Validation and standardization of virus neutralizing test using indirect immunoperoxidase technique for the quantification of antibodies to rabies virus

A virus neutralizing test using an indirect immunoperoxidase technique (VNT-IIP) for rabies has been developed for the titration of dog and cat serum samples in Japan. The VNT-IIP has the advantage that results obtained can be viewed by the naked eye. The purpose of this study was to validate the VNT-IIP and compare it with one of the international standard methods, the fluorescent antibody virus neutralization test (FAVNT). The VNT-IIP showed satisfactory repeatability, high analytical specificity and good accuracy. Regarding the comparison between the VNT-IIP and the FAVNT, the VNT-IIP showed good agreement (91.9%), high sensitivity (92.8%) as well as specificity (87.0%) and good correlation (r = 0.92). As described above, the validation of the VNT-IIP was satisfactory and the performances of the test proved to be equivalent to those of an international standard method.     

Multiple hepatic peribiliary cysts in a young pig

Histopathologic features of hepatic peribiliary cysts were described in a young slaughtered pig. The animal was an apparently healthy 6-month-old pig of mixed breed. Macroscopically, all lobes of the liver contained numerous cysts of varying size containing serous fluid in all lobes. Histopathologically, the cysts were located mainly around the large bile duct and in the connective tissue of the portal tracts. Within serial sections, these cysts were assumed to be solitary or multilocular, but they were separated from the bile duct. The cysts were lined by a single layer of columnar, cuboidal, and flattened epithelial cells. Occasionally, goblet cells were observed. The epithelial cells were stained with periodic acid-Schiff/alcian blue and high-iron diamine/alcian blue, indicating the presence of neutral mucin, sialomucin, and sulfomucin. Grimalius' method revealed the presence of endocrine cells in the lining epithelium. There was no bile pigment in the cysts by the Hall method.     

Distribution of ectomycorrhizas and ectomycorrhizal fungal inoculum with soil depth in a birch forest

The distributions of ectomycorrhizas and ectomycorrhizal fungal inoculum with soil depth (0–45 cm) were determined in a 40-year-oldBetula platyphylla var.japonica forest. Mycorrhizal and non-mycorrhizal fine roots were measured in each soil core sample that was collected at soil depths of 0–5, 5–10, 10–15, 15–20, 20–25, 30–35, and 40–45 cm. The ectomycorrhizas were mainly distributed (>50%) in the top soil (0–5 cm) of organic forest floor horizons. Below 5 cm the quantity of ectomycorrhizas decreased sharply. The percentage of fine roots which were ectomycorrhizal gradually declined with the depth of soil. The ectomycorrhizal fungal inoculum was evaluated by a bioassay method, measuring the lengths of the entire root system and of the ectomycorrhizal roots of birch seedlings planted in each soil sample. The soil samples were collected from 0–5, 10–15, 20–25, 30–35, and 40–45 cm depths of the soil profile. Ectomycorrhizal formation on birch seedling roots in the bioassay was high in both the soil depth intervals 0–5 cm and 10–15 cm, while the amount was lower in the soil depth interval from 20–45 cm. The results of these investigations show that the amount of the ectomycorrhizas in soil, and the ectomycorrhizal fungal inoculum potential as determined by bioassay, are not always consistent with each other.     

Histopathological and immunohistochemical analysis of the endocrine and exocrine pancreas in twelve cattle with insulin-dependent diabetes mellitus (IDDM).

Histological and immunohistochemical studies were carried out on the pancreas of twelve cattle of insulin-dependent diabetes mellitus (IDDM). They showed clinical signs such as persistent hyperglycemia, glycosuria and decreased glucose tolerance, and some cases accompanied with or without ketonuria. Histopathologically, eight cattle were diagnosed as chronic IDDM, while others were acute IDDM. The most characteristic lesions of the pancreas in chronic IDDM showed a decrease in the size and number of pancreatic islets, interlobular and interacinar fibrosis, mild lymphocytic insulitis, and vacuolation of a few islets. Almost all cells in the atrophied islets had a small amount of ungranulated cytoplasm. Immunohistochemical examination revealed that the atrophied islet cells did not react to anti-insulin antibody, but occasionally reacted to anti-glucagon or somatostatin antibodies. A few solitary islets with mild lymphocytic infiltration, necrotic islets with occasional calcification, and atrophied islets with mild fibrosis were also observed. A few islets consisted of many islet cells with vacuolated cytoplasm including a small number of insulin-positive granules. Accumulation of glycogen granules was occasionally observed in these islets. Islet fibrosis was due to the proliferation of collagen fibers reactive to both anti-collagen type I and type III antibodies. In acute IDDM, the major islets consisted of the cells with vacuolated cytoplasm indicating the degranulation of islet cells. These islets contained many islet cells with shrunken cytoplasm and karyorrhectic nuclei. Lymphocytic infiltration was frequently observed in the islets which consisted of many islet cells having karyorrhectic nuclei and vacuolated and severely degranulated cytoplasm. Immunohistochemically, islet cells with vacuolated cytoplasm had a small amount of insulin-positive granules, suggesting severe degranulation of beta-cells. An increase in acinar islet-cells and proliferation of ductal epithelial cells showing insulin-immunoreactivity were observed. Bovine IgG-immunoreactive islet cells were frequently seen in the vacuolated islets. In summary, pathological observations suggested that beta-cells were being destroyed by an inflammatory process which selectively affected the pancreatic islets. Lymphocytic insulitis and anti-bovine immunoreactive islet cells were thought to be the most significant changes in determining the etiology and pathogenesis of bovine IDDM, and suggested their role in anti-islet autoimmunity in this form of diabetes.     

