Antibacterial and Biofilm Production Inhibition Activity of Thymus vulgaris L. Essential Oil against Salmonella spp. Isolates from Reptiles.

Salmonellosis is an infectious disease affecting both animals and humans. Antimicrobial resistant (AMR) and biofilm-producing Salmonella spp., frequently detected in reptiles (who can then act as asymptomatic carriers for warm-blooded animals), have developed resistance to biocides; this represents a warning for the emergence of biocide/antimicrobial cross-resistance. The aim of this study was to evaluate the efficacy of Thymus vulgaris L. essential oil (TEO) in inhibiting bacterial growth and biofilm production of Salmonella spp., which had been isolated from wild reptiles housed in a Zoo in Italy. The resistance profile against different classes of antibiotics showed that all the isolates were susceptible to the tested antibiotics, despite the presence of several AMR genes. All the isolates were also tested with aqueous solutions of TEO at different dilutions (5% to 0.039%). Interestingly, TEO proved effective both in inhibiting bacterial growth at low dilutions, with MIC and MBC values ranging between 0.078% and 0.312%, and in inhibiting biofilm production, with values ranging from 0.039% to 0.156%. TEO demonstrated effective bioactivity against the biofilm producer Salmonella spp., proving to be a valid disinfectant for the prevention of salmonellosis from reptiles, a possible source of infection for humans exposed to the reptiles' environment.

Epidemic intelligence data of Crimean-Congo haemorrhagic fever, European Region, 2012 to 2022: a new opportunity for risk mapping of neglected diseases.

BackgroundThe Epidemic Intelligence from Open Sources (EIOS) system, jointly developed by the World Health Organisation (WHO), the Joint Research Centre (JRC) of the European Commission and various partners, is a web-based platform that facilitate the monitoring of information on public health threats in near real-time from thousands of online sources.AimsTo assess the capacity of the EIOS system to strengthen data collection for neglected diseases of public health importance, and to evaluate the use of EIOS data for improving the understanding of the geographic extents of diseases and their level of risk.MethodsA Bayesian additive regression trees (BART) model was implemented to map the risk of Crimean-Congo haemorrhagic fever (CCHF) occurrence in 52 countries and territories within the European Region between January 2012 and March 2022 using data on CCHF occurrence retrieved from the EIOS system.ResultsThe model found a positive association between all temperature-related variables and the probability of CCHF occurrence, with an increased risk in warmer and drier areas. The highest risk of CCHF was found in the Mediterranean basin and in areas bordering the Black Sea. There was a general decreasing risk trend from south to north across the entire European Region.ConclusionThe study highlights that the information gathered by public health intelligence can be used to build a disease risk map. Internet-based sources could aid in the assessment of new or changing risks and planning effective actions in target areas.

Pilot Study on the Action of Thymus vulgaris Essential Oil in Treating the Most Common Bacterial Contaminants and Salmonella enterica subsp. enterica Serovar Derby in Poultry Litter.

The indiscriminate use of antimicrobials in poultry farms is linked to the increase in multi-resistant bacteria. Accordingly, based on the antimicrobial properties of Thyme Essential Oil (TEO), the present study evaluated the effects of TEO on the reduction of common microbial contaminants and Salmonella on poultry litter. A litter bulk sample was collected in a broiler farm and qualitative/quantitative investigations identified Escherichia coli and Mammaliicoccus lentus. The experimental contamination with Salmonella Derby wild strain was also performed. All pathogens showed phenotypic and genotypic resistance to different classes of antibiotics. The litter, split in different units, was treated with aqueous solutions of TEO at different concentrations (5% to 1.25%), demonstrating its effectiveness in reducing the total number of bacteria. The strongest antibacterial action was observed at the lowest concentration against Enterobacteriaceae, with a growth reduction compared to the positive control of 73.3% and 77.8% against E. coli and Salmonella Derby, respectively, while towards M. lentus the reduction was 50%. Our data confirm the antimicrobial activity of TEO and suggest its possible application for the treatment of poultry litter as an effective and natural approach for the prevention of diseases caused by the most common bacteria that colonize poultry farms, counteracting the onset of antibiotic resistance.

Assessment of Paratuberculosis international official reporting in Europe using the information supplied to the WOAH by National Veterinary Services.

