Quantitative loop-mediated isothermal amplification method for the detection of <Emphasis Type="Italic">Vibrio nigripulchritudo</Emphasis> in shrimp

Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60 min at 63°C. Quantitative analysis was then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification of the initial DNA concentration with a sensitivity of 10² copy numbers equivalent to 2.3 colony forming units/ml or 0.3 fg/μl. The LAMP assay was able to specifically detect two representative strains of V. nigripulchritudo, whereas other Vibrio and non-Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time (T _t) versus the log of bacterial number. A high correlation coefficient (R ² = 0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.     

Postoperative improvement of megaesophagus and esophageal motility in a 19-day-old puppy with gastroesophageal intussusception

Canine gastroesophageal intussusception (GEI) is a rare and life-threatening condition that requires prompt diagnosis and treatment. A 19-day-old Siberian Husky with a 4-day history of regurgitation was diagnosed with GEI based on the findings of computed tomography (CT) performed without anesthesia. Endoscopic reduction of intussusception was impossible; thus, surgical reduction by traction of the duodenum was performed. CT revealed improvement of megaesophagus 82 days postoperatively. Eleven months postoperatively, fluoroscopy showed recovery to nearly normal esophageal motility. Two years postoperatively, no clinical signs were reported. CT is useful to diagnose GEI in neonate puppies with poor abdominal fat and to assess the gastric edema and the anatomical association of stomach with other organs. Fluoroscopy is helpful for evaluating postoperative esophageal motility.     

Evaluation of relationship between dispersibility of soils and charge characteristics using a modified ion adsorption method; An example of soils from Wakayama Prefecture,Japan

The relationship between the charge characteristics and the dispersibility of soils from reclaimed land (highly dispersive) and adjacent forest area (physically stable) was investigated.

To evaluate the amount of soil charges quantitatively, the measurement based on cation and anion adsorption (ion adsorption method) was attempted in two ways, where special attention was paid to the solution concentration. In the conventional ion adsorption method, the treatments with concentrated solutions (1 mol L^-1) were included in the process. In the modified method, soils were treated with dilute solutions (5 mmol L^-1), in taking account of the soil solution concentration in humid temperate regions. The amount of charges derived from the modified method was smaller than that from the conventional one, especially in the high pH zone. In a preliminary experiment, the extraction of polyvalent cations from soils was found to be significantly affected by the salt concentration. Thus, the use of solutions with an excessively high concentration was considered to result in an error in the estimation of the active charges in soils which would contribute to the dispersion behavior in the field.

The applicability of the modified method for analyzing the dispersion behavior of soils was evaluated by relating the dispersibility of silts and clays at different pHs with the charge characteristics determined under the same ionic strength. The dispersion ratio of silts of the reclaimed land reached maximum values at pH 5.5, above which the ratio decreased, whereas the ratio of clays began to increase in the pH range above 6. The dispersion ratio of silts of the forest area increased in the pH range above 6, whereas that of clays remained constant at a low level in all the pH ranges below 7. The changes in the dispersibility of silt plus clay fractions corresponded to total charge characteristics determined by the modified method. Thus, the evaluation of soil charges by the modified ion adsorption method was considered to be effective to predict soil dispersibility under field conditions, indicating that it could replace electrokinetic analyses such as electrophoresis in studies on the dispersion/flocculation behavior of soils.     

Effect of Addition of Al Hydroxides on Changes in Dispersibility and Charge Characteristics of Soils

Using a soil from a newly reclaimed area where high dispersibility of fine particles had led to poor physical properties in the field, changes in dispersibility and charge characteristics after artificial precipitation of Al hydroxides onto the soil were investigated. Al hydroxides were precipitated by neutralization with NaOH of an acidic A1C1₃ solution in which the soil samples were placed.

