Longitudinal surveillance of Coxiella burnetii following an abortion storm in domestic goats.

Q fever is a disease caused by Coxiella burnetii, which can cause serious illness in humans and abortions in goats. A Q fever outbreak among an unvaccinated goat herd led to a 65% loss of the kid crop in spring 2018. To assess the impact of the outbreak on the herd and environment, longitudinal surveillance of the ranch was conducted across three samplings in September 2018, April 2019, and May 2022. Antibodies against C. burnetii were monitored by an indirect immunofluorescence assay. Shedding was monitored through analysis of vaginal/fecal swabs and milk. Environmental swabs and bulk soil were collected from various locations around the ranch. Animal and environmental samples were analyzed for C. burnetii DNA by PCR. Herd-level seroprevalence decreased from 89% in 2018 to 84.3% in 2019, and 64.5% in 2022. Overall herd shedding was 14.4% in 2018, 7.4% in 2019, and 6.7% in 2022. The percentage of C. burnetii-positive environmental samples was 83.7% in 2018, 51.7% in 2019, and 28.6% in 2022. Serological evidence suggests that new infections were occurring in the herd 4 years post-abortion storm. This study demonstrates the presence of C. burnetii shedding and environmental contamination in a goat operation at least four kidding seasons after an outbreak. A better understanding of management practices that can improve outcomes for infected herds, particularly in areas without access to vaccines against C. burnetii, is needed to better protect operators and the public.

Coxiella burnetii in domestic doe goats in the United States, 2019-2020.

Coxiella burnetii is a bacterial pathogen capable of causing serious disease in humans and abortions in goats. Infected goats can shed C. burnetii through urine, feces, and parturient byproducts, which can lead to infections in humans when the bacteria are inhaled. Goats are important C. burnetii reservoirs as evidenced by goat-related outbreaks across the world. To better understand the current landscape of C. burnetii infection in the domestic goat population, 4,121 vaginal swabs from 388 operations across the United States were analyzed for the presence of C. burnetii by IS1111 PCR as part of the United States Department of Agriculture, Animal Plant Health Inspection Service, Veterinary Services' National Animal Health Monitoring System Goats 2019 Study. In total, 1.5% (61/4121) of swabs representing 10.3% (40/388) (weighted estimate of 7.8, 95% CI 4.4-13.5) of operations were positive for C. burnetii DNA. The quantity of C. burnetii on positive swabs was low with an average Ct of 37.9. Factors associated with greater odds of testing positive included suspected Q fever in the herd in the previous 3 years, the presence of wild deer or elk on the operation, and the utilization of hormones for estrus synchronization. Factors associated with reduced odds of testing positive include the presence of kittens and treatment of herds with high tannin concentrate plants, diatomaceous earth, and tetrahydropyrimidines. In vitro analysis demonstrated an inhibitory effect of the tetrahydropyrimidine, pyrantel pamoate, on the growth of C. burnetii in axenic media as low as 1 µg per mL. The final multivariable logistic regression modeling identified the presence of wild predators on the operation or adjacent property (OR = 9.0, 95% CI 1.3-61.6, p value = 0.0248) as a risk factor for C. burnetii infection.

Comparison of three Coxiella burnetii infectious routes in mice.

Evidence suggests that Coxiella burnetii, which is shed in the milk, urine, feces, and birth products of infected domestic ruminants, can lead to Q fever disease following consumption of unpasteurized dairy products; however, C. burnetii is not believed to be a major gastrointestinal pathogen. Most infections are associated with inhalation of aerosols generated from the excreta of domestic ruminants. We recently demonstrated that C. burnetii delivered by oral gavage (OG) resulted in dissemination and an immune response; however, it is unclear how infection via the oral route compares to other well-established routes. Therefore, we delivered three strains of C. burnetii (representing three pertinent sequence types in the United States, such as ST16, ST20, and ST8) to immunocompetent mice in four doses via aerosol challenge (AC), intraperitoneal injection (IP), or OG. Low dose (10^5) of ST16 by OG was insufficient to cause infection, yet doses 1,000- or 100-fold lower by IP or AC, respectively, induced a robust immune response and dissemination. Despite being able to induce an immune response in a dose-dependent manner, administration of C. burnetii via OG is the least efficient route tested. Not only were the immune responses and bacterial loads diminished in mice exposed by OG relative to AC or IP, the efficiency of transmission was also inferior. High doses (10^8) were not sufficient to ensure transmission to 100% of the ST20 or ST8 cohorts. These results may provide some basis for why ingestion of C. burnetii as a mode of Q fever transmission is not often reported.

