Veterinaria Italiana (Journal)
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P3-04 A new model for the synthesis of the homopolymeric O-antigen of Brucella
No full textLipopolysaccharide (LPS) is the mayor outer membrane antigen in gram-negative bacteria and, in pathogens, is a central macromolecule in the pathogenic process. In Brucella the LPS has been shown to be a virulence factor required for the successful establishment of a persistent infection and that it affects the efficient intracellular replication of the bacterium, a hallmark of the infectious cycle. To date the accepted model for the synthesis of the O-antigen of Brucella fits the canonical homopolymeric pathway: the synthesis of the complete structure is completed on one poly-prenol-phosphate that is, afterwards, translocated to the periplasmic side of the inner membrane and ligated to the lipid-A-core completing the synthesis of the LPS. In this presentation we will present evidence that the synthesis of the O-antigen in Brucella occurs through a different mechanism as the one previously proposed and that it does not adjust to the canonicalhomopolymeric model. We found that Brucella uses two decaprenol-phosphate intermediaries: one onto which the perosamines and formyl- perosamines are polymerized and a second one, primed with the trisaccharide NAc-Qui-Man-Man that serves as an acceptor for the polymerized perosamines. We have additionally identified a protein with no known function to date, that controls the length of the O-antigen linked to the LPS modulating the number of perosamines polymerized on the first lipid intermediary. This mechanism of synthesis constitutes a new model for homopolymeric O-antigens
P2-04 Whole Genome sequencing of Brucella strains isolated from water buffalo in southern Italy during 2019-2021
No full textBrucellosis is the most widespread zoonosis in the world and is responsible for significant economic losses, particularly in bovine and water buffalo herds. The causative agent of the disease is Brucella spp. which can infect humans and numerous animal species, including cattle, water buffalo, sheep and pigs. In animals the main clinical manifestation of infection is abortion. The pathogen can be transmitted through direct contact with infected animals and parturition products or through consumption of contaminated raw milk and/or dairy products. The genus Brucella is characterized by a high level of homology of DNA. For this reason, advanced molecular methods are very useful tools for the characterization of field isolates and the identification of sources of infection. The aim of this study was to characterize by whole genome sequencing (WGS) Brucella strains isolated from water buffalo farms in the Campania Region in order to perform epidemiological trace-back studies. This study was carried out on 41 strains of Brucella abortus bv.1 isolated from lymph nodes of as many water buffaloes from 40 different farms in the Campania Region. All strains were sequenced for whole genome characterization. The obtained results showed very high homogeneity of the strains under study. Indeed they exhibited a unique MLST (ST1) genetic profile and 4 distinct MLVA genetic profiles. These data indicate a very low level of genetic variability of the analyzed Brucella strains and suggest an epidemiological situation characterized by the persistence of a few bacterial strains in close geographical areas, characterized by intense inter-human and commercial exchanges. This evidence suggests an inter-herd diffusion probably due to environmental factors associated with few sources of infection, likely common to most of the farms included in this study
O4-4 Effects of age on immune response induced by Brucella abortus s19 or rb51 vaccination in heifers
No full textThe purpose of this study was to compare the immunological response induced by bovine brucellosis vaccination (S19 and RB51) in calves vaccinated in different ages between 3 and 8 months, to determine the ideal age for vaccination of calves. One hundred and five female calves aged 3 to 8 months were randomly divided in nine groups (n=12), in a 3x3 (3 vaccination regimens x 3 ages) experimental design, as follows: three groups were vaccinated with S19 at 3-4 (G1), 5-6 (G2) or 7-8 (G3) months of age, three groups were vaccinated with RB51 at 3-4 (G4), 5-6 (G5) or 7-8 (G6) months of age and three groups were inoculated with saline (controls) at 3-4 (G7), 5-6 (G8) or 7-8 (G9) months of age. Vaccination of all groups occurred at day 0 and assays to evaluate the induced immune response were performed at days 0, 28 and 56 after vaccination, from peripheral blood from all heifers at each time point. Peripheral blood mononuclear cells (PBMC) were isolated and cultured in the presence of γ irradiated Brucella abortus 2308 antigen to quantify CD4+, CD8+, CD4+CD45RO+ and CD8+CD45RO+ T lymphocytes. Culture supernatants were also tested for IFN-γ by ELISA. For each response variable, we fitted a mixed model with a fixed effect for the interaction age (3-4, 5-6 and 7-8) x treatment (S19, RB51 and control) × time (0, 28 and 56), using the packages lme4 and emmeans in the software R. The model for IFN-Ɣ production by the PBMC following vaccination exhibited a significant interaction only treatment and time, showing no effect of the age on vaccination. There was a significant higher expression of IFN-Ɣ after vaccination with S19 or RB51 at 28- and 56-days post-vaccination in comparison with control animals regardless the age of the calves, however, non-significant difference was observed between S19 and RB51 at any time or age. In conclusion, our results showed no effect of the age (3 to 8 months) on the immune response induced by vaccination of heifers with either S19 and RB51, as well as that both vaccines elicited a strong IFN-Ɣ response after vaccination
