Skip to main content
Download PDF

Ultraviolet C inactivation of Coxiella burnetii for production of a structurally preserved whole cell vaccine antigen

BMC Microbiology volume 24, Article number: 118 (2024) Cite this article

Abstract

Q fever, a worldwide-occurring zoonotic disease, can cause economic losses for public and veterinary health systems. Vaccines are not yet available worldwide and currently under development. In this regard, it is important to produce a whole cell antigen, with preserved structural and antigenic properties and free of chemical modifications. Thus, inactivation of Coxiella burnetii with ultraviolet light C (UVC) was evaluated. C. burnetii Nine Mile phase I (NMI) and phase II (NMII) were exposed to decreasing intensities in a time-dependent manner and viability was tested by rescue cultivation in axenic medium or cell culture. Effects on the cell structure were visualized by transmission electron microscopy and antigenicity of UVC-treated NMI was studied by immunization of rabbits. NMI and NMII were inactivated at UVC intensities of 250 µW/cm2 for 5 min or 100 µW/cm2 for 20 min. Reactivation by DNA repair was considered to be unlikely. No morphological changes were observed directly after UVC inactivation by transmission electron microscopy, but severe swelling and membrane degradation of bacteria with increasing severity occurred after 24 and 48 h. Immunization of rabbits resulted in a pronounced antibody response. UVC inactivation of C. burnetii resulted in a structural preserved, safe whole cell antigen and might be useful as antigen for diagnostic purposes or as vaccine candidate.

Peer Review reports

Introduction

Coxiella burnetii, a Gram-negative, obligate intracellular bacterium, is the etiological agent of the worldwide distributed zoonosis Q fever [1]. This disease is endemic in ruminants in Europe [2] and especially small ruminants are linked to human Q fever cases [3,4,5]. In animals, infections are often subclinical or cause late term abortions, weak offspring and fertility problems with a considerable economic impact [6,7,8]. The bacteria are shed within milk, urine, feces and especially in high amounts within birth products [9,10,11]. C. burnetii displays resistance to environmental stress and survives on sheep wool, in dry milk powder or in the soil for several months [12,13,14]. This is often associated with spore-like small cell variants (SCV). But data supporting this assumption are missing. The large cell variant (LCV) is metabolic active and replicates in a parasitophorous vacuole with phagolysosomal characteristics [15, 16]. Transmission to humans occurs through inhalation of contaminated aerosols, e.g. dust. Acute infection manifests in 40% of cases as a self-limiting flu-like illness presenting with fever and respiratory symptoms [17]. In 1–5% of cases the infection becomes chronic, which can be life-threatening [18, 19]. The financial burden of Q fever for public and veterinary public health was recently demonstrated for the largest ever reported Q fever outbreak in the Netherlands with over 4000 human cases and 74 Q fever related deaths [20, 21]. Intervention costs in the agricultural sector were estimated as 35,000€ per disability-adjusted life years (DALY) [20].

Vaccines and diagnostic test kits for humans and animals are currently based on formaldehyde inactivated bacteria. Use of vaccines has limitations and there is only one licensed vaccine for humans: Q-VAX (CSL Seqirus Australia Pty Ltd., Victoria, Australia), which is available in Australia only. It provides protection for a long time, but with a risk of severe side effects in pre-exposed persons [22]. Reports of Q-VAX failures exist for patients already infected during administration of the vaccine or patients which became ill after vaccination [23]. In Europe vaccination of cattle, goats and sheep is done with COXEVAC (Ceva Tiergesundheit GmbH, Düsseldorf, Germany) based on formaldehyde inactivated C. burnetii Nine Mile bacteria. Vaccination reduces shedding but can cause swelling at the injection site, a transient temperature increase and decrease in milk production in goats and cattle [24,25,26]. Swelling is more pronounced in infected animals [27]. Vaccination results in sheep are controversially discussed. One study reported no significant difference in bacterial shedding between vaccinated and non-vaccinated animals, whereas others reported a reduction of shedding [28, 29]. There is a strong need to develop new vaccines which confer protection and have limited side effects.

Veterinary diagnostic kits for antibody detection using formaldehyde inactivated Coxiellae as antigen were reported to have significantly variable specificities and sensitivities [30,31,32]. Contrary to human diagnostic assays, antibodies directed against the two antigenic variants of C. burnetii, termed phase I (smooth, full length LPS) and phase II (rough, truncated LPS) are not distinguished. Phase-specific antibodies are used in human diagnostics to differentiate between onset, acute and chronic Q fever [33]. This seems to be important for veterinary medicine also e.g. to identify chronic local infection of the udder and ongoing shedding in milk [34].

Formaldehyde inactivation is well-established and has been used for several decades for various pathogens. Known problems of this procedure are e.g. crosslinking of immunogenic structures that can lead to epitope loss or gain of new structures. This can reduce the efficiency of the vaccine and may lead to insufficient protection against subsequent infection [35]. This problem has been described for several pathogens [36, 37]. The most prominent example was the 1960ies vaccine against the respiratory syncytial virus (RSV), in which formalin inactivation has resulted in severe antibody dependent disease enhancement in infants [38, 39]. Therefore, the development of novel inactivation methods that are faster and better for antigen preservation are urgently needed. The germicidal activity of ultraviolet light C (UVC) is well established and might present an alternative to chemical inactivation. It causes mutagenic events by exciting electrons in DNA molecules and formation of pyrimidine dimers. These dimers cause replication errors and accumulation of mutations which may be lethal to the treated microorganism [40, 41]. Moreover, UVC generates reactive oxygen species (ROS) and sub-sequential oxidative damage to structural lipids and inhibits protein synthesis. These damages interfere with vital biological functions of bacteria [41]. The effectiveness of ultraviolet light (UV) inactivated vaccines was demonstrated for SARS coronavirus in mice and for vaccinia virus in macaques [42, 43]. However, many bacteria display the ability of reactivation. A process mediated by DNA repair mechanisms such as nucleotide excision repair (NER), base excision repair (BER) and homologous recombination, designated as dark repair. As a second line of defense, bacteria can express photolyases which directly rearrange bonds in a visible light depending manner [44]. This photoreactivation or light repair is more efficient than dark repair but both processes depend on adequate conditions [45, 46].

Thus, UVC exposure may be a promising method for inactivation of C. burnetii. The present study aims to determine the ability to inactivate axenic propagated C. burnetii Nine Mile phase I (NMI) and Nine Mile phase II (NMII) while preserving their antigenic structures. Thus, an antigen free of host cell materials can be gained, which is problematic to obtain for obligate intracellular bacteria.

