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The new European Union animal health law and its rule concerning brucellosis in cattle and buffaloes The authors examine the latest European Union (EU) rules concerning eradication and surveillance of brucellosis and animal infectious diseases of EU concern. The Italian rules concerning brucellosis in cattle and buffaloes Italy is included in the EU co-financed compulsory eradication and surveillance programmes for brucellosis in cattle and in buffaloes in the frame of the EU and the related Italian laws, which allowed reaching the Brucellosis Free status without vaccination (former "Officially Brucellosis Free - OBF" status) in the majority of the northern and middle Italian Regions and in some middle and southern Provinces included in Regions where the infection persists. Epidemiology of brucellosis in the EU and in Italy In the Italian Province of Caserta, the highest prevalence of brucellosis in buffaloes is reported; in 2017-2021, a total of 314 outbreaks occurred, in which 39,163 heads tested positive. Here, brucellosis is threatening not only human health and the widespread buffaloes breeding but also the important satellite activities concerning the Protected Designation of Origin (P.O.D.) cheese “Mozzarella di Bufala Campana". The authors also discuss the reemerged brucellosis in cattle in the Molise Region, which despite bordering the Province of Caserta, shows different hydrographic, orographic, and breeding characteristics. In Molise, the reemerged brucellosis had a very different epidemiological course, which allowed it to limit the adoption of One Health measures. The One Health approach to tackle brucellosis in buffaloes In order to tackle brucellosis in Campania Region and in its Province of Caserta, the One Health approach has been predisposed through strict control of animal health, human health, and the environment. The adopted model could be exportable to territories having similar characteristics.

This review was performed to gather knowledge about brucellosis in livestock and wildlife in the Brazilian Pantanal, a biome with a huge biodiversity and extensive livestock production. Following the preferred reporting items for narrative review guidelines and using the terms "Brucella" and "Pantanal," we explored the PubMed, SciELO, Jstor, Science Direct, and Scholar Google databases. Information on host species, diagnostic test, number of positive animals, and positivity rates were acquired. Articles dating from 1998 to 2022 registered 14 studies including cattle, dogs, and the following wildlife species: Ozotoceros bezoarticus, Sus scrofa, Tayassu peccari, Nasua nasua, Cerdocyon thous, Panthera onca, Dasypus novemcintus, Cabassous unicinctus, Euphractus sexcinctus, Priodontes maximus, Myrmecophaga tridactyla and Hydrochoerus hydrochaeris. Brucella occurrence in cattle was demonstrated through the serological confirmatory test 2-mercaptoetanol. Molecular diagnosis detected Brucella abortus in dogs, smooth Brucella in O. beoarticus, and Brucella spp. in T. peccari. Cattle may have a pivotal importance in maintenance and spreading of Brucella spp. due to their high population density, environmental contamination from abortion of infected cows, and eventual excretion of B. abortus S19 strain from vaccinated heifers. The occurrence of Brucella spp. in O. bezoarticus and T. peccari represent a risk for wildlife conservation. These data indicate that Brucella spp. are enzootic in the Pantanal wetland, sustained by a reservoir system including domestic and wild mammals. Due to marked seasonality and large populations of wildlife species sharing same environments with livestock, brucellosis acquires great complexity in Pantanal and, therefore, must be analyzed considering both animal production and conservation.

Background and Aim: Bovine brucellosis is a disease of global socio-economic importance caused by Brucella abortus. Diagnosis is mainly based on bacterial culture and serology. However, these methods often lack sensitivity and specificity. A range of molecular diagnostic methods has been developed to address these challenges. Therefore, this study aims to investigate the diagnostic accuracy of molecular tools, in comparison to gold standard bacterial isolation and serological assays for the diagnosis of bovine brucellosis. Materials and Methods: The systematic review and meta-analysis were conducted based on analyses of peer-reviewed journal articles published between January 1, 1990, and June 6, 2020, in the PubMed, Science Direct, Scopus, and Springer Link databases. Data were extracted from studies reporting the use of molecular diagnostic methods for the detection of B. abortus infections in animals according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. The quality of included journal articles was assessed using the quality assessment of diagnostic-accuracy studies assessment tool and meta-analysis was carried out using Review Manager. Results: From a total of 177 studies, only 26 articles met the inclusion criteria based on PRISMA guidelines. Data from 35 complete studies were included in the meta-analysis and used to construct 2 × 2 contingency tables. Improved diagnostic performance was observed when tissue (sensitivity 92.7% [95% confidence interval (CI) 82.0–98.0%]) and serum samples (sensitivity 91.3% [95% CI 86.0–95.0%]) were used, while the BruAb2_0168 locus was the gene of preference for optimal assay performance (sensitivity 92.3% [95% CI 87.0–96.0%] and specificity 99.3% [95% CI 98.0–100.0%]). Loop-mediated isothermal amplification (LAMP) had a higher diagnostic accuracy than polymerase chain reaction (PCR) and real-time quantitative PCR with sensitivity of 92.0% (95% CI 78.0–98.0%) and specificity of 100.0% (95% CI 97.0–100.0%). Conclusion: The findings of this study assign superior diagnostic performance in the detection of B. abortus to LAMP. However, due to limitations associated with decreased specificity and a limited number of published articles on LAMP, the alternative use of PCR-based assays including those reported in literature is recommended while the use of LAMP for the detection of bovine brucellosis gains traction and should be evaluated more comprehensively in future.

The pathogenic bacterium Brucella abortus invades and multiplies inside host cells. To grow inside host cells, B. abortus requires a functional histidine biosynthesis pathway. Here, we show that a B. abortus histidine auxotroph mutant also displays an unexpected chaining phenotype. The intensity of this phenotype varies according to the culture medium and is exacerbated inside host cells. Chains of bacteria consist of contiguous peptidoglycan, and likely result from the defective cleavage of peptidoglycan at septa. Genetic suppression of the chaining phenotype unearthed two essential genes with a role in B. abortus cell division: dipM and cdlP. Loss of function of dipM and cdlP generates swelling at the division site. While DipM is strictly localized at the division site, CdlP is localized at the growth pole and the division site. Altogether, the unexpected chaining phenotype of a hisB mutant allowed the discovery of new crucial actors in cell division in B. abortus.

Ovine brucellosis caused by Brucella ovis is a major cause of reproductive failure in sheep. This study aimed to evaluate transplacental infection and pathogenicity of B.ovis wild type strain ATCC 25,840 (WT B.ovis) and the candidate vaccine strain B.ovis ΔabcBA in pregnant mice. A total of 40 BALB/c mice were equally divided into 4 groups: (i) non immunized and uninfected control mice (3/10 mice became pregnant); (ii) non immunized and challenged with WT B.ovis (5/10 pregnant); (iii) inoculated only with B.ovis ΔabcBA (6/10 pregnant); (iv) immunized with B.ovis ΔabcBA and challenged with WT B.ovis (5/10 pregnant). Female mice bred, and five days after visualization of the vaginal plug, they were inoculated intraperitoneally (ip) with 100 µL of sterile PBS, 100 µL of 1 × 10

