Molecular characteristics of a strain (Salento-1) of Xylella fastidiosa isolated in Apulia (Italy) from an olive plant with the quick decline syndrome

DNA-based approaches were used to characterize a strain (Salento-1) of Xylella fastidiosa obtained from an olive plant suffering from the syndrome of quick decline in Apulia (South Italy). Salento-1 was indistinguishable from strain CoDiRO previously isolated from olive in Apulia and assigned to  X. fastidiosa subsp. pauca. Based on our results and comparative analysis with reported data, the subspecies pauca, multiplex, and fastidiosa may invade olive throughout the world (California, Italy, Argentina and Brazil). The strain Salento-1 has been deposited in the National Collection of Plant Pathogenic Bacteria (NCPPB), England, and in the Belgian Coordinated Collections of Microorganisms (BCCM), Belgium

Insights on a founder effect: the case of Xylella fastidiosa in the Salento area of Apulia, Italy

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa, causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca. In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of  14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity

Insights on a founder effect: the case of <em>Xylella fastidiosa</em> in the Salento area of Apulia, Italy

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa, causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca. In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of  14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity

Molecular characteristics of a strain (Salento-1) of Xylella fastidiosa isolated in Apulia (Italy) from an olive plant with the quick decline syndrome

DNA-based approaches were used to characterize a strain (Salento-1) of Xylella fastidiosa obtained from an olive plant suffering from the syndrome of quick decline in Apulia (South Italy). Salento-1 was indistinguishable from strain CoDiRO previously isolated from olive in Apulia and assigned to  X. fastidiosa subsp. pauca. Based on our results and comparative analysis with reported data, the subspecies pauca, multiplex, and fastidiosa may invade olive throughout the world (California, Italy, Argentina and Brazil). The strain Salento-1 has been deposited in the National Collection of Plant Pathogenic Bacteria (NCPPB), England, and in the Belgian Coordinated Collections of Microorganisms (BCCM), Belgium

Immunogenicity and reactogenicity of modified vaccinia Ankara pre-exposure vaccination against mpox according to previous smallpox vaccine exposure and HIV infection: prospective cohort study