Materials and methods

Bacterial strains and growth conditions

Coxiella burnetii Nine Mile RSA 493 phase I (NMI) and Nine Mile RSA 439 phase II (NMII) were propagated under biosafety level 3 and 2, respectively, in acidified citrate cysteine medium (ACCM-2, Sunrise Science Products, Knoxville, TN, USA) at 37 °C with 5% CO2 and 2.5% O2 [47]. Briefly, ACCM-2 was inoculated with 1 × 105 genome equivalents per milliliter (GE/ml) and incubated for 7–10 days. Bacteria were harvested by centrifugation at 10.000 x g for 20 min at 4 °C and re-suspended in sucrose-glycerol solution (270 mM sucrose, 10% (v/v) glycerol) for preservation at -80 °C.

Quantification of Coxiella burnetii by real-time PCR

Coxiella burnetii was quantified by using real-time PCR (qPCR) targeting the isocitrate dehydrogenase encoding gene icd [48, 49]. C. burnetii DNA was extracted by resuspending bacteria in phosphate-buffered saline (PBS) (BioWhittaker, Lonza, Walkersville, MD, USA) pH 7.4 and heat inactivated at 110 °C for 15 min. All qPCR reactions were performed in a 25 µl reaction mix using Maxima Probe/ROX qPCR Master Mix (ThermoFisher Scientific, Waltham, MA, USA), 300 nM each primer (icd-439 F and icd-514R), 100 nM probe (icd-464TM) and 2 µl of plasmid standard or sample [49]. The qPCR was carried out on a Mx3000P QPCR Instrument (Agilent Technologies, Santa Clara, CA, USA) using the following cycling conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 30 s at 60 °C. Data collection and analysis was carried out using the Mx Pro4 software.

UVC exposure of Coxiella burnetii

To determine the required UVC intensity for total growth inhibition, initial experiments were performed with C. burnetii NMII. Bacterial suspensions were adjusted to 1 × 1010 GE/ml in PBS pH 7.4 and for each experiment 3 ml aliquots were exposed in 6 well plates. The suspension had a filling height of 3 mm. UVC intensity was measured prior to exposure with a UVC solarmeter with a range of 0-1999 µW/cm2 (Solarmeter, Model 8.0, Glenside, PA, USA) and temperature monitored every 10 min with an Escort Junior device (Escort Messtechnik AG, Aesch bei Birmensdorf, Swizerland). NMII was exposed to different intensities, starting from 1500; 1000; 500; 250 to 100 µW/cm2 for 60; 45; 30; 20; 15; 10 and 5 min under agitation every 10 minutes. Depending on the results obtained for NMII, UVC intensities of 100 µW/cm2 and 250 µW/cm2 were chosen in a time dependent manner for total growth inhibition of NMI. Results represent the average of three independent experiments with three technical replicates each.

Viability testing of Coxiella burnetii in ACCM-2 medium

Viability of Coxiella burnetii after UVC treatment was assessed after cultivation in axenic media and compared to an untreated control by qPCR. UVC treated bacteria were serially diluted from 1:10 to 1:10000. The untreated growth control was diluted to 105 GE/ml in ACCM-2 in duplicate. From each dilution, two aliquots of 1 ml were harvested (16.000x g, 5 min, 4 °C) and resuspended in 200 µl PBS for quantification after heat inactivation (d0). After incubation for 7 days at 37 °C under 5% CO2 and 2.5% O2, bacterial cultures were harvested (16.000 x g, 5 min, 4 °C) and resuspended in 200 µl PBS for qPCR quantification after heat inactivation (d7). Viable bacteria were indicated by an increase of GE/ml on d7 compared to d0.

Determination of the surviving fraction

The surviving fraction (S/S0) was determined as the quotient of colony counts (cfu/ml) from UVC treated at 250 µW/cm2, at 100 µW/cm2 and untreated bacteria and determined using a modified soft agarose overlay method as described by Omsland et al. [47]. Briefly, 200 µl of bacteria from serial dilutions in 2x concentrated ACCM-2 were mixed with 200 µl of 1% (w/v) melted ultrapure agarose (ThermoFisher Scientific, Waltham, MA, USA) at 60 °C and then equilibrated to 37 °C. From each dilution 100 µl drops in triplicate were applied on a solidified ACCM-2 agarose base. Plates were incubated for up to 10 days and colonies counted.

Reactivation of Coxiella burnetii NMII in cell culture

Reactivation of Coxiella burnetii NMII treated with UVC was assessed by inoculation of buffalo green monkey (BGM) cells (Collection of Cell Lines in Veterinary Medicine; Friedrich-Loeffler-Institut, Greifswald Insel - Riems, Germany) and compared to an untreated control by visual assessment of Coxiella-containing vacuole (CCV) formation and qPCR. Bacteria were UVC treated as described above, incubated for 1 h in visible light and further incubated for 24 and 48 h in the dark at 37 °C at 2.5% O2 and 5% CO2. Samples were taken before, 1 h, 24 and 48 h after UVC exposure for inoculation of BGM cells (105/ml, UltraMDCK medium, Lonza Walkersville, Inc.; Walkersville, USA) and cfu/ml determination. At each time point treated and untreated bacteria were serially diluted (undiluted; 1:10 till 10–11) and 50 µl aliquots added to BGM cells in 96 well plates. Inoculated cells were incubated for seven days and medium was changed once after 24 h incubation at 37 °C with 5% CO2. CCV formation was assessed by microscopy and cell layers harvested by scraping. Bacterial load was measured by qPCR after heat inactivation. For cfu/ml determination, 5 µl aliquots in tetraplicates of undiluted and serially diluted bacteria at each time point were dropped onto ACCM-2 agarose plates and incubated for 7 to 10 days. Results represent the average of three independent experiments with three technical replicates each.

Transmission electron microscopy (TEM) analysis of UVC treated Coxiella burnetii

Triplicates of bacterial suspensions collected 1 h prior to UVC exposure, 1 h, 24 and 48 h after UVC exposure as well as 24 and 48 h without UVC exposure were fixed 1:2 (v:v) with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 1.8% glucose for 1 h at 4 °C. The fixative was replaced by cacodylate buffer. Suspensions were vortexed for 5 min and an aliquot of 100 µl was removed from each suspension for negative staining.