The present study characterizes the epidemiological situation of Paratuberculosis (PTB) in Europe during the last 24 years, using the information officially reported to the World Organisation for Animal Health (WOAH) by veterinary services of the European countries. The prevalence of PTB at country level was described during the study period. A Cox proportional hazards (PH) regression analysis was implemented to evaluate the notification behaviours. Results from this work indicate that the most affected countries are in Southern and Western Europe, whereas PTB presence appears lower in Northern and Eastern Europe. PTB was routinely declared as a notifiable disease in 65% of the countries. Less than 50% of the countries routinely implemented passive surveillance, and only 19%, reported active surveillance for disease detection. Results from the Cox PH regression indicate that the Gross National Income (GNI) per capita and the application of active surveillance significantly influence the recurrence of PTB reporting. In countries with lower and upper middle income, the hazard of recurrence is 0.13 and 0.18 times lower than in countries with high income. The hazard of recurrence in countries that infrequently and moderately applied active surveillance is 1.99 and 1.65 times higher than in countries that routinely applied active surveillance. Findings from this work highlight an important variation in reporting behaviours, disease status and surveillance across Europe.

First serological evidence of Crimean-Congo haemorrhagic fever virus in transhumant bovines in Italy.

Crimean-Congo haemorrhagic fever (CCHF) is an emerging tick-borne disease caused by the arbovirus Crimean-Congo haemorrhagic fever virus (CCHFV; family Nairoviridae). Given the public health impact, CCHF is considered a priority disease for the European Union. This study describes the first detection of anti-CCHFV antibodies in transhumant bovines in Italy. Sera from 794 cattle collected across Basilicata region (Southern Italy) were screened using a commercial ELISA kit. The animal-level and herd-level seroprevalences detected were 1.89% [95%CI: 1.12-3.1] and 29.63% [95%CI: 15.68-48.65], respectively. Results of the χ2 test for trend show that the exposure to CCHFV was significantly associated with increasing age, with the odds 5 times higher in 11-22-year old cattle than 1-4-year old cattle. The detection of antibodies against CCHFV in indigenous cattle indicates that the infection occurred in the study area and may warrant further consideration. Additionally, no significant spatial clustering of CCHF infection was detected, supporting the hypothesis that the disease is widespread in the region. Further studies at larger scale are needed to identify the areas at higher risk of zoonotic infection. A One Health approach should be implemented to better understand the disease risk and dynamics in the country, which effectively address the related public health threat.

Antimicrobial Activity of Essential Oils Evaluated In Vitro against Escherichia coli and Staphylococcus aureus.

The spread of extended-spectrum ß-lactamase-producing Escherichia coli and methicillin-resistant Staphylococcus aureus has caused a reduction in antibiotic effectiveness and an increase in mortality rates. Essential oils (EOs), known for their therapeutic efficacy, can be configured as novel broad-spectrum biocides. Accordingly, the bacteriostatic-bactericidal activity of Citrus Lemon (LEO), Pinus Sylvestris (PEO), Foeniculum Vulgaris (FEO), Ocimum Basilicum (BEO), Melissa Officinalis (MEO), Thymus Vulgaris (TEO), and Zingiber Officinalis Rosc. (GEO), at concentrations ranging from 1.25 to 40% (v/v), were tested in vitro against different E. coli and S. aureus strains using minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The chemical compositions of the EOs were analyzed using GC/MS. The major components of all seven tested oils were limonene, α-pinene, anethole, estragole, citral, thymol, and zingiberene, respectively. We found that the bacteriostatic-bactericidal activity of the EOs was related to their chemotypes and concentrations, as well as the strain of the bacteria. A dose-effect correlation was found when testing GEO against S. aureus strains, whilst FEO was found to have no activity regardless of concentration. PEO, MEO, and BEO were found to have bactericidal effect with a MIC and MBC of 1.25% (v/v) against S. aureus strains, and LEO was found to have values of 1.25% (v/v) and 5% (v/v) against ATCC and clinical isolate, respectively. Interestingly, the antimicrobial activity of TEO was not related to oil concentration and the complete inhibition of growth across all E. coli and S. aureus was observed. Although preliminary, our data demonstrate the efficacy of EOs and pave the way for further investigations on their potential synergistic use with traditional drugs in the human and veterinary fields.

Multidrug resistant Enterococcus faecium isolate from cholangitis/cholecystitis in a dog.

