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issn:1976-555
1.  Tissue-specific expression of the NOD-like receptor protein 3 in BALB/c mice 
Journal of Veterinary Science  2014;15(2):173-177.
Activation of the innate immune system requires recognition of pathogen-associated molecular patterns, such as NOD-like receptors. The NOD-like receptor protein 3 (NLRP3) inflammasome is involved in induction of the pro-inflammatory cytokine, IL-1β, and subsequent inflammatory responses. NLRP3 inflammasome plays important roles in the inflammatory and innate immune responses associated with autoimmune/inflammatory syndrome. However, analysis of the tissue distribution and expression profiles in BALB/c mice is still incomplete. In this study, we investigated the tissue distribution and expression pattern of NLRP3 in BALB/c mice to further elucidate its function in innate immunity in this commonly used laboratory animal model. NLRP3 mRNA expression levels and tissue distribution of the protein were investigated by real-time quantitative PCR and immunohistochemical analyses, respectively. NLRP3 mRNA expression was higher in the kidney and inguinal lymph nodes than in other tissues. Cytoplasmic expression of NLRP3 was detected in the epithelial reticular cells of the spleen and thymus, lymphocytes in the inguinal lymph nodes, cardiac muscle cells, cerebral cortex neurons, alveolar macrophages, renal tubule cells and liver sinusoidal endothelial cells. The results of this study will assist investigators in interpreting site-specific functions and roles of NLRP3 in inflammatory responses.
doi:10.4142/jvs.2014.15.2.173
PMCID: PMC4087217  PMID: 24690602
BALB/c mice; gene expression profile; NLRP3; tissue distribution
2.  Effects of resveratrol on the insulin signaling pathway of obese mice 
Journal of Veterinary Science  2014;15(2):179-185.
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 α/γ(Tyr467/Tyr199), PDK1 (Ser241), GSK-3α (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.
doi:10.4142/jvs.2014.15.2.179
PMCID: PMC4087218  PMID: 24675832
Akt phosphorylation; antibody array; insulin signaling pathway; obese mice; resveratrol
3.  Computed tomographic evaluation of cervical vertebral canal and spinal cord morphometry in normal dogs 
Journal of Veterinary Science  2014;15(2):187-193.
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.
doi:10.4142/jvs.2014.15.2.187
PMCID: PMC4087219  PMID: 24136210
cervical vertebra; computed tomography; dog; spinal cord; vertebral canal
4.  Accuracy of sonographic diagnosis of pneumoperitoneum using the enhanced peritoneal stripe sign in beagle dogs 
Journal of Veterinary Science  2014;15(2):195-198.
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.
doi:10.4142/jvs.2014.15.2.195
PMCID: PMC4087220  PMID: 24136212
dog; enhanced peritoneal stripe sign; pneumoperitoneum; ultrasonography
5.  Recombinant Kluyveromyces lactis expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP5 elicits mucosal and cell-mediated immune responses in mice 
Journal of Veterinary Science  2014;15(2):199-208.
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-γ 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-γ 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.
doi:10.4142/jvs.2014.15.2.199
PMCID: PMC4087221  PMID: 24378591
GP5; immune responses; Kluyveromyces lactis yeast; PRRSV; vaccine
6.  Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus 
Journal of Veterinary Science  2014;15(2):209-216.
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.
doi:10.4142/jvs.2014.15.2.209
PMCID: PMC4087222  PMID: 24378590
chimeric virus-like particle; immune response; infectious bronchitis virus
7.  Oxidative response of neutrophils to platelet-activating factor is altered during acute ruminal acidosis induced by oligofructose in heifers 
Journal of Veterinary Science  2014;15(2):217-224.
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.
doi:10.4142/jvs.2014.15.2.217
PMCID: PMC4087223  PMID: 25013355
cow; lactic acidosis; L-selectin; neutrophils; reactive oxygen species
8.  Identification of abnormal gene expression in bovine transgenic somatic cell nuclear transfer embryos 
Journal of Veterinary Science  2014;15(2):225-231.
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.
doi:10.4142/jvs.2014.15.2.225
PMCID: PMC4087224  PMID: 24675837
bovine; embryo; development; gene expression; somatic cell nuclear transfer
9.  A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis 
Journal of Veterinary Science  2014;15(2):233-239.
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.
doi:10.4142/jvs.2014.15.2.233
PMCID: PMC4087225  PMID: 24136213
DNA extraction; Map; MGIT; paratuberculosis; PCR
10.  Isolation, in vitro propagation, genetic analysis, and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil 
Journal of Veterinary Science  2014;15(2):241-248.