For negative staining, drops of 30 µl were placed on a plate of dental wax. 300-mesh copper grids that had been filmed with formvar, coated with carbon and hydrophilized by glow discharge were floated on the drops for 30 min. The grids were briefly rinsed in 3 drops of distilled water and the excess liquid drained on wet filter paper. Finally, one grid of each preparation was contrasted on a drop of 0.5% and one on a drop of 0.2% uranyl acetate for 1 min. The excess contrast medium was drained on wet filter paper. After air-drying, grids were examined by transmission electron microscopy (Tecnai 12, FEI Deutschland Gmbh, Dreieich, Germany) at 80 kV. Representative micrographs were taken with a digital camera (TEMCAM FX416, TVIPS, Gauting, Germany) from five different quadrants of each grid at 2900x and 6800x magnification. The diameter of 10 bacteria of each preparation, which equals 30 bacteria of each treatment, were measured using the EM-Measure software (TVIPS, Gauting, Germany). Mean and standard deviation of diameters were calculated with excel and the percentage of SCV and LCV was determined. For this, bacteria with a diameter ≤ 370 nm were counted as SCV and bacteria with a diameter  ≥ 400 nm were counted as LCV. Bacteria in a transition stage with a diameter between 371 and 399 nm were not included in the counts.

For the preparation of ultrathin sections, suspensions were centrifuged for 15 min at 20.000x g to obtain a bacteria pellet. The pellet was removed from the tube, evenly mixed with 20 µl of 2% molten agarose on a glass slide, allowed to cool and sectioned into 1 mm³ cubes. Cubes were post-fixed in 2% osmium tetroxide and embedded in Araldite Cy212. Relevant areas were selected in Toluidine-blue-stained semi-thin sections. Ultrathin Sect. (85 nm) were stained with uranyl acetate and lead citrate and examined by transmission electron microscopy (Tecnai 12; FEI/Thermo Fisher Scientific) at 80 kV. Representative electron micrographs were taken at magnifications of both 4800x as well as 6800x using a 4kx4k digital CMOS camera (TEMCAM FX416, TVIPS, Garching, Germany) to evaluate bacterial morphology.

Immunization of SPF-rabbits with UVC inactivated NMI

Immunization of SPF-rabbits (Zimmermann-Kaninchen, ZIKA rabbits; BioGenes) was outsourced (Assurance no. F16-00178 (A5755-01) BioGenes GmbH, Berlin, Germany). After collection of pre-immune sera, both rabbits were immunized with 200 µl of 8.3 × 109 GE/ml NMI for the first step and with 50 µl of 8.3 × 109 GE/ml NMI every 14 days for four times and subsequently every 21 days for 2 times. After a rest period of about 8-month two final boost were carried out with 100 µl of 1,1 × 109 GE/ml NMI. Final blood was taken four days after the last immunization.

Detection of Coxiella burnetii NMI and NMII reactive antibodies using indirect ELISA

Rabbit polyclonal sera were tested in tetraplicate using an indirect ELISA method. Briefly, high binding 96 well plates (Sarstedt AG & Co. KG, Nümbrecht, Germany) were coated with 2.5 × 107 GE/ml NMI or NMII in PBS overnight at 4 °C. Unbound bacterial suspension was discarded and unspecific binding sites blocked with 5% (w/v) skim milk (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) for 1 h at 37 °C. Plates were washed three times with PBS buffer containing 0.05% Tween 20 (v/v, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) prior adding of serum samples in 2-fold dilutions ranging from 1:2000 to 1:128000 in 5% skim milk overnight at 4 °C. As positive control a polyclonal serum from rabbits immunized with heat-inactivated whole cell lysates of C. burnetii NMI (1:2000, Davids Biotechnology GmbH, Regensburg, Germany) or as negative control a polyclonal serum from SPF-rabbits (1:2000, Kaneka Eurogentec S.A., Seraing, Belgium) were used. After washing, bound antibodies were detected by peroxidase-conjugated goat anti-rabbit antibodies (1:5000, Dianova GmbH, Hamburg, Germany). Substrate solution (SeramunBlau slow2 50, Seramun Diagnostica GmbH, Heidesee, Germany) was added and the reaction stopped after 30 min with 0.5 M H2SO4 (Merck KGAA, Darmstadt, Germany). The plates were analyzed spectrophotometrically at 450 nm versus 630 nm.

Rabbit polyclonal sera were diluted 1:20000 and tested using the indirect ELISA ID Screen Q Fever Indirect (ID.vet, Innovative Diagnostics, Grabels, France) in duplicate. The secondary antibody was replaced by peroxidase-conjugated goat anti-rabbit antibodies (1:5000). The ELISA results were evaluated according to the manufacturer’s guidelines. Briefly, sera with a sample to positive ratio (S/P%) ≤ 40% was considered negative, a S/P% of 40% < and ≤ 50% suspicious, a S/P% of > 50% ≤ 80% as positive or > 80% as strong positive.

Statistical analysis

All data were analyzed using GraphPad Prism version 9.4.1 (GraphPad Software, LLC, San Diego, USA). Pairwise comparisons of bacterial quantifications or diameter measurements were performed using the Kruskal-Wallis test due to invalid normality determined by the Shapiro-Wilk test. Data are presented as Tukey box plot with mean, quartiles Q1 and Q3. Statistically significant differences were indicated by p-values of < 0.05 (*), < 0.01 (**), and < 0.001 (***).

Results

Axenic propagated Coxiella burnetii NMI and NMII are effectively inactivated by UVC exposure

The effective range for UVC treatment was established with NMII. An exposure to UVC light from 1500 µW/cm2 to 500 µW/cm2 for less than 15 min resulted in complete growth reduction of NMII as shown in Table 1. The genome equivalents per ml (GE/ml) quantified by realtime PCR (qPCR) dropped from the initial 1 × 1010 GE/ml of the bacterial solution prepared for treatment to 7.03 × 104 GE/ml after exposure to 1500 µW/cm2 for 60 min or to 3,80 × 107 GE/ml with 500 µW/cm2 for 15 min. This indicates severe crosslinking of Coxiella-DNA due to UVC treatment. No increase of GE/ml, determined by qPCR, was observed after rescue cultivation in liquid ACCM-2 for 7 days. This indicates complete growth reduction of NMII at all intensities tested. Temperature monitoring could exclude a considerable effect of heat. The highest temperature of 21.7 oC was recorded using 1500 µW/cm2 for 60 min. However, no increase in GE/ml was measurable by comparing the results of qPCR quantified bacteria from day 0 with day 7 at any intensity above 500 µW/cm2 (Table 1). At a lower intensity of 100 µW/cm2 bacteria could be rescued after 5 min, 10 and 15 min indicated by the significant increase in GE/ml after incubation for 7 days in ACCM-2 medium, but not after 20 min of exposure (Fig. 1A).

