Veterinaria Italiana (Journal)
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O9-3 Brucellosis in vampire bats of Costa Rica: Brucella nosferati sp. nov.
No full textForty-two Brucella strains were isolated from the salivary glands, mammary glands, milk, placenta, fetus, uterus, brain, lung liver, kidney, intestinal content, from seventeen out of 71 vampire bats (Desmodus rotundus) and six fetuses, as described before for other mammals. Rose Bengal Test and cELISA performed in 55 vampires sera resulted in 22 seropositive animals. Immunohistopathology revealed that the bacterium extensively replicates in vampire bat tissues, including the placenta, and causes placentitis. Whole genome sequencing (WGS) and phenotypical characterization demonstrated that these isolates represent a new classical Brucella species with clear-cut genetic markers, radiating into a distinct branch from all other species. The proposed name is B. nosferati nov. Phylogenetic analysis showed that all B. nosferati strains were closely related and clustered together with Brucella sp. BCNN84.3 strain, previously isolated from an orchiepididymitis case of dog in Costa Rica. These bacteria are pathogenic, showed M type S-LPS, and, similar to other classical smooth strains, had all the virulent arsenal. The natural behaviour of D. rotundus inhabiting tropical and subtropical areas of North, Central, and South America makes this vampire bat a potential vector of brucellosis, as it is the case of rabies and bartonellosis
P3-02 How does Gefitinib inhibits Brucella infections?
No full textBrucellosis is a zoonotic disease caused by Brucella, with extensive implications in both economic and public health. To date, no human vaccine has been developed and the current treatment with antibiotics can cause severe side effects and relapses. Due to the need for new treatments to fight brucellosis, one possibility is to target host cells mechanisms to block entry and/or intracellular replication of bacteria. In addition to such host-directed therapy (HDT), we used a drug repositioning strategy, which consists in evaluating drugs already on the market. Previous findings from our lab showed that Gefitinib, a drug currently used for the treatment of a type of lung cancer, strongly inhibits the proliferation of B. melitensis, B. abortus and B. suis inside macrophages and trophoblasts in vitro, even proving a curative effect. In vivo evaluation of the drug is currently ongoing in a murine model of brucellosis in collaboration with the group of Prof. Renee Tsolis (UC Davis, CA, USA). The aim of this study is to understand the molecular mechanisms by which Gefitinib achieves the control of Brucella infection. BeWo cells (human cytotrophoblasts) were treated with Gefitinib. The activation of several intracellular signaling pathways was analysed using Western Blot (WB). In parallel, cells were infected with fluorescent B. melitensis using gentamicin protection assays and cells were analyzed using confocal microcopy. WB analyses revealed a decreased phosphorylation of EGFR upon stimulation with Gefitinib and alteration of downstream signaling pathways was detected, including FAK and MAPKs. Gefitinib treatment increased both the number and size of Lysotracker-positive vesicles (LPV). Upon infection, the number of bacteria per infected cell is significantly decreased and Brucella highly colocalized with LPV in Gefitinib treated cells. Gefitinib modifies intracellular molecular pathways and bacteria are rapidly eliminated from infected cells by an apparent increase in lysosomal activity. These results suggest Gefitinib as a promising therapeutic alternative for the treatment of human brucellosis
P3-03 Characterization of IalB family protein in Brucella abortus
No full textInvasion associated locus B (IalB) was originally described as a protein required for intra-erythrocytic parasitism in Bartonella spp.. Brucella spp. and Bartonella spp. are closely related alfa-proteobacterial pathogens that cause chronic infections in mammals. Brucella spp. genomes encode orthologous IalB proteins. One of them, encoded by locus BAB1_0368 in B. abortus, has been described as immunogenic in B. abortus and it was identified as a component of outer membrane vesicles in B. melitensis 16M. However, the contribution of IalB proteins to Brucella lifestyle is still not characterized and their biological roles remain unknown. This study is focused on characterizing and understanding the contribution of the members of IalB family protein (PF06776 in Pfam database) to B. abortus physiology and pathogenesis. To achieve this, single and multiple mutants were obtained by unmarked gene deletion in B. abortus. To evaluate the impact of these deletions in bacterial physiology, we first evaluated growth rates of these strains in rich medium Tryptic Soy Broth by using an automated growth curve analyzer. In addition, a fluorescent D-amino acid derivative was used to detect differences in bacterial size/morphology and in peptidoglycan synthesis by fluorescence microscopy of fixed cells. Finally, to address the role of IalB proteins in interaction with host cells, entry and intracellular replication of the mutant strains in non-professional phagocytic cells (HeLa) was evaluated. Single mutants in some ialB genes showed statistically significant differences to the parental strain 2308 in generation time, intracellular replication and in cell size and morphology. These preliminary results suggest a role of IalB proteins in B. abortus cell shape, as well as in vegetative and intracellular multiplication