Brucellosis is a infectious zoonosis with important health and economic impacts, both for animal production and public health. We aimed to determine the prevalence of brucellosis in beef cattle in the microregions of Araguaína and Bico do Papagaio in northern Tocantins and to verify whether there are post-mortem changes suggestive of brucellosis in the carcasses. A total of 2,871 serum samples (2,203 males and 668 females) were collected in the municipality of Araguaína between October and November 2019, from 76 beef cattle herds in 25 municipalities in the north of Tocantins. The screening for buffered acidified plate antigen test (BAPA) and confirmation test for 2-mercaptoethanol (2-ME) were conducted as recommended by the Brazilian Ministry of Agriculture, Livestock, and Supply (MAPA). Of the samples, 37.31% were reactive in the BAPA test, and of these, 26.24% were confirmed for 2-ME. Thus, 9.79% (281) of the total evaluated samples tested positive for the presence of brucellosis. The prevalence in males and females was 6.45% (142) and 20.81% (139), respectively. Among the herds evaluated, 77.6% (59) had at least one individual test positive for brucellosis, and at least one herd tested positive in each of the 25 municipalities sampled. As this study evaluated animals in a slaughterhouse, the higher prevalence of positive females may have been related to the disposal of animals with reproductive problems. There was no significant difference in the prevalence of brucellosis in slaughter cattle between the microregions evaluated (p > 0.05). No animals were reagent for brucellosis in ante-mortem documentation, and no anatomopathological changes suggestive of brucellosis were observed during post-mortem inspection. The most frequent post-mortem findings were contamination (43.91%), blood aspiration (17.36%), and pulmonary emphysema (15.98%). Considering the official results of the monitoring programs of the state of Tocantins and previous studies, post-mortem macroscopic inspection of carcasses may not be enough to detect animals with this zoonosis, and it may be necessary to take measures to promote animal and public health. A brucelose é uma doença infectocontagiosa tendo impacto sanitário e econômico, tanto na produção animal quanto na saúde pública por ser uma zoonose. O objetivo do presente trabalho foi determinar a prevalência de brucelose em bovinos de corte nas microrregiões de Araguaína e Bico do Papagaio no norte do Tocantins e verificar se havia ocorrência de alterações post mortem sugestivas de brucelose nas carcaças. Foram coletadas 2.871 amostras de soro, 2.203 de machos e 668 de fêmeas, durante a sangria em frigoríficos do município de Araguaína, entre outubro e novembro de 2019. Os animais foram oriundos de 76 rebanhos de corte de 25 municípios do norte do Tocantins. Foi realizada a prova de triagem do Antígeno Acidificado Tamponado (AAT) e a prova confirmatória do 2-mercaptoetanol (2-ME) conforme preconizado pelo Ministério da Agricultura, Pecuária e Abastecimento (MAPA). Das 2.871 amostras avaliadas, 37,31% foram reativas na prova do AAT, destas 26,24% foram confirmadas no 2-ME, o que representa 9,79% (281) do total avaliado. A prevalência em machos foi de 6,45% (142) e em fêmeas foi de 20,81% (139). Entre os rebanhos avaliados foi observada prevalência de brucelose em 77,6% (59), com pelo menos um rebanho com animais positivos em cada um dos 25 municípios amostrados. Como este estudo avaliou animais em frigorífico, a maior prevalência de fêmeas positivas pode estar relacionada com o descarte de animais com problemas reprodutivos. Não foi observada diferença significativa entre a prevalência de brucelose nos bovinos abatidos entre as microrregiões do estado de Tocantins avaliadas (p > 0,05). Durante o período de coleta de amostras nenhum animal foi notificado como positivo na documentação ante mortem e nenhuma alteração anatomopatológica sugestiva de brucelose foi observada durante a inspeção post mortem das carcaças de animais amostrados no presente estudo. Os achados post mortem mais frequentes foram contaminação (43,91%), aspiração de sangue (17,36%) e enfisema pulmonar (15,98%). Considerando os resultados oficiais dos programas de monitoramento do estado do Tocantins e estudos anteriores, foi possível observar que é preciso aprimorar e intensificar as ações para promoção de sanidade animal e, nesse caso, de saúde pública, uma vez que o processo de inspeção macroscópico post mortem das carcaças pode não ser suficiente para detectar animais com essa zoonose.

Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely, ribosomal protein L7/L12, outer membrane protein (OMP) 22, OMP25 and OMP31, was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. Four proteins were cloned, expressed and purified, and their immunocompetence was analysed. BALB/c mice were immunized subcutaneously with single subunit vaccines (SSVs) or CSV. Cellular and humoral immune responses were determined by ELISA. Results of immunoreactivity showed that these four recombinant proteins reacted with Brucella-positive serum individually but not with Brucella-negative serum. A massive production of IFN-γ and IL-2 but low degree of IL-10 was observed in mice immunized with SSVs or CSV. In addition, the titres of IgG2a were heightened compared with IgG1 in SSV- or CSV-immunized mice, which indicated that SSVs and CSV induced a typical T-helper-1-dominated immune response in vivo. Further investigation of the CSV showed a superior protective effect in mice against brucellosis. The protection level induced by CSV was significantly higher than that induced by SSVs, which was not significantly different compared with a group immunized with RB51. Collectively, these antigens of Brucella could be potential candidates to develop subunit vaccines, and the CSV used in this study could be a potential candidate therapy for the prevention of brucellosis.

This study evaluated if the milk ring test (MRT) used to the brucellosis diagnostic at dairy herd level can be done in milk samples with conservative (Bronopol®) collected to the somatic cell count (SCC) that is used to mastitis monitoring. It were analyzed 38 bulk tank milk samples (BTMS) from 19 dairy herds artisanal cheese producers and 36 BTMS from a brucellosis free dairy herd, used as negative control (NC) and positive control (PC), when was added serum from vaccinated animals. The milk samples were collected in two different bottles (with or without Bronopol®). All the 38 BTMS from artisanal cheese producers (samples with and without Bronopol®) were non-reagent to MRT, as all NC. All PC were reagent to MRT, without interference of Bronopol®. Results showed that the milk sample used to SCC presented the proportion of agreement of results in all milk samples (100%), that is, false positive and false negative results were not observed. Results indicated that Bronopol® did not interfere with the color in the time of MRT reading suggesting that the same milk samples could be used to monitoring mastitis and brucellosis. RESUMO: O objetivo deste estudo foi avaliar se o teste do anel do leite (TAL), utilizado para o diagnóstico da brucelose em nível de rebanho leiteiro, pode ser realizado em amostras de leite com conservador (Bronopol®) coletado para contagem de células somáticas (CCS) que é utilizado para monitoramento de mastite. Foram analisadas 38 amostras de leite de tanques de expansão (ALTE) de 19 rebanhos leiteiros produtores de queijo artesanal e 36 ALTE de rebanho leiteiro livre de brucelose, utilizadas como controle negativo (CN) e controle positivo (CP), quando foi adicionado soro de animais vacinados. As amostras de leite foram coletadas em dois frascos diferentes (com e sem Bronopol®). Todos as 38 ALTE de produtores artesanais de queijo (amostras com e sem Bronopol®) foram não reagentes ao TAL, como todos os CN. Todos os CP foram reagentes ao TAL, sem interferência do Bronopol®. Os resultados mostraram que a amostra de leite utilizada para CCS apresentaram a proporção de concordância dos resultados em todas as amostras de leite (100%), ou seja, não foram observados resultados falso-positivos e falso-negativos. Os resultados indicam que o Bronopol® não interferiu na cor no momento da leitura do TAL, sugerindo que as mesmas amostras de leite poderiam ser usadas para monitorar mastite e brucelose Made available in DSpace on 2022-06-14T13:26:13Z (GMT). No. of bitstreams: 1 Ring-test-in-milk-samples-preserved-with-Bronopol.pdf: 434937 bytes, checksum: 4a207aa1a41d27da10a579a8e328a8ac (MD5) Previous issue date: 2023