Growth reduction of C. burnetii NMI was evaluated accordingly. Bacteria did not grow after exposure to UVC with 250 µW/cm2 for 5 min as indicated by the significant decrease in GE/ml. At a lower UVC intensity of 100 µW/cm2 surviving bacteria could be rescued in liquid ACCM-2 after treatment for 5 min, 10 min, 15 and 20 min (Table 1; Fig. 1B).

Table 1 Growth of Coxiella burnetii NMI and NMII after UVC exposure
Full size table
Fig. 1
figure 1

UVC treatment of (A) C. burnetii NMII by exposure to 100 µW/cm2 and (B) C. burnetii NMI to 100 µW/cm2 and 250 µW/cm2 in a time dependent manner. Bacteria (1 × 1010 GE/ml) were treated with UVC and rescued in ACCM-2. A decrease in recovered GE/ml at day 7 (d7) compared to day 0 (d0) indicates inactivation. Untreated bacteria were diluted to 1 × 105 GE/ml and incubated for 7 days as growth control. The results presented as Tukey box plots represent the average of three independent experiments. Statistically significant differences are indicated by p-values of < 0.05 (*), < 0.01 (**), and < 0.001 (***)

Full size image

These results were confirmed by determination of the surviving fraction (cfu/ml). NMII bacteria were rescued after treatment at a UVC intensity of 100 µW/cm2 for 5 min, 10 and 15 min, whereas no colony forming units were detectable in suspensions treated for 20 min. The surviving fraction decreased accordingly with increasing exposure time (Fig. 2). Similar to NMII, the surviving fraction of NMI decreased with increasing exposure time at 100 µW/cm2 but without complete inactivation (Fig. 2). Taken together, growth of NMI and NMII is effectively inhibited by exposure to UVC but with different intensities and exposure times, respectively.

Fig. 2
figure 2

Surviving fraction of NMI and NMII after UVC exposure at 100 µW/cm2 for 0; 10; 15 and 20 min. Surviving fraction was determined as (S/S0) using colony forming units (cfu/ml) of UVC treated (S) and untreated (S0) bacteria. Results represent the average of three independent experiments

Full size image

Coxiella burnetii NMII does not reactivate after UVC treatment

UVC exposed bacteria were rescued after 1 h, 24 or 48 h in buffalo green monkey (BGM) cells or plated for cfu/ml counts. Independently from the given reactivation time no growth of UVC treated NMII was detectable in BGM cells. The bacterial load decreased accordingly to the serial dilution of the inoculum and no CCV formation was visible (Table 2). At all given reactivation times, the bacterial load in BGM cells after 7 days incubation decreased from approximately 1E + 05 to 1E + 03 accordingly to the dilution row. No bacteria were detectable in higher dilutions. In comparison, untreated NMII reached equal bacterial loads in BGM cells independently from the dilution of the inoculum after 7 days. CCV formation was visible in all dilutions till 10E-07 (Table 2). For UVC treated bacteria no cfu/ml could be determined. For the untreated control 2.47E + 08 cfu/ml could be detected, similar to the original suspension adjusted to 1E + 10 GE/ml by qPCR.

Table 2 Reactivation of UVC treated C. burnetii NMII
Full size table

UVC induced morphological changes

Negative contrast preparations and ultrathin sections were used to assess UVC induced morphological changes of C. burnetii NMII in a time dependent manner. Negative contrast preparation allowed to determine size and shape of Coxiella without section bias. Measurements focused on the bacterial diameter, since prior investigations had shown that large cell variants (LCV) and small cell variants (SCV) differed mainly in diameter, but not in length (personal communication).

The average diameter of C. burnetii NMII was lowest prior to UVC treatment, increased mildly within 24 h and decreased after 48 h (Fig. 3). This was associated with an increase of LCV at 24 h and a decrease at 48 h (Table 3) and may reflect the developmental cycle of C. burnetii. After UVC treatment, the average diameter of bacteria was higher compared to the untreated control and further increased significantly at 24 h and at 48 h after UVC treatment (Fig. 3). The comparatively high standard deviation in all UVC treated samples was the result of both, a few thin and a few exceedingly thick bacteria in these preparations. Based on diameter, high percentages of LCV were present after UVC treatment (Table 3). Bacteria with a large diameter were particularly electron lucent. An additional feature was the moderate amount of cellular detritus at 24 h and the large amount of cellular detritus at 48 h after UVC treatment indicating increased degradation of bacteria (Fig. 4A, B). In contrast, almost no detritus was present in the untreated controls at 24 and 48 h (Fig. 4C and D).

Fig. 3
figure 3

Effect of UVC treatment on the bacterial shape of C. burnetii NMII. Changes of the bacterial diameter (nm) are displayed for NMII bacteria before UVC treatment (-1), after 24 h (+ 24) and 48 h (+ 48) incubation as well as for UVC treated NMII 1 h (+ 1), 24 h (+ 24) and 48 h (+ 48) after UVC exposure at 100 µW/cm2 for 20 min. The results presented as Tukey box plots represent the average of three independent experiments. Statistically significant differences are indicated by p-values of < 0.05 (*), < 0.01 (**), and < 0.001 (***)

Full size image
Table 3 Distribution of C. burnetii NMII morphological forms in untreated and UVC treated suspensions
Full size table
Fig. 4
figure 4

Negative contrast preparations of UVC treated and untreated C. burnetii NMII. Bacteria were UVC treated at 100 µW/cm2 for 20 min and negative contrast preparations were done after 24 h (A) and 48 h (B) incubation. Untreated controls were prepared accordingly after 24 h (C) and 48 h (D). Most C. burnetii treated with UVC (A, B) have large diameters resembling LCV (arrows indicate examples). Small pleomorphic particles representing break-down products of degraded bacteria are present at 24 h after UVC treatment (A) and increased at 48 h after UVC treatment (B). In the untreated controls (C, D), thick LCV (arrows, examples) and thin SCV (arrowheads, examples) can be distinguished at 24 and 48 h. Bars = 1 μm

Full size image

Ultrathin sections revealed no morphological changes neither in C. burnetii NMI nor in NMII when bacteria where fixed within 60 min after UVC treatment (Fig. 5). General swelling of bacteria was observed at 24 and 48 h after UVC treatment (Fig. 6A, B). Discrimination of SCV and LCV was no longer possible because of the lysis of cytoplasm. These findings indicate that most of the bacteria identified as LCV in the negative staining preparations were most likely swollen bacteria. Different degrees of swelling were identified ranging from comparatively thin bacteria (SCV) with coarsely granular electron lucent cytoplasm to increasingly distended bacteria where only bacterial walls were left and the cytoplasm was completely dissolved. Some of these bacterial walls were intact, others were fragmented. Membrane fragments formed small vesicles. There were no marked differences in the findings between 24 and 48 h after treatment. In the time-matched untreated controls, all bacteria had cytoplasm of higher electron density, and LCV and SCV could be differentiated (Fig. 6C, D).

