Psychrobacter raelei sp. nov., isolated from a dog with peritonitis.

A strain belonging to the genus Psychrobacter, named PraFG1T, was isolated from the peritoneal effusion of a stray dog during necropsy procedures. The strain was characterized by the phylogenetic analyses based on the nucleotide sequences of 16S and 23S rRNA genes and of gyrB, which placed the strain in the genus Psychrobacter. The nucleotide sequence of the chromosome confirmed the placement, showing an average nucleotide identity of 72.1, 77.7, and 77.5 % with the closest related species, namely Psychrobacter sanguinis, Psychrobacter piechaudii, and Psychrobacter phenylpyruvicus, respectively, thus indicating a novel species. The polyphasic characterization by biochemical and fatty acid profiling as well as MALDI-TOF supported those findings. The strain was halotolerant, capable of growing within a temperature range between 4 and 37 °C, it was positive for catalase and oxidase, indole producing, nitrate reducing, and not able to use 5-keto-d-gluconic acid as a carbon source. Taken together, the data suggest that strain PraFG1T could be considered as representing a novel species, with the name Psychrobacter raelei sp. nov. (type strain PraFG1T=CIP 111873T=LMG 32233T).

Coronary Spasm Testing with Acetylcholine: A Powerful Tool for a Personalized Therapy of Coronary Vasomotor Disorders.

Coronary vasomotor disorders (CVD) are characterized by transient hypercontraction of coronary vascular smooth muscle cells, leading to hypercontraction of epicardial and/or microvascular coronary circulation. CVDs play a relevant role in the pathogenesis of ischemia, angina and myocardial infarction with non-obstructive coronary arteries. Invasive provocative testing with intracoronary Acetylcholine (ACh) administration is the gold standard tool for addressing CVD, providing relevant therapeutic and prognostic implications. However, safety concerns preclude the widespread incorporation of the ACh test into clinical practice. The purpose of this review is to shed light on the pathophysiology underlying CVD and on the clinical role of the ACh test, focusing on safety profile and prognostic implications. We will also discuss contemporary evidence on the management of CVD and the role of the ACh test in driving a personalized approach of patients with CVD.

Comprehensive Angiography-Derived Functional Assessment of Epicardial and Microvascular Coronary Disease. Correlation With Non-invasive Myocardial Stress Imaging.

Coronary angiography (CA) is poorly correlated with non-invasive myocardial stress imaging (NSI) and myocardial ischemia is often observed in patients with unobstructed coronary arteries. Moreover, the diagnostic performance of combined epicardial and microcirculatory angiography-derived physiological assessment and its correlation with NSI remains unknown. A total of 917 coronary vessels in 319 patients who underwent both CA and NSI were included in this multicenter observational retrospective analysis. Quantitative flow ratio (QFR) and angiography-derived index of microcirculatory resistance (IMRangio) analyses were performed to estimate coronary epicardial and microcirculatory function respectively. NSI demonstrated evidence of myocardial ischemia in 76% of the cases. IMRangio (36 [22 to 50] vs 29 [21 to 41], p <0.001) was significantly higher and QFR (0.92 [0.78 to 0.99] vs 0.97 [0.91 to 0.99], p <0.001) was significantly lower in vessels subtending ischemic territories. Overall, the diagnostic accuracy of QFR was moderate (area under the curve of receiver operating characteristic [AUCROC] 0.632 [95% confidence interval [CI] 0.589 to 0.674], p <0.0001) but it was higher in patients with normal microcirculatory function (AUCROC = 0.726 [95% CI 0.669 to 0.784], p <0.0001, p Value for AUCROC comparison = 0.009). Combined QFR/IMRangio assessment provided incremental diagnostic performance compared with the evaluation of epicardial or microcirculatory districts in isolation (p Value for AUC comparison <0.0001) and it was able to identify the predominant mechanism of myocardial ischemia in 77% of the patients with positive NSI. Our study suggests the value of a combined angiography-derived assessment of epicardial and microvascular function for the definition of the predominant mechanism of myocardial ischemia in patients with suspected coronary artery disease.

