Molecular monitoring and isolation of previously uncultured bacterial strains from the sheep rumen.

To estimate the contribution of uncultured bacterial groups to fiber degradation, we attempted to retrieve both ecological and functional information on uncultured groups in the rumen. Among previously reported uncultured bacteria, fiber-associated groups U2 and U3, belonging to the low-GC Gram-positive bacterial group, were targeted. PCR primers and fluorescence in situ hybridization (FISH) probe targeting 16S rRNA genes or rRNA were designed and used to monitor the distribution of targets. The population size of group U2 in the rumen was as high as 1.87%, while that of group U3 was only 0.03%. Strong fluorescence signals were observed from group U2 cells attached to plant fibers in the rumen. These findings indicate the ecological significance of group U2 in the rumen. We succeeded in enriching group U2 using rumen-incubated rice straw as the inoculum followed by incubation in an appropriate medium with an agent inhibitory for Gram-negative bacteria. Consequently, we successfully isolated two strains, designated B76 and R-25, belonging to group U2. Both strains were Gram-positive short rods or cocci that were 0.5 to 0.8 mum in size. Strain B76 possessed xylanase and alpha-l-arabinofuranosidase activity. In particular, the xylanase activity of strain B76 was higher than that of xylanolytic Butyrivibrio fibrisolvens H17c grown on cellobiose. Strain R-25 showed an alpha-l-arabinofuranosidase activity higher than that of strain B76. These results suggest that strains B76 and R-25 contribute to hemicellulose degradation in the rumen.

Eubacterium pyruvativorans sp. nov., a novel non-saccharolytic anaerobe from the rumen that ferments pyruvate and amino acids, forms caproate and utilizes acetate and propionate.

Two similar gram-positive rods were isolated from 10(-6) dilutions of ruminal fluid from a sheep receiving a mixed grass hay/concentrate diet, using a medium containing pancreatic casein hydrolysate as sole source of carbon and energy. The isolates did not ferment sugars, but grew on pyruvate or trypticase, forming caproate as the main fermentation product and valerate to a lesser extent. Acetate and propionate were utilized. One of these strains, I-6T, was selected for further study. Strain I-6T was a non-motile coccal rod, 1.2 x 0.4 microm, with a gram-positive cell wall ultrastructure and a G + C content of 56.8 mol%. No spores were visible, and strain I-6T did not survive heating at 80 degrees C for 10 min. Its rate of NH3 production was 375 nmol (mg protein)(-1) min(-1), placing it in the 'ammonia-hyperproducing' (or HAP) group of ruminal bacteria. 16S rDNA sequence analysis (1296 bases) indicated that it represents a novel species within the 'low-G + C' gram-positive group, for which the name Eubacterium pyruvativorans sp. nov. is proposed. Among cultivated bacteria, strain I-6T was most closely related (89% identity) to other asaccharolytic Eubacterium isolates from the mouth and the rumen. It was 98% identical to uncultured bacterial sequences amplified by others from ruminal digesta.

IL-2 independent transformation of a unique human T cell line, TY8-3, and its subclones by HTLV-I and -II.

Human T cell leukemia virus type I (HTLV-I) is etiologically associated with adult T cell leukemia (ATL) and chronic neurological disease, tropical spastic paraparesis (TSP). In our study, a unique IL-2 dependent human T cell line, designated TY8-3, was established from a thymoma obtained from a myasthenia gravis patient. The cells were heterogeneous and mainly consisted of those with CD4 , CD8 as well as activation markers and adhesion molecules including IL-2Ralpha,beta,gamma, CD45RO, Tf-R, HLA-DR, LFA-1alpha,beta, LFA-3, ICAM-1 and OX40 but without CD3 surface markers. Furthermore, these cells underwent an efficient and reproducible IL-2 independent transformation upon cocultivation with HTLV-I/II producing cell lines. Interestingly, although the infected cells became IL-2 independent, the growth rate of infected cells was significantly lower than those of parental TY8-3 cells. Clonal HTLV-I proviral DNA and viral particles were detected in the cells. Down-regulation of the lck and fyn genes and activation of the lyn gene was demonstrated in the IL-2 independent HTLV-positive TY8-3 cells. Subclones of TY8-3 cells were again able to be efficiently transformed and became IL-2 independent several months after coculture. Our results thus exhibit that TY8-3 cells and its subclones provide us with a very unique model whereby IL-2 independent transformation events of human T cells by HTLV-I/II in vitro can be studied at a clonal level.

Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity.

A human parvovirus B19 (B19) infectivity assay was developed using the erythroid cell line, KU812Ep6. KU812Ep6 was cloned for high efficiency infection with B19 in vitro, in the presence of erythropoietin by a limiting dilution method from the parent cell line, KU812. B19 was effectively propagated in KU812Ep6 and was detected for B19 antigens, VP1 and VP2. The titers of B19 positive sera measured with KU812Ep6 cells were in the range of 10(6) to 10(8) TCID50 ml. This KU812Ep6 infectivity assay had a 10(3)-10(4.5) higher sensitivity than the colony forming unit-erythroid (CFU-e) injury assay. It was calculated that one TCID50 needed 10(3) B19 genome copies, judging from the infectivity assay and semi-quantitative PCR. The KU812Ep6 infectivity assay was also used to determine infectivity of B19 in vitro, and to evaluate inactivation, as well as clearance of the virus. The inactivation of B19 by heating was carried out and infectivity declined from 10(4) TCID50 ml to < 10 TCID50 ml (lower limit of detection) at 60 degrees C for 3 h or at 70 degrees C for 30 min, but only decreased to 10(2.5) TCID50 ml at 50 degrees C for 8 h.

Retention behavior of very large biomolecules in ion-exchange chromatography.

Hepatitis B virus surface antigen (HBsAg) particles were efficiently adsorbed (retained) on a Sulfate-cellulose (S-C) bead column, and then desorbed with sodium chloride solutions (0.5-3.0 M). The HBsAg particles were not efficiently retained onto either sulfopropyl-agarose (SP-A) or quaternary amine-agarose (Q-A) at pH 4.5, 6 and 8. The size-exclusion curve showed that proteins of molecular mass higher than ca. 20,000 cannot penetrate into the pores of S-C beads. The dynamic binding capacity (DBC) values of lysozyme (ca. 7 mg/ml-gel) and of gamma-globulin (ca. 3 mg/ml gel) for S-C did not depend on the flow velocity while the DBC of gamma-globulin for SP-A decreased sharply with an increase in flow velocity. These results indicated that very large molecules are adsorbed only onto the surface of S-C, which resulted in fast adsorption-desorption rates although the equilibrium adsorption capacity is lower than conventional porous gel beads. Because of the rapid adsorption rate, the DBC values of gamma-globulin for S-C at high flow-rate regions are similar to those for SP-A. Bovine serum albumin was not adsorbed onto S-C. As this can not be explained by a simple electrostatic interaction mechanism, molecular recognition of S-C might be different from the agarose-based ion-exchange beads.

Rumen ciliate protozoal fauna of native sheep, friesian cattle and dromedary camel in Libya.

Rumen ciliate species and composition were surveyed on the native sheep, Friesian-cattle and dromedary (one-humped) camels kept in Libya. As a result of survey, 5 genera including 14 species with 5 formae in native sheep, 9 genera including 27 species with 6 formae in Friesian-cattle and 6 genera including 13 species and 7 formae in dromedary camels were identified. All of the ciliate species and their percentage composition detected from the Libyan sheep and cattle in this examination were similar to those found from corresponding animals in the other countries. Libyan camels lacked some peculiar ciliate species found from camels in the other countries, but had many cosmopolitan species common with those in the domestic ruminants, suggesting that ciliate faunae of camel are easily affected by the other domestic ruminants kept together. The ciliate density was estimated as 105/ml in every host species.

Establishment of monoclonal antibody, gp21-34, against HTLV-II envelope protein (p20E).