Brucellosis remains one of the most significant zoonotic diseases globally, responsible for both considerable human morbidity and economic losses due to its impacts on livestock productivity. Despite this, there remain significant evidence gaps in many low- and middle-income countries, including those of sub-Saharan Africa. Here we report the first molecular characterisation of Brucella sp. from Ethiopia. Fifteen Brucella sp. isolates from an outbreak in cattle from a herd in central Ethiopia were identified as Brucella abortus, using bacterial culture and molecular methods. Sequencing of the Ethiopian B. abortus isolates allowed their phylogenetic comparison with 411 B. abortus strains of diverse geographical origins, using whole genome single nucleotide polymorphisms (wgSNP). The Ethiopian isolates belonged to an early-branching lineage (Lineage A) previously only represented by data from two strains, both of sub-Saharan African origin (Kenya and Mozambique). A second B. abortus lineage (Lineage B), also comprised solely of strains originating from sub-Saharan Africa, was identified. The majority of strains belonged to one of two lineages of strains originating from a much broader geographical range. Further analyses based on multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA) expanded the number of B. abortus strains available for comparison with the Ethiopian isolates and were consistent with the findings from wgSNP analysis. MLST profiles of the Ethiopian isolates expanded the sequence type (ST) diversity of the early branching lineage of B. abortus, equivalent to wgSNP Lineage A. A more diverse cluster of STs, equivalent to wgSNP Lineage B, was comprised solely of strains originating from sub-Saharan Africa. Similarly, analysis of B. abortus MLVA profiles (n = 1891) confirmed that the Ethiopian isolates formed a unique cluster, similar to only two existing strains, and distinct from the majority of other strains of sub-Saharan African origin. These findings expand the known diversity of an under-represented lineage of B. abortus and suggest a potential evolutionary origin for the species in East Africa. In addition to providing information concerning Brucella species extant within Ethiopia this work serves as the basis for further studies on the global population structure and evolutionary history of a major zoonotic pathogen.

The in vitro embryo production (IVP) technology has emerged as a potential biotechnological approach to multiply genetically high yielding dairy cows. Its commercial application is increasing in many developed and developing countries over the years. Bangladesh livestock Research Institute (BLRI) adopted in vitro embryo production protocol from bovine ovaries of slaughterhouse. However, the risks of transmission of contagious diseases like Brucella abortus with embryos are not evaluated so far. Considering these facts, the present experiments were conducted to evaluate the efficiency of in vitro embryo production protocol with slaughterhouse ovaries as well as risk of contamination of produced embryos with Brucella abortus. To identify sources of contamination of embryos with Brucella abortus (if any), the laboratory water, different media used in the IVP process, semen, and follicular fluids were evaluated for confirmation of the organisms. In addition, vaginal swabs were collected from 2 buffaloes aborted due to suspected Brucella abortus infection. Molecular test were used to detect Brucella abortus contamination. Brucella abortus specific PCR product was not detected on agarose gel electrophoresis. The efficiency of IVP measured by cleavage and blastocyst development rates were 75.5±2.7% and 16.6±3.9%, respectively. The present study inferred that the in vitro produce embryos are free from Brucella abortus infection. Bang. J. Livs. Res. Vol. 27 (1&2), 2020: P. 105-112

Chang-Min Liu,1,&ast; Bing Suo,2,&ast; Yan Zhang3,&ast; 1General Hospital of Heilongjiang Province Land Reclamation Bureau Infectious Disease Department, Harbin, 150088, People’s Republic of China; 2General Hospital of Heilongjiang Province Land Reclamation Bureau Scientific Research Section, Harbin, 150088, People’s Republic of China; 3General Hospital of Heilongjiang Province Land Reclamation Bureau Clinical Laboratory, Harbin, 150088, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yan ZhangGeneral Hospital of Heilongjiang Province Land Reclamation Bureau Clinical Laboratory, No. 235, Hasuang Road, Nangang District, Harbin, Heilongjiang Province, 150088, People’s Republic of ChinaTel +86 451-55197654Fax +86 451-55197854Email zhangyanhhn@163.comObjective: To analyze the clinical features of patients with acute and chronic brucellosis in order to further improve the understanding of the disease.Methods: The clinical data of 144 patients with brucellosis who were admitted to our hospital were selected for retrospective analysis and were divided into two groups: the acute phase group (n = 86) and the chronic phase group (n = 58), and the clinical characteristics of the acute and chronic phases of the disease were analyzed. The χ2 test was used for countable data comparisons between the two groups.Results: Brucella melitensis was found as the contact organism in 61 patients (70.93%) in the acute phase group and in 12 patients (20.69%) in the chronic phase group (p < 0.01). Brucella abortus was found as the contact organism in 14 patients (16.28%) in the acute phase group and in 38 patients (65.52%) in the chronic phase group (p < 0.01). The results showed that the respective prevalence of fever, excessive sweating, splenomegaly, and lymph node enlargement were higher in the acute phase group than in the chronic phase group (p < 0.01). The respective prevalence of testicular swelling and pain were higher in the acute phase group than in the chronic phase group (p < 0.05), while the prevalence of joint and muscle pain was higher in the chronic phase group than in the acute phase group (p < 0.01).Conclusion: In Harbin, two types of clinical brucellosis, acute and chronic phase, infected sheep and cattle, respectively, are endemic at the same time, which complicates diagnosis. Besides, the clinical manifestations of brucellosis are complex and diverse, and they are often misdiagnosed and mistreated, leading to serious health injuries. Therefore, it is important to improve the understanding of disease characteristics in patients with acute and chronic brucellosis.Keywords: brucellosis, clinical characteristics, acute phase, chronic phase

Brucellosis is a widely prevalent zoonotic disease of major public health significance. A collection of Brucella melitensis and Brucella abortus field isolates of animal and human origin were subjected to MLVA-15 typing followed by phylogeography studies. The MLVA-15 analysis of B. melitensis (n = 65) field isolates resulted in 48 different profiles. The panel I marker bruce45 was found to be most conserved, while the rest of the panel I markers showed low to moderate length polymorphism. Among the panel II markers, bruce04, bruce16 and bruce30 showed a high discriminatory index. The MLVA-15 typing of 13 B. abortus field isolates revealed 13 different genotypes with panel II markers showing higher discriminatory ability vis-a-vis panel I. The minimum spanning tree analysis (MST) in comparison with isolates from the international database revealed that all B. melitensis and B. abortus isolates from this study belonged to the 'Eastern Mediterranean' and the 'abortus C' lineage, respectively. The MLVA-15 typing could differentiate field isolates of B. abortus and B. melitensis originating from different regions, reaffirming the technique's potential of high resolution and suitability for local epidemiological studies. The MLVA scheme also has the advantage of comparison of local isolates with a worldwide database, allowing for phylogeographical studies.

AIMS Treatment and preventive control strategies for Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) infection differ. A lateral flow immunoassay (LFIA) for the rapid typing and detection of brucellosis by using polychromatic dye-doped latex microspheres (LMs) as a labelling material was developed. METHODS AND RESULTS This LFIA utilizes a double-antigen sandwich method in which the BP26 protein is used as the diagnostic antigen to detect brucellosis infection and the OMP31 protein is used as the identified antigen to distinguish between bovine and sheep brucellosis. Thus, people and animals infected with brucellosis can be diagnosed according to the different colours of the signals displayed on the detection lines. The results indicated that the accuracy of this assay was found to reach 98%, and the immunochromatographic test strip is highly accurate, shows good sensitivity and can facilitate typing diagnosis, among other features. CONCLUSIONS The established LFIA can distinguish B. melitensis infection from B. abortus infection and produces results in a short period of time while retaining the advantages of LFIAs. SIGNIFICANCE AND IMPACT OF THE STUDY This technology lays a foundation for the development of multi-disease test strips and the establishment of methods for rapid, multi-specimen quantitative detection and is thus of great importance for the development of medical diagnostic technologies.

