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Concept: Brucella

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To investigate the epidemiological relationship of isolates from different Portuguese geographical regions and to assess the diversity among isolates, the MLVA16(Orsay) assay (panels 1, 2A and 2B) was performed with a collection of 126 Brucella melitensis (46 human and 80 animal isolates) and 157 B. abortus field isolates, seven vaccine strains and the representative reference strains of each species. The MLVA16(Orsay) showed a similar high discriminatory power (HGDI 0.972 and 0.902) for both species but panel 1 and 2A markers displayed higher diversity (HGDI 0.693) in B. abortus compared to B. melitensis isolates (HGDI 0.342). The B. melitensis population belong to the “Americas” (17%) and “East Mediterranean” (83%) groups. No isolate belonged to the “West Mediterranean” group. Eighty-five percent of the human isolates (39 in 46) fit in the “East-Mediterranean” group where a single lineage known as MLVA11 genotype 116 is responsible for the vast majority of Brucella infections in humans. B. abortus isolates formed a consistent group with bv1 and bv3 isolates in different clusters. Four MLVA11 genotypes were observed for the first time in isolates from S. Jorge and Terceira islands from Azores. From the collection of isolates analysed in this study we conclude that MLVA16(Orsay) provided a clear view of Brucella spp. population, confirming epidemiological linkage in outbreak investigations. In particular, it suggests recent and ongoing colonisation of Portugal with one B. melitensis lineage usually associated with East Mediterranean countries.

Concepts: Evolution, Biology, Mediterranean Sea, Portugal, Europe, Brucellosis, Brucella melitensis, Brucella

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Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level.

Concepts: DNA, Gene expression, Evolution, Species, Cellular differentiation, Brucellosis, Brucella melitensis, Brucella

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Brucellosis is the most common bacterial zoonoses worldwide. Bovine brucellosis caused by Brucella abortus has far reaching animal health and economic impacts at both the local and national levels. Alongside traditional veterinary epidemiology, the use of molecular typing has recently been applied to inform on bacterial population structure and identify epidemiologically-linked cases of infection. Multi-locus variable number tandem repeat VNTR analysis (MLVA) was used to investigate the molecular epidemiology of a well-characterised Brucella abortus epidemic in Northern Ireland involving 387 herds between 1991 and 2012.

Concepts: Epidemiology, Microbiology, Demography, Brucellosis, Variable number tandem repeat, Brucella

148

Matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) sample preparation methods including the direct, on-plate formic acid, and ethanol/formic acid tube extraction were evaluated for their ability to render highly pathogenic organisms non-viable and safe for handling in a Biosafety Level-2 laboratory. Of these, the tube extraction procedure was the most successful, with none of the tested strains surviving this sample preparation method. Tube extracts from several agents of bioterrorism and their near neighbors were analyzed in an eight laboratory study to examine the utility of the Bruker Biotyper and Vitek MS MALDI-TOF MS systems and their IVD, research use only, and Security-Relevant databases, as applicable, to accurately identify these agents. Forty-six distinct strains of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Clostridium botulinum, Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis were extracted and distributed to participating labs for analysis. A total of 35 near neighbor isolates were also analyzed.

Concepts: Bacteria, Mass spectrometry, Brucellosis, Burkholderia mallei, Burkholderia, Brucella, Burkholderia pseudomallei

137

The facultative intracellular Gram-negative bacterium Brucella melitensis causes brucellosis in domestic and wild mammals. Brucella melitensis QH61 was isolated from a yak suffering from abortion in 2015 in Qinghai, China. Here, we report the whole-genome sequence of B. melitensis strain QH61.

Concepts: Microbiology, Escherichia coli, Sequence, Brucellosis, Brucella melitensis, Brucella, Chloramphenicol, Tibet

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Erythritol has been considered as an important factor for the pathogenesis of Brucella abortus 2308 and its ability to cause abortion in ruminants. There is a lack of laboratory models to study the Brucella-erythritol relationship, as commonly used murine models do not have erythritol. We tested the effect of exogenous erythritol on the growth of Brucella in iron minimal medium (IMM), in infected macrophage culture and in infected mice to determine if these models can be used to study the relationship between Brucella and erythritol. An effect of erythritol on Brucella growth was only seen in IMM. There appear to be no effect of erythritol on Brucella growth in macrophage cell cultures or in mice. This shows that administration of erythritol to the mice or macrophages cannot mimic the environment in ruminants during pregnancy and thus cannot be used as models to understand the effect of erythritol on Brucella pathogenesis.