O3-6 Structural insights into the NyxA/B effector family
No full textBrucella spp. survival and replication inside host cells requires a type IV secretion system (T4SS named VirB. The T4SS is a molecular nanomachine that translocates effector proteins into the host cell to subvert host cell function, metabolism and defenses. Although many VirB substrates have been identified in Brucella, few of them have been characterized functionally and structurally. NyxA and NyxB are homologous and conserved proteins found to be translocated by Brucella into the host during infection 1. The proteins were found to interact with the human Sentrin specific protein SENP3 and modify its localization in the cell. In this study we have investigated the structural properties of NyxA/B. We purified NyxA and NyxB recombinantly and solved the crystal structure of NyxB at a resolution of 2.5 Å. The protein reveals a novel dimeric fold that was confirmed by Multi-angle light scattering (MALS) and Small-Angle X-ray Scattering (SAXS) experiments. Structural analysis of the NyxB surface revealed an acidic pocket delineated by several residues strictly conserved in NyxA. In the context of the dimer, these surfaces are juxtaposed to form an extended concave negatively charged. The structure enabled the design of specific mutants at a groove formed by the dimer. A structure-function study will be presented, using site- directed mutagenesis, in vitro and in cellulointeraction assays, providing insights into NyxA/B interaction with SENP3
O6-6 Preliminary study of interferon gamma test used for diagnosis of brucellosis in the water buffalo
No full textBrucellosis (BRC) is a worldwide zoonosis with a strong economic and public health impact. In water buffalo, BRC is sustained by Brucella abortus and B. melitensis. Currently, diagnosis in ruminants is based on indirect serological assays required by actual legislation, the Rapid Rose Bengal Serum Agglutination (RBT) and the Complement Fixation (CFT) tests, that mainly detected antibodies against lipopolysaccharide (S-LPS) and thus, reveal the humoral immune response. In contrast, little is known about the cell-mediated (CM) responseduring brucellosis infection in buffalo. The aim of this study is to develop a diagnostic test based on the assessment of CM immunity. To this aim we are evaluating the use of the interferon-gamma test (IFN-γ), recently introduced for the diagnosis of bovine tuberculosis also in the buffalo species. Preliminary results of a field study are herein described. Serum and heparinised blood samples, collected from buffaloes, in 11 BRC-free herds (261 samples) and in 15 brucellosis-infected herds (158 samples), that were confirmed by bacteriological test, were analyzed. RBT and CFT assays were carried out according to the WOAH official procedures. Blood samples (1 mL) for the IFN-γ test were stimulated by two different concentrations of Brucellergene OCB (Zoetis, France) (40 U/well and 100 U/well) introducing phosphate-buffered saline (PBS), as negative control for the basal value of IFN-γ. The IFN-γ production was detected by the BOVIGAM TB kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. To evaluate the relationship between nonparametric values of CFT and IFN-γ assays, Chi-square statistic test was performed. Statistical analysis highlighted a higher association rate between CFT and IFN-γ tests positivity using the antigenic stimulus at 100 U/well (phi=0.32, P<0.0001) in respect to 40 U/well (phi=0.26, P<0.001). Although previous studies in bovine have been carried out at 40 U/well, our results suggest that in buffalo the antigen stimulation should be done at 100 U/well. These preliminary results suggest that the IFN-γ test could be used in buffalo as an ancillary test as its performance are similar to CFT test. Further studies are needed to evaluate the accuracy of the test
O9-4 Pathogenesis of Brucella suis biovar 1 in the armadillo (Chaetophractus villosus)
No full textBrucella suis biovar 1 has the broadest animal host spectrum. Affects domestic animals and wildlife species. The aim of our study was to investigate the pathogenesis of B. suis biovar 1 infection in the armadillo (Chaetophractus villosus) under experimental conditions. One gravid female and three adult males were inoculated with a suspension containing 1×106 CFU/mL (colony-forming units) of B. suis biovar 1 by oral route. In addition, the gravid female and one male received the same suspension by the conjunctival route. A young male and two females not inoculated were kept in contact with the animals inoculated. The serum samples were analysed using the Buffered Plate Antigen test and Fluorescence Polarization Assay for the detection of Brucella antibodies. The inoculated armadillos showed positive antibody titres 2 weeks post-inoculation. After 55 days, the animals that tested positive for Brucella (5 animals) were euthanized under anaesthesia (tiletamine and zolazepam, 5.0 mg/kg/I.M). Of the three uninoculated animals, one female was seropositive for Brucella infection. Brucella was isolated from the spleen, liver, mesenteric lymph nodes, uterus, testes, and urine. Characteristic histologic lesions were found in the epididymis. These results suggest that armadillos can be a reservoir for the spread of B. suis infection, and the persistence of Brucella in armadillo tissues constitutes a risk for humans, because of the cultural practice of armadillo meat consumption in rural communities. The animals used in this study were handled by trained personnel, according to the standards and conditions approved by the Animal Ethics Committee of INTA (National Institute of Agricultural Technology) and the International Guiding Principles for Biomedical Research Involving Animals (Council for International Organization of Medical Sciences and the International Council for Laboratory Animal Science). It should be noted that armadillo is not an endangered species
