Veterinaria Italiana (Journal)
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P3-09 Development and evaluation of the galleria mellonella (greater wax moth) model to study brucella host pathogen interaction
No full textBrucellosis is a zoonosis caused by bacteria of the genus Brucella. These gram-negative bacteria cause long-lasting infections, a process in which Brucella PAMP-modifications ( e.g. lipopolysaccharide [LPS]) hamper a prompt and effective activation of innate immunity. Laboratory models are essential to investigate Brucella virulence and although mice and, less frequently, guinea pigs have been used, ethical and practical considerations impede their use in high-throughput screening studies. Although lacking the complexity of mammalian immune system, the insect Galleria mellonella (greater wax moth) is increasingly being used as infection model as it conserves key aspects of innate immunity with mammals and has been useful in virulence analyses of relevant pathogens such as Klebsiella, Legionella, Mycobacteria and Listeria. To assess the potential of this model for the study of Brucella virulence, we evaluated G. mellonella larva survival upon infection with B. abortus 2308W wild-type and attenuated mutants (i.e. defective in the VirB Type-IV Secretion System [T4SS] or in the LPS-O- polysaccharide [O-PS]), and B. microti CCM4915. Then, we explored whether the survival profiles were related with a differential replication of brucellae by CFU-counting of whole-larva homogenates and fluorescence microscopy of primary- phagocyte isolates. Finally, we evaluated the ability of G. mellonella immunity to efficiently recognise Brucella by quantification of the pro-phenoloxidase system and melanisation activation after infection or LPS inoculation. As compared to K. pneumoniae 52145, B. abortus and B. microti induced a delayed and less severe mortality profile. Moreover, typical- brucellae did not trigger an early-melanisation response, consistent with the low immunostimulatory properties of Brucella LPS. Finally, we observed that Brucella virulence in Galleria is influenced by VirB and O-PS, since the corresponding mutants displayed even more marked delayed and less severe mortality profiles than the parental strain. Intriguingly, bacterial replication within larvae was affected by the lack of O-PS, but not of a functional T4SS. In light of these results, the G. mellonella model may represent an alternative tool for the study of Brucella interaction with innate immune, although the suitability of this model for the study of other aspects of Brucella pathogenesis, such as Brucella intracellular life, remains to be elucidated
O7-4 Screening of FDA approved Drugs to treat Brucellosis
No full textTreatment of Brucellosis in humans for six to eight weeks with a combination of doxycycline plus an aminoglycoside and/or rifampicin has been shown to be effective in the majority of cases. But they’re a small percentage (2 to 5%) of such treated cases has been shown to relapse; relapse rates were higher with two week regimen irrespective of combination of therapeutics. Therefore, there is a need for novel therapeutics that can effectively treat brucellosis with no cases of relapse. There is no cost-effective treatment of Brucellosis in food-producing animals. Drug repurposing is an efficient way of generating novel clinical opportunities for already existing drugs with the advantage of an economical accelerated drug development timeline with reduced costs. In the current study, we screened the MedChemExpress library consisting of FDA- approved drugs and additional clinical molecules against Brucella abortus. Out of the 2,591 drugs, 87 drugs were identified that did inhibit B. abortus at a concentration of 8 μM or less. The minimum inhibitory concentration (MIC) of the eleven drugs identified in the initial screening were tested against B. abortus using commercially certified concentrations of drug set. We found that Auranofin, Cinacalcet and Setraplatin are the most potent non-antibiotic drugs, identified in the MIC assay. They were further evaluated against two clinically important species i.e., B. suis and B. melitensis. The MIC values were similar and in the range of 0.06-4 μg/ml. Synergistic activity with approved antibiotics doxycycline and gentamicin against Brucella
P2-05 Evaluation of DNA extraction methods for long-read whole genome sequencing of atypical Brucella sp. isolates
No full textIn recent decades the description of several new species of Brucella has expanded the known mammalian host diversity of the genus considerably. Of these more recently described species, B. microti, B. inopinata and B. vulpis have been described as ‘atypical’, exhibiting either atypical phenotypic traits (B. microti), or greater genetic diversity (B. inopinata and B. vulpis) than classically described Brucella species. There are also an increasing number of reports of the isolation of atypical Brucella sp. organisms from other vertebrate hosts, including fish, reptiles and particularly amphibians. Analyses based on whole genome sequencing (WGS) have indicated that, whilst such atypical isolates maintain a high degree of genetic homology with core Brucella species, they exhibit a degree of horizontal gene transfer, incorporating genomic regions with sequence identity to soil associated Alphaproteobacteria. The