A monoclonal antibody (MAb), gp21-34, specifically reactive with human T-lymphotropic virus type-II (HTLV-II) transmembranous envelope glycoprotein (p20E) was developed by immunization with a recombinant protein fused with thioredoxin moiety at the N-terminal portion. This MAb, which belongs to the IgG1 kappa subclass, reacted with HTLV-II infected cell lines (TON-1, C3-44, and Si-IIA) by IFA, but not with HTLV-I infected cell lines (TCL-Kan and MT-2). By Western blot analysis, this MAb reacted with p20E of HTLV-II lysates but not with HTLV-I lysates. Some epitope analyses with synthetic peptides were carried out to look for a plausible linear epitope in the C-terminal region of p20E.

Ciliate protozoa in the forestomach of the dromedary camel, (Camelus dromedarius), in Egypt, with description of a new species.

The composition of ciliates obtained from the forestomachs of eleven dromedary (one-humped) camels in Egypt was examined. As a result, eight genera containing 24 species with 11 forms were identified. Of them, one species was concluded to be new, then described as Dasytricha kabanii n. sp. This new species was clearly distinguished from D. ruminantium, the other species of the genus, by its lack of somatic cilia on the posterior one-fifth of the body surface. Entodinium nanellum and Epidinium ecaudatum f. caudatum were found in all camels examined. Although the percentage composition of respective species varied with the individual camel, the rate of Entodinium spp. was high in general. Total ciliate density in forestomach fluid was 1.9 x 10(5)/ml on average. Ciliate composition in Egyptian camels was similar to that in Bactrian camels, Camelus bactrianus, in China reported previously. However, more Entodinium species were detected from Egyptian camels than from Bactrian camels.

Investigations into the location of Treponema hyodysenteriae in the cecum of experimentally infected young broiler chicks by light- and electronmicroscopy.

Numerous Treponema hyodysenteriae were present both on the mucosal surface and in the deep crypts of the cecum of young broiler chicks 7 and 14 days after inoculation with the treponemes. The treponemes in the ceca of chicks inoculated with 10(8) cells were observed more frequently than those of chicks inoculated with 10(7) cells. The treponemes in the ceca were observed by light microscopy, scanning electron microscopy and transmission electron microscopy. The lesions were primarily confined to the cecum. Desquamation of epithelial cells, edema, leukocytic infiltration and hemorrhage were observed in the mucosae.

Colorimetric determination of carboxypeptidase A activity in serum.

This simple, reproducible colorimetric method for determining the activity of carboxypeptidase A (EC 3.4.17.1) is based on measuring the absorbance at 505 nm of a quinoneimine dye produced from the action of this enzyme on the new substrate p-hydroxybenzoyl-glycyl-L-phenylalanine. The enzyme acts on the substrate to produce p-hydroxybenzoyl-glycine and L-phenylalanine. The former is then hydrolyzed by hippuricase (EC 3.5.1.14) to produce p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by sodium periodate forms a quinoneimine dye. The Km for the reaction with this substrate is 3.6 mmol/L; the optimum pH is 7.8. Our within-run and between-run CVs are 4.3% and 6.6%, respectively. The activity of carboxypeptidase A in serum correlates well with that of lipase (r = 0.96) and immunoreactive elastase-1 (r = 0.76).

Experimental infection of young broiler chicks with Treponema hyodysenteriae.

In young broiler chicks which were inoculated with 10(8) cells of Treponema hyodysenteriae within 24 hr after hatching, numerous treponemes were observed by scanning electron microscopy on the surface of the cecal mucosa 7 and 14 days after the inoculation. However, in the groups inoculated with 10(7) cells, treponemes were not observed on the cecal mucosa 14 days after the inoculation, and the isolation rate from the cecal contents was lower than that from cecal contents of chicks inoculated with 10(8) cells. While the cecal mucosa of noninfected chicks had a smooth surface, that of the chicks infected with treponemes was generally roughened and the epithelium was eroded. Numerous treponemes were also observed within the eroded epithelium.