Molecular Structure and Some Physicochemical Properties of High‐Amylose Barley Starches

The molecular structure and some physicochemical properties of starches from two high‐amylose cultivars of barley, high‐amylose Glacier A (HAG‐A) and N (HAG‐N), were examined and compared with those of a normal cultivar, Normal Glacier (NG). The true amylose contents of HAG‐A, HAG‐N, and NG were 41.0, 33.4, and 23.0%, respectively. Iodine affinities before and after defatting of starch, and thermograms of differential scanning calorimetry, indicated that HAG‐A and HAG‐N starches had a higher proportion of amylose‐lipid complex than did NG starch. The amylopectins from HAG‐A and HAG‐N were similar to NG amylopectin in average chain length (18–19), β‐amylolysis limit (β‐AL 56–57%), number‐average degrees of polymerization (DP_n 6,000–7,500) and chain length distribution. Very long chains (1–2%) were found in amylopectins from all cultivars. HAG‐A amylopectin had a larger amount of phosphorus (214 ppm) than the others. The amyloses from HAG‐A and HAG‐N resembled NG amylose in DP_n (950–1,080) and β‐AL (70–74%). However, HAG‐A and HAG‐N had a larger number of chains per molecule (NC 2.4–2.7) than NG amylose (1.8) and contained the branched amylose with a higher NC (9.5–10.6) than that of NG amylose (5.8), although molar fractions of the branched amylose (15–20%) were similar.     

A pleomorphic adenoma of the lacrimal gland in a dog

A 13-year-old female mongrel dog had a pleomorphic adenoma of the lacrimal gland in the right upper orbit. The tumor measured 3.8 x 3.0 x 3.3 cm, appeared white, round, and firm, and pressed the right globe and surrounding tissues. Histopathologically, the tumor had a thin connective tissue capsule and was composed of tubules with two cell types, some resembling luminal epithelial cells making up the tubular structures and the other of myoepithelial cells. Epithelial tubules were disposed in an adenomatous fashion and separated from each other by proliferating pleomorphic myoepithelial cells. Immunohistochemically, large numbers of the luminal epithelial cells revealed an immunopositive reaction against keratin/cytokeratin (AE1/AE3), and some epithelial cells reacted against cytokeratin 14. Spindle-shaped myoepithelial cells revealed an immunopositive reaction against cytokeratin 14, alpha-smooth muscle actin, and vimentin. A small number of myoepithelial cells reacted against desmin. S-100 protein immunopositivity was frequently found in luminal epithelial cells and rarely in the pleomorphic myoepithelial cells. Glial fibrillary acidic protein positivity was commonly found in myoepithelial cells, myxoid matrices, and intracystic materials, but not in luminal epithelial cells.     

Proper reprogramming of imprinted and non‐imprinted genes in cloned cattle gametogenesis

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non‐imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non‐cloned bulls. We found no differences between cloned and non‐cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha‐satellite and Art2) in oocytes recovered from cloned and non‐cloned cows. Again, no significant differences were observed between clones and non‐clones. These results suggested that imprinted and non‐imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring.     

Polymerase chain reaction-restriction fragment length polymorphism analysis of the SzP gene of Streptococcus zooepidemicus isolated from the respiratory tract of horses

OBJECTIVE: To develop polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for molecular typing of strains of Streptococcus zooepidemicus and to use the new typing method to analyze a collection of isolates from the respiratory tract of Thoroughbreds. SAMPLE POPULATION: 10 strains of S zooepidemicus, 65 isolates from the respiratory tract of 9 yearlings following long distance transportation, and 89 isolates from tracheal aspirates of 20 foals with pneumonia. PROCEDURE: Phenotypic variations in the SzP protein were detected by western immunoblot analysis. Using PCR-RFLP analysis, genotypes were obtained with primer sets from the SzP gene, followed by restriction endonuclease digestion of the amplicons. RESULTS: Unique genotypic patterns were obtained with a primer set designed from both ends of the structural gene and the restriction endonuclease DdeI. Forty-five isolates from the lymphoid tissue within the pharyngeal recess (ie, pharyngeal tonsil) of yearlings included 10 SzP genotypes and SzP phenotypes. Isolates from the trachea of each yearling were of a single genotype that was also present among isolates from the pharyngeal tonsil of the same horses. Isolates from tracheal aspirates of foals belonged to 14 genotypes. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of the SzP gene by use of PCR-RFLP was effective for molecular typing of strains of S zooepidemicus in the study of respiratory tract disease in horses. Results of PCR-RFLP analysis indicate that a single strain of S zooepidemicus can migrate from the pharyngeal tonsil to the trachea at a high rate in horses undergoing long distance transportation.     

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC₅₀ of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC₅₀s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.     

The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The approximately 3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.