inclusion of genetic elements from outsidethe genus Brucella emphasises the need for robust and reliable WGS tools to accurately characterise the genomes of novel atypical strains. Long-read sequencing methods are particularly valuable for this, as they can be used to construct complete bacterial genomes more reliably than approaches based on the de novo assembly of short sequencing reads alone. However, the selection of suitable DNA extraction methods has been shown to be integral to the success of long-read sequencing approaches. Furthermore, as the pathogenic potential of atypical Brucella strains remains unclear, such DNA extraction methods should ideally be compatible with methods commonly used for release of inactivated bacteria from containment laboratories. Using inactivated cultures of three atypical Brucella species (B. microti, B. inopinata and B. vulpis) we evaluated DNA extraction methods for producing long-read sequencing data using Oxford Nanopore Technologies MinIon sequencing platform. We evaluated metrics of sequencing (e.g. read length N50 andread number) and compared de novo genome assemblies, in order to identify the most suitable method. The results of these analyses are presented here
O4-6 PMNs supports Brucella dispersal with reduced immune recognition
No full textNeutrophils (PMNs) are the first line of defense against bacteria entering the body. However, it has been shown that Brucella induce low PMN activation and survive within these leukocytes by resisting their microbicidal mechanisms. Brucella also induce the premature cell death of PMNs, which release chemokines and express ‘eat me’ signals. These PMNs are then phagocyted by mononuclear cells where Brucella replicate. This evidence suggests that PMNs may behave as vehicles protecting Brucella from immune recognition, favoring their dispersion to the target organs. To test this hypothesis, we analyzed the course of infection in mice intraperitoneally infected with B. abortus alone (Ba) or with Brucella-infected PMNs (Ba-PMN). We evaluated bacterial loads, histopathological analyzes in selected tissues, cytokine production in serum, anti-Brucella antibody titers, and hematological parameters. We observed that mice infected with Ba-PMN had lower bacterial loads in the spleen and bone marrow at seven days of post-infection compared to Ba. The bacterial load then became equivalent at 30 days post-infection. The pathological index demonstrated a similar trend to the bacterial load. Ba-PMN infected mice showed less granulomatous inflammation in the spleen and bone marrow at seven days post-infection than the Ba group but with similar indexes at day 30. Comparably, Ba-PMN infected mice showed less IFN-gamma and IL-6 at the beginning of the infection than the Ba group but with similar concentrations at the end of the experiment. Ba-PMN infected mice also showed fewer anti-Brucella antibody titers at day 30 than the Ba group. No significant differences were observed between infected groups in the hematological values at 7 or 30 days. Despite both groups reaching similar bacterial loads by day 30, the bacterial loads and the immunological parameter were lower at the beginning of the infection in the Ba-PMN group. We conclude that the course of infection in the Ba-PMN group was stealthier than in the Ba group. Despite the slower course of Brucella infection in the Ba-PMN, PMNs supported their dispersion to a similar extent but with reduced immune recognition
P5-04 Production and immunization of sheep using Irradiated Brucella Vaccine
No full textBrucellosis is a highly contagious disease during animal abortions due to the created aerosols which may infect animals and humans in the surrounding area. Treatment of animals is not economically feasible, while the drugs to treat human disease are expensive and take a long time. The control of disease is mainly by animal vaccination. Irradiated Brucella melitensis Rev.1 vaccine was produced. Three groups of four animals (total 12) between the ages of 2 and 5 months were vaccinated. One group of ewes received the irradiated vaccine; a second group was vaccinated with live B. melitensis Rev.1, and the control group remained unvaccinated. Samples were collected twice a week and checked for humoral and cellular immunity using RBPT, cElisa, iElisa, cytokine Elisa, flow cytometry and RT-qPCR. The animals were challenged 18 months post- vaccination by being housed with infected sheep and goats (contact transmission). In addition, samples were collected during the challenge trial and checked for humoral immunity using RBPT, SAT and cElisa and iElisa. Bacterial isolation was also conducted using PCR to identify genus and species. Cytokine analysis is ongoing. B. melitensis Rev.1 irradiated vaccine was produced and was showed no growth and good metabolic activity. All animals vaccinated using live B. melitensis Rev.1 vaccine were serologically positivefor brucellosis, while the other two groups remained negative. Furthermore qPCR and flow cytometry investigation indicated elevation of cytokines in the two groups that received either the live or irradiated vaccines, whereas unvaccinated animals indicated stable cytokine levels. The challenge trial of the vaccinated animals caused gradual appearance of brucellosis antibodies in the two vaccinated animal groups. The animal experiment was implemented using three groups of ewes; one unvaccinated as the negative control; one vaccinated using live B. melitensis Rev.1, and one vaccinated using irradiated B. melitensis Rev.1. All immunity monitoring results of the tested animal groups were compatible with the expected response when vaccinated with an efficient vaccine
