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.

Coxiella burnetii infection of a Steller sea lion (Eumetopias jubatus) found in Washington State.

A pregnant sea lion stranded in the State of Washington was found to have placentitis caused by a unique strain of Coxiella burnetii. This is the first description of coxiellosis in a sea lion and suggests that exposure to sea lions may be a risk factor for contracting Q fever.

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.

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.

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.

Coxiella burnetii infection of marine mammals in the Pacific Northwest, 1997-2010.

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Humans are commonly exposed via inhalation of aerosolized bacteria derived from the waste products of domesticated sheep and goats, and particularly from products generated during parturition. However, many other species can be infected with C. burnetii, and the host range and full zoonotic potential of C. burnetii is unknown. Two cases of C. burnetii infection in marine mammal placenta have been reported, but it is not known if this infection is common in marine mammals. To address this issue, placenta samples were collected from Pacific harbor seals (Phoca vitulina richardsi), harbor porpoises (Phocoena phocoena), and Steller sea lions (Eumetopias jubatus). Coxiella burnetii was detected by polymerase chain reaction (PCR) in the placentas of Pacific harbor seals (17/27), harbor porpoises (2/6), and Steller sea lions (1/2) collected in the Pacific Northwest. A serosurvey of 215 Pacific harbor seals sampled in inland and outer coastal areas of the Pacific Northwest showed that 34.0% (73/215) had antibodies against either Phase 1 or Phase 2 C. burnetii. These results suggest that C. burnetii infection is common among marine mammals in this region.

Virulence of pathogenic Coxiella burnetii strains after growth in the absence of host cells.

Coxiella burnetii is a gram-negative bacterium that causes the zoonotic disease Q fever. Traditionally considered an obligate intracellular agent, the requirement to be grown in tissue culture cells, embryonated eggs, or animal hosts has made it difficult to isolate strains and perform genetic studies on C. burnetii. However, it was recently demonstrated that the attenuated Nine Mile Phase 2 (NM2) C. burnetii strain will grow axenically in acidified citrate cysteine medium (ACCM) in a 2.5% oxygen environment. The current study was undertaken to determine whether more virulent C. burnetii strains could be grown in ACCM, and whether virulence would be maintained after passage. The ACCM medium supported an ?1000-fold expansion of Nine Mile Phase 1 (NM1), NM2, M44, and Henzerling strains of C. burnetii, whereas the Priscilla (Q177) strain expanded only 100-fold, and the K strain (Q154) grew poorly in ACCM. To determine if passage in ACCM would maintain the virulence of C. burnetii, the NM1 strain was grown for up to 26 weekly passages in ACCM. C. burnetii maintained in ACCM for 5 or 8 passages maintained full virulence in a mouse model, but NM1 passaged for 23 or 26 times was somewhat attenuated. These data demonstrate that virulent strains of C. burnetii can be successfully passaged in ACCM; however, some strains can lose virulence after extended passage, and other strains grow poorly in this medium. The loss of virulence in axenic culture was associated with some truncation of lipopolysaccharide chains, suggesting a possible mechanism for attenuation.