Concepts: Immune system, Pregnancy, Cell biology, Macrophage, Culture, Abortion, Brucellosis, Brucella

25

Tracking and preventing the spillover of disease from wildlife to livestock can be difficult when rare outbreaks occur across large landscapes. In these cases, broad scale ecological studies could help identify risk factors and patterns of risk to inform management and reduce incidence of disease. Between 2002 and 2014, 21 livestock herds in the Greater Yellowstone Area (GYA) were affected by brucellosis, a bacterial disease caused by Brucella abortus, while no affected herds were detected between 1990 and 2001. Using a Bayesian analysis, we examined several ecological covariates that may be associated with affected livestock herds across the region. We showed that livestock risk has been increasing over time and expanding outward from the historical nexus of brucellosis in wild elk on Wyoming’s feeding grounds where elk are supplementally fed during the winter. Although elk were the presumed source of cattle infections, occurrences of affected livestock herds were only weakly associated with the density of seropositive elk across the GYA. However, the shift in livestock risk did coincide with recent increases in brucellosis seroprevalence in unfed elk populations. As increasing brucellosis in unfed elk likely stemmed from high levels of the disease in fed elk, disease-related costs of feeding elk have probably been incurred across the entire GYA, rather than solely around the feeding grounds. Our results suggest that focused disease mitigation in areas where seroprevalence in unfed elk is high could reduce the spillover of brucellosis to livestock. We also highlight the need to better understand the epidemiology of spillover events with detailed histories of disease testing, calving, and movement of infected livestock. Finally, we recommend using case-control studies to investigate local factors important to livestock risk.

Concepts: Epidemiology, Infectious disease, Infection, Cattle, Brucellosis, Elk, Brucella, Yellowstone National Park

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Brucellosis is a well-known zoonotic disease that can cause severe economic and healthcare losses. Xinjiang, one of the biggest livestock husbandry sectors in China, has gone through increasing incidence of brucellosis in cattle and small ruminants recently. In this paper, 50 B. melitensis strains and 9 B. abortus strains collected from across Xinjiang area (from 2010 to 2015) were genotyped using multiple locus variable-number tandem-repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST). Based on 8 loci (MLVA-8), 50 B. melitensis strains were classified into three genotypes. Genotypes 42 (n=38, 76%) and 63 (n=11, 22%) were part of the East Mediterranean group, and one genotype with pattern of 1-5-3-13-2-4-3-2 represents a single-locus variant from genotype 63. MLVA-16 resolved 50 B. melitensis strains into 28 genotypes, of which 15 are unique to Xinjiang and 10 are in common with those in adjacent country Kazakhstan and neighboring provinces of China. Minimum Spanning Tree (MST) analysis implies that B. melitensis strains collected from across Kazakhstan, Xinjiang and China areas may share a common origin. Nine B. abortus strains were sorted into three genotypes by MLVA-8, genotypes 36 (n=7, 77.8%), 86 (n=1, 11.1%) and a new genotype with pattern of 4-5-3-13-2-2-3-1. Each B. abortus strain showed distinct MLVA-16 genotypes, suggesting that B. abortus species may possess more genetic diversity than B. melitensis. Using MLST, most B. melitensis strains (n=49) were identified as sequence type ST8, and most B. abortus strains (n=8) were recognized as ST2. Two new sequence types, ST37 and ST38, represented by single strain from B. melitensis and B. abortus species respectively, were also detected in this study. These results could facilitate the pathogen surveillance in the forthcoming eradication programs and serve as a guide in source tracking in case of new outbreaks occur.

Concepts: Genetics, People's Republic of China, Brucellosis, Livestock, Brucella melitensis, Brucella, Zoonosis, Kazakhstan

7

Animal reservoirs of brucellosis constitute an ongoing threat to human health globally, with foodborne, occupational and recreational exposures creating opportunities for transmission. In Australia and the United States, hunting of feral pigs has been identified as the principal risk factor for human brucellosis due to Brucella suis. Following increased reports of canine B. suis infection, we undertook a review of case notification data and veterinary records to address knowledge gaps about transmission, clinical presentation, and zoonotic risks arising from infected dogs.

Concepts: Epidemiology, Risk, United States, Brucellosis, Southern United States, Brucella, New South Wales, Zoonosis