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.

A comprehensive investigation of protein expression profiles in L. monocytogenes exposed to thermal abuse, mild acid, and salt stress conditions.

Preventing L. monocytogenes infection is crucial for food safety, considering its widespread presence in the environment and its association with contaminated RTE foods. The pathogen's ability to persist under adverse conditions, for example, in food processing facilities, is linked to virulence and resistance mechanisms, including biofilm formation. In this study, the protein expression patterns of two L. monocytogenes 1/2a strains, grown under environmental stressors (mild acidic pH, thermal abuse, and high concentration of NaCl), were investigated. Protein identification and prediction were performed by nLC-ESI-MS/MS and nine different bioinformatic software programs, respectively. Gene enrichment analysis was carried out by STRING v11.05. A total of 1,215 proteins were identified, of which 335 were non-cytosolic proteins and 265 were immunogenic proteins. Proteomic analysis revealed differences in protein expression between L. monocytogenes strains in stressful conditions. The two strains exhibited unique protein expression profiles linked to stress response, virulence, and pathogenesis. Studying the proteomic profiles of such microorganisms provides information about adaptation and potential treatments, highlighting their genetic diversity and demonstrating the utility of bioinformatics and proteomics for a broader analysis of pathogens.

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.

Stress Adaptation Responses of a Listeria monocytogenes 1/2a Strain via Proteome Profiling.

Listeria monocytogenes is a foodborne pathogen that is ubiquitous and largely distributed in food manufacturing environments. It is responsible for listeriosis, a disease that can lead to significant morbidity and fatality in immunocompromised patients, pregnant women, and newborns. Few reports have been published about proteome adaptation when L. monocytogenes is cultivated in stress conditions. In this study, we applied one-dimensional electrophoresis and 2D-PAGE combined with tandem mass spectrometry to evaluate proteome profiling in the following conditions: mild acid, low temperature, and high NaCl concentration. The total proteome was analyzed, also considering the case of normal growth-supporting conditions. A total of 1,160 proteins were identified and those related to pathogenesis and stress response pathways were analyzed. The proteins involved in the expression of virulent pathways when L. monocytogenes ST7 strain was grown under different stress conditions were described. Certain proteins, particularly those involved in the pathogenesis pathway, such as Listeriolysin regulatory protein and Internalin A, were only found when the strain was grown under specific stress conditions. Studying how L. monocytogenes adapts to stress can help to control its growth in food, reducing the risk for consumers.

Production and Characterization of Monoclonal Antibodies Against the VP7 Protein of Epizootic Hemorrhagic Disease Virus.

Monoclonal antibodies (MAbs) against epizootic hemorrhagic disease virus (EHDV) were produced by immunizing BALB/c mice with rec-VP7-EHDV2; 66 clones producing MAbs able to recognize the VP7-EHDV with a strong reaction were obtained and tested in indirect enzyme-linked immunosorbent assay (i-ELISA) against the whole epizootic hemorrhagic disease (EHD) virus serotype 2; potential cross-reactions with related orbiviruses, as Bluetongue virus (BTV) and African horse sickness virus (AHSV), were investigated as well by i-ELISA, Western blot, and immunofluorescence. Fifty-three MAbs were specific for EHDV (VP7 recombinant protein and whole virus) and 13 reacted also with the VP7 of BTV. None of the MAbs reacted with AHSV. MAbs specific for EHDV were further characterized in a competitive ELISA (c-ELISA): 20 among them were found useful to develop a c-ELISA for the detection of antibodies against EHDV in bovine sera. The availability of this extensive set of MAbs provides the opportunity to develop a c-ELISA for the serological diagnosis of EHDV and to tune new methods for the isolation and identification of the virus in biological samples and cell cultures. The experimentation protocol was approved by the Italian Ministry of Health (number 639/2018-PR, Resp. to Prot. BDF16.13#295833199#).

Immunoproteome profiling of Listeria monocytogenes under mild acid and salt stress conditions.

Listeria monocytogenes is one of the main foodborne pathogens worldwide. Although its response to stress conditions has been extensively studied, it is still present in the food processing environments and is a concern for consumers. To investigate how this microorganism adapts its proteome in mild stress conditions, a combined proteomics and bioinformatics approach was used to characterize the immunogenic protein profile of a sequence type 7 (ST7) strain that caused severe listeriosis outbreaks in central Italy. Extracted proteins were analyzed by immunoblotting using positive sera against L. monocytogenes and nLC-ESI-MS/MS, and all data were examined by five software to predict subcellular localization (SCL). A total of 226 proteins were extracted from the bands of interest, 58 of which were classified as potential immunogenic antigens. Compared to control cells grown under optimal conditions, six proteins, some of which under-described, were expressed under mild acid and salt stress conditions and/or at 12°C. In particular, adaptation and shaping of the proteome mainly involved cell motility at 12°C without acid and salt stress, whereas the combination of the same temperature with mild acid and salt stress induced a response concerning carbohydrate metabolism, oxidative stress, and DNA repair. Raw data are available via ProteomeXchange with identifier PXD033519.

SARS-CoV-2 replicates in respiratory ex vivo organ cultures of domestic ruminant species.