Bovine abortion is economically one of the most devastating problems faced by dairy farmers. Apart from non-infectious causes, several infectious pathogens are responsible for abortions, which sometimes manifests as abortion storms. Vaccine against several pathogens is available, in spite of that, abortions cause huge economic losses for the dairy sector. Timely and accurate identification of the etiological agent helps in adopting the mitigation steps to control the damage caused. In addition to the common abortion-causing pathogens such as Brucella abortus, Bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV), several emerging viral causes are being investigated for their possible role in abortion, either exclusively or as co-infection. Molecular methods are widely accepted for the identification of the involved pathogens. However, these assays require individual screening against each pathogen which is time-consuming and uneconomical, hence the multiplex format of PCR assays has been adopted by several laboratories. Multiplexing in real-time PCR is a sensitive and reliable technique, but it requires trained manpower and sophisticated equipment which is largely unavailable in regional disease diagnostic laboratories in India. Hence, in this study, a user-friendly, ready-to-use, gel-based RT-PCR multiplex assay was developed for simultaneous detection of three common pathogens (B. abortus, BHV-1, and BVDV) and two emerging pathogens; bluetongue virus (BTV) as a cause of abortions in bovine and Schmallenberg virus (SBV). After the standardization of the assay, a panel of 211 samples was screened. A high degree of concordance was observed which indicates the developed multiplex PCR assay is reliable and has the potential for screening at regional diagnostic laboratories.

The development of animal husbandry suffers various kinds of losses due to the spread of infectious diseases among animals, in particular Brucellosis. A challenge faced by Brucella researchers has been the accurate identification of new isolates within the genus while preserving sufficient, and not excessive, biosafety and biosecurity requirements. The availability of discriminatory molecular typing tools to inform and assist conventional epidemiological approaches would be invaluable in controlling these infections, but efforts have been hampered by the genetic homogeneity of the genus. In this work, for better identification of infection, for control and monitor the source of outbreaks in prosperous areas was carried out identification of Brucella spp. strains which circulating in the Kostanay region. For this was used using multilocus analysis of a variable number of tandem repeats sequenced by 16 s – PNK on a genetic analyzer (sequencer). According to the results of a study of cattle, cultures of microorganisms were infected: No. 4, 5, 7, 8. Comparison of the obtained results with similar results of domestic and foreign works by A. Shevtsov, G. Borrello, P. Le Fleche, G. Garofolo suggest that the genotyping of local strains has an importance in the molecular epizootology of the Republic of Kazakhstan.

La brucelosis bovina es una enfermedad zoonótica altamente contagiosa y produce severas pérdidas económicas, se encuentra presente en muchos países incluyendo Paraguay. El objetivo de esta investigación fue determinar la seroprevalencia de Brucella abortus en bovinos de establecimientos de pequeños productores lecheros de la colonia Nueva Alianza distrito de Yasy Cañy. El trabajo de investigación se llevó a cabo en 17 establecimientos de pequeños productores lecheros; en este estudio observacional descriptivo de corte transversal se muestreó un total de 58 (cincuenta y ocho) hembras de la especie bovina de primera aparición en adelante y sin distinción de raza. De los 58 animales en estudio el 100% ha resultado negativo al diagnóstico de brucelosis. Las pruebas utilizadas fueron la Rosa de bengala y otras pruebas confirmatorias la seroaglutinación lenta en tubo y la prueba 2 Mercaptoetanol. Siendo la prevalencia de la enfermedad del 0% en los animales muestreados.

Adjuvants are crucial components of vaccines that can enhance and modulate antigen-specific immune responses. Herein, we reported for the first time that human metallothionein-3 (MT3), a low molecular weight cysteine-rich metal-binding protein, was a novel promising adjuvant candidate that could help protein antigens to induce rapid, effective, and durable antigen-specific immune responses. In the present study, MT3 was fused to outer membrane protein 19 (Omp19) of Brucella abortus (MT3-Omp19, MO) and C fragment heavy chain (Hc) of tetanus neurotoxin (MT3-Hc, MH), respectively. The results showed that MT3 as a built-in adjuvant increased the Omp19- or Hc-specific antibody responses by 100-1000 folds in seven days after primary immunization. Compared to other commercially available adjuvants, MT3 could stimulate earlier (4 days after primary injection) and stronger (10-100 folds) antibody response with lower antigen dose, and its adjuvanticity relied on fusion to antigen. Although the mechanism was not clear yet, the fusion protein MO was observed to directly activate DCs, promote germinal center formation and improve the speed of Ig class switching. Interestingly, our subsequent study found that other members of the mammalian MT family (human MT1 or murine MT3 for examples) also had potential adjuvant effects, but their effects were lower than human MT3. Overall, this study explored a new function of human MT3 as a novel built-in adjuvant, which may have important clinical application potential in vaccine development against global pandemics.

Brucellosis is a common zoonotic disease caused by intracellular pathogens of the genusBrucella.Brucellainfects macrophages and evades clearance mechanisms, thus resulting in chronic parasitism. Herein, we studied the molecular changes that take place in human brucellosis bothin vitroandex vivo.RNA sequencing was performed in primary human macrophages (Mφ) and polymorphonuclear neutrophils (PMNs) infected with a clinical strain ofBrucellaspp. We observed a downregulation in the expression of genes involved in host response, such as TNF signaling, IL-1β production, and phagosome formation in Mφ, and phosphatidylinositol signaling and TNF signaling in PMNs, being in line with the ability of the pathogen to survive within phagocytes. Further transcriptomic analysis of isolated peripheral blood mononuclear cells (PBMCs) and PMNs from patients with acute brucellosis before treatment initiation and after successful treatment revealed a positive correlation of the molecular signature of active disease with pathways associated with response to interferons (IFN). We identified 24 common genes that were significantly altered in both PMNs and PBMCs, including genes involved in IFN signaling that were downregulated after treatment in both cell populations, andIL1R1that was upregulated. The concentration of several inflammatory mediators was measured in the serum of these patients, and levels of IFN-γ, IL-1β and IL-6 were found significantly increased before the treatment of acute brucellosis. An independent cohort of patients with chronic brucellosis also revealed increased levels of IFN-γ during relapse compared to remissions. Taken together, this study provides for the first time an in-depth analysis of the transcriptomic alterations that take place in human phagocytes upon infection, and in peripheral blood immune populations during active disease.