Mucocele and cholecystitis were diagnosed in a 10 year-old Shetland-sheepdog presenting aspecific clinical signs. Blood examinations and ultrasonography investigation were performed before to surgical approach, which allowed to collect biopsies and samples for bacteriological analyses. In addition, the patient was subjected to cholecystectomy. A multidrug resistant Enterococcus faecium was isolated from the gallbladder specimens. On the basis of antimicrobial susceptibility test, nitrofurantoin was used. The correct bacteriological diagnosis is necessary to set up effective therapy, influencing the patient's prognosis and improving the recovery time.

Genetic Diversity of Porcine Circovirus Types 2 and 3 in Wild Boar in Italy.

Porcine circovirus (PCV) infection is associated with relevant economic impact to the pig industry. To date, four species of PCV (PCV1 to 4) have been identified but only PCV2 has been associated firmly with disease in pigs. The objective of this study was to assess the prevalence of PCV2 and PCV3 in the wild boar population in Basilicata region, Southern Italy, since this region is characterized by large forested and rural areas and the anthropic pressure is lower than in other Italian regions. Liver samples from 82 hunted wild boar were collected in 2021 from 3 different hunting districts. Sixty (73%, 95%CI: 63-82) samples tested positive for PCVs by quantitative PCR. In detail, 22 (27%, 95%CI: 18-37) were positive for PCV2, 58 (71%, 95%CI: 60-79) for PCV3, and 20 (24.4%, 95%CI 16-35) for both PCV2 and PCV3. On genome sequencing, different types and sub-types of PCV2 and PCV3 were identified, remarking a genetic diversity and hinting to a global circulation for the identified PCV strains. Overall, the high prevalence suggests that PCV2 and PCV3 infections are endemic in the wild boar population, posing risks for semi-intensive and free-range pig farming, typical of this region, due to contact with PCV-infected wild boar.

Crimean-Congo Haemorrhagic Fever (CCHF) in animals: Global characterization and evolution from 2006 to 2019.

This study describes the global distribution and temporal evolution of Crimean-Congo haemorrhagic fever (CCHF) during the period 2006-2019, using the information officially reported to the World Organisation for Animal Health (OIE) by the National Veterinary Services of 210 countries. Eight per cent (CI 95% 4-12) of the countries reported the disease as present at least once during the study period, whereas 82% (CI 95% 77-87) as absent. Information on CCHF status lacked for 10% (CI 95% 6-13) of the countries. The majority of the countries (46%, CI 95% 39-53) never declared CCHF as notifiable, whereas only 27% (CI 95% 21-33) reported the disease as notifiable during the large majority (≥76%) of the study period. The percentage of countries that routinely applied some CCHF control measures were as following: 14% (CI 95% 9-18) passive surveillance, 3% (CI 95% 0.9-5) active surveillance and 1% (CI 95% -0.2-3) control of vector. The time series analysis indicates a significant decreasing trend in the percentage of countries reporting no information, whereas the percentage of countries applying disease control measures significantly increase. This finding may reflect the increased awareness on the importance of the disease and the increased efforts to monitor virus circulation in animals through the application of surveillance programmes. Out of 25 countries reporting cases in humans since 2006, only 12 report cases in animals, pointing out the lack of surveillance capacity in animal populations for some countries. The paucity of CCHF notifications in animals may also reflect the difficulty in identifying the infection due to absent or mild clinical signs. Given that the implementation of surveillance programmes by the Veterinary Services is an essential tool for monitoring the virus circulation and prevent its further spread, National Veterinary Services should keep monitoring and reporting information on CCHF, and at the same time, they should improve the quality and accuracy of the information provided.

Bayesian estimation of prevalence of Johne's disease in dairy herds in Southern Italy.

Johne's disease (JD) is a chronic granulomatous disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) causing important losses on dairy farms. In Italy, voluntary programs to control MAP infection in dairy cattle are implemented in the Northern part of the country, where several studies have been carried out. Conversely, the disease status has not been fully investigated in the Southern regions. The aims of this study were to (i) determine the herd-level true prevalence (HTP) and (ii) the conditional within herd animal-level prevalence (CWHP) of JD in selected dairy cattle herds in Southern Italy. Serum samples were taken from 27 farms and analysed using a commercial ELISA test. A Bayesian model was fitted to the data. The estimated posterior mean of HPT was 0.46 (89 % CI 0.25-0.67), while the mean CWHP was 0.03 (89 % CI: 0.012-0.045). The results presented in this study call for designing and implementing an effective JD control program at national level.

