Increase in Colorado Tick Fever Virus Disease Cases and Effect of COVID-19 Pandemic on Behaviors and Testing Practices, Montana, 2020.

In 2020, Montana, USA, reported a large increase in Colorado tick fever (CTF) cases. To investigate potential causes of the increase, we conducted a case-control study of Montana residents who tested positive or negative for CTF during 2020, assessed healthcare providers' CTF awareness and testing practices, and reviewed CTF testing methods. Case-patients reported more time recreating outdoors on weekends, and all reported finding a tick on themselves before illness. No consistent changes were identified in provider practices. Previously, only CTF serologic testing was used in Montana. In 2020, because of SARS-CoV-2 testing needs, the state laboratory sent specimens for CTF testing to the Centers for Disease Control and Prevention, where more sensitive molecular methods are used. This change in testing probably increased the number of CTF cases detected. Molecular testing is optimal for CTF diagnosis during acute illness. Tick bite prevention measures should continue to be advised for persons doing outdoor activities.

Coxiella burnetii exposure in northern sea otters Enhydra lutris kenyoni.

Valvular endocarditis has been well described in northern sea otters Enhydra lutris kenyoni of Alaska and in many cases no cause has been identified. It is also one of the most common conditions observed in people with chronic Coxiella burnetii infection. Given the high levels of C. burnetii exposure in marine mammals distributed throughout the same geographic range as the northern sea otter, and the presence of valvular lesions seen in otters, the objective of this study was to determine the level of C. burnetii exposure in otters and investigate any association between exposure, infection and valvular disease in this species. Archived serum from 75 live captured, apparently healthy otters (25 from each of 3 stocks) and 30 dead otters were tested for C. burnetii antibodies by indirect florescent antibody assay (IFA). Archived bone marrow and heart valves were tested for C. burnetii DNA by real-time PCR (qPCR). Overall, the seroprevalence in live otters was 17%, with significantly more exposed animals in the south central (40%) stock relative to the southwest (8%) and southeast (4%). The seroprevalence of animals sampled post mortem was 27%, although none of the bone marrow or heart valve samples were positive by qPCR. Results of this study failed to demonstrate a significant association between C. burnetii infection and valvular endocarditis in sea otters; however, the differing seroprevalence suggests that exposure opportunities vary geographically.

West Nile virus experimental evolution in vivo and the trade-off hypothesis.

In nature, arthropod-borne viruses (arboviruses) perpetuate through alternating replication in vertebrate and invertebrate hosts. The trade-off hypothesis proposes that these viruses maintain adequate replicative fitness in two disparate hosts in exchange for superior fitness in one host. Releasing the virus from the constraints of a two-host cycle should thus facilitate adaptation to a single host. This theory has been addressed in a variety of systems, but remains poorly understood. We sought to determine the fitness implications of alternating host replication for West Nile virus (WNV) using an in vivo model system. Previously, WNV was serially or alternately passed 20 times in vivo in chicks or mosquitoes and resulting viruses were characterized genetically. In this study, these test viruses were competed in vivo in fitness assays against an unpassed marked reference virus. Fitness was assayed in chicks and in two important WNV vectors, Culex pipiens and Culex quinquefasciatus. Chick-specialized virus displayed clear fitness gains in chicks and in Cx. pipiens but not in Cx. quinquefasciatus. Cx. pipiens-specialized virus experienced reduced fitness in chicks and little change in either mosquito species. These data suggest that when fitness is measured in birds the trade-off hypothesis is supported; but in mosquitoes it is not. Overall, these results suggest that WNV evolution is driven by alternate cycles of genetic expansion in mosquitoes, where purifying selection is weak and genetic diversity generated, and restriction in birds, where purifying selection is strong.

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.

Transmission of yellow fever vaccine virus through blood transfusion and organ transplantation in the USA in 2021: report of an investigation.

BACKGROUND: In 2021, four patients who had received solid organ transplants in the USA developed encephalitis beginning 2-6 weeks after transplantation from a common organ donor. We describe an investigation into the cause of encephalitis in these patients. METHODS: From Nov 7, 2021, to Feb 24, 2022, we conducted a public health investigation involving 15 agencies and medical centres in the USA. We tested various specimens (blood, cerebrospinal fluid, intraocular fluid, serum, and tissues) from the organ donor and recipients by serology, RT-PCR, immunohistochemistry, metagenomic next-generation sequencing, and host gene expression, and conducted a traceback of blood transfusions received by the organ donor. FINDINGS: We identified one read from yellow fever virus in cerebrospinal fluid from the recipient of a kidney using metagenomic next-generation sequencing. Recent infection with yellow fever virus was confirmed in all four organ recipients by identification of yellow fever virus RNA consistent with the 17D vaccine strain in brain tissue from one recipient and seroconversion after transplantation in three recipients. Two patients recovered and two patients had no neurological recovery and died. 3 days before organ procurement, the organ donor received a blood transfusion from a donor who had received a yellow fever vaccine 6 days before blood donation. INTERPRETATION: This investigation substantiates the use of metagenomic next-generation sequencing for the broad-based detection of rare or unexpected pathogens. Health-care workers providing vaccinations should inform patients of the need to defer blood donation for at least 2 weeks after receiving a yellow fever vaccine. Despite mitigation strategies and safety interventions, a low risk of transfusion-transmitted infections remains. FUNDING: US Centers for Disease Control and Prevention (CDC), the Biomedical Advanced Research and Development Authority, and the CDC Epidemiology and Laboratory Capacity Cooperative Agreement for Infectious Diseases.