This study investigated the contribution of lipoxygenase (LOX) inhibitors, nordihydroguaiaretic acid (NDGA), tetra

Brucellosis is considered to be a zoonotic infection with a predominant incidence in most parts of Iran that may even simply involve diagnostic laboratory personnel. In the present study, we apply matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for rapid and reliable discrimination of

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase and has been found to have protective effects against several bacterial infections. In this study, we investigate the effects of simvastatin treatment on RAW 264.7 macrophage cells and ICR mice against

Brucellosis is a zoonotic infection caused by the consumption of contaminated raw milk and dairy products. This study aims to compare survival rates of

A total of 600 serum samples from five farms were collected and the geographic coordinates of each farm were georeferenced to establish two Köppen climate classes (wet tropical and tropical rainforest). Serum samples were examined by commercially available enzyme-linked immunosorbent assay (ELISA) kits to detect antibodies against N. caninum and T. gondii, while anti-B. abortus antibodies were analysed using the Rose Bengal test and ELISA kit. The overall estimated prevalence of N. caninum, T. gondii and B. abortus was 41.2%, 30.7% and 0.5%, respectively. The highest percentages of positive buffaloes with neosporosis and toxoplasmosis were found in the states of Tabasco (46.7%, wet tropical climate) and Veracruz (32.7%, tropical rainforest climate), respectively. Multivariate logistic regression showed that N. caninum seropositivity in buffalos was associated with the normalized difference vegetation index (NDVI) (OR: 1.068; 95% CI: 1.026-1.112; P = 0.001) and tropical climate regions (OR: 1.737; 95% CI: 1.112-2.712; P = 0.015). In addition, statistical analysis revealed that sex (OR: 1.737, 95% CI: 1.685-9.737; P = 0.002), NDVI (OR: 1.382; 95% CI: 1.095-1.746; P = 0.007) and rainfall (OR: 1.478; 95% CI: 1.110-1.967; P = 0.007) were significant risk factors for T. gondii infections. These results indicate that the potential impact of the risk factors was different for each disease in buffaloes, which may be useful for developing regionally adapted control strategies and preventive measures.

Background Brucellosis is an infectious zoonotic bacterial disease of humans and other animals. In the Republic of South Africa (RSA), animal brucellosis is widespread and the current available data on the prevalence of this disease rely solely on serological testing. The primary limitation of brucellosis serology is the lack of discriminatory powers to differentiate between Brucella species and biovars as well as the cross‐reactivity observed with other Gram‐negative bacteria. Aim The aim of this study was to conduct a retrospective laboratory‐based survey on Brucella species and biovars isolated from various animal species in SA between 2008 and 2018. Material and Methods The isolation of Brucella species and biovar typing was performed using conventional microbiological techniques. Results and Discussion A total of 963 strains of Brucella species were included in this study with a frequency of detection for B. abortus (n = 883; 91.6%) followed by B. melitensis (n = 42; 4.4%), B. ovis (n = 29; 3.0%) and B. canis (n = 9; 0.9%). Of the 883 strains of B. abortus, 90.1% were typed as B. abortus biovar‐1 while 5.7% as B. abortus biovar‐2, and 3.3% and 0.5% were B. abortus S19 and B. abortus RB51 vaccine strains, respectively. Among the 42 B. melitensis strains, 71.4% were reported as B. melitensis biovar‐1 and 26.2% as B. melitensis biovar‐3 while 2.4% was B. melitensis biovar‐2. Conclusion A retrospective study, such as this one, provides useful information that can be critical in formulating policies and strategies for the control and eradication of brucellosis in animal populations in RSA., Brucellosis is an infectious zoonotic bacterial disease of humans and other animals. In the Republic of South Africa (RSA), animal brucellosis is widespread and the current available data on the prevalence of this disease rely solely on serological testing. The aim of this study was to conduct a retrospective laboratory‐based survey on Brucella species and biovars isolated from various animal species in SA between 2008 and 2018. A total of 963 strains of Brucella species were included in this study with a frequency of detection for B. abortus (n = 883; 91.6%) followed by B. melitensis (n = 42; 4.4%), B. ovis (n = 29; 3.0%) and B. canis (n = 9; 0.9%). Of the 883 strains of B. abortus 90.1 % were typed as B. abortus biovar‐1 while 5.7% as B. abortus biovar‐2, and 3.3% and 0.5% were B. abortus S19 and B. abortus RB51 vaccine strains respectively. Among the 42 B. melitensis strains, 71.4% were reported as B. melitensis biovar‐1 and 26.2% as B. melitensis biovar‐3 while 2.4% was B. melitensis biovar‐2.

Brucella is an intracellular zoonotic pathogen that can affect many hosts. Brucella melitensis Rev.1 is a live attenuated, is one of the most effective vaccine strain against brucellosis. It can be used safely in sheep, goats, and even cattle. Although many studies are available on this topic, there is no effective vaccine strain for sheep and goats that distinguishes the antibody titer produced between the field infections and vaccinations. Outer membrane protein 19 (Omp 19) is both virulent and a protective antigen found on the cell-wall of the Brucella strain. In this study, used the suicide plasmid pJQ200KS, which contained homologous region without Omp19 Open Reading Frame (ORF) that was transferred to B. melitensis Rev.1 and further transformed into spheroplasts along with penicillin, ampicillin, and glycine by electroporation. To obtain a mutant vector from Escherichia coli, we used the heat shock transformation method along with the blue-white colony screening using X-gal media, whereas for the gene transfer in Brucella, we used electroporation. A scanning electron microscope (S.E.M) was used to observe the spheroplast transformation while the mutant vector and deletion mutants were confirmed through PCR and sequence analysis. In the mouse model efficacy trials, three commercial vaccines were found to comply with the OIE standards. Although the deletion mutants 19 and 44/10 had similar efficiency as the commercial vaccines in terms of stimulation power, the ELISA test with Omp19 protein showed the same results as the negative control. The Rev.1 Omp19 deletion mutants obtained in this study contained sufficient residual virulence, and their protective immunity was similar to the commercial vaccines. The study showed that a vaccine prepared using a B. melitensis Rev.1 ΔOmp19 can act as a marker vaccine or differentiate infected from vaccinated animals (DIVA) through the ELISA test that detects the Omp19 protein.

Brucellosis, being a zoonotic disease, is difficult to control despite the availability of vaccines. Bovine brucellosis could be controlled effectively using brucellaphages. This study was conducted to isolate bacteriophages against Brucella abortus from cattle farms sewage/slurry samples (n=50). Isolation and propagation of brucellaphages was made through spot and plaque assay. Two samples (n=2) were found positive on the plates as circular, clear, and pinpoint plaques (0.3 to 0.5 mm) with 4.6×106 PFU/mL of brucellaphage titre against Brucella abortus RB51. One- step growth experiments revealed latent period of 120 min and burst size of 93 and 114 PFU for two isolated brucellaphages (ϕP1 and ϕP2) respectively. These phages were unable to lyse Staphylococcus aureus, Streptococcus spp., Salmonella spp., Escherichia coli, Bacillus subtilis and Pasteurella multocida. Isolated brucellaphages were stable up to 60°C and between 7 to 9 pH. No loss in phage titres were observed at 4°C but phage titres were reduced by one log at (-20°C) overnight. There was no effect of SM buffer, normal saline and EDTA on stability of brucellaphages and addition of divalent salts in medium showed significant increase in PFU. However, treatment with SDS and chloroform destroyed the phages in one hour exposure. The phages were found carrying double stranded DNA (~40 kb size) and two prominent protein bands of 45 kDa and 70 kDa. Biocontrol activity of brucellaphages showed average Brucella abortus count reduction to 4.5×103 from 5.0×108 CFU/ g of soil sample within 48 h when treated with maximum phage titre of 5.0×1011 PFU/ mL. Hence, this study revealed that brucellaphages are present in our environment and can potentially be consider for their cost-effective practical applications in inactivation of Brucella abortus after comprehensive experimental evaluations. © 2021 Friends Science Publishers

