Determination of bacteria constituting ruminal fibrolytic consortia developed on orchard grass hay stem

To determine the relationship between Fibrobacter succinogenes and other rumen bacteria, the bacterial community structure on fiber was analyzed by using two different materials. These were ruminally incubated orchard grass hay stems without and with preincubation with F. succinogenes (natural and artificial consortia, respectively). The natural consortium mainly consisted of Firmicutes (56.6%) and Bacteroidetes (33.1%), while the artificial consortium showed a significantly higher proportion of Firmicutes (85.5%) and a lower proportion of Bacteroidetes (4.6%). At species or genus level, Butyrivibrio fibrisolvens, the U2 group, Ruminococcus albus and Lachnospiraceae incertae sedis made up a higher proportion in the artificial consortium. The most dominant bacterial group was the Butyrivibrio‐Pseudobutyrivibrio‐Lachnospiraceae incertae sedis group, which accounted for 19.7% in the natural and 29.5% in the artificial consortium. Within the genus Butyrivibrio, the phylogenetic groups SA and VA2 and phylogeny‐undefined Butyribivrio, but not VA1, were detected at high frequency in the artificial consortium. These results suggest that ecological and possibly functional relationships exist in the rumen among F. succinogenes, a subset of B. fibrisolvens, the U2 group, R. albus and Lachnospiraceae incertae sedis.     

Characterization of bullet tuna myoglobin with reference to the thermostability-structure relationship

Myoglobin (Mb) was isolated from bullet tuna (Auxis rochei) skeletal muscle and characterized from the viewpoint of the thermostability-structure relationship. Differential scanning calorimetry (DSC) measurement showed that the thermostability of bullet tuna Mb was the lowest among all the scombridae fish Mbs so far examined. The highest value (72.8 degrees C) of melting temperature (Tm) was obtained at pH 6.52. alpha-Helical content at 10 degrees C was 34.5%, clearly lower than that of horse Mb (55.3%). The amino acid sequence was then deduced by cloning cDNA which encodes bullet tuna Mb. Bullet tuna Mb consisted of 147 amino acids, and the sequence identity was very close to that of skipjack (Katsuwonus pelamis) Mb (91.8%). A few amino acid substitutions, which could be involved in stability difference of Mb, were recognized. By mass spectrometry of lysyl endoproteinase digest of Mb, the N-terminus was found to be acetylated like that of other fish Mbs.     

Erythrocyte migration and gap formation in rabbit blood clots in vitro

Thrombolytic agents must be carried by the blood circulation to thrombi to exert their functions. Structural gaps exist between blood vessels and thrombi or in the area surrounding thrombi. Therefore, information about fundamental gap formation at thrombotic areas is critically important for thrombolytic therapy. We previously reported that t-PA accelerates the activities of bovine erythrocytes and hemoglobin (Hb) towards bovine plasminogen activation. Here, we examined gap generation by observing morphological changes during thrombolytic processes in rabbit blood clots deformation of erythrocytes from blood clots and Hb transfer from erythrocytes to serum in vitro. Rabbit venous blood samples (1 ml) were stored under sterile conditions in glass tubes at 37°C for 2, 24, 48 h, 1, and 2 weeks. We examined clot diameter, erythrocyte diameter and number as well as Hb volume in the serum, as well as histological changes in the clots. The diameter of blood clots did not change until 2 weeks after sampling. Erythrocyte diameter decreased within 48 h and at 2 weeks after sampling at the clot surface (p < 0.001) and interior (p < 0.001). The number of erythrocytes in the serum started to increase starting from 24 h after sampling (p < 0.01). Serum Hb volume also gradually increased from 24 h until 2 weeks after sampling (p < 0.01). The erythrocyte envelope became disrupted and cytoplasm started to flow through pores into the serum at 24 h. The results indicated that blood clots are reduced due to clot retraction, erythrocyte dissociation and cytoplasm leakage without a distinct fibrinolytic reaction. These results indicated that gaps start to form between 2 and 24 h after blood clotting.     