Long-term persistence of neutralizing SARS-CoV-2 antibodies in pets.

We monitored the severe acute respiratory syndrome coronavirus 2 antibody response in seven dogs and two cats by using two multispecies ELISA tests, plaque reduction neutralisation test and virus neutralization. SARS-CoV-2 neutralizing antibodies in pets persisted up to 10 months since the first positive testing, thus replicating observations in COVID-19 human patients.

Basic knowledge and misconceptions on antibiotic use: a comparative survey between Veterinary College and High School students in Bari (Italy).

Misconceptions about the use and effectiveness of antibiotics contribute to the persistence of antimicrobial resistance. The aim of this study was to gather information on appropriate use of antibiotics in students from the Veterinary Medicine College (G1, n = 119) and from High School (G2, n = 220), from Bari (Italy) through a questionnaire. The response rate was 89% in G1 and 89.5% in G2. Fifty­five % of college students and 79% of high­school students had taken antibiotics in the last 12 months. Unsurprisingly, high­school students had more misconceptions about antibiotics than G1. The majority of misconceptions stated that i) antibiotics kill viruses (OR 8.4, CI 4.8­14.7, p < 0.001); ii) they are active against cold and flu (OR 4.6, CI 2.6­8.1, p < 0.001); iii) it is possible to purchase antibiotics without a medical prescription (OR 7.3, CI 4.3­12.5, p < 0.001). Information campaigns among young people are urgently needed to reduce misuse and to improve knowledge on antibiotic.

A systematic review and meta-analysis of hepatitis E virus (HEV) in wild boars.

This systematic review and meta-analysis summarize the available information on Hepatitis E virus (HEV) -specific antibody seroprevalence and HEV RNA prevalence in wild boar, one of the most abundant game species worldwide. A literature search (CAB Abstracts, Web of Science, Embase and Scopus) was performed to find relevant peer-reviewed works published during the period 1990-2020. A random-effect model was carried out to calculate the pooled HEV-specific antibody seroprevalence and HEV RNA prevalence estimates with 95% confidence intervals, and I2 statistic was used to assess the heterogeneity of the data. Values by subgroups were compared according to the geographical area, age class (≤ 12 months old and > 12 months old), and sample type (bile, faeces, liver, meat/muscle, serum). Sixty-nine publications were selected, with the majority of the studies from Southern Europe (n = 27). The pooled HEV-specific antibody seroprevalence in wild boar was 28% (CI95% 23-34) and the HEV RNA prevalence 8% (CI95% 6-10). The analysis highlighted a significant heterogeneity among the estimates from the included studies (I2 = 98% and I2 = 95% for HEV-specific antibody seroprevalence and viral prevalence respectively). The moderator analysis indicated a statistically significant difference (p-value = 0.03) for the HEV RNA prevalence according to the sample type, with the highest value in bile (17%, CI95% 9-27), followed by liver (10%, CI95% 7-14), serum (7%, CI95% 4-10), faeces (5%, CI95% 2-9), and meat/muscle (3%, CI95% 0.04-10). Finally, the HEV RNA prevalence in Europe (8.7, CI95% 6.7-11) was significantly (p-value = 0.04) higher than in Asia (4, CI95% 0.6-8). The analysis highlights the important role of wild boar in the epidemiology of HEV.

Fatal Calf Pneumonia Outbreaks in Italian Dairy Herds Involving Mycoplasma bovis and Other Agents of BRD Complex.

Mycoplasma bovis is increasingly recognized worldwide as an important cause of disease with major welfare and production impairments on cattle rearing. Although it was detected in veal calves and beef cattle, little is known on the infection impact and on its temporal morbidity pattern in Italian dairy herds. Thus, this study aimed to investigate the involvement of M. bovis on fatal calf pneumonia outbreaks that occurred during 2009-2019 in 64 Italian dairy farms. Furthermore, a deeper diagnostic workup of concurrent infection with other viral and bacterial respiratory pathogens was assessed. Out of the investigated fatal pneumonia cases, M. bovis was frequently detected (animal prevalence, 16.16%; 95%CI, 11.82-21.33; herd prevalence, 26.56; 95%CI, 16.29-39.08) either as the single agent of the disease in more than half of the positive samples (20/37) or in concurrent infections with Histophilus somni (9/37, 24.3%), Mannheimia haemolytica (6/37, 16.621%), Trueperella pyogenes (1/37, 2.70%), Pasteurella multocida (1/37, 2.70%), bovine respiratory syncytial virus (5/37, 13.51%), and bovine viral diarrhea virus (2/37, 5.55%). Based on time-series analysis, M. bovis was recorded in the area since 2009 with outbreaks displaying a clear morbidity seasonal pattern with peaks in April (43.21%) and in September (13.51%). This might be due to the stressing conditions during spring and late summer periods. Results of this study highlight that M. bovis infection warrants consideration, and control measures are needed given its involvement in lethal pneumonia outbreaks in dairy herds from an extended area.