Fig. 5
figure 5

Ultrathin sections of C. burnetii NMII and NMI directly after UVC treatment. UVC treated (A) NMII at 100 µW/cm2 for 20 min and (B) NMI at 250 µW/cm2 for 5 min as well as untreated controls of (C) NMII and (D) NMI and were fixed for preparation of ultrathin sections within 60 min after exposure. Large cell variants (LCV; L, examples) and small cell variants (SCV; S, examples) are present in all bacterial suspensions. Distinct bacterial walls (arrowheads, examples) are clearly visible in the majority of Coxiella. There is no visible damage to the cell wall or cytoplasm and all bacteria appear undamaged. Bars = 500 nm

Full size image
Fig. 6
figure 6

Ultrathin section of C. burnetii NMII after UVC treatment. C. burnetii NMII (A) 24 h and (B) 48 h after UVC treatment at 100 µW/cm2 for 20 min and untreated controls (C) after 24 h and (D) 48 h. Swelling and degradation of Coxiella is prominent 24 and 48 h after UVC treatment (A, B). Thin bacteria with coarsely granular electron lucent cytoplasm (arrowheads, examples) were interpreted as swollen SCV. Different degrees of swelling occurs in bacteria where the bacterial wall encloses lysed cytoplasm ( ↑ = mild, ↑↑ = moderate, ↑↑↑ = severe). Bacterial walls are occasionally fragmented (open arrows). LCV (L, examples) and SCV (S, examples) without visible damage are present in the untreated controls (C, D). Bars = 500 nm

Full size image

Antibody response after immunization with UVC inactivated Coxiella burnetii NMI

The antibody titer of two SPF-rabbits immunized with UVC inactivated C. burnetii NMI was analyzed by titration against viable C. burnetii NMI. Pre-immune sera of both rabbits were tested and resulted in an OD450 value below 0.04. Titers of both rabbits were above 1:128000 after the second and fourth booster immunization (Fig. 7). Prediluted rabbit sera (1:20000) collected after the fourth booster immunization were tested strong positive with the commercial ID Screen Q Fever Indirect ELISA Test Kit with a S/P% of 94.5 and 94.3, respectively. This indicates that the antigenic properties of UVC inactivated C. burnetii are intact since generated antibodies react with untreated C. burnetii NMI as well as with a bovine derived C. burnetii antigen presented in the used commercial ELISA.

Fig. 7
figure 7

Reactivity of sera from two SPF-rabbits (Zimmermann Kaninchen, ZIKA) immunized with UVC inactivated C. burnetii NMI after the second and fourth booster. Plates were coated with 2.5 × 107 GE/ml of NMI. Serum samples from two rabbits were serially diluted and traced with a horseradish-conjugated goat anti-rabbit antibody. As positive control (PC) a polyclonal rabbit serum against heat-inactivated whole cell lysate of C. burnetii NMI and as negative control (NC) a polyclonal SPF-rabbit serum was used. Results represent the average of two independent experiments

Full size image

Discussion

The effect of UVC exposure on the viability of Coxiella burnetii was studied in the early 1950ties, but those results are difficult to interpret. Ransom and Huber (1951) reported inactivation of C. burnetii yolk sac preparations after 0.3 s exposure to a Levinson-Oppenheimer lamp of unknown intensity [50]. Another study reported that dried Coxiella survived exposure to a “bacterial lamp” with 30 W at 75 cm distance to the sample [51]. Also exposure of C. burnetii-suspensions prepared from spleens of mice exposed to UV at 25 W in 1 m distance was not effective and viable bacteria were detected by inoculation of mice [52]. In these studies, neither the quantity of bacteria used nor the applied intensities of light are specified. In a more recent study, 108 NMI or NMII bacteria in suspension or cultured in macrophages were inactivated by UV treatment with 600 µW/cm2 at 10 cm distance for 15 s [53]. These results confirm the here reported data, that C. burnetii NMI and NMII are completely inactivated at any UVC intensity above 500 µW/cm2 likely due to extensive DNA damage. This was indicated by the significant decrease of genome equivalents after UVC treatment. Contrary to all studies mentioned above, a UVC solarmeter was used to determine the UVC intensity at sample level. A complete inactivation was achieved even at low intensities of 250 µW/cm2 and 100 µW/cm2 for suspensions of both, the virulent strain NMI as well as the laboratory strain NMII.

UVC has a low penetration depth and is quenched by turbid or protein-containing liquids [54, 55]. The suspensions used in this study were prepared with PBS and had a filling height of 3 mm. Additionally, every 10 min the plates were agitated for mixing. Despite these precautions for equal exposure of the bacteria to UVC, complete inactivation should be always tested carefully for each batch, since bacteria display the ability to reverse UVC induced DNA damage by DNA repair mechanisms and start to replicate again. C. burnetii has an efficient DNA repair system which is constitutively expressed [56]. Further it encodes for a photolyase (phrB, CBU_1176, UniProt Q83CE4) of 38% amino acid sequence identity to that of Escherichia coli (phrB, UniProt P00914). Photoreactivation is the most efficient repair system and depends on light and environmental factors such as temperature or salinity [46]. It was shown to rescue E. coli after 99.9% UVC inactivation but effectivity is highly variable in different strains [57, 58]. Photo repair activity is measurable in less than 20 min after UVC treatment. Therefore, reactivation was investigated 1 h, 24 and 48 h after UVC exposure by determination of cfu/ml, bacterial loads in BGM cells by qPCR or CCV formation [46]. The lack of C. burnetii growth indicated that there was no reactivation.

