Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

An Unexpected Case of Opisthorchis felineus Infection Revealed during Liver Transplantation.

A man with hepatitis B infection was admitted to Pisa University Hospital for hepatological evaluation, which revealed multiple cystic lesions and suggested a cirrhotic evolution. Treatment with Entecavir 0.5 mg/day was started, resulting in rapid viral load suppression and alanine aminotransferase normalization. After 10 years, imaging documented a single nodule of hepatocellular carcinoma (HCC), and a robot-assisted nodule resection was performed. One year later, HCC recurrence prompted orthotopic liver transplantation, during which the patient died because of the sudden rupture of the donor's organ and rapid multiorgan deterioration before retransplantation. During post-mortem liver examination, adult worms were evidenced within large biliary ducts, suggesting infection with Opisthorchis or Clonorchis spp. flukes. Sequencing of the ITS2 locus, following PCR amplification of DNA extracted from liver tissue, revealed 100% identity with the reference sequence of O. felineus. Infection of the patient with O. felineus was confirmed by the presence of specific IgG detected by ELISA in the patient's sera. Two major alkaline phosphatase serum levels peaks observed during the first two years of antiviral therapy support the hypothesis that O. felineus infection worsened liver function. This case report highlights the importance of a very careful screening of parasitic infections in solid organ transplantation candidates.

Randomized Clinical Trial of Conventional versus Indoor Daylight Photodynamic Therapy for Treatment of Actinic Cheilitis.

INTRODUCTION: Actinic cheilitis (AC) is the biologic precursor of invasive squamous cell carcinoma (SCC) of the vermilion, and different treatment options have been investigated, but their efficacy is hampered by local inflammation, pain and slow recovery. Daylight photodynamic therapy (dl-PDT) has been demonstrated to represent a valuable treatment option for AC, but its feasibility is limited by weather conditions and latitude. METHODS: Our study proposed to compare the efficacy and tolerability of conventional photodynamic therapy (c-PDT) and indoor daylight photodynamic therapy (idl-PDT) with a white LED lamp for the treatment of AC. Sixteen patients were enrolled in the study: 8 (50%) treated with c-PDT and 8 (50%) treated with idl-PDT. All patients completed the study protocol. RESULTS: Both idl-PDT and c-PDT were demonstrated to be highly effective in terms of reduction of the cumulative lesional area and severity of the clinical score. Neither treatment was inferior to the other. The inflammatory reaction and the pain scores were milder with idl-PDT, whereas the cosmetic outcome was not different. CONCLUSION: The present findings confirm that idl-PDT can represent a valid therapeutic strategy for AC patients as well, despite the procedural difficulties and the risk of poor tolerability related to the body site.

CSPG4-specific immunity and survival prolongation in dogs with oral malignant melanoma immunized with human CSPG4 DNA.

PURPOSE: Due to the many similarities with its human counterpart, canine malignant melanoma (cMM) is a valuable model in which to assess the efficacy of novel therapeutic strategies. The model is herein used to evaluate the immunogenicity, safety, and therapeutic efficacy of a human chondroitin sulfate proteoglycan-4 (hCSPG4) DNA-based vaccine. The fact that homology between hCSPG4 and cCSPG4 amino-acidic sequences stands at more than 80% provides the rationale for using an hCSPG4 DNA vaccine in the cMM model. EXPERIMENTAL DESIGN: Dogs with stage II-III surgically resected CSPG4-positive oral MM were subjected to monthly intramuscular plasmid administration, which was followed immediately by electroporation (electrovaccination) for at least 6, and up to 20, months. The immunogenicity, safety, and therapeutic efficacy of the vaccine have been evaluated. RESULTS: hCSPG4 electrovaccination caused no clinically relevant local or systemic side effects and resulted in significantly longer overall and disease-free survival times in 14 vaccinated dogs as compared with 13 nonvaccinated controls. All vaccinated dogs developed antibodies against both hCSPG4 and cCSPG4. Seven vaccinated dogs were also tested for a cCSPG4-specific T-cell response and only two gave a detectable interferon (IFN)γ response. CONCLUSION: Xenogeneic electrovaccination against CSPG4 is able to overcome host unresponsiveness to the "self" antigen and seems to be effective in treating cMM, laying the foundation for its translation to a human clinical setting.

Glottic and skull indices in canine brachycephalic airway obstructive syndrome.