Risk of Crimean Congo haemorrhagic fever virus (CCHFV) introduction and spread in CCHF-free countries in southern and Western Europe: A semi-quantitative risk assessment.

Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne viral zoonotic disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV). The disease is usually asymptomatic in domestic and wild animals, both of which may act as reservoirs of the virus. CCHF is endemic in parts of Africa, Asia, the Middle East and Eastern Europe. During the last decade, the emergence or re-emergence of CCHF was described in several countries in the Eastern Mediterranean Region, with an increasing risk of extension into new areas. Given the public health importance, this study undertakes a semi-quantitative risk assessment to analyse the likelihood of entry and exposure of CCHFV into 9 CCHF-free countries in Southern and Western Europe. Based on a framework outlining the probability of the virus entry and exposure, the risk estimates were assessed for each individual country. The risk assessment was performed using information from public databases and the available scientific literature. The likelihood of entry was conducted considering 3 main pathways: infected tick vectors, wildlife and livestock. The likelihood of exposure was assessed considering the probability of survival of the infected ticks once introduced in CCHF-free countries (depending on abiotic and biotic factors), and the exposure of resident uninfected susceptible ticks to infected imported wildlife and livestock. The risk estimates (combined CCHFV introduction and exposure) were low for the majority of the countries (Austria, Belgium, Germany, Luxembourg, Netherlands, Slovenia and Switzerland) and medium for France and Italy, if accounting only for animal health consequences. Considering the public health consequences only, the risks were rated low for all the countries, except for Italy where it was assessed to be medium.

Salmonella enterica Subsp. houtenae Associated with an Abscess in Young Roe Deer (Capreolus capreolus).

BACKGROUND: S. enterica subsp. houtenae has been rarely documented, and very limited genomic information is available. This report describes a rare case of primary extraintestinal salmonellosis in a young roe deer, associated with Salmonella enterica subsp. houtenae. Methods: A traditional cultural-based analysis was carried out from the contents of a neck abscess; biochemical identification and PCR assay were performed to isolate and identify the pathogen. Through whole-genome sequencing (WGS), multilocus sequence typing (MLST), core genome MLST (cgMLST), and the Salmonella pathogenicity islands (SPIs) survey, resistome and virulome genes were investigated to gain insight into the virulence and antimicrobial resistance of S. houtenae. RESULTS: Biochemical identification and PCR confirmed the presence of Salmonella spp. in the swelling. The WGS analysis identified Salmonella enterica subspecies houtenae serovar 43:z4,z23:- and ST 958. The virulence study predicted a multidrug resistance pattern with resistance shown against aminoglycosides, tetracycline, beta-lactamase, fluoroquinolones, fosfomycin, nitroimidazole, aminocoumarin, and peptide. Fifty-three antibiotic-resistant genes were identified. No plasmids were detected. CONCLUSION: This study demonstrates the importance of continuous surveillance of pathogenic salmonellae. Biomolecular analyses combined with epidemiological data can provide important information about poorly described Salmonella strains and can help to improve animal welfare.

Possible Human-to-Dog Transmission of SARS-CoV-2, Italy, 2020.

We detected severe acute respiratory syndrome coronavirus 2 in an otherwise healthy poodle living with 4 family members who had coronavirus disease. We observed antibodies in serum samples taken from the dog, indicating seroconversion. Full-length genome sequencing showed that the canine and human viruses were identical, suggesting human-to-animal transmission.

Detection of multi-drug resistance and AmpC ß-lactamase/extended-spectrum ß-lactamase genes in bacterial isolates of loggerhead sea turtles (Caretta caretta) from the Mediterranean Sea.