Background and Aim: The present serodiagnosis of brucellosis in livestock is based on the whole cell or smooth lipopolysaccharide of the Brucella organism in which specificity is hampered by the cross-reactivity, especially with the antibodies against Yersinia enterocolitica O:9 organism. The problem can be addressed by screening for better immunodominant antigens. Hence, the present study was undertaken to screen protein antigens of Brucella abortus for their diagnostic potential in cattle brucellosis. Materials and Methods: Protein antigens of B. abortus (n=10) non-reactive to antibodies against Y. enterocolitica O:9 were selected, expressed in Escherichia coli, assessed the reactivity of expressed recombinant proteins by Western blot, standardized indirect-enzyme-linked immunosorbent assay (ELISA) for detecting Brucella antibodies in cattle serum, and comparative evaluation was done. Results: All the selected protein antigens were expressed and in the Western blot with Brucella antibodies positive cattle serum, six recombinant (Brucella protein 26 [BP26], Cu-Zn Superoxide dismutase [SodC], B. abortus I-1885, Serine protease, Bacterioferritin, and Brucella Lumazine Synthase [BLS]) proteins showed reaction whereas none of the proteins showed reactivity with Brucella negative cattle serum. ELISA has been done using known Brucella positive and negative cattle sera samples (n=113 each) in which the performance of recombinant proteins in diagnosing brucellosis was in the order of BP26 > BLS > SodC followed by rest of the proteins. BP26 based ELISA was found to be better with area under the curve as 0.953, and diagnostic sensitivity, diagnostic specificity, and Youden's index of 90.27%, 95.58%, and 0.8584, respectively, with the excellent agreement (k=0.85). Conclusion: BP26 could be a potential diagnostic antigen among the immunodominant proteins of B. abortus in ruling out Y. enterocolitica O:9 infection while diagnosing brucellosis in cattle herds.

Heat shock proteins are molecular chaperones that are immunogens as well as potent inducers of an antigen-specific immunological response. In this study, we aimed to evaluate if co-immunization of Brucella rOmp22 and rDnaK proteins had boosted immunogenic activity as compared to rOmp22 immunization alone in mice. For this, gene-encoding DnaK of B. abortus was cloned, expressed in E. coli and purified using Ni-NTA agarose. Immuno-modulatory effect of rDnaK protein was evaluated in mice when co-immunized with Brucella rOmp22. Four groups of mice (n = 6 per group) were used in the study. The control group was immunized with rOmp22 alone, while rOmp22 emulsified with conventional adjuvants (Freund's complete and incomplete adjuvants) and rOmp22 mixed with rDnaK were injected to group I and group II in mice, respectively. Group III mice were immunized with rDnaK alone. IgG class switching (IgG1 and IgG2a) response to immunization was assessed by enzyme-linked immunosorbent assay and expression of IL-4 and IL-12 mRNA was assessed by real-time PCR to evaluate the immune response in mice. The ratio of IgG1-IgG2a was less than 1 in mice co-immunized with rOmp22 and rDnaK, indicating that the immune response was directed towards CMI arm in this group of mice. Moreover, IL-12 mRNA expression was also up-regulated to a greater extent in mice co-immunized with rOmp22 and rDnaK as compared to those immunized with rOmp22 along with the conventional adjuvants, or rOmp22 alone. Our data suggest that rDnaK could be responsible for modulating the immune response, specifically the CMI response.

Objectives: Brucellosis is a zoonosis with severe complications for both humans and animals. In this work, we intended to examine the Brucella infection in dromedary camels in Qatar by using different analysis. Materials and Methods: A total of 203 samples of dromedary camels were randomly collected from the nearby farms in Qatar. Real-time PCR for the genus specific Brucella cell surface salt extractable bcsp31 kDa protein gene were performed on DNA extracted from camel samples. Rose Bengal and rivenol tests were performed to detect the Brucella species. The milk samples were collected from the camels and utilized for the milk ring test. Results: The outcomes of RT-PCR analysis illustrate the presence of Brucella spp. in 170 samples (83.74%) out of 203 samples. The findings of immunological assays also proved the presence of Brucella spp. such as Rose Bengal (67.14%), ELISA (71.42%), and precipitation assay (65.71) in both serum and blood samples of the dromedary camels, which were collected from the Qatar. Conclusions: In conclusion, it was clear that the incidence of the brucellosis in camels is significantly rising in Qatar region and there is a need to control the spread of the disease from camels to camels as well as from camels to humans.

To better understanding Brucella abortus infection, serum metabolites of B. abortus -infected and -uninfected mice were analyzed and twenty-one metabolites were tentatively identified at 3 and 14 days post-infection (d.p.i.). Level of most lysophosphatidylcholines (LPCs) was found to increase in infected mice at 3 d.p.i., while it was decreased at 14 d.p.i. as compared to uninfected mice. In contrast, acylcarnitines were initially reduced at 3 d.p.i then elevated after two-weeks of infection, while hydroxysanthine was increased at 14 d.p.i. in infected mice. Our findings suggest that the significant changes in LPCs and other identified metabolites may serve as potential biomarkers in acute phase of B. abortus infection.

Brucellosis in cattle is a disease caused by Brucella abortus due to the reduction in livestock population caused by abortion, stillbirth, weak birth, infertility and sterility. Brucella abortus has several potential virulence factors, i.e. virB11 gene that encodes VirB11 protein is an important virulence factor acts as an ATPase for assembling organelles when the bacteria replicate, helping to complete the bacterial cycle and agress to another cells. The aim of this study are to re-identification Brucella abortus and detect virB11 gene as encoding of B. abortus VirB11 protein in local isolates from Pinrang, NTT, strain vaccines S19 and RB51. The isolates Brucella abortus were re-cultured in Brucella agar base and re-identification is followed by microscopic with Gram staining and biochemical tested with urease, citrat, indol and TSIA test. virB11gene was detected with PCR method. The PCR result showed virB11 gene have DNA band 720 bp. virB11 gene are present in local isolates from Pinrang, NTT, strain vaccines S19 and RB51.

Background: Human brucellosis is an important zoonotic disease of public health and often remains neglected owing to lack of sensitive and efficient diagnostic methods. This study evaluates diagnostic utility of in-house designed enzyme-linked immunosorbent assay (ELISA) using whole-cell antigens of Brucella abortus (B. abortus) S19 against the commercially available kits. Methods: A prospective cohort study involving different populations within the Vidarbha regions of Maharashtra, India was conducted through camps organised from May 2009 to October 2015. A total of 568 serum samples were collected from high-risk people recruited as study cohorts based on inclusion criteria, additional risk factors and clinical symptoms. Samples were evaluated by indirect ELISA using the whole-cell antigens of B. abortus. The results were compared with the commercially available IgG detection ELISA kit to ascertain the specificity and sensitivity of the developed test. Results: Fever, body ache, joint pain, lower back pain, loss of appetite and weight loss were major symptoms associated with the disease. With the cut-off of > 0.8, the positivity of brucellosis infection was at 12.32% (70/568) compared to 9.33% (53/568) as detected by the commercial kit. The in-house developed ELISA method yielded a sensitivity of 87.5% and specificity of 99.18% as compared to the commercial kits (sensitivity –80.30% and specificity –99.6%). Discussion: The B. abortus S19-derived whole-cell protein-based ELISA is rapid and cost- effective and can be used for screening brucellosis infection in lieu of the commercially available ELISA kits.

