Beyond the spore, the exosporium sugar anthrose impacts vegetative Bacillus anthracis gene regulation in cis and trans.

The Bacillus anthracis exosporium nap is the outermost portion of spore that interacts with the environment and host systems. Changes to this layer have the potential to impact wide-ranging physiological and immunological processes. The unique sugar, anthrose, normally coats the exosporium nap at its most distal points. We previously identified additional mechanisms rendering B. anthracis anthrose negative. In this work, several new ant - B. anthracis strains are identified and the impact of anthrose negativity on spore physiology is investigated. We demonstrate that live-attenuated Sterne vaccines as well as culture filtrate anthrax vaccines generate antibodies targeting non-protein components of the spore. The role of anthrose as a vegetative B. anthracis Sterne signaling molecule is implicated by luminescent expression strain assays, RNA-seq experiments, and toxin secretion analysis by western blot. Pure anthrose and the sporulation-inducing nucleoside analogue decoyinine had similar effects on toxin expression. Co-culture experiments demonstrated gene expression changes in B. anthracis depend on intracellular anthrose status (cis) in addition to anthrose status of extracellular interactions (trans). These findings provide a mechanism for how a unique spore-specific sugar residue affects physiology, expression and genetics of vegetative B. anthracis with impacts on the ecology, pathogenesis, and vaccinology of anthrax.

Spatial and phylogenetic patterns reveal hidden infection sources of Bacillus anthracis in an anthrax outbreak in Son La province, Vietnam.

Bacillus anthracis, the bacterial cause of anthrax, is a zoonosis affecting livestock and wildlife often spilling over into humans. In Vietnam, anthrax has been nationally reportable since 2015 with cases occurring annually, mostly in the northern provinces. In April 2022, an outbreak was reported in Son La province following the butchering of a water buffalo, Bubalus bubalis. A total of 137 humans from three villages were likely exposed to contaminated meat from the animal. Early epidemiological investigations suggested a single animal was involved in all exposures. Five B. anthracis isolates were recovered from human clinical cases along with one from the buffalo hide, another from associated maggots, and one from soil at the carcass site. The isolates were whole genome sequenced, allowing global, regional, and local molecular epidemiological analyses of the outbreak strains. All recovered B. anthracis belong to the A.Br.001/002 lineage based on canonical single nucleotide polymorphism analysis (canSNP). Although not previously identified in Vietnam, this lineage has been identified in the nearby countries of China, India, Indonesia, Thailand, as well as Australia. A twenty-five marker multi-locus variable number tandem repeat analysis (MLVA-25) was used to investigate the relationship between human, soil, and buffalo strains. Locally, four MLVA-25 genotypes were identified from the eight isolates. This level of genetic diversity is unusual for the limited geography and timing of cases and differs from past literature using MLVA-25. The coupled spatial and phylogenetic data suggest this outbreak originated from multiple, likely undetected, animal sources. These findings were further supported by local news reports that identified at least two additional buffalo deaths beyond the initial animal sampled in response to the human cases. Future outbreak response should include intensive surveillance for additional animal cases and additional molecular epidemiological traceback to identify pathogen sources.

Characterization of Bacillus anthracis replication and persistence on environmental substrates associated with wildlife anthrax outbreaks.

Anthrax is a zoonosis caused by the environmentally maintained, spore-forming bacterium Bacillus anthracis, affecting humans, livestock, and wildlife nearly worldwide. Bacterial spores are ingested, inhaled, and may be mechanically transmitted by biting insects or injection as occurs during heroin-associated human cases. Herbivorous hoofstock are very susceptible to anthrax. When these hosts die of anthrax, a localized infectious zone (LIZ) forms in the area surrounding the carcass as it is scavenged and decomposes, where viable populations of vegetative B. anthracis and spores contaminate the environment. In many settings, necrophagous flies contaminate the outer carcass, surrounding soils, and vegetation with viable pathogen while scavenging. Field observations in Texas have confirmed this process and identified primary browse species (e.g., persimmon) are contaminated. However, there are limited data available on B. anthracis survival on environmental substrates immediately following host death at a LIZ. Toward this, we simulated fly contamination by inoculating live-attenuated, fully virulent laboratory-adapted, and fully virulent wild B. anthracis strains on untreated leaves and rocks for 2, 5, and 7 days. At each time point after inoculation, the number of vegetative cells and spores were determined. Sporulation rates were extracted from these different time points to enable comparison of sporulation speeds between B. anthracis strains with different natural histories. We found all B. anthracis strains used in this study could multiply for 2 or more days post inoculation and persist on leaves and rocks for at least seven days with variation by strain. We found differences in sporulation rates between laboratory-adapted strains and wild isolates, with the live-attenuated strain sporulating fastest, followed by the wild isolates, then laboratory-adapted virulent strains. Extrapolating our wild strain lab results to potential contamination, a single blow fly may contaminate leaves with up to 8.62 x 105 spores per day and a single carcass may host thousands of flies. Replication outside of the carcass and rapid sporulation confirms the LIZ extends beyond the carcass for several days after formation and supports the necrophagous fly transmission pathway for amplifying cases during an outbreak. We note caution must be taken when extrapolating replication and sporulation rates from live-attenuated and laboratory-adapted strains of B. anthracis.

Use of Recombinant Escherichia coli Strains in Immunofluorescence Assays for Melioidosis Diagnosis.