P5-03 Development of a B. melitensis Rev1 mutant lacking streptomycin resistance
No full textBrucella melitensis is the etiological agent of ovine and caprine brucellosis. Since this species is the major cause of human brucellosis, vaccination of small ruminants is essential for its control and eradication avoiding brucellosis in humans. However, B. melitensis Rev.1 (the only vaccine available) is abortifacient when applied to pregnant sheep and goats, virulent to humans and resistant to streptomycin (Strp), one of the antibiotics of choice for treating human brucellosis. The goals of this work were to study the streptomycin resistance mechanisms in Rev.1 strain and to correct genetically this resistance to develop a potentially safer vaccine. In bacteria, Strp resistance is associated with mutations in the 16SrRNA, in proteins of the 30S ribosomal subunit and in enzymes modifying the 16SrRNA. Thus, we compared the corresponding genes in Rev.1 and B. melitensis 16M (reference strain of the biovar 1 and Strp-sensitive [Strps]). Whereas the 16Sr-RNA sequences were identical, we identified point mutations in Rev.1 rpsL (encoding the 30S S12 protein) and rsmG (encoding a methyltransferase acting on N7 of G527 of 16SrRNA) leading to P91L (mutation previously described) and P81R changes, respectively. Consistently with rpsL essentiality, we could not obtain Rev.1rpsL mutants. However, we demonstrate the involvement of rpsL in Rev.1 Strp resistance by introducing the 16M rpsL gene in Rev.1. The rsmG deletion in 16M (BmeΔrsmG mutant) conferred Strp resistance, whilersmG deletion in Rev.1 (Rev.1ΔrsmG) did not affect Strp resistance. Introduction of 16M rsmG in Rev.1ΔrsmG (Rev.1ΔrsmG::Tn7BmersmG -abbreviated as Rev.1StrpS-) resulted in increased Strp sensitivity. Rev.1StrpS showed an attenuated profile both in BeWo trophoblasts and THP-1 monocyte-derived-macrophages. In mice, Rev.1StrpS conferred similar protection against B. suis and resulted in lower residual virulence and abortifacient effects than Rev.1. The mutant was proven safe in pregnant sheep (see the results of the work presented by, P. M. Muñoz.). Protective efficacy experiments are in progress in sheep
P3-08 Suppression of B. melitensis Rev1 erythritol catabolism as a strategy to avoid genital tropism to develop a safe brucellosis vaccine
No full textSmall ruminant brucellosis by Brucella melitensis is manifested mostly as abortion and infertility, symptoms that result from the marked tropism and intense multiplication of these bacteria within the cells of the genital organs and placenta. Animal mass vaccination is critical to control brucellosis and lessen human infection in countries with high prevalence. However, Rev.1, the only available vaccine for small ruminants, keeps genital tropism and thus induces abortion in pregnant females. An obvious strategy for minimizing the abortifacient effects is the suppression of the tropism for the placenta, a phenomenon that has been postulated to be connected to the abundance of erythritol in this organ and the preferential use of this polyol by B. melitensis. Based on this hypothesis, we obtained non-polar deletion mutants in ery1 and ery2, genes of the recently unraveled erythritol catabolic pathway. Rev.1∆ery1 was unable to metabolize erythritol and its growth was not affected by the presence of this polyol in rich medium. Rev.1∆ery2 was also unable to use erythritol but its growth was inhibited by this polyol. Studies in THP-1 monocyte-derived-macrophages and BeWo trophoblasts showed that while both mutants multiplied in macrophages like Rev.1, Rev.1∆ery2 multiplication in trophoblasts was significantly lower. In our pregnant mouse model, the deletion of both genes resulted in a decrease in abortions and reduced bacterial replication in the placenta and vertical transmission to the fetuses. The virulence (splenic multiplication curves) and protection assays in mice confirmed the attenuated profile of Rev.1∆ery2. In contrast, Rev.1∆ery1 showed a multiplication profile similar to Rev.1 and optimum protection against the B. melitensis H38 challenge. These results led us to assess the safety of mutant Rev.1∆ery1 in pregnant sheep (work O5-2 presented by Muñoz, P. M. et al in this Conference)
P7-04 Evaluation of a Real-Time PCR for the diagnosis of brucellosis in humans