Environmental relationships and anthrax epidemiology: field experiences of host resistance as opposed to dose-dependent experiments.

Even though anthrax is a disease of antiquity that has been studied for centuries, serious concerns have been raised about our understanding of its epidemiology. Since the 1960s, we have based the epidemiology of anthrax on the results of dose-dependent experiments, especially those involving cattle at that time. In this species the experiments demonstrated that the severity of infection was dependent upon the numbers of Bacillus anthracis spores ingested. The opinion was that ingesting only a few spores would be insufficient to cause an apparent infection; any infection that resulted would be latent (i.e., unrecognized). Based on the results of these experiments, it was accepted that the ingestion of large numbers of spores was the source of infection for hundreds of anthrax outbreaks. However, many investigations of both human and animal anthrax outbreaks have failed to identify sources of large numbers of spores, suggesting that these outbreaks are only rarely a consequence of ingestion or inhalation of large quantities of spores. This opinion piece builds upon the indirect evidence previously presented in an article focused on the existence of latent infections. Much of the evidence for the existence of latent infections was predicated upon a reduction of host resistance, which revealed how latent infections could be a source of more severe forms of the infection. That is, a latent infection can be the source of a severe infection, but the cause of the severe infection is the reduced host resistance. That first article concentrated on the arguments for latent infections, while this article concentrates on the arguments for host resistance. Host resistance is virtually impossible to measure objectively in the field. To provide a subjective measure of host resistance during anthrax outbreaks, we suggest the use of the opinions of livestock owners and or their veterinary practitioners and or field workers during investigations of anthrax outbreaks. When veterinary personal work in the field they are much like field biologists. In some ways field biologists better appreciate environmental factors, population ecology and other perspectives that are of use to epidemiologists. The more diverse the information the better the epidemiology is understood. To this effect we present our personal anecdotal and theoretical ideas from our experiences as well as a collection of bibliographic observations from others'. Our conclusions are that a combination of latent infections and reduced host resistance based on the host's relationship with its environment would better explain the epidemiology of severe infections in anthrax outbreaks for which large quantities of spores have not been located. This applies especially if the area has a history of the disease and/or if necropsies have shown the presence of latent infections in otherwise normal animals in the area and/or if environmental conditions are considered stressful and include intense insect activity.

"The INOCA-IT: Rationale and design of a multicenter registry investigating ischemia in patients with non-obstructive coronary artery (INOCA) disease in Italy".

BACKGROUND: Ischemia with non-obstructive coronary artery (INOCA) disease is being progressively acknowledged as one of the pathophysiological mechanisms of chronic coronary syndrome (CCS) in an increasingly wide range of clinical pictures. Although the research has already begun to move towards a defined diagnostic pathway and a specific medical therapy for this disease, at present it remains a clinical challenge, especially if not thoroughly investigated. METHODS AND RESULTS: The INOCA IT Multicenter Registry RF-2019-12369486 is a prospective, multicentric, non-randomized, single-arm, open label clinical study which aims to evaluate the efficacy of a stratified diagnostic and therapeutic approach on adverse events prevention and symptom relief in Italian patients with INOCA disease. The study population includes patients with a clinical presentation of CCS for angina and/or positive stress test for myocardial ischemia and evidence of non-obstructive coronary artery disease (CAD) at coronary angiography. In these patients a complete invasive coronary physiology assessment is performed with the guidewire-based measurement of coronary flow reserve (CFR) and index of microvascular resistance (IMR), followed by acetylcholine (ACh) spasm provocation test. On the basis of the results of coronary function testing, patients are stratified into different INOCA endotypes (coronary microvascular disease, vasospastic angina, microvascular spasm, non-cardiac chest pain) and given a tailored medical therapy in accordance with the latest scientific evidence. At one year follow-up the impact of such a target therapy on angina class and quality of life, as well as on cardiovascular adverse events (hospitalization and coronary revascularization) is evaluated. CONCLUSIONS: The INOCA-IT Multicenter Registry will inform clinicians on sex-specific prevalence of INOCA in Italy and will show the impact of a stratified diagnostic and therapeutic approach on symptoms burden and prognosis of INOCA patients.