Coxiella burnetii infections in mice: Immunological responses to contemporary genotypes found in the US.

Coxiella burnetii is an obligate intracellular bacterium that causes the human disease Q fever, which can manifest as an acute flu-like illness or a long-term chronic illness, such as endocarditis. Three genotypes (ST8, ST16, and ST20) of Coxiella burnetii are commonly found in the contemporary US and are associated with specific animal hosts. Although all three genotypes have been isolated from humans with Q fever, studies comparing virulence between C. burnetii sequence types have been rare. Here, groups of mice were infected via aerosol inoculation with isolates derived from cow's milk, environmental, animal, and human samples. Mice were monitored for weight loss and blood samples were takenweekly. Animals were euthanized at 2- and 12-weeks post-infection, and bacterial burden was determined for tissues by real-time PCR. The levels of anti-Coxiella antibodies and selected inflammatory cytokines were determined for serum samples. Weight loss and splenomegaly were observed in mice infected with ST20 and ST16 isolates but were absent in the mice infected with ST8 isolates. Bacterial concentrations in the tissues were lower in the ST8 isolates at 2 weeks post-infection relative to all other isolates. ST16 and ST20 isolates induced robust antibody and cytokine responses, while ST8 isolates produced significantly lower anti-C. burnetii titers early in the infection but saw increased titers in some animals several weeks post-infection. The data suggest that the ST8 isolates are less virulent in this mouse model, as they produce less robust antibody responses that are slow to develop, relative to the ST16 and ST20 isolates.

SARS-CoV-2 Infection and Mitigation Efforts among Office Workers, Washington, DC, USA.

Despite mitigation efforts, 2 coronavirus disease outbreaks were identified among office workers in Washington, DC. Moderate adherence to workplace mitigation efforts was reported in a serologic survey; activities outside of the workplace were associated with infection. Adherence to safety measures are critical for returning to work during the pandemic.

Q Fever: A troubling disease and a challenging diagnosis.

Q fever is a disease caused by the bacterial pathogen Coxiella burnetii. This hardy organism can easily spread long distances in the wind, and only a few infectious aerosolized particles are necessary to cause serious illness. Presentations of Q fever disease can be wide-ranging, allowing it to masquerade as other illnesses and highlight the importance of laboratory testing for diagnosis and treatment. This review summarizes Q fever's epidemiology and clinical presentations and presents classical laboratory diagnostic assays and novel approaches to detecting this troubling disease.

Phylogenetic inference of Coxiella burnetii by 16S rRNA gene sequencing.

Coxiella burnetii is a human pathogen that causes the serious zoonotic disease Q fever. It is ubiquitous in the environment and due to its wide host range, long-range dispersal potential and classification as a bioterrorism agent, this microorganism is considered an HHS Select Agent. In the event of an outbreak or intentional release, laboratory strain typing methods can contribute to epidemiological investigations, law enforcement investigation and the public health response by providing critical information about the relatedness between C. burnetii isolates collected from different sources. Laboratory cultivation of C. burnetii is both time-consuming and challenging. Availability of strain collections is often limited and while several strain typing methods have been described over the years, a true gold-standard method is still elusive. Building upon epidemiological knowledge from limited, historical strain collections and typing data is essential to more accurately infer C. burnetii phylogeny. Harmonization of auspicious high-resolution laboratory typing techniques is critical to support epidemiological and law enforcement investigation. The single nucleotide polymorphism (SNP) -based genotyping approach offers simplicity, rapidity and robustness. Herein, we demonstrate SNPs identified within 16S rRNA gene sequences can differentiate C. burnetii strains. Using this method, 55 isolates were assigned to six groups based on six polymorphisms. These 16S rRNA SNP-based genotyping results were largely congruent with those obtained by analyzing restriction-endonuclease (RE)-digested DNA separated by SDS-PAGE and by the high-resolution approach based on SNPs within multispacer sequence typing (MST) loci. The SNPs identified within the 16S rRNA gene can be used as targets for the development of additional SNP-based genotyping assays for C. burnetii.

Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Genotyping and Axenic Growth of Coxiella burnetii Isolates Found in the United States Environment.

Coxiella burnetii is a gram-negative bacterium that is the etiologic agent of the zoonotic disease Q fever. Common reservoirs of C. burnetii include sheep, goats, and cattle. These animals shed C. burnetii into the environment, and humans are infected by inhalation of aerosols. A survey of 1622 environmental samples taken across the United States in 2006-2008 found that 23.8% of the samples contained C. burnetii DNA. To identify the strains circulating in the U.S. environment, DNA from these environmental samples was genotyped using an SNP-based approach to derive sequence types (ST) that are also compatible with multispacer sequence typing methods. Three different sequence types were observed in 31 samples taken from 19 locations. ST8 was associated with goats and ST20 with dairy cattle. ST16/26 was detected in locations with exposure to various animals and also in locations with no direct animal contact. Viable isolates were obtained for all three sequence types, but only the ST20 and ST16/26 isolates grew in acidified citrate cysteine medium (ACCM)-2 axenic media. Examination of a variety of isolates with different sequence types showed that ST8 and closely related isolates did not grow in ACCM-2. These results suggest that a limited number of C. burnetii sequence types are circulating in the U.S. environment and these strains have close associations with specific reservoir species. Growth in ACCM-2 may not be suitable for isolation of many C. burnetii strains.

Coxiella burnetii Infection in a Community Operating a Large-Scale Cow and Goat Dairy, Missouri, 2013.

Coxiella burnetii is a zoonotic pathogen that causes Q fever in humans and is transmitted primarily from infected goats, sheep, or cows. Q fever typically presents as an acute febrile illness; however, individuals with certain predisposing conditions, including cardiac valvulopathy, are at risk for chronic Q fever, a serious manifestation that may present as endocarditis. In response to a cluster of Q fever cases detected by public health surveillance, we evaluated C. burnetii infection in a community that operates a large-scale cow and goat dairy. A case was defined as an individual linked to the community with a C. burnetii phase II IgG titer ≥ 128. Of 135 participants, 47 (35%) cases were identified. Contact with or close proximity to cows, goats, and their excreta was associated with being a case (relative risk 2.7, 95% confidence interval 1.3-5.3). Cases were also identified among individuals without cow or goat contact and could be related to windborne spread or tracking of C. burnetii on fomites within the community. A history of injection drug use was reported by 26/130 (20%) participants; follow-up for the presence of valvulopathy and monitoring for development of chronic Q fever may be especially important among this population.

Massive dispersal of Coxiella burnetii among cattle across the United States.

Q-fever is an underreported disease caused by the bacterium Coxiella burnetii, which is highly infectious and has the ability to disperse great distances. It is a completely clonal pathogen with low genetic diversity and requires whole-genome analysis to identify discriminating features among closely related isolates. C. burnetii, and in particular one genotype (ST20), is commonly found in cow's milk across the entire dairy industry of the USA. This single genotype dominance is suggestive of host-specific adaptation, rapid dispersal and persistence within cattle. We used a comparative genomic approach to identify SNPs for high-resolution and high-throughput genotyping assays to better describe the dispersal of ST20 across the USA. We genotyped 507 ST20 cow milk samples and discovered three subgenotypes, all of which were present across the entire country and over the complete time period studied. Only one of these sub-genotypes was observed in a single dairy herd. The temporal and geographic distribution of these sub-genotypes is consistent with a model of large-scale, rapid, frequent and continuous dissemination on a continental scale. The distribution of subgenotypes is not consistent with wind-based dispersal alone, and it is likely that animal husbandry and transportation practices, including pooling of milk from multiple herds, have also shaped the patterns. On the scale of an entire country, there appear to be few barriers to rapid, frequent and large-scale dissemination of the ST20 subgenotypes.