When the titration rates were changed, no significant differences in dispersibility were observed in the resultant soils. In the systems with the addition of 2 g kg^-1 or less of A1(OH)₃, dispersion ratios of clays were almost identical with those of blank samples although the specific surface areas were reduced. In the systems with 5 g kg^-1 or more of A1(OH)₃, considerable effects of Al hydroxides on reduction of clay dispersibility were recognized (e.g. 10 g kg^-1 addition yielded less than one-third of the original ratio of the clay dispersion). A slightly larger decrease in dispersion was observed by the addition of 15 g kg^-1 or more. As the amount of added Al increased, the amount of negative charges of the soil decreased while that of positive charges increased. The charge characteristics of the system with the addition of 20 g kg^-1 of A1(OH)₃ were closer to those of the adjacent forest soil which was characterized by a very low dispersibility. It was inferred that added Al hydroxides polymerized to form Al polycation species that were not readily exchangeable, neutralizing negative charges of clays, and acting as interparticle bonding between the clays. On the other hand, forest soils were considered to have acquired a physical stability against the dispersion of fine particles as free Al oxides had been accumulated in the process of natural weathering. It was concluded that charge characteristics primarily determined the dispersion and flocculation behavior of soils and that Al hydroxides were important modifiers of charge characteristics of soils.     

Influence of ventral fixation techniques on atlantoaxial joint fusion in canine models with dens partial resection

We evaluated the completeness of bony fusion of the atlantoaxial joint (AAJ) through polymethylmethacrylate fixation (PMF) and atlantoaxial plate fixation (APF) using six canine models with dens partial resection. In both groups, the hydroxyapatite content at the AAJ was measured up to 7 months postoperatively using quantitative computed tomography. Histological assessment revealed fibrous fusion in the PMF group. Meanwhile, in the APF group, only one dog achieved fibrous fusion, whereas the remaining three showed bony fusion. To our knowledge, this study was the first to evaluate AAJ fusion histologically after PMF and APF. The present study demonstrates that PMF and APF may stabilize the AAJ without clinical complications. Therefore, PMF and APF are clinically useful fixation methods for atlantoaxial instability.     

Minimum alveolar concentration for blunting adrenergic responses (MAC-BAR) of sevoflurane in dogs

It is well known that heart rate or arterial blood pressure may increase in response to surgical stimulation despite the absence of a purposeful movement. However, there is limited information regarding anesthetic requirement for blunting adrenergic response in dogs. This study was designed to compare the minimum alveolar concentrations of sevoflurane required to prevent autonomic response (MAC-BAR) and purposeful movement (MAC) in dogs. Sevoflurane MAC-BAR and MAC were determined in 5 beagle dogs by judging dogs' response to a noxious electrical stimulus applied to the gingiva. The sevoflurane MAC-BAR was significantly higher than MAC (3.33 ± 0.48 vs 2.10 ± 0.28%, P=0.005). These results suggested that autonomic responses occurred at sevoflurane anesthetic concentrations at which purposeful movements were absent.     

Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan

A Lancefield serological group C Streptococcus sp. was isolated from cultured amberjack, Seriola dumerili Risso, and yellowtail, Seriola quinqueradiata Temminck and Schlegel, immunized with Lactococcus garvieae commercial vaccines in Japan. The isolated bacteria were Gram-positive cocci, auto-aggregating in saline, morphologically long chains in growth medium, catalase negative and alpha-haemolytic on blood agar. An almost complete gene sequence of the 16S rDNA of two isolates was determined and compared with that of bacterial strains in the database. The isolates were identified as Streptococcus dysgalactiae based on the results of the 16S rDNA sequence, the bacteriological properties and the Lancefield serological grouping. Oligonucleotide primers specifically designed for the 16S-23S rDNA intergenic spacer region of S. dysgalactiae amplified a gene from all the fish isolates, as well as the type strains alpha-haemolytic S. dysgalactiae subsp. dysgalactiae ATCC430738 and beta-haemolytic S. dysgalactiae subsp. equisimilis ATCC35666, but not those of S. equi ATCC33398, Lactococcus garvieae ATCC43921 and L. garvieae KG9408. The severe necrotic lesions of the caudal peduncle seen in experimentally infected fish were similar to those seen in naturally infected fish.