Minor Variants of Orf1a, p33, and p23 Genes of VT Strain Citrus Tristeza Virus Isolates Show Symptomless Reactions on Sour Orange and Prevent Superinfection of Severe VT Isolates.

The control of tristeza quick decline (QD) of citrus is based on the use of rootstocks that are tolerant or resistant to the Citrus tristeza virus (CTV), but some of them show bio-agronomic limits. The application of cross-protection (CP) has been insufficiently explored. The present study examined the possibility of QD control by cross-protection (CP) following reports showing the dependence of the CP strategy on the close genetic relationships between the protective and challenging CTV isolates. Taking advantage of deep sequencing technologies, we located six naturally infected trees harboring no-seedling yellow (no-SY) and no QD decline (mild) VT isolates and used these for challenge inoculation with three QD VT isolates. Symptom monitoring showed that all six Sicilian mild no-SY isolates, based on their genomic relatedness and mild symptoms reactions, provide effective protection against the three severe local VT isolates. The differences between the six mild and three severe isolates were confined to just a few nucleotide variations conserved in eight positions of three CTV genes (p23, p33, and Orf1a). These results confirm that the superinfection exclusion (SIE mechanism) depends on close genetic relatedness between the protective and challenging severe VT strain isolates. Ten years of investigation suggest that CP could turn into an efficient strategy to contain CTV QD infections of sweet orange trees on SO rootstock.

Development of a real-time PCR method for the specific detection of the novel pear pathogen Erwinia uzenensis.

Erwinia uzenensis is a plant-pathogenic bacterium, recently described in Japan, which infects pear trees, causing the 'bacterial black shoot disease of European pear' (BBSDP). Like other Erwinia pear pathogens, E. uzenensis causes damp, black lesions on young shoots resembling those of E. amylovora, but not blossom blight, fruitlet blight or wilting of the shoot tip. The distribution of E. uzenensis seems restricted to the country where it was reported up to now, but it may spread to other countries and affect new hosts, as is the current situation with E. piriflorinigrans and E. pyrifoliae. Fast and accurate detection systems for this new pathogen are needed to study its biology and to identify it on pear or other hosts. We report here the development of a specific and sensitive detection protocol based on a real-time PCR with a TaqMan probe for E. uzenensis, and its evaluation. In sensitivity assays, the detection threshold of this protocol was 101 cfu ml-1 on pure bacterial cultures and 102-103 cfu ml-1 on spiked plant material. The specificity of the protocol was evaluated against E. uzenensis and 46 strains of pear-associated Erwinia species different to E. uzenensis. No cross-reaction with the non-target bacterial species or the loss of sensitivity were observed. This specific and sensitive diagnostic tool may reveal a wider distribution and host range of E. uzenensis initially considered restricted to a region and will expand our knowledge of the life cycle and environmental preferences of this pathogen.

Genotyping Citrus tristeza virus Isolates by Sequential Multiplex RT-PCR and Microarray Hybridization in a Lab-on-Chip Device.

Citrus tristeza virus (CTV) is the largest known plant RNA virus (ca. 20 Kb), with a plethora of isolates conventionally categorized into six main genotypic groups (T36, VT, T3, RB, T68, T30). Each group includes many isolates with different phenotype profiles. Several techniques and protocols, mostly based on RT-PCR analysis of different regions of specific genes, have been developed for managing the diseases caused by CTV. However, more accurate genomic information would help to plan a correct strategy. This chapter describes a pilot protocol based on a sequential multiplex RT-PCR reaction and microarray hybridization in a miniaturized silicon lab-on-chip (LoC) device. The system comprises a set of 12 primers and 44 probes (× 2 replicates), designed on variable genomic regions of 6 genes: 5'UTR, ORF1a, ORF1b (RdRp), p33, p20, and p23. The system can rapidly analyze any genotype diversity associated with field isolates and distinguish the endemic from the non-endemic isolates. The identification of CTV strains is based on a number of probe hybridizations, which varies according to the genotypes present in the isolates and the differences among the genotypes.

Deep sequencing and analysis of small RNAs in sweet orange grafted on sour orange infected with two citrus tristeza virus isolates prevalent in Sicily.

Two representative isolates of a citrus tristeza virus population in Sicily, SG29 (aggressive) and Bau282 (mild), were sequenced via viral small RNAs (vsRNA) produced in budlings of sweet orange grafted on sour orange. Phylogenetic relationships with Mediterranean and exotic isolates revealed that SG29 clustered within the "VT-Asian" subtype, whereas Bau282 belonged to the cluster T30. The study confirms that molecular data need to be integrated with bio-indexing in order to obtain adequate information for risk assessment.

Rare localizations of echinococcosis: personal experience.