Epizootiological investigation of a Q fever outbreak and implications for future control strategies.

OBJECTIVE: To describe the epizootiological investigation of an outbreak of Q fever (Coxiella burnetii infection). DESIGN: Epidemiological study. ANIMALS: 17 goat herds in Washington, Montana, and Oregon. PROCEDURES: In April 2011, an abortion storm at a commercial goat farm in Washington was determined to be caused by C burnetii. A joint epidemiological investigation by public health and veterinary professionals was subsequently performed to assess the extent of the outbreak by performing a trace-forward of goats sold from the index farm, to determine risk factors associated with infection, and to implement control measures. A herd management plan was developed to control the outbreak and reduce risk of human exposure. Quarantine and temporary holds preventing the sale or movement of goats allowed time for trace-forward investigation, education of farmers regarding disease risk, and testing to determine the scope of the outbreak. RESULTS: 17 farms were affected; 21 human Q fever cases were identified. Bacterial shedding in feces, vaginal fluid, or milk was confirmed in 156 of 629 (25%) goats tested by PCR assay. Seroprevalence of antibodies against C burnetii in goats, determined by ELISA, was 12%. The risk for C burnetii infection in goats was highest among females, those on farms associated with human Q fever, and those on Washington farms. A protective effect was observed for goats at farms where the primary form of goat carcass disposal was burial. CONCLUSIONS AND CLINICAL RELEVANCE: This outbreak illustrated the importance of a joint investigation for zoonotic pathogens and the need to expand and strengthen relationships between medical, public health, and veterinary partners. Heightened awareness and enhanced veterinary diagnostic capabilities for C burnetii are needed to identify and control outbreaks expediently.

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.

Serological Evidence of Coxiella burnetii Infection in Cattle and Goats in Bangladesh.

We tested 1149 ruminant sera conveniently collected from three districts of Bangladesh to identify the serological evidence of Coxiella burnetii infection in cattle and goats by enzyme-linked immunosorbent assay. We found that 0.7% (8/1149) of ruminants had detectable immunoglobulin G for C. burnetii: 0.65% (4/620) in cattle and 0.76% (4/529) in goats. A sub-set of ruminant samples was retested and confirmed by immunofluorescence assay (18/112). Although we cannot rule out false-positive reactions, our study suggests the presence of C. burnetii in cattle and goats in Bangladesh. Further studies are required to estimate disease burden at the population level and identify risk factors for Q fever in ruminants in Bangladesh.

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

BACKGROUND: Coxiella 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. RESULTS: We 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). CONCLUSIONS: The 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.].

Presence and persistence of Coxiella burnetii in the environments of goat farms associated with a Q fever outbreak.

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.

Stability of Coxiella burnetii in stored human blood.

BACKGROUND: Coxiella burnetii, an obligate intracellular organism, is the causative agent of the zoonotic disease Q fever. The seroprevalence rate for Q fever in the United States is 3.1%, suggesting a high number of infections each year. However, fewer than 200 cases of Q fever are reported to the CDC annually. This discrepancy is likely the result of underutilized diagnostics and a high percentage (>50%) of asymptomatic infections. The detection of C. burnetii in patient blood during the first 2 to 3 weeks of infection raises the possibility that the organism could be present in donated human blood. The purpose of this study was to determine if extracellular C. burnetii would be stable in blood under normal storage conditions. STUDY DESIGN AND METHODS: Donated human blood was separated into whole blood, leukoreduced whole blood, red blood cells, and plasma. Each component was spiked with purified, extracellular C. burnetii strain Nine Mile Phase 1, and the viability and infectivity of the organisms were tested weekly. RESULTS: C. burnetii did not decrease in viability or the ability to infect cells after storage in any of the blood products, even after 6 weeks of storage at 1 to 6 °C. CONCLUSIONS: Extracellular C. burnetii can survive and remain infectious in donated blood products.

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.

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.