The activity of repair mechanisms might have been influenced by the fact that a suspension of C. burnetii in PBS was exposed to UVC and this environment does not resemble the interior of the Coxiella-containing vacuole (CCV). Further experiments using axenic media or cell culture for assessment of enzymatic activity need to be carried out to examine if photo repair or dark repair are active mechanisms and play a role after UVC treatment for rescue of intracellular C. burnetii. The dependence of C. burnetii on the pH gradient between the lumen of the CCV and the bacterial cytoplasm was discovered a long time ago and lead to the designation of metabolic acid activation. The gradient is utilized for transport and utilization of nutrients and generation of ATP using proton motive force [59,60,61]. Medium with slightly neutral or neutral pH leads to decreased metabolic activity and reduced replication [62]. This pH gradient is not present in PBS used for preparation of bacterial suspension and may have additionally reduced the ability of C. burnetii to repair UVC induced damage on the DNA and protein level.

UVC exposure leads to rearrangements of the phospholipid bilayer, pore formation, causes lipid oxidation and induces changes in the amount and composition of fatty acids [63,64,65,66]. This is not immediately reflected in the cellular structure resulting in morphologically intact LCV and SCV directly after UVC treatment. This allows to obtain structurally intact antigen of inactivated Coxiellae. Severe effects of UVC exposure on the morphology of Coxiellae became visible at 24 h after exposure which explains the inactivation. They were characterized by swelling of the bacteria, rarefaction of cytoplasm and finally fragmentation of the bacterial cell wall. The swelling made a discrimination between LCV and SCV impossible. The high number of LCV identified in the negative contrast preparation most likely represent swollen bacteria and are not an indication of changes in the C. burnetii life cycle. Swelling is a fundamental expression of acute cellular injury. Failure of energy dependent membrane pumps to control ion gradients and pore formation may result in an influx of water and dilution of cytoplasm. Similar morphological changes were reported for Shigella flexneri after UVC treatment [67]. The different degrees of swelling observed may also reflect differences in the reaction of LCV and SCV which differ in membrane structure [68]. There was no indication of recovery at 48 h after UVC exposure which further supports the absence of reactivation.

The necessary UVC intensity was higher for total growth inhibition of NMI than for NMII. The only difference between these two isogenic strains is the severely truncated lipopolysaccharide (LPS) in NMII [69, 70]. The LPS is the dominant component of the outer leaflet of the outer membrane (OM) and plays an important role as barrier against antibiotics or host defense factors. It stabilizes the OM due to the interaction of the O-polysaccharide side chains and binding of divalent cations. Bacteria with a deep rough LPS accumulate phospholipid patches which result in higher membrane fluidity and higher permeability [71, 72]. It is known that UVC exposure produces reactive oxygen species which attack nucleic acids and proteins. Additionally, UVC leads to lipid oxidation and pore formation [66, 73]. Interestingly, the amount of free endotoxin increases upon UV irradiation of Escherichia coli [74]. This might indicate that the LPS confers some kind of protection against UVC exposure for NMI and NMII is more sensitive for irradiation.

Bacterial vaccines are often produced by chemical inactivation with ß-propiolactone or formaldehyde [75]. Despite several effective vaccines based on formaldehyde inactivated pathogens e.g. poliovirus or cholera, negative effects of the crosslinking nature of formaldehyde on effectiveness of the preparations are known. Induced structural and antigenic changes may cause a lack of specific epitopes or generation of new unspecific epitopes and thus have a decreased efficiency [36, 76, 77]. An alternative strategy for gentle inactivation is low-energy electron irradiation leaving the bacterial cell structure intact which has been proven for bacterial and viral pathogens [78, 79]. UVC inactivation of pathogens provides a rapid and low-cost method when vaccines are urgently demanded, e.g. during the SARS-CoV pandemic. This technique is feasible in many settings. The generated antigen contains all relevant structures such as quaternary structures and glycosylation of proteins relevant for immunogenicity. The UVC derived C. burnetii NMI antigen resulted in a pronounced antibody response in rabbits. Furthermore, these rabbit sera reacted strongly with viable NMI and NMII as well as in a commercial ELISA kit for detection of C. burnetii-specific antibodies in sera from ruminants. This implies that UVC inactivated NMI bacteria display highly similar antigenic properties as viable bacteria. To confirm this, the antigenic nature of UVC treated bacteria needs to be analyzed in more detail. Similar results for immunization with UVC inactivated pathogens were reported for SARS-CoV virus. Immunized mice produced a strong IgG response with neutralizing activities [43, 80]. Application of UV inactivated vaccinia virus elicits a strong antibody response in macaques and reduced the viral load [42]. Taken together, the antibody response in SPF rabbits imply that UVC inactivated bacteria may confer protection but this needs to be evaluated in further studies.

Conclusions

UVC treatment is suitable for inactivation of virulent C. burnetii in translucent liquids under laboratory settings. After each treatment the effective inactivation has to be confirmed for safety reasons. The structural integrity of the NMI bacteria is preserved at 250 µW/cm2 for 5 min. This antigen is safe and free of host cell materials originating from classical propagation of C. burnetii. It provokes a strong antibody response in rabbits and might be suitable for the development of new serological tests or as a component of a vaccine.

Data availability

The datasets supporting the conclusions of this article are available in the Zenodo repository after publication, DOI: 10.5281/zenodo.10217973.

References

  1. Angelakis E, Raoult D. Q, fever. Vet Microbiol. 2010;140(3–4):297–309. https://doi.org/10.1016/j.vetmic.2009.07.016.

    Article  CAS  PubMed  Google Scholar 

  2. (EFSA) EFSA. Scientific opinion on Q fever. EFSA J 2010;8(5).

  3. Dobos A, Balla E. Industrial Dairy Cattle Farms in Hungary Source of Coxiella burnetii infection in humans. Vector Borne Zoonotic Dis. 2021;21(7):498–501. https://doi.org/10.1089/vbz.2020.2749.

    Article  PubMed  Google Scholar 

  4. Boden K, Brasche S, Straube E, Bischof W. Specific risk factors for contracting Q fever: lessons from the outbreak Jena. Int J Hyg Environ Health. 2014;217(1):110–5. https://doi.org/10.1016/j.ijheh.2013.04.004.

    Article  PubMed  Google Scholar 

  5. Schimmer B, Morroy G, Dijkstra F, Schneeberger PM, Weers-Pothoff G, Timen A, et al. Large ongoing Q fever outbreak in the south of the Netherlands, 2008. Euro Surveill. 2008;13:31.

    Article  Google Scholar 

  6. Bernard H, Brockmann SO, Kleinkauf N, Klinc C, Wagner-Wiening C, Stark K, Jansen A. High seroprevalence of Coxiella burnetii antibodies in veterinarians associated with cattle obstetrics. Bavaria 2009 Vector Borne Zoonotic Dis. 2012;12(7):552–7. https://doi.org/10.1089/vbz.2011.0879.