BACKGROUND: Pre-exposure vaccination with MVA-BN has been widely used against mpox to contain the 2022 outbreak. Many countries have defined prioritized strategies, administering a single dose to those historically vaccinated for smallpox, to achieve quickly adequate coverage in front of low supplies. Using epidemiological models, real-life effectiveness was estimated at approximately 36%–86%, but no clinical trials were performed. Few data on MVA-BN immunogenicity are currently available, and there are no established correlates of protection. Immunological response in PLWH in the context of the 2022 outbreak was also poorly described. METHODS: Blood samples were collected from participants eligible for pre-exposure MVA-BN vaccination before (T1) receiving a full course of vaccine (single-dose for vaccine-experienced or smallpox-primed and two-dose for smallpox vaccine-naïve or smallpox non-primed) and one month after the last dose (T2 and T3, respectively). MPXV-specific IgGs were measured by in-house immunofluorescence assay, using 1:20 as screening dilution, MPXV-specific nAbs by 50% plaque reduction neutralization test (PRNT50, starting dilution 1:10), and IFN-γ-producing specific T cells to MVA-BN vaccine, by ELISpot assay. Paired or unpaired t-test and Wilcoxon or Mann–Whitney test were used to analyse IgG and nAbs, and T-cell response, as appropriate. The probability of IgG and nAb response in vaccine-experienced vs. vaccine-naïve was estimated in participants not reactive at T1. The McNemar test was used to evaluate vaccination's effect on humoral response both overall and by smallpox vaccination history. In participants who were not reactive at T1, the proportion of becoming responders one month after full-cycle completion by exposure groups was compared by logistic regression and then analysed by HIV status strata (interaction test). The response was also examined in continuous, and the Average Treatment Effect (ATE) of the difference from baseline to schedule completion according to previous smallpox vaccination was estimated after weighting for HIV using a linear regression model. Self-reports of adverse effects following immunization (AEFIs) were prospectively collected after the first MVA-BN dose (T1). Systemic (S-AEFIs: fatigue, myalgia, headache, GI effects, chills) and local (L-AEFIs: redness, swelling, pain) AEFIs were graded as absent (grade 0), mild (1), moderate (2), or severe (3). The maximum level of severity for S-AEFIs and L-AEFIs ever experienced over the 30 days post-dose by vaccination exposure groups were analysed using a univariable multinomial logistic regression model and after adjusting for HIV status; for each of the symptoms, we also compared the mean duration by exposure group using an unpaired t-test. FINDING: Among the 164 participants included, 90 (54.8%) were smallpox vaccine-experienced. Median age was 49 years (IQR 41–55). Among the 76 (46%) PLWH, 76% had a CD4 count >500 cells/μL. There was evidence that both the IgG and nAbs titers increased after administration of the MVA-BN vaccine. However, there was no evidence for a difference in the potential mean change in humoral response from baseline to the completion of a full cycle when comparing primed vs. non-primed participants. Similarly, there was no evidence for a difference in the seroconversion rate after full cycle vaccination in the subset of participants not reactive for nAbs at T1 (p = 1.00 by Fisher's exact test). In this same analysis and for the nAbs outcome, there was some evidence of negative effect modification by HIV (interaction p-value = 0.17) as primed people living with HIV (PLWH) showed a lower probability of seroconversion vs. non-primed, and the opposite was seen in PLWoH. When evaluating the response in continuous, we observed an increase in T-cell response after MVA-BN vaccination in both primed and non-primed. There was evidence for a larger increase when using the 2-dose vs. one-dose strategy with a mean difference of −2.01 log2 (p ≤ 0.0001), after controlling for HIV. No evidence for a difference in the risk of developing any AEFIs of any grade were observed by exposure group, except for the lower risk of grade 2 (moderate) fatigue, induration and local pain which was lower in primed vs. non-primed [OR 0.26 (0.08–0.92), p = 0.037; OR 0.30 (0.10–0.88), p = 0.029 and OR 0.19 (0.05–0.73), p = 0.015, respectively]. No evidence for a difference in symptom duration was also detected between the groups. INTERPRETATION: The evaluation of the humoral and cellular response one month after the completion of the vaccination cycle suggested that MVA-BN is immunogenic and that the administration of a two-dose schedule is preferable regardless of the previous smallpox vaccination history, especially in PLWH, to maximize nAbs response. MVA-BN was safe as well tolerated, with grade 2 reactogenicity higher after the first administration in vaccine-naïve than in vaccine-experienced individuals, but with no evidence for a difference in the duration of these adverse effects. Further studies are needed to evaluate the long-term duration of immunity and to establish specific correlates of protection

Immunogenicity and reactogenicity of modified vaccinia Ankara pre-exposure vaccination against mpox according to previous smallpox vaccine exposure and HIV infection. Prospective cohort study