There is strong evidence that severe acute respiratory syndrome 2 virus (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, originated from an animal reservoir. However, the exact mechanisms of emergence, the host species involved, and the risk to domestic and agricultural animals are largely unknown. Some domestic animal species, including cats, ferrets, and minks, have been demonstrated to be susceptible to SARS-CoV-2 infection, while others, such as pigs and chickens, are not. Importantly, the susceptibility of ruminants to SARS-CoV-2 is unknown, even though they often live in close proximity to humans. We investigated the replication and tissue tropism of two different SARS-CoV-2 isolates in the respiratory tract of three farm animal species - cattle, sheep, and pigs - using respiratory ex vivo organ cultures (EVOCs). We demonstrate that the respiratory tissues of cattle and sheep, but not of pigs, sustain viral replication in vitro of both isolates and that SARS-CoV-2 is associated to ACE2-expressing cells of the respiratory tract of both ruminant species. Intriguingly, a SARS-CoV-2 isolate containing an amino acid substitution at site 614 of the spike protein (mutation D614G) replicated at higher magnitude in ex vivo tissues of both ruminant species, supporting previous results obtained using human cells. These results suggest that additional in vivo experiments involving several ruminant species are warranted to determine their potential role in the epidemiology of this virus.

Humoral immune response and delayed-type hypersensitivity in rabbits infected with Trypanosoma equiperdum.

Trypanosoma equiperdum is the causative agent of dourine, a parasitic venereal disease of equids. In this work, rabbits were infected with T. equiperdum strain OVI; serological tests (complement fixation test, ELISA and immunoblotting), used for the diagnosis of dourine in horses, were applied to study rabbit humoral immune response and to characterise T. equiperdum antigen pattern recognised by antibodies from infected rabbits. Moreover a protein extract of T. equiperdum strain OVI was produced and tested in skin tests on infected rabbits to detect the cell-mediated response induced by T. equiperdum, in order to evaluate its use in the field diagnosis of dourine. Sera of infected rabbits recognized in immunoblotting Trypanosoma protein bands with molecular weight below 37 kDa, providing a serological response comparable with that already observed in dourine infected horses. Moreover the trypanosome protein extract was capable to produce in vivo delayed-type hypersensitivity (DHT Type IV) in rabbits and proved itself to be non-toxic and non-sensitizing.

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.

Pro-Inflammatory Response of Bovine Polymorphonuclear Cells Induced by Mycoplasma mycoides subsp. mycoides.

Mycoplasma mycoides subsp. mycoides (Mmm) is the etiological agent of contagious bovine pleuropneumonia (CBPP), one of the major diseases affecting cattle in sub-Saharan Africa. Some evidences suggest that the immune system of the host (cattle) plays an important role in the pathogenic mechanism of CBPP, but the factors involved in the process remain largely unknown. The present study aimed to investigate the cell response of bovine polymorphonuclear neutrophils (PMNs) after Mmm in vitro exposure using one step RT-qPCR and Western blotting. Data obtained indicate that gene and protein expression levels of some pro-inflammatory factors already change upon 30 min of PMNs exposure to Mmm. Of note, mRNA expression level in Mmm exposed PMNs increased in a time-dependent manner and for all time points investigated; targets expression was also detected by Western blotting in Mmm exposed PMNs only. These data demonstrate that when bovine PMN cells are triggered by Mmm, they undergo molecular changes, upregulating mRNA and protein expression of specific pro-inflammatory factors. These results provide additional information on host-pathogen interaction during CBPP infection.

Trypanosoma equiperdum Low Molecular Weight Proteins As Candidates for Specific Serological Diagnosis of Dourine.

The diagnosis of dourine can be difficult because the clinical signs of this disease in horses are similar to those of surra, caused by Trypanosoma evansi. Moreover, T. equiperdum and T. evansi are closely related and, so far, they cannot be distinguished using serological tests. In a previous work, the T. equiperdum protein pattern recognized by antibodies from dourine-infected horses and the humoral immune response kinetics were investigated by immunoblotting assay; a total of 20 sera from naturally and experimentally infected horses and from healthy animals were tested. Immunoblotting analysis showed that antibodies from infected horses specifically bind T. equiperdum low molecular weight proteins (from 16 to 35 kDa), which are not recognized by antibodies from uninfected horses. In this work, we tested other 615 sera (7 from naturally infected horses and 608 sera from healthy horses and donkeys): results confirmed the data obtained previously. In addition, six SDS-PAGE bands with molecular weight ranging from 10 to 37 kDa were analyzed by mass spectrometry, in order to identify immunogenic proteins that could be used as biomarkers for the diagnosis of dourine. A total of 167 proteins were identified. Among them, 37 were found unique for T. equiperdum. Twenty-four of them could represent possible candidate diagnostic antigens for the development of serological tests specific for T. equiperdum.

Selection of a monoclonal antibody by ELISA, immunoblotting and Quartz Crystal Microbalance technology for immunohistochemical detection of Mycoplasma mycoides subsp. mycoides.

An immunohistochemical (IHC) technique was optimised using a monoclonal antibody (MAb) to detect Mycoplasma mycoides subsp. mycoides (Mmm), the agent of Contagious Bovine Pleuropneumonia (CBPP), in sections of lung tissue. A panel of MAbs was produced and screened for Mmm speci city and for cross-reactivity against other mycoplasmas belonging and not belonging to the Mycoplasma mycoides cluster, using in parallel indirect ELISA (i-ELISA) and Immunoblotting (IB). Based on i-ELISA and IB characterization data, 1 MAb (clone 3G10E7) was selected and its highest a nity vs Mmm was con rmed by the Quartz Crystal Microbalance (QCM) technology. Afterwards, IHC analyses were conducted to compare MAb 3G10E7 vs rabbit Mmm speci c hyperimmune serum using lung tissue sections of CBPP infected and CBPP negative animals. Results suggest that screening of MAbs using in parallel ELISA, IB, and QCM technology enables to select high a nity target speci c MAbs. Immunohistochemical results demonstrated that MAb 3G10E7 improved IHC performances, showing reduced background staining and no cross-reactivity against Mycoplasma bovis, which is responsible of pneumonia in cattle.

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host-pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host-pathogen interactions.