Rachael A. Priestley

Inability of a Variant Strain of Anaplasma phagocytophilum to Infect Mice

Nymphal Ixodes scapularis ticks were collected from several sites in Rhode Island. Polymerase chain reaction and DNA sequencing were used to determine the presence and prevalence of Anaplasma phagocytophilum human agent (AP-ha) and a genetic variant not associated with human disease (AP-variant 1). The remaining ticks from each cohort were allowed to feed to repletion on either white-footed (Peromyscus leucopus) or DBA/2 (Mus musculus) mice. The engorged ticks and murine blood samples were evaluated for the presence of AP-ha and AP-variant 1. Although a high percentage of the infecting ticks harbored AP-variant 1, only AP-ha was amplified from the murine blood samples. Additional ticks were fed on immunocompromised SCID mice, and, again, only AP-ha was capable of establishing an infection, and only AP-ha could be detected by xenodiagnosis. These data suggest that AP-variant 1 cannot establish an infection in mice, and we propose that AP-variant 1 has an alternative natural reservoir, possibly white-tailed deer.

Presence of Coxiella burnetii DNA in the environment of the United States, 2006 to 2008.

ABSTRACT Coxiella burnetii is an obligate intracellular bacterium that causes the zoonotic disease Q fever. Because C. burnetii is highly infectious, can survive under a variety of environmental conditions, and has been weaponized in the past, it is classified as a select agent and is considered a potential bioweapon. The agent is known to be present in domestic livestock and in wild animal populations, but the background levels of C. burnetii in the environment have not been reported. To better understand the amount of C. burnetii present in the environment of the United States, more than 1,600 environmental samples were collected from six geographically diverse parts of the United States in the years 2006 to 2008. DNA was purified from these samples, and the presence of C. burnetii DNA was evaluated by quantitative PCR of the IS1111 repetitive element. Overall, 23.8% of the samples were positive for C. burnetii DNA. The prevalence in the different states ranged from 6 to 44%. C. burnetii DNA was detected in locations with livestock and also in locations with primarily human activity (post offices, stores, schools, etc.). This study demonstrates that C. burnetii is fairly common in the environment in the United States, and any analysis of C. burnetii after a suspected intentional release should be interpreted in light of these background levels. It also suggests that human exposure to C. burnetii may be more common than what is suggested by the number of reported cases of Q fever.

Rapid typing of Coxiella burnetii.

Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels) among multispacer sequence typing (MST) loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies.

Presence and Persistence of Coxiella burnetii in the Environments of Goat Farms Associated with a Q Fever Outbreak

ABSTRACT Q fever is a zoonotic disease caused by inhalation of the bacterium Coxiella burnetii. Ruminant livestock are common reservoirs for C. burnetii, and bacteria present in aerosols derived from the waste of infected animals can infect humans. The significance of infection from material deposited in the environment versus transmission directly from infected animals is not known. In 2011, an outbreak of Q fever cases on farms in Washington and Montana was associated with infected goats. A study was undertaken to investigate the quantity and spatial distribution of C. burnetii in the environment of these goat farms. Soil, vacuum, and sponge samples collected on seven farms epidemiologically linked to the outbreak were tested for the presence of C. burnetii DNA by quantitative PCR. Overall, 70.1% of the samples were positive for C. burnetii. All farms had positive samples, but the quantity of C. burnetii varied widely between samples and between farms. High quantities of C. burnetii DNA were in goat housing/birthing areas, and only small quantities were found in samples collected more than 50 m from these areas. Follow-up sampling at one of the farms 1 year after the outbreak found small quantities of C. burnetii DNA in air samples and large quantities of C. burnetii persisting in soil and vacuum samples. The results suggest that the highest concentrations of environmental C. burnetii are found in goat birthing areas and that contamination of other areas is mostly associated with human movement.

Detection of Coxiella burnetii in commercially available raw milk from the United States.