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 Uberlândia, 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 Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São 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.
doi:10.4142/jvs.2014.15.2.241
PMCID: PMC4087226  PMID: 24136211
Brazil; Ehrlichia canis; genetic characterization; isolation; serology
11.  Equine hyperimmune serum protects mice against Clostridium difficile spore challenge 
Journal of Veterinary Science  2014;15(2):249-258.
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 107 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.
doi:10.4142/jvs.2014.15.2.249
PMCID: PMC4087227  PMID: 24136208
Clostridium difficile; colitis; equine; immunotherapy
12.  Extent of Mycobacterium bovis infection in dairy cattle herds subject to partial culling as determined by an interferon-gamma assay 
Journal of Veterinary Science  2014;15(2):259-265.
The interferon-gamma (IFN-γ) assay is employed as a complementary diagnostic test for bovine tuberculosis (BTB) in many countries. To simplify this assay, we established a 96-well plate format using the ESAT-6 and CFP-10 antigens and then employed it to determine the extent of Mycobacterium (M.) bovis infection in dairy herds with a history of BTB outbreaks in a country where only selective culling is practiced. The sensitivity and specificity of this IFN-γ assay were 85.9% and 100%, respectively, based on comparison with the conventional single intradermal tuberculin test (SIDT). The IFN-γ assay was also positive in 30.4% and 36.8% of SIDT-negative animals from herds with recent and remote BTB outbreaks, respectively. Of 14 SIDT-negative, IFN-γ positive cattle, five (35.7%) were culture positive and an additional six were positive based on a polymerase chain reaction-based test for M. bovis. Therefore, the IFN-γ assay has the potential to serve as a specific and sensitive test for M. bovis infection in dairy cattle. Further, the results indicated that a substantial portion of SIDT-negative animals in herds with previous BTB outbreaks were actually infected with M. bovis. Accordingly, the present selective-culling strategy may require modifications to include this more sensitive assay.
doi:10.4142/jvs.2014.15.2.259
PMCID: PMC4087228  PMID: 24378586
bovine tuberculosis; IFN-gamma assay; Mycobacterium bovis
13.  Use of aglepristone for the treatment of P4 induced insulin resistance in dogs 
Journal of Veterinary Science  2014;15(2):267-271.
Insulin resistance (IR) in dogs is suspected when hyperglycemia is present despite administration of insulin doses greater than 1.0 to 1.5 UI/kg. IR is caused by increases in counter regulatory hormones concentrations (glucagon, glucocorticoids, catecholamines and growth hormone). This study was conducted to investigate the use of aglepristone (RU 46534), a P4 receptor antagonist, for the treatment of IR diabetes mellitus in bitches during the luteal phase. All animals were treated with porcine insulin zinc suspension (Caninsulin) and aglepristone (Alizin) 10 mg/kg subcutaneously at day 1, 2, 9 and 17 from diagnosis. At day 5, no significant variation in glycemia was shown. At day 12 and 20, serum glucose concentrations were significant lower (p < 0.05). From day 12 the insulin dose was reduced to 0.8 IU BID. Insulin was reduced in the following weeks and glycemia was controlled.
doi:10.4142/jvs.2014.15.2.267
PMCID: PMC4087229  PMID: 24378588
aglepristone; bitches; diabetes mellitus; diestrus; insulin resistance
14.  Effect of superovulation on uterine and serum biochemical parameters and its potential association with transferable embryos in Holstein dairy cows 
Journal of Veterinary Science  2014;15(2):273-281.
The objective of this study was to determine the effects of superovulation (SOV) on serum and uterine biochemical parameters, uterine bacteriology and cytology and number of transferable embryos (TE). Dairy cows were placed on a Presynch/CIDR Synch protocol. The SOV group was superovulated, induced in estrus, and inseminated, whereas the control group was induced in estrus and inseminated without SOV. Uterine bacteriology and cytology and uterine and serum biochemical parameters were measured at day 7 of the estrous cycle to start the SOV protocol, as well as on the day of embryo recovery (DER). The SOV group produced 7.5 ± 6.7 oocytes/embryos, of which 3.4 ± 4.7 were TE. Serum urea and E2 and uterine Glu, CK, LDH, TP, P4 and PGFM in the control group and serum P4 and PGFM and uterine LDH and PGFM in the SOV group were significantly higher (p < 0.01) at DER than day 7. At DER, uterine urea, LDH, PGFM and TP and serum urea, LDH, PGFM, and P4 concentrations were higher (p < 0.01) in the SOV group than the control. There was no significant variation in uterine bacteriology or cytology. Overall, these results infer that SOV affects both serum profile and uterine secretions, and that these changes may influence the number of TE.
doi:10.4142/jvs.2014.15.2.273
PMCID: PMC4087230  PMID: 24675828
biochemical parameters; dairy cows; superovulation; transferable embryos
15.  Retrospective surveillance of metabolic parameters affecting reproductive performance of Japanese Black breeding cows 
Journal of Veterinary Science  2014;15(2):283-288.