Pathology of canine oral malignant melanoma with cartilage and/or osteoid formation

Of 197 cases of canine oral malignant melanoma, 29 cases with myxoid, cartilage, and osteoid formation were studied pathologically and immunohistochemically. Tumor tissues were classified into spindle cell type (13 cases), epithelioid cell type (1 case), and mixed type (15 cases). Myxoid matrixes (29 tumors) were formed mainly in the tissues of spindle cell type and were positive for Alcian blue (pH 2.5). Cartilaginous matrixes (12 tumors) were formed in the myxoid tumor tissues. The morphology of atrophied neoplastic cells, which were embedded in the cartilage cavities, significantly differed from that of spindle cells proliferating in surroundings. There were reticular areas in the process of transitioning from myxoid to cartilaginous matrixes. Osteoid matrixes were not continuous with myxoid or cartilaginous matrixes, and arose as eosinophilic trabeculae in the dense collagenous connective tissues. A calcified bone trabecula was present among the osteoid trabeculae in a case. Melanin-producing melanocytes were proliferating in the collagenous matrixes, while amelanotic cells were in the osteoid matrixes. Immunohistochemistry demonstrated proliferating neoplastic cells as melanocytes. All cells in/out of these three matrixes were positive for Melan-A, S-100 protein, NSE, and vimentin. From these results, it is suggested that cartilage and osteoid matrixes are produced by dedifferentiated melanocytes.     

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk‐3)

Reduced expression in immortalized cells (REIC/Dkk‐3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk‐3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk‐3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk‐3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk‐3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk‐3 represents a potential molecular target for the development of therapies against feline mammary cancers.     

Pneumonia in horses induced by intrapulmonary inoculation of Streptococcus equi subsp. zooepidemicus

To evaluate the possibility that Streptococcus equi subsp. zooepidemicus (S.z) the causative bacterial agent of equine shipping fever pneumonia (ESFP), as well as to investigate its pathogenesis, 10 horses (seven Thoroughbreds and three Anglo-Arab species, ranging from 2-4 years in age) were experimentally inoculated, via an endoscope, into bronchus of the lung lobe with a dose of 30 ml of 1-7 x 10(8) CFU/ml of S.z. After inoculation, autopsy and pathological examinations were sequentially conducted 30 min, 1, 2, 3, 4, 17, 20 hr and 2 weeks later. Pneumonia induced by the intrapulmonary inoculation of S.z was characterized by small purulent pneumonic foci in the inoculated areas. With the lapse of time, these foci developed into serous hemorrhagic pneumonia, hemorrhagic purulent pneumonia, and then purulent, coagulation necrotic pneumonia. These pathomorphological characteristics of experimental pneumonia closely resemble those naturally occurring ESFP. There is strong evidence that S.z. is implicated as a causal factor in ESFP. S.z. grew in the mucus, exudate, and pulmonary effusions. Further, the bacteria showed resistance against phagocytosis by pulmonary alveolar macrophages (PAM) and neutrophils. Inhibition of PAM and neutrophil function is considered to be important in the development of pneumonia. With the progression of the disease, the neutrophils often adhered to the endothelial surface of the alveolar capillary lumen and played a role in generating coagulation necrosis of lung tissues.     

Ultrasonographic monitoring of nuclear transferred fetal weight during the final stage of gestation in Holstein cows

Dystocia or stillbirth accompanied by Large Offspring Syndrome (LOS) occurs rather frequently in Holstein nuclear transferred calves. In regard to prophylaxes, nuclear transferred Holstein fetuses were monitored with ultrasonography during the final stage of gestation. Fetal weight was estimated weekly based on the fetal metacarpal width using ultrasonography. Fourteen Holstein cows pregnant with Holstein nuclear transferred fetuses were the subjects of this experiment. The fetal weight was estimated by measuring the fetal metacarpal width during the last month of gestation according to the expected date of parturition. Measurements were performed on a weekly basis. The ultrasound-estimated metacarpal width and body weight of 13 of the fetuses in the last week of gestation (30.2+/-2.2 mm, 50.0+/-4.7 kg) were similar to the actual measurement immediately after birth (30.0+/-2.1 mm, 51.2+/-5.5 kg). These results indicate that ultrasonographic monitoring within a week of parturition to is accurate for estimating fetal weight. Prediction of LOS with ultrasonography contributes to reliable a diagnostic method that minimizes syndrome-related gyneco-obstetric complications at parturition with the aid of appropriate treatments.     

Ultrastructure of aortic elastic fibers in copper-deficient Sika deer (Cervus nippon Temminck)

Light microscopic and transmission and scanning electron microscopic observations were performed on the aortas of two 4- and 6-year-old deer affected with cervine ataxia and two 6-month- and 4-year-old healthy deer. Examination of the aortas from affected deer by transmission electron microscopy revealed the absence of distinct elastic laminae in the internal elastic lamina and tunica media, but discontinuous and irregular clumps of elastin were present. Scanning electron microscopy disclosed immature architecture of elastic fibers in the aortas from the copper-deficient deer, and the architecture was similar to that of a 6-month-old healthy deer.