Application of Fourier Transform Infrared Spectroscopy to Discriminate Two Closely Related Bacterial Species: Bacillus anthracis and Bacillus cereus Sensu Stricto.

Fourier transform infrared spectroscopy (FTIRS) is a diagnostic technique historically used in the microbiological field for the characterization of bacterial strains in relation to the specific composition of their lipid, protein, and polysaccharide components. For each bacterial strain, it is possible to obtain a unique absorption spectrum that represents the fingerprint obtained based on the components of the outer cell membrane. In this study, FTIRS was applied for the first time as an experimental diagnostic tool for the discrimination of two pathogenic species belonging to the Bacillus cereus group, Bacillus anthracis and Bacillus cereus sensu stricto; these are two closely related species that are not so easy to differentiate using classical microbiological methods, representing an innovative technology in the field of animal health.

Prognostic value of combined fractional flow reserve and pressure-bounded coronary flow reserve: outcomes in FFR and Pb-CFR assessment.

BACKGROUND: Coronary flow reserve (CFR) has an emerging role to predict outcome in patients with and without flow-limiting stenoses. However, the role of its surrogate pressure bounded-CFR (Pb-CFR) is controversial. We investigated the usefulness of combined use of fractional flow reserve (FFR) and Pb-CFR to predict outcomes. METHODS: This is a sub-study of the PROPHET-FFR Trial, including patients with chronic coronary syndrome and functionally tested coronary lesions. Patients were divided into four groups based on positive or negative FFR (cut-off 0.80) and preserved (lower boundary ≥2) or reduced (upper boundary <2) Pb-CFR: Group1 FFR≤0.80/ Pb-CFR <2; Group 2 FFR≤0.80/Pb-CFR≥2; Group 3 FFR >0.80/Pb-CFR<2; Group 4 FFR>0.80/Pb-CFR≥2. Lesions with positive FFR were treated with PCI. Primary endpoint was the rate of major adverse cardiac events (MACEs), defined as a composite of death from any cause, myocardial infarction, target vessel revascularization, unplanned cardiac hospitalization at 36-months. RESULTS: A total of 609 patients and 816 lesions were available for the analysis. At Kaplan-Meier analysis MACEs rate was significantly different between groups (36.7% Group 1, 27.4% Group 2, 19.2% Group 3, 22.6% Group 4, P=0.019) and more prevalent in groups with FFR≤0.80 irrespective of Pb-CFR. In case of discrepancy, no difference in MACEs were observed between groups stratified by Pb-CFR. FFR≤0.80 was associated with an increased MACEs rate (30.2% vs. 21.5%, P<0.01) while Pb-CFR<2 was not (24.5% vs. 24.2% Pb-CFR≥2 P=0.67). CONCLUSIONS: FFR confirms its ability to predict outcomes in patients with intermediate coronary stenoses. Pb-CFR does not add any relevant prognostic information.

[Ischemia with non-obstructive coronary artery disease: state-of-the-art review]. / L'ischemia miocardica in assenza di coronaropatia ostruttiva: stato dell'arte.