Abortions cause tremendous economic losses in food-producing animals and may lead to food insecurity.This study aimed to characterize Brucella spp. and other abortigenic pathogens from aborted tissues of cattle.For cattle, aborted tissues (n = 19) were cultured, and Brucella spp. were detected using the genus-specific 16S-23S ribosomal DNA interspacer region (ITS) assay and speciated using Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR assays. Brucella negative samples were screened using the eight abortigenic pathogens PCR panel. Samples from an abortion outbreak that occurred within a goat tribe were included in this investigation. Sera of females (n = 8) and males (n = 2) were analyzed using the Rose Bengal Test (RBT) and indirect enzyme-linked immunosorbent assay (i-ELISA), while vaginal swabs (n = 3) and aborted tissues (n = 1) were cultured and characterized.The ITS-PCR detected Brucella DNA in cultures from two aborted tissues of cattle (10.5%, [2/19]), which were identified as B. melitensis (n = 1), and B. abortus (n = 1) using AMOS and Bruce-ladder PCR assays. Campylobacter fetus (n = 7) and Leptospira spp. (n = 4) including co-infections (n = 2) of C. fetus and Leptospira spp. were identified from the Brucella negative samples of cattle. Goats (100.0%, 10/10) were brucellosis seropositive on RBT and i-ELISA. Mixed infections caused by B. melitensis and B. abortus were isolated from the vaginal swabs (n = 3) and aborted tissues (n = 1).This is the first identification of abortion-associated pathogens in aborted cattle indicating the enormous financial losses and a threat to public health. It is therefore essential to include these identified pathogens in the surveillance scheme of veterinary and human services.

Research on Brucella pathogenesis has focused primarily on its ability to cause persistent intracellular infection of the mononuclear phagocyte system. At these sites, Brucella abortus evades innate immunity, which results in low-level inflammation and chronic infection of phagocytes. In contrast, the host response in the placenta during infection is characterized by severe inflammation and extensive extracellular replication of B. abortus. Despite the importance of reproductive disease caused by Brucella infection, our knowledge of the mechanisms involved in placental inflammation and abortion is limited. To understand the immune responses specifically driving placental pathology, we modeled placental B. abortus infection in pregnant mice. B. abortus infection caused an increase in the production of tumor necrosis factor alpha (TNF-α), specifically in the placenta. We found that placental expression levels of Tnfa and circulating TNF-α were dependent on the induction of endoplasmic reticulum stress and the B. abortus type IV secretion system (T4SS) effector protein VceC. Blockade of TNF-α reduced placental inflammation and improved fetal viability in mice. This work sheds light on a tissue-specific response of the placenta to B. abortus infection that may be important for bacterial transmission via abortion in the natural host species.

Fifty-two, Hereford heifers were obtained from brucellosis-free herds and randomly assigned to Brucella abortus strain RB51 (RB51) vaccination (n = 32) or control (n = 20) treatments. Vaccinates received 10

We present the case of an 18-year-old female with a 1-month history of fever, headache, and double vision, whose examination revealed papilledema and cranial nerve VI palsy. Blood cultures grew Brucella abortus cattle vaccine strain RB51, which is inherently resistant to rifampin. We discuss the management of the first known case of neurobrucellosis by this strain.

We evaluated serologic responses of cattle, bison, elk, and swine representing negative control, early vaccination (4–8 wk), late vaccination (21–28 wk) or booster vaccination, early after-experimental challenge (2–4 wk), and late after-experimental challenge (8–21 wk), in a brucellosis fluorescence polarization assay (FPA; n = 10 sera per species per treatment) using negative control sera from cattle, bison, elk, and swine ( n = 5 per species). Sera from cattle shedding Brucella abortus strain RB51 in milk were also evaluated against the 20 negative control sera. The species of negative control sera used in the FPA could increase ( p < 0.05) delta millipolarization (mP; delta mP = sample mP − negative control mP) results. In general, the species of negative control sera did not alter the interpretation of FPA results in control, vaccinated, or infected animals. Even after repeated RB51 vaccinations in bison, cattle, or elk, or in cattle shedding RB51 in milk, serologic results from the FPA remained negative. Species differences in FPA results were noted; elk developed robust humoral responses very quickly after infection that resulted in strong positive FPA results. In cattle and bison, humoral responses appeared to develop over a longer period of time, and greater delta mP values were detected at later times after infection. Sensitivity of the FPA for detecting infected animals was greatest for elk in early challenge samples and bison in late challenge samples. Our data suggest that species of origin of negative control sera does not influence interpretation of the FPA in natural hosts of Brucella abortus.

This initial retrospective study presents information on bacterial and fungal causes of abortion in domestic ruminants in South Africa over 10 years (2006-2016). A complete set of data was collected at the Faculty of Veterinary Science through a keyword search of pathology and bacteriology laboratory registers. Additional electronic data were received from an external laboratory. A total of 288 cases were recorded from six provinces. Overall diagnostic rate was 35.1%. In 14.6% of cases, histological evidence of an infectious process was found, but no aetiological agent was detected. Several cases did not include aetiological diagnoses because applicable diagnostic techniques were not available or not applied when necessary. Increased submission of placenta, as well as additional conventional and molecular diagnostic methods, can contribute to an improved diagnostic rate. In addition, the study highlights the superior significance of Brucella abortus as a major bovine pathogen in South Africa.

Brucellosis is one of the most contagious and infectious diseases with high incidence rates of cattle and humans in Kazakhstan. Using modern biotechnology techniques to develop vaccines that are reliable and affordable for farmers is an alternative solution to the problem. Plant viruses are often used as a vector for obtaining the expression of antigens of the pathogen. The grape virus A (BAB) is widely used among plant viruses. Brucella membrane proteins are the main objects of this research for futher development of vaccines or diagnostic texts against brucellosis, Membrane proteins (OMPs) are cell specific surface antigens that are immunogenic. OMPs are ideal candidates for the production of recombinant brucellosis vaccines. The object of the study was the outer membrane protein (Omp16), which plays an important role in the suppression of TNF-α production in macrophages. In this study, molecular cloning and analysis of the expression of the Omp16 gene, which was used to express the recombinant protein in plants, was carried out. We selected brucella from the vaccine strain of Brucella abortus 19, and the plant Nicotiana benthamiana, as the subjects for our research, since they widely used for the production of recombinant proteins, and they both appropriate for molecular genetic research. A viral vector was constructed to express the brucellosis antigen Omp16 in Nicotiana benthamiana plants. Nineteen explants were used for the regeneration of transgenic plants. As a result of this studies, the introduced gene of Omp16 was under the subgenomic promoter control of the ORF4 and was successfully expressed while maintaining the efficiency of expression in transgenic plants. The efficiency of viral vectors was evaluated at the level of transcription during expression of the protein Omp16 with viral proteins. The entire leaf blade was infiltrated; the density of Agrobacteria was 0.7. We were able to obtained transgenic plants Nicotiana benthamiana carrying the gene of capsid protein BAB, and the expression of the membrane antigen Omp16 in the viral vector was achieved by replacing the ORF4 with the Omp16 gene. The development of transgenic plants was carried out using agrobacterial transformation.

Introduction. Brucellosis belongs to zooantroponoses, the relevance of which is connected with widespread in several regions of the Russian Federation and the unfavorable employment outlook for cases of persons. Material and methods. Based on updated data, the modern trends of the sanitary-epidemiological situation on the incidence of occupational brucellosis in the Federal districts of Russia, including occupational cohorts, conditions, diagnosis, and occupational prognosis were analyzed. Results. In the period from 2011 to 2017, 412 cases of occupational brucellosis (OB) were shown to be registered in rural regions. OB was proved to rank third in the nosological structure of accumulated occupational pathology (14.6%). Despite the downward linear trend in the frequency of detection of OB, its prevalence in the nosological structure of the primary occupational morbidity of the working rural population did not undergo statistically significant changes and was within the range of 10.28 - 17.16%. Most often, the disease was detected in workers in occupations as follows: animal breeder, veterinarian, milker/operator of machine milking, and veterinary paramedic, infected during industrial contact with cattle. The North-Caucasus, Siberian, and Southern Federal districts were the most disadvantaged regions of the Russian Federation in terms of the OB incidence. In 96.6% of cases, OB was represented by a residual form, diagnosed in 88.5% of cases with self-treatment. The late diagnosis of OB and the high percentage of workers with primary established loss of performance (59.5%) could be related to both the virulence of Brucella abortus and the low quality of periodic medical examinations and limited availability of primary occupational pathology care in rural regions. Conclusion. Reducing the level of the OB incidence is not possible without strictly controlling the timeliness, completeness of coverage and regulated volume of periodic medical examinations, increasing the level of awareness and awareness of occupational risks, compliance with sanitary and veterinary rules for the prevention and control of infectious diseases common to humans and animals.

