Serological detection of Grapevine rupestris stem pitting-associated virus (GRSPa V) by a polyclonal antiserum to recombinant virus coat protein

The coat protein gene of Grapevine rupestris stem pitting-associated virus (GRSPaV) was amplified with primers based on the completely sequenced Californian GRSPaV isolate, The protein expressed in Escherichia coli was used to raise an antiserum in rabbit. This antiserum was successfully used to detect virus coat protein in infected grapevine extracts either spotted on polyvinyl difluoride membranes (dot immunobinding) or blotted on membranes after gel separation (Western blot). The antiserum titre was 1:5,000 in Western blot. GRSPaV was detected in leaf petioles and cortical scrapings from dormant canes during the whole vegetative season. Several accessions of Vitis rupestris, currently used as presumptive virus-free indicators of Rupestris stem pitting, were found to be infected by this virus. While the application of the antiserum in ELISA was ineffective, the availability of similarly simple and effective serological tools, such as dot immunobinding, may allow a wide survey for GRSPaV

Surface Plasmon Resonance Assay for Label-Free and Selective Detection of Xylella Fastidiosa

Xylella fastidiosa is among the most dangerous plant bacteria worldwide causing a variety of diseases, with huge economic impact on agriculture and environment. A surveillance tool, ensuring the highest possible sensitivity enabling the early detection of X. fastidiosa outbreaks, would be of paramount importance. So far, a variety of plant pathogen biomarkers are studied by means of surface plasmon resonance (SPR). Herein, multiparameter SPR (MP-SPR) is used for the first time to develop a reliable and label-free detection method for X. fastidiosa. The real-time monitoring of the bioaffinity reactions is provided as well. Selectivity is guaranteed by biofunctionalizing the gold transducing interface with polyclonal antibodies for X. fastidiosa and it is assessed by means of a negative control experiment involving the nonbinding Paraburkholderia phytofirmans bacterium strain PsJN. Limit of detection of 105 CFU mL 1 is achieved by transducing the direct interaction between the bacterium and its affinity antibody. Moreover, the binding affinity between polyclonal antibodies and X. fastidiosa bacteria is also evaluated, returning an affinity constant of 3.5   107m 1, comparable with those given in the literature for bacteria detection against affinity antibodies

Serological detection of Grapevine rupestris stem pitting-associated virus (GRSPaV) by a polyclonal antiserum to recombinant virus coat protein

The coat protein gene of Grapevine rupestris stem pitting-associated virus (GRSPaV) was amplified with primers based on the completely sequenced Californian GRSPaV isolate, The protein expressed in Escherichia coli was used to raise an antiserum in rabbit. This antiserum was successfully used to detect virus coat protein in infected grapevine extracts either spotted on polyvinyl difluoride membranes (dot immunobinding) or blotted on membranes after gel separation (Western blot). The antiserum titre was 1:5,000 in Western blot. GRSPaV was detected in leaf petioles and cortical scrapings from dormant canes during the whole vegetative season. Several accessions of Vitis rupestris, currently used as presumptive virus-free indicators of Rupestris stem pitting, were found to be infected by this virus. While the application of the antiserum in ELISA was ineffective, the availability of similarly simple and effective serological tools, such as dot immunobinding, may allow a wide survey for GRSPaV

Isolation of recombinant antibodies (scFvs) to grapevine virus B

A panel of 15 recombinant single chain antibodies (scFv) specific to grapevine virus B (GVB) were recovered from a human combinatorial scFv antibody library using the phage display technique against purified virus particles. Two selected scFv-encoding genes were expressed in recombinant Escherichia coli cells as dimeric antibodies. Successful detection of GVB in tissues of herbaceous hosts and grapevine was obtained in a direct binding assay using dimeric scFvs. This reagent was also shown to substitute efficiently for a GVB polyclonal serum in standard DAS-ELISA test used routinely for diagnosis

Strategies for preventing group B streptococcal infections in newborns: A nation-wide survey of Italian policies

Background: There are no Italian data regarding the strategies for preventing neonatal group B streptococcal (GBS) infection. We conducted a national survey in order to explore obstetrical, neonatal and microbiological practices for the GBS prevention. Methods: Three distinct questionnaires were sent to obstetricians, neonatologists and microbiologists. Questionnaires included data on prenatal GBS screening, maternal risk factors, intrapartum antibiotic prophylaxis, microbiological information concerning specimen processing and GBS antimicrobial susceptibility. Results: All respondent obstetrical units used the culture-based screening approach to identify women who should receive intrapartum antibiotic prophylaxis, and more than half of the microbiological laboratories (58%) reported using specimen processing consistent with CDC guidelines. Most neonatal units (89 out of 107, 82%) reported using protocols for preventing GBS early-onset sepsis consistent with CDC guidelines. Conclusions: The screening-based strategy is largely prevalent in Italy, and most protocols for preventing GBS early-onset sepsis are consistent with CDC guidelines. However, we found discrepancies in practices among centers that may reflect the lack of Italian guidelines issued by public health organizations

Strategies for preventing group B streptococcal infections in newborns: A nation-wide survey of Italian policies

Background: There are no Italian data regarding the strategies for preventing neonatal group B streptococcal (GBS) infection. We conducted a national survey in order to explore obstetrical, neonatal and microbiological practices for the GBS prevention. Methods: Three distinct questionnaires were sent to obstetricians, neonatologists and microbiologists. Questionnaires included data on prenatal GBS screening, maternal risk factors, intrapartum antibiotic prophylaxis, microbiological information concerning specimen processing and GBS antimicrobial susceptibility. Results: All respondent obstetrical units used the culture-based screening approach to identify women who should receive intrapartum antibiotic prophylaxis, and more than half of the microbiological laboratories (58%) reported using specimen processing consistent with CDC guidelines. Most neonatal units (89 out of 107, 82%) reported using protocols for preventing GBS early-onset sepsis consistent with CDC guidelines. Conclusions: The screening-based strategy is largely prevalent in Italy, and most protocols for preventing GBS early-onset sepsis are consistent with CDC guidelines. However, we found discrepancies in practices among centers that may reflect the lack of Italian guidelines issued by public health organizations