A new Brucella canis species-specific PCR assay for the diagnosis of canine brucellosis

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis. The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6 pg/μl by DNA dilution, or 3 × 10³ colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis. Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis, and the new assay will provide a valuable tool for the diagnosis of B. canis infection.     

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.     

Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus

The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.     

Pharmacokinetics of tramadol and its major metabolite after intramuscular administration in piglets

Tramadol (T) is a centrally acting atypical opioid used for treatment of dogs. Piglets might experience pain following castration, tooth clipping and tail docking and experimental procedures. The aim of this study was to assess the pharmacokinetics of T and its active metabolite M1 in male piglets after a single intramuscular injection. Six healthy male piglets were administered T (5 mg/kg) intramuscularly. Blood was sampled at scheduled time intervals and drug plasma concentrations evaluated by a validated HPLC method. T plasma concentration was quantitatively detectable from 0.083 to 8 h. M1 was quantified over a shorter time period (0.083–6 h) with a T_max at 0.821 h. The study demonstrated that piglets produce a larger amount of M1 compared with dogs, horses and goats. The human minimum effective concentration of M1 (40 ng/mL) was exceeded for over 3 h in piglets. If it is assumed to also apply to piglets, it could be speculated that the drug efficacy might exert its action over 3 h or longer. This assumption has to be confirmed by further specific pharmacokinetic/pharmacodynamic studies.     

Determination of Feeder Cell‐Based Cellular Niches Supporting the Colonization and Maintenance of Spermatogonial Stem Cells from Prepubertal Domestic Cat Testes

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.     

Effects of lactulose supplementation on performance,blood profiles,excreta microbial shedding of Lactobacillus and Escherichia coli,relative organ weight and excreta noxious gas contents in broilers

This study was to evaluate the effects of lactulose supplementation on performance, blood profiles, excreta microbial shedding of Lactobacillus and Escherichia coli, relative organ weight and excreta noxious gas contents in broilers. A total of 720 ROSS 308 broilers with a body weight of 46 ± 0.1 g (1 day of age) were used in a 28‐d experiment. Broilers were randomly allotted to 4 experiment diets with 12 replicate pens and 15 birds per pen. Dietary treatments were as follows: NC, negative control (without antibiotic); PC, NC + 0.1% tiamulin; L1, NC + 0.1% lactulose; and L2, NC + 0.2% lactulose. Broilers were fed with phase 1 (1–8 day), phase 2 (9–18 day) and phase 3 (19–28 day) diets in the form of mash. During day 1–8, broilers fed the PC and L2 diets had higher (p < 0.05) body weight gain than those fed the NC diet. During day 19–28, broilers fed the L1 and L2 diets had lower (p < 0.05) feed intake than those fed the NC diet. The feed conversion ratio (FCR) was decreased (p < 0.05) in L1 treatment compared with NC treatment. Overall, the FCR was improved (p < 0.05) in all supplementation treatments compared with NC treatment. The apparently metabolizable nitrogen in L1 treatment was higher (p < 0.05) than that in NC treatment at day 28. The excreta Lactobacillus was increased and E. coli was decreased in PC and L2 treatments compared with NC treatment at day 28 (p < 0.05). The excreta NH₃, H₂S and acetic acid contents were decreased (p < 0.05) in L1 and L2 treatments compared with NC treatment. The relative weight of abdominal fat of broilers fed the PC diet was lowest (p < 0.05) compared with other treatments. In conclusion, this study indicated that dietary supplementation of 0.1% or 0.2% lactulose could improve growth performance, decrease excreta E. coli and excreta NH₃ and H₂S contents.     

First discovery of a cave-dwelling Tineid moth （Lepidoptera,Tineidae） from East Asia

In this study, we report Monopis crocicapitella （Clemens, 1859） （Tineidae）, which was collected from bat guano in a cave in the southern region of Korea, for the first time from East Asia. We briefly redescribe and illustrate the external morphology and genital structures of both sexes. Also, we discuss the local habitat characteristics and some of the ecological information that was observed during our field investi-gation.     