Brucellosis is an important occupational disease, mainly among veterinarians, because of their frequent contact with sick animals, contaminated secretions and live attenuated anti-Brucella vaccines. This study aimed to determine the prevalence of accidental exposure to S19 and RB51 vaccine strains and occupational brucellosis among veterinarians registered to administer vaccinations in Minas Gerais, Brazil, as well as to identify the risk factors associated with accidental exposure to anti-Brucella abortus vaccines. Data were collected through an online questionnaire. Three hundred and twenty-nine veterinarians were included in the analyses using stratified random sampling. A multivariate logistic regression analysis was used to evaluate the predictors of accidental exposure to S19 and RB51 strains. Nearly one third of the veterinarians registered to administer bovine brucellosis vaccination in Minas Gerais, 32.83% (108/329) (95% confidence interval [CI]: 27.78-38.19%), reported having been accidentally exposed to S19 or RB51 vaccine strains. The exposure factors associated with this outcome included a score of personnel protective equipment (PPE) use during work (odds ratio [OR], 0.94; 95% CI: 0.89-0.98) and a score of knowledge about brucellosis symptoms, classified as poor (base category), intermediate (OR, 0.26; 95% CI: 0.07-0.87) or good (OR, 0.22; 95% CI: 0.07-0.62). In addition, 4.56% (15/329) (95% CI: 2.57-7.41%) of veterinarians reported that they had brucellosis, of which 46.67% (7/15) considered that the disease was due to accidental exposure to anti-B. abortus live attenuated vaccine. The prevalence of accidental exposure to B. abortus vaccine strains among veterinarians from Minas Gerais enrolled in the control of bovine brucellosis was high. The reduced knowledge about human brucellosis symptoms and lack of appropriate PPE use were risk factors from unintentional contact with S19 and RB51 vaccine strains.

Brucellosis, which is caused by Brucella spp., is an important zoonotic infectious disease that can cause great hazard to public health and safety. However, the current vaccines have several drawbacks, including residual virulence for animals and humans. Bacterial ghost is the empty envelopes of bacteria, which emerge as a proper vaccine candidate. With the purpose of generating B. abortus ghosts and investigating the immunogenicity of bacterial ghosts as vaccine candidate, we used homologous recombination and bacterial ghost technologies to construct 2308ΔgntR ghost strain. Mice were injected with 2308ΔgntR ghost and the safety and immunogenicity of ghost were further evaluated. The mice inoculated with ghost showed no splenomegaly. The 2308ΔgntR ghost induced high protective immunity in BALB/c mice against challenge with S2308, and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of interferon gamma (IFN-γ) and interleukin-4 (IL-4). Additionally, 2308ΔgntR ghost demonstrated strong spleen CD4+ and CD8+ T cell responses. These results suggest that 2308ΔgntR ghost is a potential vaccine candidate and may represent a promising new approach for vaccination against Brucella infection.

Highlights • Oral co-administration of dmLT with U-Omp19 increases dmLT immunogenicity. • U-Omp19 oral co-delivery with dmLT induces anti-LT antibody responses. • U-Omp19 co-administered with dmLT protects against oral challenge with LT. • U-Omp19 can allow antigen dose sparing by oral route. • U-Omp19 can be used as adjuvant in an oral vaccine formulation against ETEC., Acute diarrhea disease caused by bacterial infections is a major global health problem. Enterotoxigenic Escherichia coli (ETEC) is one of the top causes of diarrhea-associated morbidity and mortality in young children and travelers to low-income countries. There are currently no licensed vaccines for ETEC. Induction of immunity at the site of entry of the bacteria is key to prevent infection. Current approaches to ETEC vaccines include a less toxic mutant form of E. coli heat-labile toxin (double-mutant heat-labile enterotoxin -dmLT-) with both antigenic and immunostimulatory properties. U-Omp19 is a protease inhibitor from Brucella spp. with immunostimulatory properties that has been used as oral adjuvant. In this work, we use U-Omp19 as adjuvant in an oral vaccine formulation against ETEC containing dmLT in outbred and inbred mice. To evaluate antigen dose sparing by U-Omp19 three different immunization protocols with three different doses of dmLT were evaluated. We demonstrated that U-Omp19 co-delivery increases anti-LT IgA in feces using a mid-dose of dmLT following a prime-boost protocol (after one or two boosts). Oral immunization with U-Omp19 induced protection against LT challenge when co-formulated with dmLT in CD-1 and BALB/c mice. Indeed, there was a significant increase in anti-LT IgG and IgA avidity after a single oral administration of dmLT plus U-Omp19 in comparison with dmLT delivered alone. Interestingly, sera from dmLT plus U-Omp19 vaccinated mice significantly neutralize LT effect on intestine inflammation in vivo compared with sera from the group immunized with dmLT alone. These results demonstrate the adjuvant capacity of U-Omp19 to increase dmLT immunogenicity by the oral route and support its use in an oral subunit vaccine formulation against ETEC.

Seroprevalence of the antibodies of Brucella canis and Brucella abortus in dogs was assessed using a cross-sectional survey in Anambra and Enugu States, Nigeria. A total of 123 Companion dogs made up of 65 clinic dogs, 34 slaughter dogs and 24 household dogs were screened. For B. abortus antibody assay, the collected serum was used for Rose Bengal plate test (RBPT), Serum agglutination test (SAT) and Solid Phase Immunoassay technique with Immunocomb® Canine Brucellosis Antibody Test Kit was used. Out of the 123 dogs screened, none was positive for Brucella abortus antibodies while 34 (27.7%) of the dogs screened were positive for B. canis antibodies. There was a significant association (P0.05) between infection and antibody titre levels in the different categories of dogs. However, there was significant association (P0.05) entre la infección y los niveles de titulación de anticuerpos en las diferentes categorías de perros. Sin embargo, hubo una relación significativa (P

This study measured total serum immunoglobulin A (IgA), immunoglobulin G (IgG)1, IgG2a response against whole cell antigen (WCA), outer membrane protein (OMP), periplasmic protein (PP), cytoplasmic...

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

The RB51 vaccine strain of Brucella abortus, which confers safe and effective protection of cattle from B. abortus infection, was originally generated via serial passage of B. abortus 2308 to generate spontaneous, attenuating mutations. While some of these mutations have been previously characterized, such as an insertional mutation in the wboA gene that contributes to the rough phenotype of the strain, a comprehensive annotation of genetic differences between RB51 and B. abortus 2308 genomes has not yet been published. Here, the whole genome sequence of the RB51 vaccine strain is compared against two available 2308 parent sequences, with all observed single nucleotide polymorphisms, insertions, and deletions presented. Mutations of interest for future characterization in vaccine development, such as mutations in eipA and narJ genes in RB51, were identified. Additionally, protein homology modeling was utilized to provide in silico support for the hypothesis that the RB51 capD mutation is the second contributing mutation to the rough phenotype of RB51, likely explaining the inability of wboA-complemented strains of RB51 to revert to a smooth phenotype.