Chest pain affects more than 100 million people globally, however up to 70% of patients undergoing invasive angiography do not have obstructive coronary artery disease and ischemia with non-obstructive coronary artery disease (INOCA) is often a cause of the clinical picture. The symptoms reported by INOCA patients are very heterogeneous and often misdiagnosed as non-cardiac leading to under-diagnosis/investigation and under-treatment. The underlying pathophysiological mechanisms of INOCA are multiple and include coronary vasospasm and microvascular dysfunction. Most importantly, this condition must not be considered benign: compared to asymptomatic individuals, INOCA patients present an increased incidence of cardiovascular events, rehospitalizations, as well as impaired quality of life, with increasing costs for healthcare systems. The aims of this review are to describe the pathophysiological and clinical characteristics of INOCA and to provide guidance to the medical community on the diagnostic approaches and management of INOCA, also via a series of clinical case reports.

Retrospective Analysis of Official Data on Anthrax in Europe with a Special Reference to Ukraine.

Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis that mostly affects grazing livestock and wildlife. Furthermore, B. anthracis is considered one of the most important biological agents of bioterrorism that could also be potentially misused in biological weapons. The distribution of anthrax in domestic animals and wildlife in Europe with a particular focus on Ukraine as a country of war was analyzed. Between 2005 and 2022, 267 anthrax cases were registered at the World Organization of Animal Health (WOAH) in animals in Europe, including 251 cases in domestic animals and 16 in wildlife. The highest numbers of cases were recorded in 2005 and 2016 followed by 2008, and the highest numbers of registered cases were reported from Albania, Russia, and Italy. In Ukraine, anthrax is currently a sporadic infection. Since 2007, 28 notifications were registered, with isolates mainly from soil samples. The highest number of confirmed anthrax cases was registered in 2018, and Odesa, which is close to Moldova, had the highest number of cases, followed by the Cherkasy region. The presence of thousands of biothermal pits and burial grounds of fallen cattle nationwide favors the re-emergence of new foci. Most confirmed cases were in cattle; however, single cases were confirmed in dogs, horses, and pigs. Further investigation of the disease in wildlife and in environmental samples is needed. The genetic analysis of isolates, investigation of susceptibility to antimicrobial compounds, and determination of virulence and pathogenicity factors are required in this volatile region of the world for awareness raising and preparedness.

Intracoronary physiology-guided percutaneous coronary intervention in patients with diabetes.

OBJECTIVE: The risk of vessel-oriented cardiac adverse events (VOCE) in patients with diabetes mellitus (DM) undergoing intracoronary physiology-guided coronary revascularization is poorly defined. The purpose of this work is to evaluate the risk of VOCE in patients with and without DM in whom percutaneous coronary intervention (PCI) was performed or deferred based on pressure-wire functional assessment. METHODS: This is a retrospective analysis of a multicenter registry of patients evaluated with fractional flow reserve (FFR) and/or non-hyperaemic pressure ratio (NHPR). Primary endpoint was a composite of VOCE including cardiac death, vessel-related myocardial infarction (MI), and ischemia-driven target vessel revascularization (TVR). RESULTS: A large cohort of 2828 patients with 3353 coronary lesions was analysed to assess the risk of VOCE at long-term follow-up (23 [14-36] months). Non-insulin-dependent-DM (NIDDM) was not associated with the primary endpoint in the overall cohort (adjusted Hazard Ratio [aHR] 1.18, 95% CI 0.87-1.59, P = 0.276) or in patients with coronary lesions treated with PCI (aHR = 1.30, 95% CI 0.78-2.16, P = 0.314). Conversely, insulin-dependent diabetes mellitus (IDDM) demonstrated an increased risk of VOCE in the overall cohort (aHR 1.76, 95% CI 1.07-2.91, P = 0.027), but not in coronary lesions undergoing PCI (aHR 1.26, 95% CI 0.50-3.16, P = 0.621). Importantly, in coronary lesions deferred after functional assessment IDDM (aHR 2.77, 95% CI 1.11-6.93, P = 0.029) but not NIDDM (aHR = 0.94, 95% CI 0.61-1.44, P = 0.776) was significantly associated with the risk of VOCE. IDDM caused a significant effect modification of FFR-based risk stratification (P for interaction < 0.001). CONCLUSION: Overall, DM was not associated with an increased risk of VOCE in patients undergoing physiology-guided coronary revascularization. However, IDDM represents a phenotype at high risk of VOCE.