No full textDiagnosis of brucellosis is made by culture and serological tests. Microbiological isolation is considered the gold standard; however, it requires a minimum of viable bacteria in the sample and an incubation period of up to 4 weeks. The time limitation led to the evaluation of a new molecular biology method based on real-time PCR for rapid and more sensitive detection of Brucella. To evaluate a molecular biology test for the detection of Brucella spp. in humans. The design of primers and probes was carried out using the Beacon Designer v8.1 (BD8) program, using the sequence of the 31 kDa outer membrane protein (BSCP31) for genus, and for the detection of specie the alkB gene regions and BMI1162 for B. abortus and B. melitensis, respectively. An internal reaction control (IPC) was added, with the purpose of positive validation of samples. The PCR test was evaluated using 64 samples of blood from individuals with suspected brucellosis. These samples were also processed by serology with Rose Bengal, standard agglutination and agglutination tests in the presence of 2-mercaptoethanol. Statistical analysis was performed with a 2×2 contingency table using EPIDAT 3.0 software. The designed oligonucleotides were successfully tested, a detection limit of 5 fg was obtained, which is equivalent to 1 CFU in the serum sample. The diagnostic sensitivity and specificitywere 97.2 and 96% respectively. In this work the identification of Brucella spp. at the level of genus and species was performed by a highly sensitive real-time PCR test. This test considerably is reducing the identification time for Brucella species whereby we can recommend it as a support tool for the timely diagnosis of human brucellosis
P8-10 The One Health Integrated investigation for brucellosis suspected cases - Shargelneel locality, Khartoum State. Sudan, February 2016
No full textOn 2nd February, 2016, cases of human brucellosis were reported from Sharg elneel locality, they suspected milk bought from nearby dairy farms to be the source of their infection. An outbreak investigation was done jointly to verify the existence of an outbreak, identify the source of infection and to raise awareness of health cadres. It was decided to study patients who fulfill criteria of case definition of brucellosis, from the 1st of January 2015 to 2nd February, 2016. Five patients were investigated; simultaneously random blood samples were collected from two dairy farms and awareness posters were distributed. Medical records of nine health facilities were reviewed. 27 health cadres were interviewed. Two awareness workshops were conducted. The effect of this intervention was assessed. All five patients investigated were positive for brucellosis. Three of five had a history of unpasteurized milk consumption, and one had direct contact to animals. 20 blood samples were randomly selected from two dairy farms, six were positive for Rose Bengal Test and the results was confirmed by ELISA. Nine physicians were interviewed two of them considered brucellosis priority in diagnosis of fevers and diagnosed cases, one of nine is not aware about treatment protocol. Nine lab technicians were interviewed for six of them the diagnostic kids is available, two registered positive cases, all of them are well aware and trained. All nine statistician were didn’t register cases of brucellosis. Consumption of milk was the main cause of the outbreak. The burden of brucellosis is not clearly defined. Brucellosis is neglected in clinical diagnosis. Awareness of community is weak.We recommended assessing the effect of intervention within six months period, raising awareness of medical cadres and study prevalence of human brucellosis in Sharg elneel locality. Integration with veterinarians is important in prevention and control
O7-3 Whole Genome Sequencing for Tracing Geographical Origin of Imported Cases of Human Brucellosis in Sweden
No full textHuman infections with Brucella melitensis are occasionally reported in Sweden, despite the fact that the national flocks of sheep and goats are officially free from brucellosis. The aim of our study was to analyze 103 isolates of B. melitensis collected from patients in Sweden between 1994 and 2016 and determine their putative geographic origin using whole genome sequencing (WGS)-based tools. The majority of the strains were assigned to East Mediterranean and African lineages. Both in silico Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) and core genome Multilocus Sequence Typing (cgMLST) analyses identified countries of the Middle East as the most probable source of origin of the majority of the strains. Isolates collected from patients with travel history to Iraq or Syria were often associated with genotypes from Turkey, as the cgMLST profiles from these countries clustered together. Sixty strains were located within a distance of 20 core genes to related genotypes from the publicly available database, and for eighteen isolates, the closest genotype was different by more than 50 loci. Our study showed that WGS based tools are effective in tracing back the geographic origin of infection of patients with unknown travel status, provided that public sequences from the location of the source are available