This retrospective study was conducted to confirm the relationship between pre- and postpartum metabolic parameters and postpartum reproductive performance and to clarify seasonal characteristics of the metabolic parameters by using our metabolic profile test (MPT) database of Japanese Black breeding herds. In evaluation 1, MPT databases of blood samples from multiparous cows collected prepartum and postpartum were divided into two groups according to calving interval, and each MPT parameter was compared. In evaluation 2, the same MPT databases used in evaluation 1 were divided into two groups according to the sampling period. Significant differences were found in the prepartal total protein and postpartal γ-glutamyltransferase in evaluation 1. In evaluation 2, significant differences were found in the prepartal and postpartal total protein, albumin/globulin ratio, and glucose. Clear seasonal differences in MPT results emphasized the usefulness of the MPT in breeding cattle herds fed home-pasture roughage and suggest that unsatisfactory reproductive performance during hot periods reflects inadequate nutritional content of the diet and possible reduced feed intake due to heat stress.
doi:10.4142/jvs.2014.15.2.283
PMCID: PMC4087231  PMID: 24675835
Japanese Black cattle; metabolic parameters; reproductive performance; retrospective surveillance
16.  Comparable bone healing capacity of different bone graft matrices in a rabbit segmental defect model 
Journal of Veterinary Science  2014;15(2):289-295.
We compared the bone healing capacity of three different demineralized bone matrix (DBM) products applied using different carrier molecules (hyaluronic acid [HA] vs. carboxymethylcellulose [CMC]) or bone compositions (cortical bone vs. cortical bone and cancellous bone) in a rabbit segmental defect model. Overall, 15-mm segmental defects in the left and right radiuses were created in 36 New Zealand White rabbits and filled with HA-based demineralized cortical bone matrix (DBX), CMC-based demineralized cortical bone matrix (DB) or CMC-based demineralized cortical bone with cancellous bone (NDDB), and the wound area was evaluated at 4, 8, and 12 weeks post-implantation. DBX showed significantly lower radiopacity, bone volume fraction, and bone mineral density than DB and NDDB before implantation. However, bone healing score, bone volume fraction, bone mineral density, and residual bone area at 4, 8, and 12 weeks post-implantation revealed no significant differences in bone healing capacity. Overall, three DBM products with different carrier molecules or bone compositions showed similar bone healing capacity.
doi:10.4142/jvs.2014.15.2.289
PMCID: PMC4087232  PMID: 24675830
bone graft substitutes; bone regeneration; carboxymethylcellulose; hyaluronic acid; rabbit
17.  Evaluation of endoscopically obtained duodenal biopsy samples from cats and dogs in an adapter-modified Ussing chamber 
Journal of Veterinary Science  2014;15(2):297-307.
This study was conducted to evaluate an adapter-modified Ussing chamber for assessment of transport physiology in endoscopically obtained duodenal biopsies from healthy cats and dogs, as well as dogs with chronic enteropathies. 17 duodenal biopsies from five cats and 51 duodenal biopsies from 13 dogs were obtained. Samples were transferred into an adapter-modified Ussing chamber and sequentially exposed to various absorbagogues and secretagogues. Overall, 78.6% of duodenal samples obtained from cats responded to at least one compound. In duodenal biopsies obtained from dogs, the rate of overall response ranged from 87.5% (healthy individuals; n = 8), to 63.6% (animals exhibiting clinical signs of gastrointestinal disease and histopathological unremarkable duodenum; n = 15), and 32.1% (animals exhibiting clinical signs of gastrointestinal diseases and moderate to severe histopathological lesions; n = 28). Detailed information regarding the magnitude and duration of the response are provided. The adapter-modified Ussing chamber enables investigation of the absorptive and secretory capacity of endoscopically obtained duodenal biopsies from cats and dogs and has the potential to become a valuable research tool. The response of samples was correlated with histopathological findings.
doi:10.4142/jvs.2014.15.2.297
PMCID: PMC4087233  PMID: 24378587
canine; feline; short circuit current; transport physiology; Ussing chamber
18.  Accuracy of capillary blood 3-β-hydroxybutyrate determination for the detection and treatment of canine diabetic ketoacidosis 
Journal of Veterinary Science  2014;15(2):309-316.
