Analysis of Trypanosoma equiperdum Recombinant Proteins for the Serological Diagnosis of Dourine.

The significance of Trypanosoma equiperdum as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with Trypanosoma equiperdum. This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, putative (A0A1G4I740) (UniProt accession numbers SCU68469.1, SCU66661.1 and SCU67727.1), were identified as unique to T. equiperdum by bioinformatics analysis. The proteins were expressed as recombinant proteins and tested using an indirect ELISA and immunoblotting test with a panel of horse positive and negative sera for dourine. The diagnostic sensitivity, specificity and accuracy of the i-ELISAs were 86.7%, 53.8% and 59.0% for A0A1G4I8N3; 53.3%, 58.7% and 57.9% for A0A1G4I464; and 73.3%, 65.0% and 66.3% for A0A1G4I740, respectively, while the diagnostic sensitivity, specificity and accuracy of immunoblotting were 86.7%, 92.5% and 91.6% for A0A1G4I8N3; 46.7%, 81.3% and 75.8% for A0A1G4I464; and 80.0%, 63.8% and 66.3% for A0A1G4I740. Among the three proteins evaluated in the present work, A0A1G4I8N3 provided the best results when tested by immunoblotting; diagnostic application of this protein should be further investigated using a greater number of positive and negative sera.

Evaluation of Three Serological Tests for Diagnosis of Canine Brucellosis.

Canine brucellosis caused by Brucella canis, is an infectious disease affecting dogs and wild Canidae. Clinical diagnosis is challenging, and laboratory testing is crucial for a definitive diagnosis. Various serological methods have been described, but their accuracy is uncertain due to limited validation studies. The present study aimed to evaluate the performances of three serological tests for the diagnosis of B. canis in comparison with bacterial isolation (gold standard), in order to establish a protocol for the serological diagnosis of canine brucellosis. A panel of sera from naturally infected dogs (n = 61), from which B. canis was isolated, and uninfected dogs (n = 143), negative for B. canis isolation, were tested using microplate serum agglutination (mSAT), complement fixation performed using the Brucella ovis antigen (B. ovis-CFT), and a commercial immunofluorescence assay (IFAT). The sensitivity and specificity of the three serological methods were, respectively, the following: 96.7% (95% CI 88.8-98.7%) and 92.3 (95% CI 86.7-95.1%) for mSAT; 96.7% (95% CI 88.8-98.7%) and 96.5 (95% CI 92.1-98.2%) for B. ovis-CFT; 98.4% (95% CI 91.3-99.4%) and 99.3 (95% CI 96.2-99.8%) for IFAT. The use in of the three methods in parallel, combined with bacterial isolation and molecular methods, could improve the diagnosis of the infection in dogs.

Proteomics and bioinformatics investigations to improve serological diagnosis of canine brucellosis.

PURPOSE: Brucella canis is pathogenic for dogs and humans. Serological diagnosis is a cost-effective approach for disease surveillance, but a major drawback of current serological tests is the cross-reactivity with other bacteria that results in false positive reactions. Development of indirect tests with improved sensitivity and specificity that use selected B. canis proteins instead of the whole antigen remain a priority. EXPERIMENTAL DESIGN: A western blotting assay was developed to define the serum antibody patterns associated to infection using a panel of positive and negative dog sera. B. canis positive sera recognized immunogenic bands ranging from 7 to 30 kDa that were then submitted to ESI-LC-MS/MS and analyzed by bioinformatics tools. RESULTS: A total of 398 B. canis proteins were identified. Bioinformatics tools identified 16 non cytoplasmic immunogenic proteins predicted as non-homologous with the most important Brucella cross-reactive bacteria and nine B. canis proteins non-homologous to B. ovis; among the latter, one resulted non-homologous to B. melitensis. Data are available via ProteomeXchange with identifier PXD042682. CONCLUSIONS AND CLINICAL RELEVANCE: The western blotting test developed was able to distinguish between infected and non-infected animals and may serve as a confirmatory test for the serological diagnosis of B. canis. The mass spectrometry and in silico results lead to the identification of specific candidate antigens that pave the way for the development of more accurate indirect diagnostic tests.

