The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.

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Based on the results of GC content determination and 16S rRNA sequence analysis among the type strains of Achromobacter xylosoxidans, 4 Alcaligenes species, 5 Bordetella species, and 12 species of 4 other genera, the separation of genus Achromobacter Yabuuchi and Yano 1981, with the type species Achromobacter xylosoxidans, is confirmed. Alcaligenes ruhlandii (Packer and Vishniac) Aragno and Schlegel 1992 is a distinct species and not a senior synonym of Achromobacter xylosoxidans. Alcaligenes ruhlandii and Alcaligenes piechaudii Kiredjian et al 1986 are transferred to genus Achromobacter. Thus 2 new combinations, Achromobacter ruhlandii (Packer and Vishniac) and Achromobacter piechaudii (Kiredjian et al) are proposed; their type strains are ATCC 15749 and ATCC 43552, respectively. Alcaligenes denitrificans Rüger and Tan 1983 is also transferred to genus Achromobacter and ranked down to the subspecies of Achromobacter xylosoxidans. Thus a new subspecies name, Achromobacter xylosoxidans subsp. denitrificans (Rüger and Tan) is proposed. The type strain of the subspecies is ATCC 15173. This proposal automatically creates type subspecies, Achromobacter xylosoxidans subsp. xylosoxidans, with type strain ATCC 27061. An emended description of genus Achromobacter and of type species Achromobacter xylosoxidans are given.

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The Yuri strain of small round structured virus (SRSV) was cloned from a fecal specimen in which virus particles were observed by electron microscopy. The most common RT-PCR protocol in Japan, however, using 35/36 and NV81/NV82/SM82 nested primer pairs, could not detect the SRSV genome in this specimen. Nucleotide and amino acid sequence analysis revealed that the Yuri strain is genetically close to the genotype II of SRSV. A novel procedure using primer sets designed from the nucleotide sequence of the Yuri strain was applied to the screening of 119 stool samples obtained from subjects with sporadic diarrhea and 46 samples obtained during seven foodborne gastroenteritis outbreaks. Using this novel procedure, PCR bands were detected in 44% and 52% of the samples, respectively. These detection rates were approximately twice those obtained with the 35/36 and NV81/NV82/SM82 nested primers. In particular, more than 40% of positive samples could be detected by using only the Yuri primer sets. Furthermore, three improvements were made in the RNA preparation, cDNA synthesis, and amplification steps to save materials and time. The background, or extra bands, in the amplification reaction resulting from DNA in the fecal specimens was completely removed by DNase I treatment just before cDNA synthesis. Random nonamers were used as universal primers in the reverse transcription. No difference in sensitivity or specificity was noted in the final results when either random nonamers or specific primers were used. The use of a preamplification step under low stringent conditions before standard amplification under highly stringent conditions compensated for any mismatched bases in the primers with respect to the target sequences. Thus our novel procedure using Yuri primer sets may be useful for the screening of SRSV in the recent SRSV outbreaks in Japan.

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When C57BL/6 (B6) mice were irradiated (9Gy) and received bone marrow (BM) cells of B6-lpr/lpr mouse origin (i.e., lpr→B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3^- IL-2Rβ⁺ NK cells and IL-2Rβ⁺ CD3^int cells (i.e., CD3^int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1⁺ and NK1.1^-, among the IL-2Rβ⁺ CD3^int cells. However, the NK1.1⁺ subset (i.e., NK1.1⁺ T cells) was much more radioresistant, and the majority of CD3^int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6-lpr/lpr mice injected with both BM cells of B6-lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti-NK1.1mAb or anti-asialoGM₁ antibody to eliminate NK cells alone or both NK cells and NK1.1⁺ T cells. When irradiated B6 mice were pretreated with anti-NK1.1mAb, the mice could survive. These results suggest that intact NK1.1⁺ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double-negative CD4^- 8^- cells, in B6-lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.

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The immunostimulatory effect of intragastrically or parenterally administered β-(1→3; 1→4) glucan, extracted from oats (OβG), on disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Multiple administrations of OβG by intragastric or subcutaneous mutes reduced fecal oocyst shedding compared to the non-treated control group. The administration of OβG by subcutaneous route resulted in higher levels of total serum immunoglobulins and antigen (sporozoite and merozoite)-specific immunoglobulins as compared with the non-treated group. To evaluate the effect of a single subcutaneous dose, groups of mice were treated with OβG 2 days before E. vermiformis infection, at the time of infection and at 2 or 6 days after infection. From day 11 post-infection the oocyst discharge was significantly diminished (P<0.05-0.01) in the OβG-treated groups, except in those treated 6 days after infection, as compared to the non-treated control group. The proliferative responses to E. vermiformis sporozoite antigen of lymphocytes isolated from the spleen were significantly increased (P<0.05) when OβG was administered 2 days before or at the time of E. vermiformis infection. Lymphocyte proliferative responses to merozoite antigen were not influenced by treatment. In conclusion, OβG appeared to up-regulate immune mechanisms and provide enhanced resistance against eimerian coccidiosis in mice.

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The presence of shuttle plasmid pTS749 in the oral bacterium Streptococcus mutans resulted in growth inhibition under heat stress conditions. This effect was dependent upon the growth stage of the inoculum used to initiate growth. Likewise, the introduction of plasmids containing distinct ori sequences into Escherichia coli also resulted in growth inhibition at elevated temperatures and could be correlated with the plasmid levels within the cells. These results suggest that these plasmids in bacteria render the cells to be more susceptible to heat stress conditions.

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The short-term kinetics of infectious HIV titers, HIV copy numbers and p24-antigen during the first 28 days of AZT monotherapy were evaluated. In three of four patients, infectious HIV was culturable and infectious titers rose 2- and 4-fold compared to baseline values. This increase was neither associated with mutations conferring resistance to AZT nor a switch from NSI to SI phenotypes. Two patients showed an increase of plasma infectivity associated with a reduction of HIV copies and p24-antigen. We conclude that transient dissociations of plasma infectivity and HIV copy numbers occur during early AZT monotherapy.

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