Unpasteurized (raw) milk can be purchased in 39 U.S. states, with direct consumer purchase for human consumption permitted in 29 of those 39 states. Raw milk (n=21; cow, 14; goat, 7) was purchased in 12 states, and Coxiella burnetii, the agent of Q fever, was detected in 9 of 21 (42.9%) samples tested by polymerase chain reaction. Viability of the pathogen was demonstrated by isolation of the agent in tissue culture. The demonstration of viable C. burnetii in commercially available raw milk poses a potential public health risk.

High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk

BackgroundCoxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years.ResultsWe detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8).ConclusionsThe high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes.

When Outgroups Fail; Phylogenomics of Rooting the Emerging Pathogen, Coxiella burnetii

Rooting phylogenies is critical for understanding evolution, yet the importance, intricacies and difficulties of rooting are often overlooked. For rooting, polymorphic characters among the group of interest (ingroup) must be compared to those of a relative (outgroup) that diverged before the last common ancestor (LCA) of the ingroup. Problems arise if an outgroup does not exist, is unknown, or is so distant that few characters are shared, in which case duplicated genes originating before the LCA can be used as proxy outgroups to root diverse phylogenies. Here, we describe a genome-wide expansion of this technique that can be used to solve problems at the other end of the evolutionary scale: where ingroup individuals are all very closely related to each other, but the next closest relative is very distant. We used shared orthologous single nucleotide polymorphisms (SNPs) from 10 whole genome sequences of Coxiella burnetii, the causative agent of Q fever in humans, to create a robust, but unrooted phylogeny. To maximize the number of characters informative about the rooting, we searched entire genomes for polymorphic duplicated regions where orthologs of each paralog could be identified so that the paralogs could be used to root the tree. Recent radiations, such as those of emerging pathogens, often pose rooting challenges due to a lack of ingroup variation and large genomic differences with known outgroups. Using a phylogenomic approach, we created a robust, rooted phylogeny for C. burnetii. [Coxiella burnetii; paralog SNPs; pathogen evolution; phylogeny; recent radiation; root; rooting using duplicated genes.]

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.

Transmission efficiency of the AP-variant 1 strain of Anaplasma phagocytophila.

Abstract: Nymphal Ixodes scapularis ticks were collected from several sites in Rhode Island. DNA was extracted from a subset of these ticks, and PCR and DNA sequencing of the 16S rRNA gene were used to determine the ratio of Anaplasma phagocytophila‐human agent (AP‐ha) to a genetic variant not associated with human disease (AP‐Variant 1). The remaining ticks were allowed to feed to repletion on either white‐footed (Peromyscus leucopus) or DBA/2 (Mus musculus) mice. The engorged ticks, and blood samples drawn from each mouse at one‐week intervals, were evaluated by PCR and DNA sequencing for the presence of AP‐ha and Variant 1. Although a high percentage of the infecting ticks harbored AP‐Variant 1, only AP‐ha was amplified from the mouse blood samples. Because the A. phagocytophila variant did not establish an infection either in the natural reservoir of AP‐ha, the white‐footed mouse, or in a common research laboratory mouse (DBA/2), AP‐Variant 1 may have an alternative natural reservoir, possibly the white‐tailed deer.

Molecular and Biological Characterization of a Novel Coxiella-like Agent from Carios capensis

Abstract: The genus Coxiella is currently defined by a single monotypic species, Coxiella burnetii. Novel Coxiella spp. have been detected in ticks throughout the world. These bacteria have not been cultured or named, and their evolutionary relationships to C. burnetii are poorly known. A novel Coxiella‐like agent was detected by PCR amplification and sequencing of DNA extracted from 64 pelican ticks, Carios capensis, from Devoux Bank, South Carolina, USA. PCR was used to amplify and characterize genes from the new bacterium. Sequences from some metabolic and housekeeping genes shared a 92–98% similarity to C. burnetii, but other genes such as the IS1111 transposon, com1, and 5S and 16S rRNA genes were not amplified by conventional PCR. Transovarial and transtadial transmission and environmental shedding of the agent were detected by PCR.