Recirculating immunomagnetic separation and optimal enrichment conditions for enhanced detection and recovery of low levels of Escherichia coli O157:H7 from fresh leafy produce and surface water.

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (C(T) values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42 degrees C for 5 h provided better detection than enrichment at 37 degrees C. Extended incubation of surface water for 20 h at 42 degrees C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

Molecular Subtyping of Shiga Toxin-Producing Escherichia coli Using a Commercial Repetitive Sequence-Based PCR Assay.

PCR-based typing methods, such as repetitive sequence-based PCR (rep-PCR), may facilitate the identification of Shiga toxin-producing Escherichia coli (STEC) by serving as screening methods to reduce the number of isolates to be processed for further confirmation. In this study, we used a commercial rep-PCR typing system to generate DNA fingerprint profiles for STEC O157 (n = 60) and non-O157 (n = 91) isolates from human, food, and animal samples and then compared the results with those obtained from pulsed-field gel electrophoresis (PFGE). Fifteen serogroups were analyzed using the Kullback Leibler or extended Jaccard statistical method, and the unweighted pair group method of averages algorithm was used to create dendrograms. Among the 151 STEC isolates tested, all were typeable by rep-PCR. Among the non-O157 isolates, rep-PCR clustered 79 (88.8%) of 89 isolates according to serogroup status, with peak differences ranging from 1 (96.4% similarity) to 12 (58.7% similarity). The genetic relatedness of the non-O157 serogroups mirrored the branching of distinct clonal groups elucidated by other investigators. Although the discriminatory power of rep-PCR (Simpson's index of diversity [SID] = 0.954) for the O157 isolates was less than that of PFGE (SID = 0.993), rep-PCR was able to identify 29 pattern types, suggesting that this method can be used for strain typing, although not to the same level as PFGE. Similar results were obtained from analysis of the non-O157 isolates. With rep-PCR, we assigned non-O157 isolates to 46 pattern types with a SID of 0.977. By PFGE, non-O157 STEC strains were divided into 77 pattern types with a SID of 0.996. Together, these results indicate the ability of the rep-PCR typing system to distinguish between and within O157 and non-O157 STEC groups. Rapid PCR-based typing methods could be invaluable tools for use in outbreak investigations by excluding unrelated STEC isolates within 24 h.

Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain.

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.

Species tropism of chimeric SHIV clones containing HIV-1 subtype-A and subtype-E envelope genes.

To analyze HIV-1 genes in a nonhuman primate model for lentivirus infection and AIDS, recombinant SIV/HIV-1 (SHIV) clones were constructed from two HIV-1 subtype-A isolates (HIV-1(SF170) and HIV-1(Q23-17) from individuals in Africa) and two HIV-1 subtype-E isolates (HIV-1(9466) and HIV-1(CAR402) from AIDS patients in Thailand and Africa), respectively. These four SHIV clones, designated SHIV-A-170, SHIV-A-Q23, SHIV-9466.33, and SHIV-E-CAR, contain envelope (env) genes from the subtype-A or -E viruses. Interestingly, SHIV-A-170, SHIV-A-Q23, and SHIV-9466.33 were restricted for replication in cultures of macaque lymphoid cells, whereas SHIV-E-CAR replicated efficiently in these cells. Additional studies to define the block to replication in macaque cells were focused on the subtype-E clone SHIV-9466.33. A SHIV intragenic env clone, containing sequence-encompassing V1/V2 regions of HIV-1(CAR402) and V3/V4/V5 regions of SHIV-9466.33, infected and replicated in macaque lymphoid cells. These results indicated that the sequence-encompassing V1/V2 region of HIV-1(9466) was responsible for the block of the SHIV-9466.33 replication in macaque cells. Analysis of viral DNA in acutely infected macaque cells revealed that SHIV-9466.33 was blocked at a step at/or before viral DNA synthesis, presumably during the process of virion entry into cells. In a fluorescence-based cell-cell fusion assay, fusion pore formation readily took place in cocultures of cells expressing the SHIV-9466.33 env glycoprotein with macaque T-lymphoid cells. Taken together, these results demonstrated that the block of SHIV-9466.33 replication in macaque cells is at an early step after fusion pore formation but before reverse transcription.