P6-06 The seroprevalence and geographic distribution of camel brucellosis in Kordofan States, Western Sudan
No full textThis work has been formulated to determine the special geographical distribution of brucellosis in camels in Sudan’s Kordofan States (North, West and South) and to compare different diagnostic tests for their sensitivity, specificity and agreement. In this study we used competitive enzyme-linked immunosorbent assay (cELISA) as the gold standard as compared with the modified Rose Bengal test (mRBPT), Buffer Acidified Plate Antigen (BAPA), and Serum Agglutination Test (SAT). The study area was chosen according to movement of camels and high densities of camels and ruminants. A total of 388 apparently healthy camels were sampled during the period from May to December 2018 to determine the seroprevalence of brucellosis. Within the collected samples, 43 (11.08%), 41 (10.56%) and 30 (7,73%) were found positive by mRBPT, BAPA, and SAT respectively. The 43 mRBPT positive samples were tested by cELISA where 38/43 (88.4%) were confirmed positive. Using cELISA as the gold standard, a sensitivity of 52.4% and a specificity of 100% were recorded for BAPA and a sensitivity of 58.8% and a specificity of 83.3% for SAT. KAPPA coefficient agreement between cELISA and BAPA (16%) and between cELISA and SAT (31%) mean there is poor agreement between cELISA as a golden standard test and the other two tests (≤40 consider poor). The results of this study revealed that brucellosis occurs in camels from the Western States, which confirms and extends earlier findings regarding the widespread infection in camels in the Western Sudan. Accordingly with a map of camel brucellosis distribution, we acknowledge that control measures can be more effectively targeted in high prevalence areas like the Skiekan locality. Brucella organisms that infect camels are contagious for humans, and the disease is considered to have an economic impact as it affects reproduction, production, and camel trade; and it needs much more attention to attain successful control
P8-11 Brucella canis serological survey in kennel dogs in North of Italy: case reports and preliminary data
No full textCanine brucellosis is a worldwide infection and zoonotic disease caused by Brucella canis (Bc), which major public health concern is due to close contact between dogs and humans. In dogs, brucellosis most commonly manifests with reproductive symptoms, lymphadenopathy, osteoarticular and neurological disorders, although the occurrence of asymptomatic and chronic infections are frequently reported. The present study aims to assess the seroprevalence in 227 kennel/free-ranging dogs, 30% female and 70% male dogs, average age 5 years old (range 1-16 Y/o), enrolled within the Ricerca Corrente IZSVE 12/19. The dogs were screened for Bc specific antibodies through a microplate serum agglutination tests (mSAT). Dog’s sera were tested using two-fold dilutions (from 1:20 to 1:640) and incubated for 48 hours at 37°C. In case of serologic titers ≥1:20, samples were submitted to the National and WOAH Reference Laboratory for Brucellosis, wherecomplement fixation test (CFT), immunofluorescence test (IF) and bacterial isolation from blood and urine were attempted. Moreover, PCR targeting Brucella spp. genes was performed with DNA extracted from the clinical samples. Results reported an overall serologic prevalence of 1.77 % (4/227). Among the positive animals, one dog (entire male, 7 Y/o, mixed breed) with a concurrent parasitic disease (Trichuris spp.) was found positive for B. canis antibodies (mSAT 1:40) and negative for the bacterial isolation: this dog was found seronegative one month later. The second significant case was a dog (entire male, 5 Y/o, Rottweiler) with testicles decreased in volume, reporting a positive serology (mSAT 1:20, CTF 1:10, IF 1:80) and negative bacterial isolation. The dog was re-tested after one month: mSAT 1:20, CTF negative, IF 1:40, negative bacterial culture (blood and urine), and borderline positivity for real time PCR Brucella spp. This dog was consequently neutered and further investigations are ongoing. Currently, prevention and control of canine brucellosis are not easily achieved, especially due to the difficulty in identifying infected dogs: the development of novel diagnostic methods and hopefully of specific regulations will represent a crucial point for surveillance of canine brucellosis, and raising awareness among human health in a One-Health perspective. Funding: Ricerca Corrente IZSVE 12/19, Italian Ministry of Health
P2-07 Study of the genetic variability of Brucella melitensis isolated from goats and sheep of San Luis province in Argentina