    Article  PubMed  Google Scholar 

  7. Agerholm JS. Coxiella burnetii associated reproductive disorders in domestic animals–a critical review. Acta Vet Scand. 2013;55:13. https://doi.org/10.1186/1751-0147-55-13.

    Article  PubMed  PubMed Central  Google Scholar 

  8. De Biase D, Costagliola A, Del Piero F, Di Palo R, Coronati D, Galiero G, et al. Coxiella burnetii in infertile dairy cattle with chronic endometritis. Vet Pathol. 2018;55(4):539–42. https://doi.org/10.1177/0300985818760376.

    Article  PubMed  Google Scholar 

  9. Bauer B, Prufer L, Walter M, Ganter I, Frangoulidis D, Runge M, Ganter M. Comparison of Coxiella burnetii Excretion between Sheep and Goats naturally infected with one cattle-Associated genotype. Pathogens. 2020;9(8). https://doi.org/10.3390/pathogens9080652.

  10. Roest HJ, van Gelderen B, Dinkla A, Frangoulidis D, van Zijderveld F, Rebel J, van Keulen L. Q fever in pregnant goats: Pathogenesis and Excretion of Coxiella burnetii. PLoS ONE. 2012;7(11):e48949. https://doi.org/10.1371/journal.pone.0048949. PONE-D-12-20240 [pii].

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Boarbi S, Mori M, Rousset E, Sidi-Boumedine K, Van Esbroeck M, Fretin D. Prevalence and molecular typing of Coxiella burnetii in bulk tank milk in Belgian dairy goats, 2009–2013. Vet Microbiol. 2014;170(1–2):117–24. https://doi.org/10.1016/j.vetmic.2014.01.025.

    Article  CAS  PubMed  Google Scholar 

  12. Kersh GJ, Fitzpatrick KA, Self JS, Priestley RA, Kelly AJ, Lash RR, et al. Presence and persistence of Coxiella burnetii in the environments of goat farms associated with a Q fever outbreak. Appl Environ Microbiol. 2013;79(5):1697–703. https://doi.org/10.1128/AEM.03472-12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Alvarez-Alonso R, Basterretxea M, Barandika JF, Hurtado A, Idiazabal J, Jado I, et al. A Q fever outbreak with a high rate of abortions at a dairy Goat Farm: Coxiella burnetii Shedding, Environmental Contamination, and viability. Appl Environ Microbiol. 2018;84(20). https://doi.org/10.1128/AEM.01650-18.

  14. von Sprockhoff H. Zur Tenazität Von Chlamydien Und Coxiella burnetii. Dtsch Tierärztliche Wochenschr. 1980;87:39.

    Google Scholar 

  15. Coleman SA, Fischer ER, Cockrell DC, Voth DE, Howe D, Mead DJ, et al. Proteome and antigen profiling of Coxiella burnetii developmental forms. Infect Immun. 2007;75(1):290–8. https://doi.org/10.1128/IAI.00883-06. doi: IAI.00883-06 [pii].

    Article  CAS  PubMed  Google Scholar 

  16. McCaul TF, Williams JC. Developmental cycle of Coxiella burnetii: structure and morphogenesis of vegetative and sporogenic differentiations. J Bacteriol. 1981;147(3):1063–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kampschreur LM, Wegdam-Blans MC, Thijsen SF, Groot CA, Schneeberger PM, Hollander AA, et al. Acute Q fever related in-hospital mortality in the Netherlands. Neth J Med. 2010;68(12):408–13.

    CAS  PubMed  Google Scholar 

  18. Kampschreur LM, Wever PC, Wegdam-Blans MC, Delsing CE, Bleeker-Rovers CP, Sprong T, et al. Defining chronic Q fever: a matter of debate. J Infect. 2012;65(4):362–3. https://doi.org/10.1016/j.jinf.2012.08.002.

    Article  CAS  PubMed  Google Scholar 

  19. Kampschreur LM, Delsing CE, Groenwold RH, Wegdam-Blans MC, Bleeker-Rovers CP, de Jager-Leclercq MG, et al. Chronic Q fever in the Netherlands 5 years after the start of the Q fever epidemic: results from the Dutch chronic Q fever database. J Clin Microbiol. 2014;52(5):1637–43. https://doi.org/10.1128/JCM.03221-13.

    Article  PubMed  PubMed Central  Google Scholar 

  20. van Asseldonk MA, Prins J, Bergevoet RH. Economic assessment of Q fever in the Netherlands. Prev Vet Med. 2013;112(1–2):27–34. https://doi.org/10.1016/j.prevetmed.2013.06.002.

    Article  PubMed  Google Scholar 

  21. Dijkstra F, van der Hoek W, Wijers N, Schimmer B, Rietveld A, Wijkmans CJ, et al. The 2007–2010 Q fever epidemic in the Netherlands: characteristics of notified acute Q fever patients and the association with dairy goat farming. FEMS Immunol Med Microbiol. 2012;64(1):3–12. https://doi.org/10.1111/j.1574-695X.2011.00876.x.

    Article  CAS  PubMed  Google Scholar 

  22. Woldeyohannes SM, Perkins NR, Baker P, Gilks CF, Knibbs LD, Reid SA. Q fever vaccine efficacy and occupational exposure risk in Queensland, Australia: a retrospective cohort study. Vaccine. 2020;38(42):6578–84. https://doi.org/10.1016/j.vaccine.2020.08.006.

    Article  PubMed  Google Scholar 

  23. Bond KA, Franklin LJ, Sutton B, Firestone SM. Q-Vax Q fever vaccine failures, Victoria, Australia 1994–2013. Vaccine. 2017;35(51):7084–7. https://doi.org/10.1016/j.vaccine.2017.10.088.

    Article  PubMed  Google Scholar 

  24. Arricau-Bouvery N, Souriau A, Bodier C, Dufour P, Rousset E, Rodolakis A. Effect of vaccination with phase I and phase II Coxiella burnetii vaccines in pregnant goats. Vaccine. 2005;23(35):4392–402. https://doi.org/10.1016/j.vaccine.2005.04.010.

    Article  CAS  PubMed  Google Scholar 

  25. Bauer BU, Knittler MR, Prufer TL, Wolf A, Matthiesen S, Runge M, Ganter M. Humoral immune response to Q fever vaccination of three sheep flocks naturally pre-infected with Coxiella burnetii. Vaccine. 2021;39(10):1499–507. https://doi.org/10.1016/j.vaccine.2021.01.062.