Fire performance of carbonized medium density fiberboard manufactured at different temperatures

Authors established a new manufacturing technology for crack-free carbonized boards by pressing and carbonizing the medium-density fiberboard. Industrialization of new functional carbon materials was performed by investigating the fundamental properties of the carbonized boards. To be used as a construction material, the carbonized board needs to satisfy the fire performance regulation. In this study, the carbonized boards were manufactured from medium-density fiberboard (c-MDF) at different temperatures and then fire performance including flame retardancy and smoke toxicity was analyzed using a cone calorimeter and noxious gas analyzer. The results show that as the carbonization temperature increases, weight loss ratio decreases and flame retardancy increases. In the c-MDF at 800 and 1000 °C, no external damage was observed after combustion. These c-MDFs satisfy the total heat release (standard below 8 MJ/m²) and heat release rate (standard below 200 kW/m²) regulations according to the Building Standard Law of Korea and Japan. In addition, the c-MDFs showed the lower total smoke release (TSR, 0.213 m²/m²) than that of virgin MDF (94.281 m²/m²). The c-MDF at 800 and 1000 °C were, therefore, classified as a class III flame retardancy material and can be used as indoor finishing material.     

The study on the cytology and histochemistry of lymphoid organ spheroids in Penaeus chinensis

The lymphoid organ spheroids (LOS) were investigated by cytological and histochemical techniques in Penaeus chinensis. The results showed that the LOS only appeared in the individuals with relatively large lymphoid organ (long diameter>2 mm.). The LOS were mainly made up of two types of cells, one of which had a low cytoplasm to nuclear volumetric ratio, developed marginated chromatin and 1–2 nucleoli, while the other had large cytoplasm to nuclear volumetric ratio, homogeneous chromatin, and no or only one nucleolus. These two kinds of cells all contained mitochondria and rough endoplasmic reticulum. The necrotic cells could usually be observed. The LOS showed negative PAS reaction, weak positive phenoloxidase, and negative -naphthyl-acetate esterase (-NAE).     

Reevaluation of the Dietary Protein Requirement of Japanese Flounder Paralichthys olivaceus

An experiment was conducted to determine the dietary protein requirement by different analysis methods and to study the effects of dietary protein levels on growth performance and body composition in Japanese flounder Paralichthys olivaceus fed white fish meal and casein-based diets for 8 wk. After a 1-wk conditioning period, one of six isocaloric diets containing 30, 36, 42, 48, 54, and 60% crude protein (CP) was fed to fish at approximately 4–5% of wet body weight on a dry matter basis to triplicate groups of 15 fish averaging 13.3 ± 0.06 g (mean ± SD). After 8 wk of the feeding trial, weight gain (WG) and feed efficiency (FE) from fish fed 48% CP diet were similar to those from fish fed 42% and 54% CP diets, and were significantly higher than those from fish fed 30, 36 and 60% CP diets ( P < 0.05). Fish fed 48 and 54% CP diets had a significant higher specific growth rate (SGR) than did fish fed 30 and 36% CP diets ( P 0.05). Protein efficiency ratio (PER) was inversely related to the dietary protein level. No significant differences existed in hematocrit (PCV) and survival rate among the dietary treatments. Broken-line model analysis indicated that the optimum dietary protein level could be 44.0 ± 3.0% for maximum WG in Japanese flounder. Polynomial regression analysis of the dose-response showed that maximum WG occurred at 50.2% ( R² = 0.94) based on WG, and the second-order polynomial regression analysis with 95% confidence limits revealed that the range of minimum protein requirement was between 38.9% and 40.3% based on WG. Therefore, these findings suggest that the optimum dietary protein requirement for maximum growth of Japanese flounder is greater than 40%, but less than 44% CP in the fish meal and casein-based diets containing 17.0 kJ/g of energy.