneal stripe sign (EPSS) can verify cases of pneumoperitoneum if more than 0.2 mL of intra-abdominal free gas is present The EPSS is a reliable and specific ultrasonographic characteristic for diagnosing pneumoperitoneum in dogs.
Animals ; Dog Diseases/*ultrasonography ; Dogs ; Female ; Male ; Pneumoperitoneum/ultrasonography/*veterinary ; Reproducibility of Results ; Species Specificity ; Ultrasonography/standards/veterinary

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Computed tomographic evaluation of cervical vertebral canal and spinal cord morphometry in normal dogs.

Eunjeong SEO ; Jihye CHOI ; Mincheol CHOI ; Junghee YOON

Journal of Veterinary Science.2014;15(2):187-193. doi:10.4142/jvs.2014.15.2.187

The height, width, and cross-sectional area of the vertebral canal and spinal cord along with the area ratio of spinal cord to vertebral canal in the cervical vertebra were evaluated in images obtained using computed tomography (CT). Measurements were taken at the cranial, middle, and caudal point of each cervical vertebra in eight clinically normal small breed dogs (two shih tzu, two miniature schnauzers, and four mixed breed), 10 beagles, and four German shepherds. CT myelography facilitated the delineation of the epidural space, subarachnoid space, and spinal cord except at the caudal portion of the 7th cervical vertebra. The spinal cord had a tendency to have a clear ventral border in the middle portion of the vertebral canal and lateral borders near both end plates. The height, width, and area of the vertebral canal and spinal cord in the cervical vertebra were increased as the size of dog increased. However, the ratio of the spinal cord area to vertebral canal area in the small dogs was higher than that of the larger dogs. Results of the present study could provide basic and quantitative information for CT evaluation of pathologic lesions in the cervical vertebra and spinal cord.
Animals ; Body Size ; Cervical Vertebrae/*anatomy & histology/radiography ; Dogs/*anatomy & histology/growth & development ; Reference Values ; Spinal Canal/*anatomy & histology/radiography ; Spinal Cord/*anatomy & histology/radiography ; Tomography, X-Ray Computed/*veterinary

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Effects of resveratrol on the insulin signaling pathway of obese mice.

Hyun Ju HONG ; Wonyoung KANG ; Dong Geon KIM ; Dae Ho LEE ; Youngjae LEE ; Chang Hoon HAN

Journal of Veterinary Science.2014;15(2):179-185. doi:10.4142/jvs.2014.15.2.179

The present study was conducted to investigate the effects of resveratrol on the insulin signaling pathway in the liver of obese mice. To accomplish this, we administered resveratrol to high fat diet-induced obese mice and examined the levels of protein phosphorylation in the liver using an antibody array. The phosphorylation levels of 10 proteins were decreased in the high fat diet and resveratrol (HFR) fed group relative to the levels in the high fat diet (HF) fed group. In contrast, the phosphorylation levels of more than 20 proteins were increased in the HFR group when compared with the levels of proteins in the HF group. Specifically, the phosphorylation levels of Akt (The308, Tyr326, Ser473) were restored to normal by resveratrol when compared with the levels in the HF group. In addition, the phosphorylation levels of IRS-1 (Ser636/Ser639), PI-3K p85-subunit alpha/gamma(Tyr467/Tyr199), PDK1 (Ser241), GSK-3alpha (S21) and GSK-3 (Ser9), which are involved in the insulin signaling pathway, were decreased in the HF group, whereas the levels were restored to normal in the HFR group. Overall, the results show that resveratrol restores the phosphorylation levels of proteins involved in the insulin signaling pathway, which were decreased by a high fat diet.
Animals ; Anti-Inflammatory Agents/*pharmacology ; Fluorescent Antibody Technique ; Insulin/*physiology ; Liver/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Obese ; Phosphorylation ; Proteins/metabolism ; Signal Transduction/*drug effects ; Stilbenes/*pharmacology

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