Nonconsensus West Nile virus genomes arising during mosquito infection suppress pathogenesis and modulate virus fitness in vivo.

West Nile virus (WNV) is similar to other RNA viruses in that it forms genetically complex populations within hosts. The virus is maintained in nature in mosquitoes and birds, with each host type exerting distinct influences on virus populations. We previously observed that prolonged replication in mosquitoes led to increases in WNV genetic diversity and diminished pathogenesis in mice without remarkable changes to the consensus genome sequence. We therefore sought to evaluate the relationships between individual and group phenotypes in WNV and to discover novel viral determinants of pathogenesis in mice and fitness in mosquitoes and birds. Individual plaque size variants were isolated from a genetically complex population, and mutations conferring a small-plaque and mouse-attenuated phenotype were localized to the RNA helicase domain of the NS3 protein by reverse genetics. The mutation, an Asp deletion, did not alter type I interferon production in the host but rendered mutant viruses more susceptible to interferon compared to wild type (WT) WNV. Finally, we used an in vivo fitness assay in Culex quinquefasciatus mosquitoes and chickens to determine whether the mutation in NS3 influenced fitness. The fitness of the NS3 mutant was dramatically lower in chickens and moderately lower in mosquitoes, indicating that RNA helicase is a major fitness determinant of WNV and that the effect on fitness is host specific. Overall, this work highlights the complex relationships that exist between individual and group phenotypes in RNA viruses and identifies RNA helicase as an attenuation and fitness determinant in WNV.

Q fever, spotted fever group, and typhus group rickettsioses among hospitalized febrile patients in northern Tanzania.

BACKGROUND: The importance of Q fever, spotted fever group rickettsiosis (SFGR), and typhus group rickettsiosis (TGR) as causes of febrile illness in sub-Saharan Africa is unknown; the putative role of Q fever as a human immunodeficiency virus (HIV) coinfection is unclear. METHODS: We identified febrile inpatients in Moshi, Tanzania, from September 2007 through August 2008 and collected acute- and convalescent-phase serum samples. A ≥4-fold increase in immunoglobulin (Ig) G immunfluorescence assay (IFA) titer to Coxiella burnetii phase II antigen defined acute Q fever. A ≥4-fold increase in IgG IFA titer to Rickettsia conorii or Rickettsia typhi antigen defined SFGR and TGR, respectively. RESULTS: Among 870 patients, 483 (55.5%) were tested for acute Q fever, and 450 (51.7%) were tested for acute SFGR and TGR. Results suggested acute Q fever in 24 (5.0%) patients and SFGR and TGR in 36 (8.0%) and 2 (0.5%) patients, respectively. Acute Q fever was associated with hepato- or splenomegaly (odds ratio [OR], 3.1; P = .028), anemia (OR, 3.0; P = .009), leukopenia (OR, 3.9; P = .013), jaundice (OR, 7.1; P = .007), and onset during the dry season (OR, 2.7; P = .021). HIV infection was not associated with acute Q fever (OR, 1.7; P = .231). Acute SFGR was associated with leukopenia (OR, 4.1; P = .003) and with evidence of other zoonoses (OR, 2.2; P = .045). CONCLUSIONS: Despite being common causes of febrile illness in northern Tanzania, Q fever and SFGR are not diagnosed or managed with targeted antimicrobials. C. burnetii does not appear to be an HIV-associated co-infection.

Practical method for extraction of PCR-quality DNA from environmental soil samples.

Methods for the extraction of PCR-quality DNA from environmental soil samples by using pairs of commercially available kits were evaluated. Coxiella burnetii DNA was detected in spiked soil samples at <1,000 genome equivalents per gram of soil and in 12 (16.4%) of 73 environmental soil samples.

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

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.

The genome of deep-sea vent chemolithoautotroph Thiomicrospira crunogena XCL-2.

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

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.

Early cytokine and antibody responses against Coxiella burnetii in aerosol infection of BALB/c mice.