O8-1 T-Racing: a contact tracing tool for supporting epidemiological investigation during livestock disease outbreaks
No full textA user-friendly web application (T-Racing) was developed in an R environment to support epidemiological investigations through interacting maps, network graphs, and tables. The application relies on Temporal Network Analysis techniques and accounts for the dynamic nature of animal trade, to perform a quick and efficient back and forward tracing activity. T-Racing makes use of web services to retrieve data from diverse Official National Databases, through the plumber package and has been distributed using Shinyproxy. Movement data underlying the spatial-temporal tracing pattern can be explored according to time frame, animal species, movement to slaughterhouses, and movements in and out of the selected farm. The forward/backward search can be performed from one or more farms, selected among those which had moved animals or notified outbreak of disease within a selected time frame and or falling in a user-defined spatial buffer. Considering search results may often involve thousands of farms/movements (which would not be possible to visualize or use, when many), the T- Racing application is able to provide tools that may interactively and iteratively handle huge networks. Users can add or remove links/nodes according to epidemiological information and/or spatial location, node’s property, and network deepness,to get the most relevant results, including paths among outbreaks caused by genetically similar pathogens. Moreover, the result can be restricted to paths followed by single identified animals linked to the seeds. This tool may strongly support animal disease control measures since it can rapidly organize essential contact information from large datasets, and identify connections in a multi-area outbreak investigation thus supporting the epidemiological investigation
P9-03 Brucellosis in terrestrial and marine wildlife species from the European perspective
No full textBrucellosis is a major zoonosis worldwide. The disease infects humans and a wide range of domestic animal and wildlife species resulting in substantial economic losses in the livestock industry and a significant public health impact. The disease is endemic in the Mediterranean basin, including the European parts. Brucellosis has been extensively investigated in humans and domestic animals in several European Union (EU) countries. The disease has been eradicated in livestock, and brucellosis remains a rare event in domestic animals in many EU countries. However, the situation in wildlife is still not obvious. Therefore, the current study aims to clarify the situation of brucellosis in terrestrial and marine wildlife species in Europe. All published articles on wildlife brucellosis in EU countries in the last twenty years have been analyzed. In the last two decades, brucellosis was reported in terrestrial and marine wildlife species in 20 and 10 European countries, respectively. Wild boars and brown hares were the most studied terrestrial wildlife species. Poland, Croatia, and Belgium showed the highest seroprevalences among wild boar caused by B. suis biovar 2. The most investigated marine wildlife species were seals and porpoises. Most samples were collected from dead carcasses, and brucellosis was mainly due to B. ceti and B. pinnipedialis. The highly pathogenic B. melitensis and B. abortus have been reported from terrestrial and marine wild animals, pointing to a zoonotic threat to wild animal handlers and hunters. Countries reporting brucellosis in both terrestrial and marine wildlife species are Germany, Croatia, Norway, Sweden, Italy, and the Netherlands. Most reports detected anti-Brucella antibodies by serology, and B. suis biovar 2 was the main isolates found in wild pigs. Currently, no vaccine is available for wild animals. Culling infected wildlife and developing specific diagnostic criteria for wildlife brucellosis remains a challenge
P10.1 Time trends and forecasting of antimicrobial use and minimum inhibitory concentration for Salmonella spp. in chicken production
No full textAntimicrobial resistance (AMR) is a major concern for “One Health”. Any use of antimicrobials can potentially contribute to the spread of multidrug-resistant bacteria. Antimicrobial use (AMU) in food animals is projected to increase over time. Thus, it is important to monitor AMU and changes in the efficacy of antimicrobials. The objectives of this study were to determine (i) AMU over time in chicken production in Canada, (ii) to examine the advantages of using minimum inhibitory concentration (MIC) data to characterize temporal trends in AMR and (iii) to forecast the AMR to commonly used antimicrobials in Salmonella spp. from chicken production.