In human medicine, diagnosis of diabetic ketoacidosis (DKA) is usually based on measurement of capillary 3-β-hydroxybutyrate (3-HB) with a hand held ketone sensor. This study was conducted to determine if measurement of capillary 3-HB could be useful for the diagnosis and monitoring of canine DKA. Fifteen dogs with diabetic ketosis and 10 with DKA were evaluated. Paired measurements of 3-HB of capillary and venous blood samples were analysed by the electrochemical sensor and reference method. Use of capillary 3-HB measurement during DKA management was then evaluated through simultaneous measurements of capillary 3-HB, urinary AcAc and venous blood gas analysis. Good agreement between capillary and venous 3-HB measurement was detected by the electrochemical sensor and reference method. Monitoring treatment of DKA revealed a significant correlation between capillary 3-HB and acidosis markers, while no significant correlation was observed between AcAc and acidosis markers. A cut-off value of capillary blood 3-HB >3.8 mmol/L for diagnosis of DKA resulted in 70% and 92% sensitivity and specificity. The electrochemical sensor accurately measures 3-HB concentration in both capillary and venous blood samples, is accurate in diagnosing canine DKA, and appears to reflect the patient's metabolic status during DKA treatment.
doi:10.4142/jvs.2014.15.2.309
PMCID: PMC4087234  PMID: 24675829
acetoacetate; diabetes mellitus; dog; ketone bodies
19.  Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle 
Journal of Veterinary Science  2014;15(2):317-325.
A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on Tm values of 85.03 ± 0.54℃ for stx1 and 87.47 ± 0.35℃ for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/µL), and quantifiable (R2 = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.
doi:10.4142/jvs.2014.15.2.317
PMCID: PMC4087235  PMID: 24675834
cattle farm; E. coli O157; LAMP; shiga toxin; stx
20.  Distribution, quantitative load and characterization of Salmonella associated with swine farms in upper-northern Thailand 
Journal of Veterinary Science  2014;15(2):327-334.
This study was conducted to analyze the prevalence and quantitative loads of Salmonella spp. on pig farms in Chiang Mai, Lamphun, Thailand to assess loading levels before slaughtering. The serotype diversity, antimicrobial-resistance pattern and pulse-field type of Salmonella spp. were also characterized to assess the dynamic propagation of the pathogen. The Salmonella-positive prevalence was 246/805 (30.56%), and the quantitative loads varied from 1.48~4.04 Log10MPN/g, with a mean ± standard deviation of 2.11 ± 0.57. AMP/S/TE (ampicillin/streptomycin/tetracycline) was the highest frequency antimicrobial resistance pattern found in this study. In addition, Salmonella Rissen was the primary serotype in this region. PFGE results indicated the occurrence of infection by cross contamination among pig farms. Our study showed that pork is easily contaminated with this pathogen. Farm control programs must be based on strict biosecurity and hygienic measures, which could further reduce the contamination pressure at slaughterhouses or retail shops.
doi:10.4142/jvs.2014.15.2.327
PMCID: PMC4087236  PMID: 24675839
characterization; pig; prevalence; quantitative load; Salmonella
21.  An overview of calf diarrhea - infectious etiology, diagnosis, and intervention 
Journal of Veterinary Science  2014;15(1):1-17.
Calf diarrhea is a commonly reported disease in young animals, and still a major cause of productivity and economic loss to cattle producers worldwide. In the report of the 2007 National Animal Health Monitoring System for U.S. dairy, half of the deaths among unweaned calves was attributed to diarrhea. Multiple pathogens are known or postulated to cause or contribute to calf diarrhea development. Other factors including both the environment and management practices influence disease severity or outcomes. The multifactorial nature of calf diarrhea makes this disease hard to control effectively in modern cow-calf operations. The purpose of this review is to provide a better understanding of a) the ecology and pathogenesis of well-known and potential bovine enteric pathogens implicated in calf diarrhea, b) describe diagnostic tests used to detect various enteric pathogens along with their pros and cons, and c) propose improved intervention strategies for treating calf diarrhea.
doi:10.4142/jvs.2014.15.1.1
PMCID: PMC3973752  PMID: 24378583
calf diarrhea; etiology; intervention
22.  Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells 
Journal of Veterinary Science  2014;15(1):111-115.