Sea turtles are useful sentinels to monitor the dissemination of antimicrobial resistance (AMR) in the marine coastal ecosystems. Forty Gram negative bacteria were isolated from wounds of 52 injured Caretta caretta, living in the Mediterranean Sea. Bacteria were identified using 16S rRNA gene sequencing and tested for susceptibility to 15 antibiotics. In addition, NGS amplicon sequencing was performed to detect the presence of AmpC ß-lactamase genes (blaAmpC) and extended-spectrum ß-lactamase (ESBL) genes (blaCTX-M,blaSHV,blaTEM). Seventy-five percent of the isolates (30/40 isolates) exhibited multidrug resistance (MDR) phenotypes and 32.5% (13/40 isolates) were confirmed to be positive for at least one gene. The variants of ESBLs genes were blaCTX-M-3,blaTEM-236 and blaSHV-12. Variants of the blaAmpCß-lactamase gene i.e., blaACT-24, blaACT-2, blaACT-17, blaDHA-4 and blaCMY-37, were also detected. In addition, 4 isolates were found simultaneously harboring CTX and AmpC genes while 2 strains harbored 3 genes (blaACT-2+TEM-236+SHV-12, and blaCTX-M-3+ACT-24+TEM-236).

Paratuberculosis at European scale: an overview from 2010 to 2017.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (PTB), a disease affecting domestic and wild ruminants. MAP may also play a zoonotic role in Crohn's disease. Although both governments and industries are carrying out programmes to prevent and control the infection, there is a lack of harmonization across Europe. Moreover, the success of these programmes is influenced by the current lack of sensitivity of the diagnostic tests used. For these reasons, it is complex to evaluate the overall epidemiological situation of this disease. This study describes the European distribution of PTB from 2010 to 2017 using the information reported by Member Countries to the OIE. Countries were classified in three categories ('Absent', 'Epizootic', 'Enzootic') depending on the disease epidemiology, and the trend of countries reporting the disease presence was computed throughout the study period. A multilevel model with random slope was built for twelve countries, with complete reporting history. Most of the countries (57.44%) were classified as 'Enzootic'. The percentage of countries reporting the disease presence slightly increased along the study period, probably due to the improvement of PTB monitoring, rather than to a deterioration of the epidemiological situation of the disease in Europe. Results of the model account for different dynamics in the number of outbreaks reported by 'Enzootic' and 'Epizootic' countries.

Brucella melitensis B115-based ELISA to unravel false positive serologic reactions in bovine brucellosis: a field study.

BACKGROUND: Brucellosis is a zoonosis whose incidence is not declining worldwide despite the global effort to control the disease. Accurate and precise diagnosis is a crucial step in any prophylaxis program but single tests to unequivocally detect animals infected with Brucella spp. are currently unavailable. In Italy, serological diagnosis of bovine brucellosis is performed with two official tests: a rapid agglutination test (i.e., Rose Bengal Plate test, RBPT) and a complement fixation test (CFT) that detect antibodies directed mainly to the smooth lipopolysaccharide (S-LPS). Neither of the two tests is able to avoid the detection of false positive serological reactions (FPSRs) caused by bacteria sharing S-LPS components with Brucella spp. and responsible for the single reactors (SR) phenomenon. A B. melitensis R strain-based ELISA showed a good diagnostic performance in unravelling FP animals; however, since a limited number of animals were analyzed in that study, a large field study was conducted here to discriminate between Brucella-infected from FP animals, with the final aim of reducing the unnecessary slaughter of the latter. An ELISA based on a R strain of Brucella, i.e., Brucella melitensis B115, was employed to measure specific IgG responses in a collection of bovine sera (n = 648). Sera were obtained from 180 farms (either officially brucellosis-free or not brucellosis-free) recruited during an extended period of time (2014-2018) and were preliminarily assayed with the official tests by the Italian Reference Centers and then subjected to the ELISA. RESULTS: Negative sera, when subjected to the ELISA, gave O.D. values below the cutoff; SR sera, i.e. RBPT positive and CFT negative, as well as double positive (DP) sera, i.e. RBPT and CFT positive, gave O.D. values that were below the cutoff. All positive sera, i.e. from Brucella-infected animals, were RBPT positive and CFT positive (ICFTU ranging from 20 to 1280) and gave ELISA O.D. values above the cutoff. CONCLUSIONS: The B. melitensis B115-based ELISA systematically unravelled all false positive (FP) sera while confirming the diagnosis in Brucella-infected animals. Thus, the test employed in the present study may complement the official assays to avoid the costly slaughter of FP animals.