BACKGROUND: Forty dogs presented for brachycephalic airway obstructive syndrome with laryngeal collapse not over 1st degree (saccule eversion) underwent glottis endoscopic and radiographic skull measurements before surgery. Fifteen Pugs, fifteen French and ten English Bulldogs were included. The goals were prospectively to compare three common brachycephalic breeds for anatomical differences regarding glottis and skull measurements, and to assess if any correlation between glottis and skull measurements was present. Linear measurements were used to obtain glottis and skull indices. Correlations between glottis and skull indices and glottic measurements were evaluated. Finally, glottis indices were compared among the three breeds. RESULTS: No correlation was found for glottis and skull indices. The glottic index differed among the three breeds (smaller in Pugs and higher in English Bulldogs), ultimately representing a morphologic indicator of the different larynx shape in the three breeds (more rounded in English Bulldogs, more elliptical in Pugs and in-between in French Bulldogs). CONCLUSIONS: The lack of correlation between skull/glottic indices does not support skull morphology as predictor of glottic morphology. As Pugs had the lowest glottic index, it may be speculated that Pugs' original narrow glottic width may predispose to further progressive respiratory deterioration more easily than in the other two breeds.

Genetic variability of Echinococcus granulosus sensu stricto in Europe inferred by mitochondrial DNA sequences.

The genetic diversity of Echinococcus granulosus sensu stricto (s.s.) metacestodes from four European countries was evaluated by the DNA sequence analysis of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. Of the 312 organisms investigated, 132 were from Bulgaria, 35 from Hungary, 89 from Italy and 56 from Romania. Considerable intraspecific variation was observed in the mitochondrial cox1 sequences: 24 haplotypes were detected in the Eastern European population and seven in the Italian population. The Eastern European population parsimony network displayed a star-like features consisting of the most common haplotype EG1 (G1 genotype) and the three major haplotypes: EG2, EG3 and EG4. The EG1 was also the major haplotype in the Italian population network, though with a higher prevalence (73%) compared to the Eastern European network. The percentage of the population constituted by the G1 genotype was used as an indirect index to evaluate the genetic diversity within E. granulosus s.s. populations of Eurasia. A clinal correlation between the percentage of the G1 genotype and the geographical regions of Eurasia was observed: the G1 genotype is highly represented in the Mediterranean Basin; it decreases in Eastern Europe and South-West Asia and increases in China. This clinal correlation could reflect the spreading of livestock domestication from Southern-Western Asia during the Neolithic period, beginning around 12,000 BC.

Pleural omentalisation with en bloc ligation of the thoracic duct and pericardiectomy for idiopathic chylothorax in nine dogs and four cats.

Conventional treatment of idiopathic chylothorax (IC) involves thoracic duct (TD) ligation (with/without lymphagiography) combined with subphrenic pericardiectomy. Nine dogs and four cats with IC, which received intrathoracic omentalisation with TD en bloc ligation (not preceded by lymphangiography) and subphrenic pericardiectomy, were evaluated retrospectively. Seven of nine dogs and 3/4 cats were still alive and disease-free at the time of reporting (range 10-53 and 19-31 months, respectively). Clinical signs of IC did not decrease after the first surgery in one cat and one dog; in another dog clinical signs recurred after 5 months. Overall efficacy rate of this one-stage combined procedure was 77% (6 months), 73% (12 months), and 57% (24 months). Where a second surgery was performed in case of failure, the success rate in dogs was 89% (6 months) and 80% (24 months). Addition of pleural omentalisation to TD en bloc ligation and subphrenic pericardiectomy does not seem to improve results when compared with published data and at present does not seem advisable as a first choice.

Echinococcus ortleppi and E. granulosus G1, G2 and G3 genotypes in Italian bovines.

To increase the knowledge on Echinococcus genotypes infesting cattle and water buffaloes (Bubalus bubalis) born and bred in Italy, the germinal layer of hydatid cysts was collected from the liver and the lungs of 80 animals slaughtered in 2007. Two mitochondrial genes (the cytochrome c oxidase subunit I and the NADH subunit I) were tested by PCR. Four genotypes were identified: G1 (sheep strain), G2 (Tasmanian sheep strain), G3 (buffalo strain), and G5 (cattle strain). Fertile cysts were detected only in the lungs of 4.5% of the total G1 lung cysts, of 9.4% of the total G3 lung cysts, and in the only G5 infected animal. This is the first report of Echinococcus ortleppi (genotype G5) in Italy.