Identification of Universally Applicable and Species-Specific Marker Peptides for Bacillus anthracis.

Anthrax is a zoonotic infection caused by the bacterium Bacillus anthracis (BA). Specific identification of this pathogen often relies on targeting genes located on two extrachromosomal plasmids, which represent the major pathogenicity factors of BA. However, more recent findings show that these plasmids have also been found in other closely related Bacillus species. In this study, we investigated the possibility of identifying species-specific and universally applicable marker peptides for BA. For this purpose, we applied a high-resolution mass spectrometry-based approach for 42 BA isolates. Along with the genomic sequencing data and by developing a bioinformatics data evaluation pipeline, which uses a database containing most of the publicly available protein sequences worldwide (UniParc), we were able to identify eleven universal marker peptides unique to BA. These markers are located on the chromosome and therefore, might overcome known problems, such as observable loss of plasmids in environmental species, plasmid loss during cultivation in the lab, and the fact that the virulence plasmids are not necessarily a unique feature of BA. The identified chromosomally encoded markers in this study could extend the small panel of already existing chromosomal targets and along with targets for the virulence plasmids, may pave the way to an even more reliable identification of BA using genomics- as well as proteomics-based techniques.

Safety and effectiveness of post percutaneous coronary intervention physiological assessment: Retrospective data from the post-revascularization optimization and physiological evaluation of intermediate lesions using fractional flow reserve registry.

Background: While the importance of invasive physiological assessment (IPA) to choose coronary lesions to be treated is ascertained, its role after PCI is less established. We evaluated feasibility and efficacy of Physiology-guided PCI in the everyday practice in a retrospective registry performed in a single high-volume and "physiology-believer" center. Materials and methods: The PROPHET-FFR study (NCT05056662) patients undergoing an IPA in 2015-2020 were retrospectively enrolled in three groups: Control group comprising patients for whom PCI was deferred based on a IPA; Angiography-Guided PCI group comprising patients undergoing PCI based on an IPA but without a post-PCI IPA; Physiology-guided PCI group comprising patients undergoing PCI based on an IPA and an IPA after PCI, followed by a physiology-guided optimization, if indicated. Optimal result was defined by an FFR value ≥ 0.90. Results: A total of 1,322 patients with 1,591 lesions were available for the analysis. 893 patients (67.5%) in Control Group, 249 patients (18.8%) in Angiography-guided PCI Group and 180 patients (13.6%) in Physiology-guided PCI group. In 89 patients a suboptimal functional result was achieved that was optimized in 22 cases leading to a "Final FFR" value of 0.90 ± 0.04 in Angiography-Guided PCI group. Procedural time, costs, and rate of complications were similar. At follow up the rate of MACEs for the Physiology-guided PCI group was similar to the Control Group (7.2% vs. 8.2%, p = 0.765) and significantly lower than the Angiography-guided PCI Group (14.9%, p < 0.001), mainly driven by a reduction in TVRs. Conclusion: "Physiology-guided PCI" is a feasible strategy with a favorable impact on mid-term prognosis. Prospective studies using a standardized IPA are warrant to confirm these data.

Toxigenic Genes, Pathogenic Potential and Antimicrobial Resistance of Bacillus cereus Group Isolated from Ice Cream and Characterized by Whole Genome Sequencing.

Bacillus cereus is isolated from a variety of foods where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature. In this study, we identified members of B. cereus groups in 65% of the ice cream samples analyzed, which were characterized based on multi locus variable number tandem repeats analysis (MLVA) and whole genome sequencing (WGS). The MLVA revealed that 36 strains showed different allelic profiles. Analyses of WGS data enabled the identification of three members of the B. cereus group: B. cereus sensu stricto, B. mosaicus and B. thuringiensis. Based on the multi locus sequence typing (MLST) scheme, the strains were classified in 27 sequence types (STs), including ST26 that causes food poisoning. Toxin genes' detection revealed the presence of the genes encoding nonhemolytic enterotoxin (NHE), hemolysin BL (HBL), cytotoxin K (cytK) and cereulide (ces) in 100%, 44%, 42% and 8% of the strains, respectively. The identification of eleven antimicrobial resistance (AMR) genes predicted the resistance to five different antimicrobials, and the resistance to beta-lactam antibiotics was confirmed with a phenotypic antimicrobial test. Taken together, the results showed that the B. cereus strains isolated from ice cream were a potential hazard for consumer safety.

Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method.

Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller-Hinton without additives for the AST of B. melitensis.

Isolation and characterization of Yersinia enterocolitica from foods in Apulia and Basilicata regions (Italy) by conventional and modern methods.

Yersiniosis is the third most reported food-borne zoonosis in Europe. The aim of the present study was to perform the search for Yersinia enterocolitica in food samples collected from Apulia and Basilicata regions (Southern Italy) and to characterize any isolates by classical and modern analytical methods. A total of 130 samples were analyzed between July 2018 and July 2019: most of them were raw milk and dairy products made from it. Furthermore, 8 out of 130 samples were individual milk samples collected from bovines reared in a Brucella-free farm which showed false positive serological reaction for brucellosis due to the presence of pathogenic Y. enterocolitica O:9 biotype 2 in faeces. The Real Time PCR targeting the ail gene and the culture method were performed to detect pathogenic Y. enterocolitica. Isolates were subjected to API 20E (Biomerieux) and MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time-of-Flight) for species identification. All samples were negative for the ail gene. The culture method allowed to isolate suspicious colonies from 28 samples. The API 20E system and the MALDI-TOF MS technique identified 20 Y. enterocolitica and 1 Y. intermedia in a concordant way. The remaining 7 strains were all identified as Y. enterocolitica by the API 20E system, while the MALDI-TOF MS recognized 4 Y. intermedia, 1 Y. bercovieri and 2 Y. massiliensis. Genotypic characterization of the discordant strains was performed by rMLST and it confirmed the MALDI-TOF MS' results. Only non-pathogenic Y. enterocolitica biotype 1A strains were found, although with a non-negligible prevalence (P = 0.15 with CI 95% = ± 0.06). This study indicates a poor circulation of pathogenic Y. enterocolitica in food products made and marketed in the investigated areas. However, the small number of samples, insufficient for some food categories such as meat and vegetable, does not allow to exclude the presence of pathogenic strains at all.

An Outbreak of Human Systemic Anthrax, including One Case of Anthrax Meningitis, Occurred in Calabria Region (Italy): A Description of a Successful One Health Approach.

In this report, three cases of human cutaneous anthrax are described, one complicated by meningitis, and all were linked to a single infected bullock. A 41-year-old male truck driver, along with two male slaughterhouse workers, 45 and 42, were hospitalized for necrotic lesions of the arm associated with edema of the limb and high fever. All three patients were involved in transporting a bullock to the slaughterhouse. Microbiological examination on the prescapular lymph node and a piece of muscle from the bullock carcass showed the presence of Bacillus anthracis. The three patients underwent a biopsy of the affected tissues, and all samples tested positive for B. anthracis DNA using PCR. Furthermore, the truck driver also complained of an intense headache, and a CSF sampling was performed, showing him positive for B. anthracis by PCR, confirming the presumptive diagnosis of meningitis. Fast diagnosis and appropriate treatment are crucial for the management of human anthrax. Cooperation between human and veterinary medicine proved successful in diagnosing and resolving three human anthrax cases, confirming the reliability of the One Health approach for the surveillance of zoonoses.

SARS-CoV-2 Gamma and Delta Variants of Concern Might Undermine Neutralizing Activity Generated in Response to BNT162b2 mRNA Vaccination.