Coxiella burnetii, a Gram-negative intracellular bacterium, can give rise to Q fever in humans and is transmitted mainly by inhalation of infected aerosols from animal reservoirs. Serology is commonly used to diagnose Q fever, but the early cellular immune response-i.e., C. burnetii-specific interferon γ (IFN-γ) production in response to antigen challenge-might be an additional diagnostic. Detection of IFN-γ responses has been used to identify past and chronic Q fever infections, but the IFN-γ response in acute Q fever has not been described. By challenging immunocompetent BALB/c mice with aerosols containing phase I C. burnetii, the timing and extent of IFN-γ recall responses were evaluated in an acute C. burnetii infection. Other cytokines were also measured in an effort to identify other potential diagnostic markers. The data show that after initial expansion of bacteria first in lungs and then in other tissues, the infection was cleared from day 10 onwards as reflected by the decreasing number of bacteria. The antigen-induced IFN-γ production by splenocytes coincided with emergence of IgM phase II antibodies at day 10 postinfection and preceded appearance of IgG antibodies. This was accompanied by the production of proinflammatory cytokines including interleukin (IL) 6, keratinocyte-derived cytokine, and IFN-γ-induced protein 10, followed by monocyte chemotactic protein 1, but not by IL-1ß and tumor necrosis factor α, and only very low production of the anti-inflammatory cytokine IL-10. These data suggest that analysis of antigen-specific IFN-γ responses could be a useful tool for diagnosis of acute Q fever. Moreover, the current model of C. burnetii infection could be used to give new insights into immunological factors that predispose to development of persistent infection.

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.

Long-Term immune responses to Coxiella burnetii after vaccination.

Q fever is a zoonotic disease caused by infection with the bacterium Coxiella burnetii. Infection with C. burnetii results in humoral and cellular immune responses, both of which are thought to contribute to protection against subsequent infection. Whole-cell formalin-inactivated vaccines have also been shown to induce both humoral and cellular immunity and provide protection. Whether measurement of cellular or humoral immunity is a better indicator of immune protection is not known, and the duration of immunity induced by natural infection or vaccination is also poorly understood. To better understand the measurement and duration of C. burnetii immunity, 16 people vaccinated against Q fever (0.2 to 10.3 years before analysis) and 29 controls with a low risk of Q fever exposure were tested for immune responses to C. burnetii by an indirect fluorescent-antibody test (IFA) to measure circulating antibody and by a gamma interferon release assay (IGRA) to measure cellular immunity. Among vaccinated subjects, the IFA detected antibodies in 13/16, and the IGRA also detected positive responses in 13/16. All of the vaccinated subjects had a positive response in at least one of the assays, whereas 8/29 control subjects were positive in at least one assay. There was not a correlation between time since vaccination and responses in these assays. These results show that IFA and IGRA perform similarly in detection of C. burnetii immune responses and that Q fever vaccination establishes long-lived immune responses to C. burnetii.

Survey of laboratory animal technicians in the United States for Coxiella burnetii antibodies and exploration of risk factors for exposure.

Little is known about the prevalence of zoonotic infections among laboratory animal care technicians (LAT). Q fever, a disease caused by Coxiella burnetii, is a known occupational hazard for persons caring for livestock. We sought to determine the seroprevalence of C. burnetii antibodies among LAT and to identify risk factors associated with C. burnetii seropositivity. A survey was administered and serum samples collected from a convenience sample of 97 LAT. Samples were screened by using a Q fever IgG ELISA. Immunofluorescent antibody assays for phase I and phase II IgG were used to confirm the status of samples that were positive or equivocal by ELISA; positive samples were titered to endpoint. Antibodies against C. burnetii were detected in 6 (6%) of the 97 respondents. In our sample of LAT, seropositivity to C. burnetii was therefore twice as high in LAT as compared with the general population. Age, sex, and working with sheep regularly were not associated with seropositivity. Risk factors associated with seropositivity included breeding cattle within respondent's research facility, any current job contact with waste from beef cattle or goats, and exposure to animal waste during previous jobs or outside of current job duties. Only 15% of responding LAT reported being aware that sheep, goats, and cattle can transmit Q fever. Research facilities that use cattle or goats should evaluate their waste-management practices and educational programs in light of these findings. Additional efforts are needed to increase awareness among LAT regarding Q fever and heightened risk of exposure to infectious materials. Physicians should consider the risk of infection with C. burnetii when treating LAT with potential occupational exposures.

Coxiella burnetii in northern fur seal (Callorhinus ursinus) placentas from St. Paul Island, Alaska.

The decline in the number of northern fur seal (NFS; Callorhinus ursinus) pups on St. Paul Island, Alaska, has led to multidisciplinary research, including investigation into issues of reproductive health and success. Given the recent identification of Coxiella burnetii in the placenta of two other marine mammal species, NFS placentas were collected from Reef rookery on St. Paul Island, Alaska, during the 2010 pupping season, examined histologically, and tested for C. burnetii using polymerase chain reaction (PCR). Of 146 placentas examined, gram-negative intratrophoblastic bacteria that were positive for C. burnetii on immunohistochemistry were observed in 5 (3%) placentas. Placental infection was usually devoid of associated inflammation or significant ancillary pathology. One hundred nine (75%) of the placentas were positive for C. burnetii on PCR. C. burnetii is globally distributed and persists for long periods in the environment, providing ample opportunity for exposure of many species. The significance of this finding for the declining fur seal population, potential human exposure and infection, and impact on other sympatric marine mammal or terrestrial species is unclear; further investigation into the epidemiology of Coxiella in the marine ecosystem is warranted.

Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice.

Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts.