Ixodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins.

The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures. Recently, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., 2000; J. Biol. Chem. 275, 18717-18723). Here, we isolated two different sequences, similar to Isac, from the transcriptome of I. ricinus salivary glands. Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins, called I. ricinus anticomplement (IRAC) protein I and II, are coexpressed constitutively in I. ricinus salivary glands and are upregulated during blood feeding. Also, we demonstrated that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses demonstrate that ticks belonging to the Ixodes ricinus complex encode a family of relatively small anticomplement molecules undergoing diversification by positive Darwinian selection.

Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning.

Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.

Risk factors for Clostridium difficile-associated diarrhoea in HIV-infected patients.

OBJECTIVE: To identify risk factors associated with a first episode of Clostridium difficile-associated diarrhoea (CDAD) in patients with HIV infection. DESIGN: A case-control study. SETTING: University teaching hospital HIV inpatient unit. PATIENTS AND METHODS: Nineteen HIV-infected patients with CDAD, defined as diarrhoea with positive stool culture for Clostridium difficile (CD) and positive stool cytotoxin B assay, were compared with 38 randomly selected controls (HIV-infected patients hospitalized on the ward on the day the matched case was diagnosed). CD isolates were phenotyped by electrophoretic protein patterns. RESULTS: The incidence of CDAD among HIV-infected patients was 4.1/100 of patient-admissions. On univariate analysis, cases were more likely to have used clindamycin [11 out of 19 compared with four out of 38; odds ratio (OR) 19; 95% confidence interval (CI), 2-160; P = 0.0007], and pyrimethamine (14 out of 19 compared with 13 out of 38; OR, 4.8; 95% CI, 1.4-16, P = 0.02) in the month before diagnosis, and to have had cerebral toxoplasmosis (12 out of 19 compared with 13 out of 38; OR, 2.8; 95% CI, 0.9-8.6; P = 0.09). There was also a significant increase of the risk of CDAD as duration of hospitalization in the ward increased (chi 2 for trend, P = 0.007). Multivariate models associated two risk factors with CDAD: clindamycin use (OR, 42; 95% CI, 2-813; P = 0.01), and prolonged hospitalization in the ward (OR, 3.6 per week in the ward; 95% CI, 1-13, P = 0.048). Of 18 available CD isolates, 15 (83%) had identical electrophoretic protein pattern. CONCLUSIONS: Clindamycin use and prolonged hospitalization in the ward were the main risk factors associated with CDAD in this study. These observations, together with the occurrence of one major phenotype of CD, suggest nosocomial transmission of CD in the ward.