O8-3 Risk factors for the persistence and spread of Brucellosis in buffaloes in the province of Caserta (2015-2020)
No full textDespite the fact that brucellosis has been eradicated in many countries, it is still present in various geographical areas and causes considerable economic and health damages. In Campania, the infection is present in the provinces of Salerno and Caserta, where in the latter province a drastic increase in the prevalence and incidence of infection in buffalo has been observed in the last years. Campania is the area with the highest concentration of buffalo farms in Italy. About 80% of Campania’s buffalo population is present in Caserta. This zootechnical activity is characterized by the presence of over 1400 farms and about 300000 heads, occupying a prominent role in the agricultural production system and representing an important component of the regional economy as part of buffalo mozzarella from Campania DOP. The results of official controls carried out from 2015 to 2020 in the buffalo farms of the Caserta Province were analysed. The data was extracted from the National Veterinary Information Systems and from the Laboratory Management System of the Experimental Zooprophylactic Institute of Southern Italy. The statistical analysis was carried out through the software R version 4.1.0. The dataset consists of 4,583 serologically controls in buffalo farms, of which 353 were positive over the six years considered. The association (Chi Square and Wilcoxon Tests) between a series of covariates was examined and the response variable “presence / absence” of the disease in the company was evaluated. A mixed effects logistic regression model was carried out to evaluate the contribution of possible factors over time and quantify the risks for the development of the disease. The infection is concentrated in the geographic areas with the highest density of animals and farms for Km2. More than 50% of the positive farms were repeatedly positive over time. From the analysis of the model, it emerged that the presence of abortions, a higher number of animal movements and herds density are significantly associated with the detection of infection in herds. The presence of infection’s geographic clusters suggests the presence of environmental factors that may have facilitated the spread of brucellosis between neighbouring farms
O4-2 Antibody response elicited by Brucella abortus strain RB51 vaccine in young water buffaloes (Bubalus bubalis) using a triple dose and the vaccination schedule authorised in Caserta province, Italy
No full textEradication of animal brucellosis is still a priority in Italy and the disease has a relevant impact on water buffaloes in the province of Caserta, Campania Region, Italy. In order to reduce the prevalence of infection, vaccination with the live attenuated vaccine B. abortus strain RB51 has been authorized by Central Veterinary Authority (Ministry of Health) in water buffalo herds in endemic territories, under controlled conditions. Namely, vaccination of young animal only (6-8 months old females, before sexual maturity), using a dose 3 times higher than the one used for cattle, followed by a second injection of same dose, 1 month after the first administration. In fact, when used on adult animals RB51 can be excreted in milk or induce abortion in pregnant animals. The aim of the study was to acquire information on development and persistence of RB51 specific antibodies induced by the vaccination schedule authorized from the time of vaccination (6-8 months old) to the stage of adult (26-28 months old). 22 buffaloes 7 - 8 months old were vaccinated with the commercial vaccine RB51 CZ Veterinaria, S.A. (Spain) using a triple dose. Four buffaloes were included as controls, inoculated with placebo and immediately mixed with vaccinated animals. A booster vaccination was administered after 30 days. Blood samples were collected regularly after vaccination. Sera were tested using a specific Complement Fixation test performed using RB51 antigen (RB51-CFT). RB51- CFT identified as positive 80% of the animals 9 days after first vaccination. Following booster vaccination, all vaccinated animals tested positive for approximately 2 months. Compared to our previous studies, results suggest that age of vaccination influences antibody response and RB51-CFT results. Two months after booster vaccination, antibody titers and numbers of vaccinated animals testing positive for RB51-CFT was variable and decreased in a non-progressive trend. Actually, we observed that several animal alternated positive and negative results to RB51-CFT up to 18 months after booster vaccination. This information represent a relevant element to develop diagnostic protocols to survey and detect the possible unauthorised application of RB51 vaccination on adult animals