Molecular mechanisms underlying the effects of Fyn on cell morphology, pseudopodium movement, and cell migration were investigated. The Fyn gene was subcloned into pEGFP-N1 to produce pEGFP-N1-Fyn. Chinese hamster ovary (CHO) cells were transfected with pEGFP-N1-Fyn. The expression of Fyn mRNA and proteins was monitored by reverse transcription-PCR and Western blotting. Additionally, transfected cells were stained with 4',6-diamidino-2-phenylindole and a series of time-lapse images was taken. Sequences of the recombinant plasmids pMD18-T-Fyn and pEGFP-N1-Fyn were confirmed by sequence identification using National Center for Biotechnology Information in USA, and Fyn expression was detected by RT-PCR and Western blotting. The morphology of CHO cells transfected with the recombinant vector was significantly altered. Fyn expression induced filopodia and lamellipodia formation. Based on these results, we concluded that overexpression of mouse Fyn induces the formation of filopodia and lamellipodia in CHO cells, and promotes cell movement.
doi:10.4142/jvs.2014.15.1.111
PMCID: PMC3973753  PMID: 24378585
filopodia; Fyn; lamellipodia; time-lapse
23.  Photodynamic hyperthermal chemotherapy with indocyanine green: a novel cancer therapy for 16 cases of malignant soft tissue sarcoma 
Journal of Veterinary Science  2014;15(1):117-123.
Sixteen cases of malignant soft tissue sarcoma (STS; 10 canines and six felines) were treated with a novel triple therapy that combined photodynamic therapy, hyperthermia using indocyanine green with a broadband light source, and local chemotherapy after surgical tumor resection. This triple therapy was called photodynamic hyperthermal chemotherapy (PHCT). In all cases, the surgical margin was insufficient. In one feline case, PHCT was performed without surgical resection. PHCT was performed over an interval of 1 to 2 weeks and was repeated three to 21 times. No severe side effects, including severe skin burns, necrosis, or skin suture rupture, were observed in any of the animals. No disease recurrence was observed in seven out of 10 (70.0%) dogs and three out of six (50.0%) cats over the follow-up periods ranging from 238 to 1901 days. These results suggest that PHCT decreases the risk of STS recurrence. PHCT should therefore be considered an adjuvant therapy for treating companion animals with STS in veterinary medicine.
doi:10.4142/jvs.2014.15.1.117
PMCID: PMC3973754  PMID: 24136207
cancer; chemotherapy; hyperthermia; PDT; soft tissue sarcoma
24.  Prostaglandin F receptor expression in intrauterine tissues of pregnant rats 
Journal of Veterinary Science  2014;15(1):125-131.
In this investigation, we studied the expression and localization of rat prostaglandin F (FP) receptor in uterine tissues of rats on gestational Days 10, 15, 18, 20, 21, 21.5 and postpartal Days 1 and 3 using Western blotting analysis, real-time PCR, and immunohistochemistry. A high level of immunoreactivity was observed on gestational Days 20, 21, and 21.5 with the most significant signals found on Day 20. FP receptor protein was expressed starting on gestational Day 15, and a fluctuating unsteady increase was observed until delivery. Uterine FP receptor mRNA levels were low between Days 10 and 18 of gestation (p < 0.05). The transcript level increased significantly on Day 20 and peaked on Day 21.5 just before labor (p < 0.05). There was a positive correlation between FP receptor mRNA expression and serum estradiol levels (rs = 0.78; p < 0.01) along with serum estradiol/progesterone ratios (rs = 0.79; p < 0.01). In summary, we observed an increase FP receptor expression in rat uterus with advancing gestation, a marked elevation of expression at term, and a concominant decrease during the postpartum period. These findings indicate a role for uterine FP receptors in the mediation of uterine contractility at term.
doi:10.4142/jvs.2014.15.1.125
PMCID: PMC3973755  PMID: 24136214
immunohistochemistry; pregnancy; prostaglandin F receptor; rat; uterus
25.  Regulation of matrix metalloproteinase-9 protein expression by 1α,25-(OH)2D3 during osteoclast differentiation 
Journal of Veterinary Science  2014;15(1):133-140.
To investigate 1α,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1α,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1α,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1α,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.
doi:10.4142/jvs.2014.15.1.133
PMCID: PMC3973756  PMID: 24136216
1α,25-(OH)2D3; bone lacunar resorption; MMP-9; osteoclast; TRAP

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