A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.     

Immunohistochemical evaluation of a malignant intestinal carcinoid in a dog

An intestinal carcinoid with multiple metastases was identified in a 5-year-old male Shih Tzu with a clinical history of anemia, fatigue, anorexia, vomiting, intermittent diarrhea, intestinal bleeding, and progressive emaciation. There was a yellowish-white mass 15 mm in diameter in the anterior jejunum and white nodules consistent with metastases in many organs. Histopathologically, the mass consisted of neoplastic cells arranged in lobules, trabeculae, or closely interdigitating islands of cells. Neoplastic cells were generally polygonal with round hyperchromatic nuclei, modest amounts of eosinophilic cytoplasm, and eosinophilic cytoplasmic granules. Mitoses were common. Rosette formations of tumor cells were apparent in metastatic tumors. Immunohistochemically, tumor cells stained positive for cytokeratin 13, synaptophysin, protein gene product 9.5, neuron-specific enolase, chromogranin A, calcitonin gene-related peptide, serotonin (5-HT), and Leu-7. Serum 5-HT concentrations for this dog were increased 10-fold compared with those of normal dogs. All findings were consistent with a diagnosis of a malignant intestinal carcinoid.     

Kinetics of endotoxin concentration and tumor necrosis factor-alpha,interleukin-1beta,and interleukin-6 activities in the systemic and portal circulation during small intestinal ischemia and reperfusion in dogs

OBJECTIVE: To determine whether small intestinal ischemia and reperfusion induces bacterial translocation and proinflammatory cytokine response in either the systemic or portal circulation in dogs. ANIMALS: 17 healthy adult Beagles. PROCEDURE: The superior mesenteric artery (SMA) was occluded for 0 (group-3 dogs), 30 (group-1 dogs), or 60 (group-2 dogs) minutes, followed by reperfusion for 180 minutes; serum lactate and endotoxin concentrations and tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 activities in the systemic and portal circulation and intramucosal pH were measured at various time points. RESULTS: In group-2 dogs, TNFalpha activity was found to be significantly increased in the portal circulation, peaking at 60 minutes of reperfusion; TNF-alpha activity, in the systemic circulation, gradually increased from 60 minutes of reperfusion to the end of the experiment; however, the increase was not significant. In group-1 and -2 dogs, IL-6 activities significantly and gradually increased in the systemic and portal circulation during the reperfusion phase, and the magnitude of these increases was dependent on the duration of the ischemic phase. There were no significant changes in IL-1beta activity or endotoxin concentration in any dog group. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the our study indicate that intestinal ischemia and reperfusion leads to significant increases of the circulating TNF-alpha and IL-6 activities, depending on the duration of the ischemia phase, in the absence of detectable endotoxin in the circulation. This finding suggests that intestinal ischemia and reperfusion induces a systemic proinflammatory cytokine response in dogs.     

Eimeria organisms develop in the epithelial cells of equine small intestine

Histopathologic and immunohistochemical examinations were performed to determine the origin of host cells parasitized by Eimeria in the small intestines collected from five foals. Eimeria organisms at various stages (mainly microgametes and macrogametes) were frequently found in the cytoplasm of hypertrophied host cells in the lamina propria at the tips of villi of the jejunum and ileum. The cytoplasm of the host cell was immunohistochemically positive for cytokeratin AE1/AE3 and cytokeratin 13 and was negative for vimentin, desmin, alpha-smooth muscle actin, chromogranin A, neuron-specific enolase, and factor VIII. The host cells parasitized by Eimeria species had the immunostaining characteristics of epithelial cells but not of mesenchymal cells, endothelial cells of lacteals or capillaries, smooth muscle cells or neuroendocrine cells. These results suggest that the host cell of Eimeria species is possibly derived from intestinal epithelial cells and then displaced into the lamina propria of the small intestine.     

Properties of acetolactate synthase from sulfonylurea-resistant Scirpus juncoides Roxb. var. ohwianus T. Koyama

Properties of acetolactate synthase (EC 4.1.3.18; ALS) from sulfonylurea-resistant (SUR) Scirpus juncoides Roxb. var. ohwianus T. Koyama were studied biochemically and physiologically in comparison with those from sulfonylurea-susceptible weed (SUS). GR₅₀ values for growth inhibition and I₅₀ values for ALS inhibition by imazosulfuron were determined for both SUR and SUS. Imazosulfuron controlled the SUS above 80% at the dosage more than 10 g a.i./ha but did not control the SUR at the even great dosage of 1000 g a.i./ha. The rates required for 50% growth inhibition of the SUR relative to the SUS (R/S ratio) were 271-fold. The I₅₀ value for inhibition of ALS from the SUS was 15 nM, compared to I₅₀ of >3000 nM for inhibition of ALS from the SUR. These results suggest that a resistance may due to an altered ALS that is insensitive to imazosulfuron. The K_m (pyruvate) value of ALS from the SUR was similar to the K_m for ALS from the SUS, suggesting that a mutation resulting in resistance does not change the affinity of the enzyme for pyruvate. The specific activity of the SUR ALS was similar to that of the SUS ALS, which indicates that resistance is not an over-expression of the enzyme. ALS activity from both biotypes was inhibited by isoleucine, valine, and leucine in this order. However, the SUR ALS was less sensitive to inhibition by valine than the SUS ALS.     

Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.     

Effect of volume of non-surgical embryo transfer medium on ability of porcine embryos to survive to term

Non-surgical embryo transfer is a promising method for improving efficiency in the pork industry and also for biotechnology applications, such as in vitro embryo production, transgenesis and cloning. Several groups have reported successful piglet production using an artificial insemination catheter or flexible catheter designed for this procedure; however, the efficiency of the technique is still low. The critical points that need to be addressed in order to improve this procedure are (1) the embryo deposition site and (2) volume of transfer medium associated with the embryos; however, the latter has not yet been examined systematically. In the present study, we evaluated the effect of the volume of non-surgical embryo transfer medium on the ability of porcine embryos to survive to term by using a recently produced flexible catheter. The catheter consists of a guide and an injector. Blastocysts 200-230 mum in diameter were collected from donor gilts and transferred to recipient gilts. The time required for the completion of embryo transfer using this catheter was 14.6 +/- 3.9 min. The tip of the injector was determined by laparotomy to be located in a uterine horn 20-30 cm anterior from the branching point of the uterus body. We transferred 17.0-17.3 embryos with different volumes of medium (1.6, 3.2 and 10 ml) into each of 5, 4 and 4 recipients, respectively, and pregnancy was confirmed in 4, 3 and 1 of these recipients, respectively. Three recipients in the 1.6 ml group farrowed a total of 19 piglets (4, 5 and 10 piglets, respectively). These results suggest that successful non-surgical embryo transfer is affected by the volume of transfer medium.