Isolation and characterization of I5B2, a new phosphorus containing inhibitor of angiotensin I converting enzyme produced by Actinomadura sp.

A new inhibitor of angiotensin I converting enzyme, I5B2, was isolated from the culture broth of Actinomadura sp. No. 937ZE-1. This compound contains N-methylvaline, tyrosine and 1-amino-2-(4-hydroxyphenyl)ethylphosphonic acid. The microorganism also produced another inhibitor, I5B1, which is identical with K-4 isolated from Actinomadura sp. as an antihypertensive agent.

Cellular fatty acid composition of Haemophilus equigenitalis.

The cellular fatty acid composition of eight Haemophilus equigenitalis strains was determined by gas-liquid chromatography. All strains showed a grossly similar pattern characterized by large amounts of 18:1 and 16:0. The amounts of 16:1, 18:2, 18:0, 3-OH 14:0, 3-OH 16:0, and 3-OH 18:1 were relatively small.

Cellular fatty acid composition in staphylococci isolated from bovine milk.

Cellular fatty acid composition was examined in 61 strains of coagulase-negative staphylococci of bovine milk origin and 19 strains of Staphylococcus aureus and S. epidermidis serving as reference strains. The 61 strains had been divided into 9 species in accordance with the classification of Kloos & Schleifer. When S. aureus and the coagulase-negative staphylococci were examined, they contained iso-C15:0, anteiso-C15:0, C18:0, and C20:0 as main fatty acids. There was no marked difference in the cellular fatty acid composition between any two staphylococcal species. The percent cellular fatty acid composition of each species was regarded as one of the quantities of continuity and subjected to the analysis of variance by the unary configuration method. As a result, S. capitis, S. cohnii, S. epidermidis, and S. xylosus seemed to have a cellular fatty acid composition a little different from that of any other species. When similarity values among all the strains were calculated, they were 0.89 or more and made it possible to divide all the strains into two large groups at the similarity value of 0.90. In the one group the iso-C15:0 was a characteristic fatty acid. In the other group the anteiso-C15:0 was a characteristic fatty acid. To the first group belonged S. hyicus and S. lentus. To the second group belonged S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, and S. warneri. In the two groups were distributed S. aureus, S. capitis, S. simulans, and S. xylosus.

Occurrence of free ceramides in Bacteroides fragilis NCTC 9343.

The occurrence of free ceramides at high concentrations was demonstrated in the chloroform-methanol extractable lipids of Bacteroides fragilis NCTC 9343. The long-chain bases were isolated from the free ceramides and identified as branched and normal saturated dihydroxy bases with carbon chains consisting of 17, 18, and 19 atoms. The major fatty acid was 3-hydroxy 15-methylhexadecanoic acid. The major molecular species of the ceramides were identified by gas chromatography-mass spectrometry and gas chromatography of the cleaved products as LCB-d-iso17: 0-3-OH iso17: 0 FA, LCB-d-anteiso17: 0-3-OH iso17: 0 FA, LCB-d-iso18: 0-3-OH iso17: 0 FA, and LCB-d-anteiso19: 0-3-OH iso17: 0 FA.

Isolation of Selenomonas spp. from lesions and non-digestive organs of cows, pigs and man.

Selenomonas spp. were isolated for the first time from lesions and non-digestive organs which were apparently normal in 2 cows, 6 pigs, and 1 human being. Identification as Selenomonas was based firstly on electron microscopical observation and secondly on fermentation products. They were divided into 3 major groups by biological properties, as well as by the patterns of these products. It has been confirmed that the habitats of organisms of the genus Selenomonas are generally digestive organs, including the rumen of the ruminant, the cecum of the guinea pig, and the oral cavity of man. The existence of these organisms in lesions and non-digestive organs in such animals and man, however, has been unknown as yet. Moreover, it has been completely unknown about the habitat of these organisms in swine. The findings obtained suggested the possibility of invasion of Selenomonas into other parts than the digestive organ in some animals and the presumable existence of the organism in the swine digestive organs. The role of Selenomonas as a secondary invader into some animals was proposed.