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis in humans and animals in the tropics. The clinical manifestations of melioidosis are diverse, ranging from localized infections to whole-body sepsis. The effective serological method is crucial for the point-of-care diagnosis of melioidosis. The aim of this study was to develop indirect immunofluorescence assay (IFA)-based methods for detecting immunoglobulin G (IgG) antibodies in melioidosis patients. These methods use whole-cell antigens made from recombinant E. coli strains that express major B. pseudomallei antigens, including TssM, OmpH, AhpC, BimA, and Hcp1. A total of 271 serum samples from culture-confirmed melioidosis patients (n = 81), patients with other known infections (n = 70), and healthy donors (n = 120) were tested. Our study showed that the recombinant TssM strain had the highest performance, with 92.6% sensitivity, 100% specificity, 100% positive predictive value, 96.9% negative predictive value, 97.8% efficiency, 97.0% accuracy, and no cross-reactivity. The method agreement analysis based on k efficiency calculations showed that all five IFA methods perfectly agreed with the standard culturing method, while the traditional indirect hemagglutination (IHA) method moderately agreed with the culture. In summary, our investigations showed that the TssM-IFA method could be used for melioidosis diagnosis.

Humoral Immune Responses to Burkholderia pseudomallei Antigens in Captive and Wild Macaques in the Western Part of Java, Indonesia.

Burkholderia pseudomallei, the Gram-negative bacterium which causes melioidosis, is a threat to human and a wide range of animal species. There is an increased concern of melioidosis in Indonesian primate facilities, especially following case reports of fatal melioidosis in captive macaques and orangutans. Our preliminary serosurveillance of immunoglobulin G (IgG) to B. pseudomallei lipopolysaccharide showed that a significant number of captive and wild macaques in the western part of Java, Indonesia, have been exposed to B. pseudomallei. To better characterize the humoral immune response in those animals, a panel of assays were conducted on the same blood plasma specimens that were taken from 182 cynomolgus macaques (M. fascicularis) and 88 pig-tailed macaques (M. nemestrina) reared in captive enclosures and wild habitats in the western part of Java, Indonesia. The enzyme-linked immunosorbent assays (ELISAs) in this study were conducted to detect IgG against B. pseudomallei proteins; alkyl hydroperoxide reductase subunit C (AhpC), hemolysin-coregulated protein (Hcp1), and putative outer membrane porin protein (OmpH). The performances of those immunoassays were compared to ELISA against B. pseudomallei LPS, which has been conducted previously. Seropositivity to at least one assay was 76.4% (139/182) and 13.6% (12/88) in cynomolgus macaques and pig-tailed macaques, respectively. Analysis of demographic factors showed that species and primate facility were significant factors. Cynomolgus macaques had higher probability of exposure to B. pseudomallei. Moreover, macaques in Jonggol facility also had higher probability, compared to macaques in other facilities. There were no statistical associations between seropositivity with other demographic factors such as sex, age group, and habitat type. There were strong positive correlations between the absorbance results of AhpC, HcpI, and OmpH assays, but not with LPS assay. Our analysis suggested that Hcp1 assay would complement LPS assay in melioidosis serosurveillance in macaques.

Investigation of Melioidosis Outbreak in Pig Farms in Southern Thailand.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially life-threatening infection that can affect humans and a wide variety of animals in the tropics. In December 2017, a swine melioidosis case was discovered during a meat inspection at a privately-owned slaughterhouse in Nakhon Si Thammarat Province in southern Thailand. The infection, which continued for several months, caused a dispute about where the disease began. An environmental investigation into two farms-both involved in raising the first infected pig-ensued. Through genetic analysis, the investigation revealed that a contaminated water supply at one farm was the probable source of infection. The three local sequence types identified in the investigation were types 51, 298 and 392.

Apoptosis and abundance of Bcl-2 family and transforming growth factor ß1 signaling proteins in canine myxomatous mitral valves.

OBJECTIVES: To determine the percentage of cells undergoing apoptosis within canine myxomatous valves and to evaluate whether TGFß1 can be implicated as an anti-apoptosic signal through the Bcl-2 family of signaling proteins. ANIMALS: Post-mortem mitral valve leaflets harvested from 5 normal dogs, 5 dogs with early-stage myxomatous mitral valve disease (MMVD), and 5 dogs with late-stage MMVD. MATERIALS AND METHODS: The number of cells expressing cleaved caspase-3, DNA fragmentation (TUNEL marker) and apoptotic bodies were evaluated as a measure of apoptosis. To evaluate the relationship between TGFß1 signaling and apoptosis, the abundance of activated TGFß1 signaling protein, phosphorylated Smad 2/3 (p-Smad 2/3), and Bcl-2 family proteins (pro-apoptotic Bax and anti-apoptotic Bcl-2) was determined by immunohistochemistry. RESULTS: Cells in normal and both stages of MMVD expressed the TUNEL marker and cleaved caspase-3, but not apoptotic bodies. The percentage of TUNEL marker and cleaved caspase-3 positive nuclei was not significantly different between groups of dogs (p > 0.05). P-Smad 2/3 and Bax were more abundant in myxomatous mitral valves while Bcl-2 was less abundant. P-Smad 2/3 primarily increased in the atrialis layer and was abundantly increased only in late-stage MMVD. CONCLUSIONS: These data suggest that interstitial cells in MMVD are in a pro-apoptotic condition; however, they do not execute apoptosis. Thus, apoptosis does not explain differences in cellular density in canine MMVD. TGFß1 signaling through the canonical SMAD pathway is increased in myxomatous mitral valves, but does not apparently mediate interstitial cell apoptosis in canine MMVD.