Antibody-mediated protection against MERS-CoV in the murine model.

Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBD-Fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.

Detection of Xanthomonas fragariae and presumptive detection of Xanthomonas arboricola pv. fragariae, from strawberry leaves, by real-time PCR.

Real-time (TaqMan) PCR assays were developed to detect the strawberry angular leaf spot pathogen Xanthomonas fragariae (Xf) and the strawberry bacterial blight pathogen Xanthomonas arboricola pv. fragariae (Xaf). The Xf PCR (Xf gyrB) was designed within regions of the gyraseB gene, unique to Xf, after generating gyraseB DNA sequence data from Xf and other closely related strains. The Xaf PCR (Xaf pep) was designed within regions of the pep prolyl endopeptidase gene that were unique to Xaf, after generating pep DNA sequence data from Xf and Xaf strains. The Xf gyrB PCR detected only Xf strains amongst a panel of 20 Xanthomonas-related spp. and pathovars. The Xaf pep PCR assay detected all Xaf strains tested plus two other (of three tested) X. arboricola pathovars. An existing genomic DNA extraction protocol was modified to facilitate detection of both pathogens to 10(3) cells per strawberry leaf disc.

Strain discrimination of Yersinia pestis using a SERS-based electrochemically driven melting curve analysis of variable number tandem repeat sequences.

Strain discrimination within genetically highly similar bacteria is critical for epidemiological studies and forensic applications. An electrochemically driven melting curve analysis monitored by SERS has been utilised to reliably discriminate strains of the bacterial pathogen Yersinia pestis, the causative agent of plague. DNA amplicons containing Variable Number Tandem Repeats (VNTRs) were generated from three strains of Y. pestis: CO92, Harbin 35 and Kim. These amplicons contained a 10 base pair VNTR repeated 6, 5, and 4 times in CO92, Harbin 35 and Kim respectively. The assay also included a blocker oligonucleotide comprising 3 repeats of the 10-mer VNTR sequence. The use of the blocker reduced the effective length of the target sequence available to bind to the surface bound probe and significantly improved the sensitivity of the discrimination. The results were consistent during three replicates that were carried out on different days, using different batches of PCR product and different SERS sphere segment void (SSV) substrate. This methodology which combines low cost, speed and sensitivity is a promising alternative to the time consuming current electrophoretic methods.

Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR.

AIMS: The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains. METHODS AND RESULTS: A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain. One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids. The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR. CONCLUSIONS: The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis. The technique is therefore a rapid and cost-effective detection method. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen. The method can be used as a model system for the detection of other Agrobacterium pathogens.

Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat.

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).

Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay.

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.

Irradiation of clinically uninvolved cervical lymph nodes.

Of 402 patients with cancers of the oral cavity, oropharynx, and supraglottic larynx treated at Stanford between 1957 and 1972, 164 had clinically uninvolved cervical lymph nodes prior to the initiation of radiation therapy. Lymph node metastases developed later in 38 per cent of patients with primary oral cavity carcinomas who were treated with interstitial radium implants alone. No late cervical lymph node involvement was found in those patients who received high dose external irradiation to at least the primary site and first echelon lymph nodes. Lymph node failures were ultimately noted in 20 of the 140 patients (14 per cent), who received partial or complete neck irradiation, but 18 of these occurred in patients with uncontrolled primary lesions, suggesting that re-seeding of cervical lymph nodes had taken place rather than failure of the initial irradiation to control subclinical metastases. Our present policy is to treat the primary lesion and adjacent lymph nodes with high dose megavoltage techniques, combined with interstitial irradiation if possible. Bilateral supplemental inferior neck radiation ports are added for patients with advanced primary neoplasms and for those with clinically involved cervical lymph nodes. All other patients undergoing radiation therapy for stage T1 primary lesions and clinically negative necks also receive ipsilateral low neck irradiation. In addition, cervical lymph nodes are electively irradiated when the primary lesion has been resected. When these policies are adopted, the incidence of cervical lymph node failures is extremely low in patients whose primary sites remain controlled, and morbidity from the cervical radiation fields is negligible.

Carcinoma of the oropharynx. Results of megavoltage radiation therapy in 305 patients.

Three hundred and five patients with oropharyngeal carcinomas received linear accelerator radiation therapy at the Stanford University Medical Center between 1956 and 1973. All were staged by the TNM system, using the UICC Classification of 1962. Actuarial five year survival for patients with tonsil cancers ranged from 50% to 18% for those with T1 and T3 lesions, respectively. Disease-free survival was higher than actuarial survival for patients with T1 and T2 lesions, reflecting an alteration of the latter curves by deaths from intercurrent diseases and second primary malignancies. Actuarial survival for patients with base of the tongue carcinomas was approximately 35% for those with T1 and T2 primary lesions and 22% for patients with T3 carcinomas. Considering all oropharyngeal sites of involvement together, clinically involved cervical lymph nodes were controlled by radiation therapy alone in 60 of 91 patients with N1 nodal involvement (67%), in twelve of 25 with N2 neck disease (48%) and in 46% of those patients with N3 involvement (34 of 74). Eighty-nine percent of patients whose lymph nodes were not controlled by radiation therapy alone also had uncontrolled primary cancers. Over 90% of the patients whole cervical lymph nodes were initially uninvolved remained free of late nodal metastases if at least the regional (first echelon) lymph nodes were included in the primary treatment fields. The results of a randomized trial which compared surgery and radiation therapy alone and the two modalities in combination for the treatment of a limited number of patients with advanced cancers of the oropharynx, supraglottic larynx and hypopharynx suggest that surgery alone is not the treatment of choice.

Initial relapses in previously treated Hodgkin's disease. I. Results of second treatment.

Two hundred forty-three consecutive patients with Hodgkin's disease who relapsed after an initial course of treatment at the Stanford University Division of Radiation Therapy underwent subsequent systematic evaluation and retreatment. An analysis of the influence of numerous parameters, including sex, histopathology, original stage, relapse site, and original and second therapy, on actuarial survival and on relapse-free survival was undertaken. Most relapses (87%) occurred within 3 years of the initial treatment course. The 5-year relapse-free survival measured from the time of second treatment increased from 14% before to 39% after the introduction of multiple agent chemotherapy (MOPP) for relapsing disease. Patients treated with MOPP chemotherapy for nodal relapses showed increased subsequent relapse-free survival (61%) when compared with patients treated only with radiotherapy for nodal relapses. Based on the combined findings of this analysis, recommendations are made regarding the management of patients with Hodgkin's disease who have suffered a relapse.