Background: Pre-exposure vaccination with MVA-BN has been widely used against mpox to contain the 2022 outbreak. Many countries have defined prioritized strategies, administering a single dose to those historically vaccinated for smallpox, to achieve quickly adequate coverage in front of low supplies. Using epidemiological models, real-life effectiveness was estimated at approximately 36%-86%, but no clinical trials were performed. Few data on MVA-BN immunogenicity are currently available, and there are no established correlates of protection. Immunological response in PLWH in the context of the 2022 outbreak was also poorly described. Methods: Blood samples were collected from participants eligible for pre-exposure MVA-BN vaccination before (T1) receiving a full course of vaccine (single-dose for vaccine-experienced or smallpox-primed and two-dose for smallpox vaccine-naïve or smallpox non-primed) and one month after the last dose (T2 and T3, respectively). MPXV-specific IgGs were measured by in-house immunofluorescence assay, using 1:20 as screening dilution, MPXV-specific nAbs by 50% plaque reduction neutralization test (PRNT50, starting dilution 1:10), and IFN-γ-producing specific T cells to MVA-BN vaccine, by ELISpot assay. Paired or unpaired t-test and Wilcoxon or Mann-Whitney test were used to analyse IgG and nAbs, and T-cell response, as appropriate. The probability of IgG and nAb response in vaccine-experienced vs. vaccine-naïve was estimated in participants not reactive at T1. The McNemar test was used to evaluate vaccination's effect on humoral response both overall and by smallpox vaccination history. In participants who were not reactive at T1, the proportion of becoming responders one month after full-cycle completion by exposure groups was compared by logistic regression and then analysed by HIV status strata (interaction test). The response was also examined in continuous, and the Average Treatment Effect (ATE) of the difference from baseline to schedule completion according to previous smallpox vaccination was estimated after weighting for HIV using a linear regression model. Self-reports of adverse effects following immunization (AEFIs) were prospectively collected after the first MVA-BN dose (T1). Systemic (S-AEFIs: fatigue, myalgia, headache, GI effects, chills) and local (L-AEFIs: redness, swelling, pain) AEFIs were graded as absent (grade 0), mild (1), moderate (2), or severe (3). The maximum level of severity for S-AEFIs and L-AEFIs ever experienced over the 30 days post-dose by vaccination exposure groups were analysed using a univariable multinomial logistic regression model and after adjusting for HIV status; for each of the symptoms, we also compared the mean duration by exposure group using an unpaired t-test. Findings: Among the 164 participants included, 90 (54.8%) were smallpox vaccine-experienced. Median age was 49 years (IQR 41-55). Among the 76 (46%) PLWH, 76% had a CD4 count &gt;500&nbsp;cells/μL. There was evidence that both the IgG and nAbs titers increased after administration of the MVA-BN vaccine. However, there was no evidence for a difference in the potential mean change in humoral response from baseline to the completion of a full cycle when comparing primed vs. non-primed participants. Similarly, there was no evidence for a difference in the seroconversion rate after full cycle vaccination in the subset of participants not reactive for nAbs at T1 (p&nbsp;=&nbsp;1.00 by Fisher's exact test). In this same analysis and for the nAbs outcome, there was some evidence of negative effect modification by HIV (interaction p-value&nbsp;=&nbsp;0.17) as primed people living with HIV (PLWH) showed a lower probability of seroconversion vs. non-primed, and the opposite was seen in PLWoH. When evaluating the response in continuous, we observed an increase in T-cell response after MVA-BN vaccination in both primed and non-primed. There was evidence for a larger increase when using the 2-dose vs. one-dose strategy with a mean difference of&nbsp;-2.01 log2 (p ≤ 0.0001), after controlling for HIV. No evidence for a difference in the risk of developing any AEFIs of any grade were observed by exposure group, except for the lower risk of grade 2 (moderate) fatigue, induration and local pain which was lower in primed vs. non-primed [OR 0.26 (0.08-0.92), p&nbsp;=&nbsp;0.037; OR 0.30 (0.10-0.88), p&nbsp;=&nbsp;0.029 and OR 0.19 (0.05-0.73), p&nbsp;=&nbsp;0.015, respectively]. No evidence for a difference in symptom duration was also detected between the groups. Interpretation: The evaluation of the humoral and cellular response one month after the completion of the vaccination cycle suggested that MVA-BN is immunogenic and that the administration of a two-dose schedule is preferable regardless of the previous smallpox vaccination history, especially in PLWH, to maximize nAbs response. MVA-BN was safe as well tolerated, with grade 2 reactogenicity higher after the first administration in vaccine-naïve than in vaccine-experienced individuals, but with no evidence for a difference in the duration of these adverse effects. Further studies are needed to evaluate the long-term duration of immunity and to establish specific correlates of protection. Funding: The study was supported by the National Institute for Infectious Disease Lazzaro Spallanzani IRCCS "Advanced grant 5&nbsp;×&nbsp;1000, 2021" and by the Italian Ministry of Health "Ricerca Corrente Linea 2"