Farm-level data, including AMU and other farm management practices, were collected by the Canadian Integrated Program for Antimicrobial Resistance (CIPARS). Using CLSI guidelines, Salmonella spp. were isolated and susceptibility tested using broth microdilution. Statistical analysis was performed to determine the effect of AMU and management factors on MIC over time. Important risk factors included in the generalized linear model were selected by LASSO regression. The ability to predict subtle changes on MIC over time was compared using two different models (i) logistic and (ii) multinomial regression. The best model fit was determined by Akaike information criteria. Forecasting for the effect of AMU on AMR over time in Salmonella spp. was performed using ARIMA model.
Overall, AMU in chickens has decreased over the last decade. However, the use of some antimicrobials has fluctuated over time. The effect of time was most evident on the use of streptomycin, sulfamethoxazole, and tetracycline. In terms of AMR, there was decrease in the MIC’s of streptomycin and sulfamethoxazole and an increase in tetracycline over time. Both logistic and multinomial regression models showed similar results on the effect of AMU and time on MIC, however, the multinomial model was more sensitive to subtle MIC time trends. We found that the use of lincosamides-aminocyclitols or third generation cephalosporins contributed to tetracycline resistance. Forecasting model results suggest that if antimicrobials continue to be used at the current rate tetracycline resistance will continue to increase in Salmonella spp. isolated from Canadian chicken farms in the next 4 years.
Reduction in AMU in chicken production mitigates AMR in Salmonella spp., an important pathogen for human and animal health. Analyzing the AMR surveillance data by multinomial model is more accurate than logistic regression in detecting early subtle changes in MIC’s. Continued monitoring of AMU and AMR is necessary to evaluate the effect of measures to reduce AMU on food production and safety and to preserve the efficacy of antimicrobials to treat infections
R09.3 Unravelling the epidemiology of Mycobacterium bovis in North Cameroon using genomic, environmental and demographic data
No full textIn recent years, the increased availability, efficiency and reliability of genomic reading techniques, such as whole-genome sequencing (WGS), has significantly increased the amount of information we can use to study infectious diseases. Consequently, it has improved the precision of epidemiological inferences for pathogens like M. bovis. As a consequence, novel methods have been developed to combine spatial explicit data and pathogen’s genome to understand the impact of the landscape on epidemic spread in space and time (Dellicour et al., 2016).
In this study, we used WGS to gain insights into the epidemiology of M. bovis in Cameroon, a developing country where the pathogen has been reported for decades. Ninety-one high-quality sequences were obtained from tissue samples collected in four abattoirs located in the North of the country (Egbe et al., 2017). Sixty-four of these had complete metadata including, information on the geolocation of the herd the cattle were coming from. We tested the maximum credibility trees generated with the software Beast 1.10.4 against several spatial explicit factors: land cover, road network, population and cattle density, altitude.
Our findings suggested that M. bovis in Cameroon is slowly expanding its epidemiological range over time, therefore endemic stability is unlikely, as it was previously hinted by other studies in the literature (Awah-Ndukum et al., 2012). The most recent common ancestor was dated in the 1950s, and after an initial slow spread, a sudden jump in space to cover the entire study area was dated during the late 1960s. As previously reported (Egbe et al., 2017), most of the M. bovis genetic diversity was detected in the Adamawa and North region. The simultaneous prevalence of M. bovis in co-located cattle and humans highlights the risk of zoonotic transmission.
The spatial analysis suggested that percentage of land covered by forest and the altitude might have played a role in facilitating M. bovis spread in the area. While the former could be a proxy for wild species presence, the latter result could be due caused by the presence of agropastoral communities living in these regions uplands. Thanks to such rearing practices, cattle from different herds mingle together, increasing the number of potential infectious contacts, thus facilitating spread.
In conclusion, we showed how using genomic tools combined with geographical information can improve our understanding of livestock diseases ecology and spread patterns. This is of paramount importance in contexts where data about animal populations and their movements are scarce, which could hamper the usefulness of tools like epidemiological mechanistic models. Adopting genomic tools as part of surveillance would vastly improve our understanding of disease transmission processes and, therefore, control strategies