Brucella abortus Strain RB51 Administered to Prepubescent Water Buffaloes, from Vaccination to Lactation: Kinetics of Antibody Response and Vaccine Safety.

Brucella RB51 is a live modified vaccine. Its use in water buffalo has been proposed using a vaccination protocol different to that used for cattle, but knowledge of the long-term effects of RB51 vaccination in this species remains incomplete. The aim of the study was to evaluate the safety and kinetics of antibody responses in water buffaloes vaccinated according to the protocol described for the bovine species in the WOAH Manual, modified with the use of a triple dose. Water buffaloes were vaccinated with the vaccine RB51. A booster vaccination was administered at 12 months of age. When turning 23-25 months old, female animals were induced to pregnancy. RB51-specific antibodies were detected and quantified using a CFT based on the RB51 antigen. Vaccinated animals showed a positive serological reaction following each vaccine injection, but titers and the duration of the antibody differed among animals. For 36 weeks after booster vaccination, the comparison of CFT values between vaccinated and control groups remained constantly significant. Afterwards, antibody titers decreased. No relevant changes in antibody response were recorded during pregnancy or lactation. In conclusion, results indicated that the vaccination schedule applied is safe and allows for vaccinated and unvaccinated controls to be discriminated between for up to 8 months after booster vaccination.

Reemergence of an atypical bluetongue virus strain in goats, Sardinia, Italy.

Bluetongue virus (BTV) is the etiologic agent of bluetongue, a WOAH (founded as Office International des Épizooties, OIE)-notifiable economically important disease of ruminants. BTV is transmitted by Culicoides biting midges and 24 different "classical" serotypes have been reported to date. In recent years, several putative novel BTV serotypes, often referred to as "atypical" BTVs, have been documented. These are characterized by unusual biological characteristics, most notably avirulence and vector-independent transmission. Here, we describe the recurrence of such an atypical virus strain BTV-X ITL2021 detected in goats six years after its first discovery in Sardinia, Italy. Combined serological and genome analysis results clearly suggest that the two strains belong to the same BTV serotype. However, unlike the 2015 strain, BTV-X ITL2021 was successfully isolated in BSR cell-culture allowing further serological characterization. Lastly, seropositivity for BTV-X ITL2021 was detected by virus-neutralization in approximately 74% of animals tested, suggesting that this atypical BTV serotype has been circulating undetected in asymptomatic animals for years.

Infection sustained by lineage B.1.1.7 of SARS-CoV-2 is characterised by longer persistence and higher viral RNA loads in nasopharyngeal swabs.

Following the announcement on December 2020 about the emergence of a new variant (VOC 202012/ 01, B.1.1.7 lineage) in the United Kingdom, a targeted surveillance was put in place in the Abruzzo region (Italy), which allowed detection of 313 persons affected by lineage B.1.1.7, up to the 20th of February 2021. We investigated the results of RT-PCR on nasopharyngeal swabs tested from December 2020 to February 2021 to verify any difference on the viral load and persistence between people infected by lineage B.1.1.7 and others. Statistically significant lower values of CT associated with the detection of the N protein encoding gene (CT N) were observed in persons with lineage B.1.1.7 infection (median CT N = 15.8)in comparison to those infected by other lineages (median CT N = 16.9). A significantly longer duration of the persistence of SARS-CoV-2 RNA in nasopharyngeal swabs was observed in persons with lineage B.1.1.7 infection (16 days) in comparison to those infected by other lineages (14 days).

Proteomic analysis of Brucella melitensis and Brucella ovis for identification of virulence factor using bioinformatics approachs.