BACKGROUND: The rare localizations of hydatidosis represent an interesting topic, especially with reference to epidemiological and diagnostic problems. Hydatid disease, common in Mediterranean countries, still remains endemic in some regions of Italy. METHODS: In the period April 1983-October 2003 73 patients with hydatid disease were observed. RESULTS: Seven patients (9.5%) presented a primary rare localization (thyroid, kidney, sacrospinal muscle, peritoneum and spleen). The main peculiarity of primary rare hydatidosis localizations concerns diffusion of the infecting embryo; the most reliable hypothesis is that liver and lung can be bypassed through precapillary anastomosis between pre- and post-parenchymal circulation. The new imaging techniques allow a correct topographic evaluation and so a radical surgical treatment. The chemotherapy, complementary to surgery, produced degenerative modifications of parasite in 75.8% of the present cases and reduction of recurrence from 23.3% to 6.7%. At follow up of 1-5 years no patients had recurrence. CONCLUSION: It is important to be aware, especially in countries where echinococcosis is endemic, that it is possible to find an echinococcosis cyst in a rare localization.

Novel use of a T-tube access to perform an internal/external biliary drainage.

We report a case of post-surgical temporary functional stenosis of the sphincter of Oddi and biliary leak in a patient with a previous Billroth II reconstruction who had undergone cholecystectomy, surgical choledochotomy and sphincterotomy for biliary calculi. The patient was treated by creation of an internal/external biliary drainage using the T-tube access with an unreported technique.

Ischemic colitis following colonoscopy in a systemic lupus erythematosus patient: report of a case.

BACKGROUND: Ischemic colitis is an uncommon complication in patients with systemic lupus erythematosus but may be precipitated by colonoscopy. CASE REPORT: A 43-year-old female with systemic lupus erythematosus under treatment with immunosuppressive drugs and prednisone was submitted to colonoscopy because of a change in bowel habit. Apart from the presence of a small metaplastic polyp, colonoscopy showed only a few erythematosus areas in the sigma and left colon. Four hours after colonoscopy, the patient developed lower colic abdominal pain and mucous diarrhea followed by rectal bleeding from ischemic colitis. The patient was successfully treated with fluids, spasmolytic drugs, sodic heparin, antibiotics and enteral feeding. CONCLUSIONS: Awareness of the risk of this potential complication, secondary to colonoscopy, in patients with connective tissue disorders may lead to a prompt diagnosis and effective treatment, with a successful outcome.

Complex anal fistula surgery: personal experience.

Personal experience with the treatment cryptogenic complex anal fistulas over the 10-year period from 1993 to 2002 is reported. Such fistulas accounted for 54 out of 255 fistulas observed (21.1%). Accurate anatomo-pathological classification, based on the connections between the fistulas and the sphincter and the musculature of the pelvic floor, is mandatory, as is thorough preoperative evaluation of ano-rectal function and of the risk of faecal incontinence. The surgical strategies used, in relation to the different kinds of complex fistulas treated, are schematically reported. A mixed technique consisting in fistulectomy-fistulotomy with setons was particularly preferred, because of the risk related to immediate dissection of the sphincter, especially when concurrent risk factors are present. As regards the results obtained, the surgical outcome consisted in healing in 49/54 cases (90.7%) as against recurrence or persistence of the fistula in 5/54 (9.3%). Minor complications occurred in 6/54 (11.1%); no major complications were observed and no cases of permanent faecal incontinence were reported. In conclusion, the surgical choice in cases of complex fistulas must lead to definitive, radical treatment of the lesion, at the same time avoiding irreversible anal incontinence due to severe lesions to the sphincter.

Epithelioid hemangioendothelioma of the stomach: clinical-pathological features, nosologic setting, and surgical therapy. Report of a case.

Vascular tumors of the stomach represent 0.9%-3.3% of all gastric neoplasms. A 41-year-old man was admitted to our department with a 3-month history of early postprandial epigastric pain, sluggish digestion, nausea, asthenia, and occasional alimentary emesis. Preoperative staging detected a submucosal neoformation in the prepyloric zone, which narrowed the lumen, without any infiltrative features; a wedge gastric resection was performed and the definitive diagnosis was an epithelioid hemangioendothelioma of stomach. An 8-month follow up did not show any relapse of the disease. The term hemangioendothelioma is controversial because of disagreements regarding the nosologic setting and treatment. As a result, the latest WHO classification calls such neoplasms "borderline." The correct diagnosis depends on the histological findings supported by immunohistochemistry. Surgery represents the treatment of choice; however, a conservative approach is preferred whenever possible. However, due to the borderline biological behavior of this neoplasm, it is important that detailed clinical evaluations be carried out for such patients along with a thorough follow-up.