The Delta variant raised concern regarding its ability to evade SARS-CoV-2 vaccines. We evaluated a serum neutralizing response of 172 Italian healthcare workers, three months after complete Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer) vaccination, testing their sera against viral isolates of Alpha, Gamma and Delta variants, including 36 subjects with a previous SARS-CoV-2 infection. We assessed whether IgG anti-spike TRIM levels and serum neutralizing activity by seroneutralization assay were associated. Concerning Gamma variant, a two-fold reduction in neutralizing titres compared to the Alpha variant was observed, while a four-fold reduction of Delta virus compared to Alpha was found. A gender difference was observed in neutralizing titres only for the Gamma variant. The serum samples of 36 previously infected SARS-CoV-2 individuals neutralized Alpha, Gamma and Delta variants, demonstrating respectively a nearly three-fold and a five-fold reduction in neutralizing titres compared to Alpha variant. IgG anti-spike TRIM levels were positively correlated with serum neutralizing titres against the three variants. The Comirnaty vaccine provides sustained neutralizing antibody activity towards the Alpha variant, but it is less effective against Gamma and even less against Delta variants.

Production of a Bacillus anthracis Secretome with Suitable Characteristics as Antigen in a Complement Fixation Test.

In this study, we cultured the Bacillus anthracis vaccine strain Sterne 34F2 in a medium containing EDTA, and we assessed the best conditions to inhibit the activity of zinc-dependent metalloproteases to obtain a secretome containing a high concentration of non-degraded PA (PA83), as evaluated by the SDS-PAGE analysis. Then, we used this secretome as the antigen in a Complement Fixation Test (CFT) to monitor the production of antibodies against PA83 in the sera of rabbits vaccinated with Sterne 34F2 and then infected with a B. anthracis virulent strain to evaluate the potency of the vaccine. The PAS-based CFT results were compared with those obtained by using a commercial ELISA kit. The two serological tests gave similar results in terms of specificity and sensitivity, as the kinetics of the antibodies production was very similar. The Sterne 34F2 vaccine induced an antibody response to PA83, whose titer was not inferior to 1:8 in PAS-based CFT and 42 kU/mL in PA83-based ELISA, respectively, in all vaccinated rabbits. Our opinion is that the PAS-based CFT can be successfully employed in humans and in animals for epidemiological retrospective studies or post-vaccination monitoring. We also suggest the use of our method to test the efficacy of veterinary anthrax vaccines.

Evaluation of a Real-time PCR in Combination with a Cultivation Method for the Detection of Brucella Abortus in Organs of Infected Cattle in Southern Italy.

Introduction: Brucellosis is a widespread zoonosis of great economic importance for livestock farming in many areas of the world. It is a highly infectious disease which is diagnosed using conventional serological and microbiological methods. The aim of this study was to assess the efficiency of a specific real-time PCR in combination with broth cultivation in detecting Brucella spp. in organs of infected cattle, in order to compare the sensitivity of the two approaches and the time needed in them until a correct diagnosis is made. Material and Methods: We examined 67 organs collected from 10 cattle slaughtered following a brucellosis outbreak which occurred in February 2016 in southern Italy. The research was carried out by enrichment broth cultivations in combination with a real-time PCR every week for six weeks. Results: Brucella strains were isolated by cultivation from 44 enrichment broths of organs. All isolates were later identified as Brucella abortus by real-time PCR. Using this method in combination with cultivation made it possible to identify the same percentage of infected animals faster than by cultivation alone. Moreover, the same diagnostic results were obtained, on average two weeks before they would have been using only cultivation. In almost all cases, Brucella was detected by real-time PCR after the first week of cultivation in pre-enrichment Brucella broth, while the bacterial growth was evident usually after 2 or 3 weeks. Conclusion: Real-time PCR has allowed results to be obtained faster than in the classical microbiological method, reducing the response times to identify positive animals by half.