The genus Brucella includes several genetically monomorphic species but with different phenotypic and virulence characteristics. In this study, proteins of two Brucella species, B. melitensis type strain 16 M and B. ovis REO198 were compared by proteomics approach, in order to explain the phenotypic and pathophysiological differences among Brucella species and correlate them with virulence factors. Protein extracts from the two Brucella species were separated by SDS-PAGE and 5 areas, which resulted qualitatively and quantitatively different, were analyzed by nLC-MS/MS. A total of 880 proteins (274 proteins of B. melitensis and 606 proteins of B. ovis) were identified; their functional and structural features were analyzed by bioinformatics tools. Four unique peptides belonging to 3 proteins for B. ovis and 10 peptides derived from 7 proteins for B. melitensis were chosen for the high amount of predicted B-cell epitopes exposed to the solvent. Among these proteins, outer-membrane immunogenic protein (N8LTS7) and 25 kDa outer-membrane immunogenic protein (Q45321), respectively of B. ovis and B. melitensis, could be interesting candidates for improving diagnostics tests and vaccines. Moreover, 8 and 13 outer and periplasmic non homologue proteins of B. ovis and B. melitensis were identified to screen the phenotypic differences between the two Brucella strains. These proteins will be used to unravel pathogenesis and ameliorate current diagnostic assays.

The prevalence, characterisation, and antimicrobial resistance of Yersinia enterocolitica in pigs from Central Italy.

Widely spread in nature, Yersinia enterocolitica (YE) is a foodborne pathogen of major health and economic significance in developed countries. The aim of this study is to analyse YE strains isolated from 400 slaughtered pigs from the Abruzzo region, Italy, using biochemical tests and a multiplex polymerase chain reaction PCR detecting 6 chromosomal genes (ystA, irp2, 16s, ail, inv, hemR) and one plasmid-borne virulence gene (yadA). Antimicrobial susceptibility was evaluated and pulsed-field gel electrophoresis (PFGE) was also performed in order to assess phylogenetic diversity. In total, 56 samples of porcine tonsils (14%) were found to be positive for the presence of pathogenic YE. All YE belonged to the pathogenic bioserotype 4/O:3. All YE samples were positive for the chromosomal virulence genes ystA, ail, and inv, whereas results for the presence of yadA and hemR were variable. This study found that YE isolates were resistant to ampicillin (100%), streptomycin (26.79%), sulfisoxazole (19.65%), tetracycline (16.08%), nalidixic acid (14.30%), and chloramphenicol (10.72%). The strains characterised by PFGE showed a high similarity. This study demonstrates the usefulness of multiplex polymerase chain reaction (PCR) compared with conventional phenotypic assays for the identification of pathogenic YE isolates and the limitations of PFGE for the molecular typing of YE bioserotype 4/O:3.

Study of the toxic effects of flame retardant PBDE-47 on the clam Chamelea gallina (Linnaeus, 1758).

The purpose of the study is to evaluate the effects of 2,2',4,4'-tetrabromodiphenylether (PBDE-47) on the Chamelea gallina clam (according to current commercial regulations: Venus gallina). PBDEs, which are used as flame retardants in various industrial products, are classed as hazardous substances by Directive 2011/65/EU. They are bioaccumulative compounds, considered to be endocrine disruptors, genotoxic, neurotoxic and practically ubiquitous, and their concentration in the environment has considerably increased in recent years. The aim of this study is to establish the effects of PBDE-47 on Chamelea gallina: toxic power and any harmful effects on the gonads, bioaccumulation capacity in the tissues, and possible entry into the food chain. The research used 96-hour and 21-day experimental tests on clams housed in filtered seawater. The tests were preceded by a period of acclimatisation of the molluscs lasting five to seven days. The clams were fed on seaweed (Dunaliella tertiolecta). The choice of the toxic compound PBDE-47 was based on the high concentration, among the congeners of PBDE, found in some aquatic species. The study demonstrated that the concentration of the contaminant used did not alter the vital functions, cause significant levels of mortality or lead to evident alteration in the gonads of Chamelea gallina. However, the research demonstrated the bioaccumulation capacity of the bivalve mollusc, allowing PBDE-47 to enter the food chain.