No full textThe objective of this work was the analysis of the genetic variability of Brucella melitensis in the departments of Libertador General San Martín, Ayacucho and Belgrano that belong to the province of San Luis, Argentina. For this purpose, flocks with different prevalence values of brucellosis were identified by conventional serological techniques as Buffered Plate Antigen Test (BPAT), Fluorescence Polarization Assay (FPA) and Complement Fixation Test (CFT). Samples of spleen, lymph nodes, liver, testis, epididymis, semen, milk, and synovial fluid and abomasal contents of abortions from a total of 104 animals were processed, of which 98 were goats and 6 were sheep. 32 isolates of B. melitensis biovar 1 were obtained and identified by classical bacteriological methods. Also, the presence of the pathogen in tissues samples was evidenced by PCR. An animal was considered positive for PCR for B. melitensis when a specific band was obtained in at least one of the tissues from the same animal. After the molecular detection of B. melitensis in tissues with isolation, DNA samples from tissues from which it was not possible to isolate were analyzed and it was possible to demonstrate the presence of the agent in 62 animals (including 4 sheep). As genotyping method, the MLVA 16 scheme was used, which uses the variability of 16 molecular markers to generate an unequivocal individualization of the genomes of B. melitensis from different animals. The genotypes were analyzed to determine the phylogenetic relationship between them and their georeferenced data were added to have complete information on the presence and circulation of different genotypes in each department. This study allowed identifying 21 circulating genotypes in the goats from the different departments. The phylogenetic relationship model found some of the genotypes described as potential candidate founders of the lineage that is distributed in different areas of the province. The discriminatory ability of the technique in a real epidemiological situation was confirmed, including discriminating genotypes within the same herd. The genotypes from this work were no previously described in any international data base
K9 Brucellosis in wildlife and livestock
No full textBrucellosis is a highly contagious disease caused by bacteria in the genus Brucella. This disease causes devastating losses to commercial and small-scale farmers in endemic regions. However, the control of this disease is challenging as it is mainly focused on livestock but also occurs in wildlife. This presentation highlights the multidisciplinary approach to detecting and characterising brucellosis as the biology of Brucella infection and detection is essential. Accurate diagnosis of brucellosis using serological tests is an existing shortcoming complicating brucellosis control, which is further complicated by brucellosis in wildlife using tests developed for livestock. The isolations of Brucella species from cattle and wildlife that could be due to spillover in livestock or between wildlife and livestock were further investigated. Multiple transmission patterns were observed such as spillover in livestock, spread within a herd to the introduction of infected animals. This research emphasised vaccination and testing as basic control measures which are more challenging in wildlife than livestock. Despite the large body of research on brucellosis, breaking the chain of transmission and controlling this disease remains challenging and underscores the interdisciplinary and collaborative approach required to reduce the impact of this disease on livestock and wildlife
O5-7 Brucella abortus RB51 Vaccine Strain and Raw Milk Consumption: An Emerging Public Health Risk
No full textThe RB51 vaccine is an attenuated live strain of B. abortus and has been used in the United States since 1996 as part of the State-Federal Brucellosis Eradication Program. It is estimated that 4,000,000 calves are vaccinated annually with RB51 and an unknown proportion of these vaccinated animals may become chronic shedders of RB51. Since August 2017, the U.S. Centers for Disease Control and Prevention (CDC) has reported three confirmed human cases of brucellosis in Texas, New Jersey and New York due to B. abortus RB51. These cases were each associated with consumption of domestically acquired unpasteurized (raw) dairy products. The epidemiological investigation of a human RB51 case from New York led to a dairy farm in Pennsylvania (PA) as the potential source of infection. Laboratory testing by USDA’s National Veterinary Services Laboratory (NVSL) on animal samples collected from the PA farm identified one cow to be shedding RB51 in milk. Whole- genome sequencing (WGS) analysis of the RB51 strains cultured from the PA cow identified two distinct RB51 strain types that match the genetic profiles of RB51 strains isolated from two human RB51 brucellosis cases- one reported in October 2017 from New Jersey and another reported from New York in November 2018. CDC acquired the PA cow under an approved research protocol to investigatecurrently unknown questions related to the molecular diversity of RB51 in a host, patterns of shedding, host factors associated with chronic infection, and exposure risk to humans. From March to September 2019, we collected samples of quarter milk, blood, urine, rectal and vaginal swabs for culture, bacterial quantification and sequencing as well as antibiotic susceptibilty tesitng. Findings from this testing show intermittent shedding patterns of RB51 in the milk, genetic and phenotypic variations of the RB51 vaccine strain isolated in the mammary system and host immune response to infection with the RB51 vaccine strain