Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep

Aims: To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Methods and Results: Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis‐specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1·2% (2/162) and 17·2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27·7% (45/162) of blood and 29·0% (47/162) of lymphoid tissue samples. Conclusions: The species‐specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0·01 in blood PCR, P < 0·001 in tissue PCR) and serologically negative (P < 0·001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. Significance and Impact of the Study: The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes.     

Sequential immunization with V3 peptides from primary human immunodeficiency virus type 1 produces cross-neutralizing antibodies against primary isolates with a matching narrow-neutralization sequence motif

An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the "PGR" motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.     

Cervical leiomyoma in a dairy cow during pregnancy

In the present report we describe a case of cervical leiomyoma that was diagnosed at parturition in a Holstein cow. The tumor mass, which measured 25.5 cm x 21.5 cm x 14.5 cm in size and weighed 4.5 kg, was removed surgically. The tumor was solid, well circumscribed, whitish-pink colored, and encapsulated. The tumor was diagnosed as leiomyoma. The leiomyoma had no adverse effects on pregnancy. This is the first report of a bovine cervical leiomyoma during parturition.     

Serum IL-18 levels in patients with type 1 diabetes: relations to metabolic control and microvascular complications

The study was designed to examine serum IL-18 level and its relation to metabolic control parameters and microvascular complications in type 1 diabetes mellitus (DM). Sixty two patients with type 1 DM and 30 healthy individuals were enrolled in the study. Serum IL-18 levels of patients with type 1 DM were significantly increased compared to controls (293.4+/-133.4 vs 211.2+/-63.9 pg/ml, P=0.003). Patients with poor glycemic control had higher levels of IL-18 than patients with well glycemic control (329.9+/-141.0 vs 226.3+/-89.6 pg/ml, P=0.02). There was no significant difference between the serum IL-18 levels of patients with microvascular complications and those of patients without microvascular complications (307.6+/-127.6 vs 293.2+/-145.6 pg/ml, P>0.05). IL-18 correlated positively with HbA(1c) (r=0.32, P=0.01) and postprandial blood glucose (PPBG) (r=0.26, P=0.02); and negatively with HDL-cholesterol (HDL-C) (r=-0.38, P=0.007). By linear regression analysis, PPBG was determined as the most explanatory parameter for the alterations in serum IL-18 levels (P=0.02). High levels of IL-18 in patients with type 1 DM is related to short and long term glycemic control and HDL-C levels but not to microvascular complications.     

Protective Effects of <Emphasis Type="Italic">Mekabu</Emphasis> Aqueous Solution Fermented by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Sanriku-SU7 on Human Enterocyte-Like HT-29-luc Cells and DSS-Induced Murine IBD Model

Most wakame Undaria pinnatifida, a brown algae, products are made from the frond portion. In this study, the polysaccharide content and antioxidant property of aqueous extract solutions (AESs) of the four parts (frond: wakame, stem of the frond: kuki-wakame, sporophyll: mekabu, and kuki-mekabu) of wakame were investigated. Polysaccharide content was high in both the wakame and mekabu. Superoxide anion (O₂ ^?) radical-scavenging capacities were high in the mekabu. These AESs could be fermented by Lactobacillus plantarum Sanriku-SU7. The O₂ ^? radical-scavenging activity of the kuki-wakame, mekabu, and kuki-mekabu were increased by the fermentation. Fermented mekabu clearly showed a protective effect on human enterocyte-like HT-29-luc cells and in a mouse model of dextran sodium sulphate-induced inflammatory bowel disease (IBD). These results suggest that the mekabu fermented by L. plantarum Sanriku-SU7 has anti-IBD effect related to O₂ ^? radical-scavenging.     

Protein-based complex medium design for recombinant serine alkaline protease production

This work reports on the design of a complex medium based on simple and complex carbon sources, i.e. glucose, sucrose, molasses, and defatted-soybean, and simple and complex nitrogen sources, i.e. (NH₄)₂HPO₄, casein, and defatted-soybean, for serine alkaline protease (SAP) production by recombinant Bacillus subtilis carrying pHV1431::subC gene. SAP activity was obtained as 3050 U cm⁻³ with the initial defatted-soybean concentration C_soybean^o=20 kg m⁻³ and initial glucose concentration C_G^o=8 kg m⁻³; whereas, addition of the inorganic nitrogen source (NH₄)₂HPO₄ decreased SAP production considerably. Further increase in SAP production (3850 U cm⁻³) was obtained when sucrose was replaced with glucose at C_sucrose^o=15 kg m⁻³ and C_soybean^o=20 kg m⁻³. Nevertheless, when molasses was replaced with sucrose, the maximum activity was obtained with molasses having 10 kg m⁻³ initial sucrose concentration and C_soybean^o=15 kg m⁻³as 2130 U cm⁻³; moreover, when casein was replaced with defatted-soybean SAP production decreased considerably (ca. 250 U cm⁻³). Thereafter, the effects of inorganic ionic compounds were investigated; and except phosphate, inorganic compounds supplied from defatted-soybean were found to be sufficient for the bioprocess. The highest SAP activity was obtained as 5350 U cm⁻³ in the medium that contained (kg m⁻³): C_soybean^o=20, C_sucrose^o=15, C_Na2HPO4^o=0.021, and C_NaH2PO4^o=2.82, that was 6.5-fold higher than that of the SAP produced in the defined medium. By using the designed complex medium, oxygen transfer characteristics of the bioprocess were investigated; and, Damköhler number that is the oxygen transfer limitation increases with the cultivation time until t=14 h; and, at t>20 h both mass transfer and biochemical reaction resistances were effective. Overall oxygen transfer coefficient varied between 0.010 and 0.044 s⁻¹; volumetric oxygen uptake rate varied between 0.001 and 0.006 mol m⁻³ s⁻¹; and specific oxygen uptake rate varied between 0.0001 and 0.0022 mol kg⁻¹ DW s⁻¹ throughout the bioprocess.     

Density gradient fractionation of effector cells in human natural cell-mediated cytotoxicity

In order to separate and characterize cytotoxic effector cells in natural cell-mediated cytotoxicity (NCMC), human lymphocytes were fractionated by Percoll continuous density gradient centrifugation (Pharmacia, Piscataway, N.J.). Lymphocytes from normal donors were fractionated through a 35-ml gradient and 2- or 3-ml aliquots were collected, counted, and grouped into three or more fractions in order to obtain sufficient cells for testing. Fractions of cells were tested for cytotoxicity in a 4-hr chromium release test and/or a 40-hr [³H]proline assay. Cell markers were assessed by testing for cells forming E rosettes, EA rosettes, and for cells with surface membrane immunoglobulin (SMIg). The lightest fraction contained larger cells and also usually contained the highest concentrations of cells with receptors for the Fc portion of IgG (FcR + cells), although slight variations were seen among individual donors. Results of cytotoxicity tests showed that cells from the top portions of the Percoll gradient had consistently greater cytotoxic activity on a per cell basis than the denser cells sedimenting lower. Estimation of cytotoxic activity in lytic units showed that 54–75% of the activity was recovered in the top 26–29% of the cells. This approach to investigating cell-mediated cytotoxicity should yield useful information regarding cellular interaction in, and regulation of, cytotoxic activities.