    Article  CAS  PubMed  Google Scholar 

  26. Schulze LS, Borchardt S, Ouellet V, Heuwieser W. Effect of a phase I coxiella burnetii inactivated vaccine on body temperature and milk yield in dairy cows. J Dairy Sci. 2016;99(1):541–50. https://doi.org/10.3168/jds.2015-9628.

    Article  CAS  PubMed  Google Scholar 

  27. Bauer B, Runge M, Campe A, Henning K, Mertens-Scholz K, Boden K, et al. Coxiella burnetii: Ein Übersichtsartikel Mit Fokus auf das Infektionsgeschehen in Deutschen Schaf- Und Ziegenherden. Berliner Und Münchner Tierärztliche Wochenzeitschrift. 2020;133. https://doi.org/10.2376/0005-9366-19030.

  28. Astobiza I, Barandika JF, Hurtado A, Juste RA, Garcia-Perez AL. Kinetics of Coxiella burnetii excretion in a commercial dairy sheep flock after treatment with oxytetracycline. Vet J. 2010;184(2):172–5. https://doi.org/10.1016/j.tvjl.2009.01.017.

    Article  CAS  PubMed  Google Scholar 

  29. Eibach R, Bothe F, Runge M, Ganter M. Long-term monitoring of a Coxiella burnetii-infected sheep flock after vaccination and antibiotic treatment under field conditions. Berl Munch Tierarztl Wochenschr. 2013;126(1–2):3–9.

    PubMed  Google Scholar 

  30. Hogerwerf L, Koop G, Klinkenberg D, Roest HI, Vellema P, Nielen M. Test and cull of high risk Coxiella burnetii infected pregnant dairy goats is not feasible due to poor test performance. Vet J. 2014;200(2):343–5. https://doi.org/10.1016/j.tvjl.2014.02.015.

    Article  PubMed  Google Scholar 

  31. Muleme M, Stenos J, Vincent G, Campbell A, Graves S, Warner S, et al. Bayesian validation of the indirect immunofluorescence assay and its superiority to the enzyme-linked immunosorbent assay and the complement fixation test for detecting antibodies against Coxiella burnetii in Goat serum. Clin Vaccine Immunol. 2016;23(6):507–14. https://doi.org/10.1128/CVI.00724-15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lurier T, Rousset E, Gasqui P, Sala C, Claustre C, Abrial D, et al. Evaluation using latent class models of the diagnostic performances of three ELISA tests commercialized for the serological diagnosis of Coxiella burnetii infection in domestic ruminants. Vet Res. 2021;52(1):56. https://doi.org/10.1186/s13567-021-00926-w.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Vaca DJ, Dobler G, Fischer SF, Keller C, Konrad M, von Loewenich FD, et al. Contemporary diagnostics for medically relevant fastidious microorganisms belonging to the genera Anaplasma, Bartonella, Coxiella, Orientia, and Rickettsia. FEMS Microbiol Rev. 2022. https://doi.org/10.1093/femsre/fuac013.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Bottcher J, Frangoulidis D, Schumacher M, Janowetz B, Gangl A, Alex M. The impact of Q fever-phase-specific milk serology for the diagnosis of puerperal and chronic milk shedding of C. Burnetii in dairy cows. Berl Munch Tierarztl Wochenschr. 2013;126(9–10):427–35.

    PubMed  Google Scholar 

  35. Amanna IJ, Raue HP, Slifka MK. Development of a new hydrogen peroxide-based vaccine platform. Nat Med. 2012;18(6):974–9. https://doi.org/10.1038/nm.2763.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Fan YC, Chiu HC, Chen LK, Chang GJ, Chiou SS. Formalin inactivation of Japanese Encephalitis Virus Vaccine alters the Antigenicity and immunogenicity of a Neutralization Epitope in envelope protein domain III. PLoS Negl Trop Dis. 2015;9(10):e0004167. https://doi.org/10.1371/journal.pntd.0004167.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Brown F. Formaldehyde as an inactivant. Vaccine. 1995;13(2):231. https://doi.org/10.1016/0264-410x(95)93142-v.

    Article  CAS  PubMed  Google Scholar 

  38. Acosta PL, Caballero MT, Polack FP. Brief history and characterization of enhanced respiratory Syncytial Virus Disease. Clin Vaccine Immunol. 2015;23(3):189–95. https://doi.org/10.1128/CVI.00609-15.

    Article  CAS  PubMed  Google Scholar 

  39. Florencia Delgado M, Irusta PM, Polack FP. Reply to: antibody affinity maturation and respiratory syncytial virus disease. Nat Med. 2009;15(7):725–6. https://doi.org/10.1038/nm0709-725b.

    Article  CAS  Google Scholar 

  40. Azimi Y, Allen D, Farnood R. Kinetics of UV inactivation of wastewater bioflocs. Water Res. 2012;46(12):3827–36.

    Article  CAS  PubMed  Google Scholar 

  41. Rastogi R, Richa, Kumar A, Tyagi M, Sinha R. Molecular mechanisms of Ultraviolet Radiation-Induced DNA damage and repair. 2010;J Nucleic Acids.

  42. Jones BG, Sealy RE, Zhan X, Freiden PJ, Surman SL, Blanchard JL, Hurwitz JL. UV-inactivated Vaccinia virus (VV) in a multi-envelope DNA-VV-protein (DVP) HIV-1 vaccine protects macaques from lethal challenge with heterologous SHIV. Vaccine. 2012;30(21):3188–95. https://doi.org/10.1016/j.vaccine.2012.03.001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Takasuka N, Fujii H, Takahashi Y, Kasai M, Morikawa S, Itamura S, et al. A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice. Int Immunol. 2004;16(10):1423–30. https://doi.org/10.1093/intimm/dxh143.

    Article  CAS  PubMed  Google Scholar 

  44. Jones DL, Baxter BK. DNA repair and photoprotection: mechanisms of overcoming environmental Ultraviolet Radiation exposure in Halophilic Archaea. Front Microbiol. 2017;8:1882. https://doi.org/10.3389/fmicb.2017.01882.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Quek PH, Hu J. Influence of photoreactivating light intensity and incubation temperature on photoreactivation of Escherichia coli following LP and MP UV disinfection. J Appl Microbiol. 2008;105(1):124–33. https://doi.org/10.1111/j.1365-2672.2008.03723.x.

    Article  CAS  PubMed  Google Scholar 

  46. Salcedo I, Andrade JA, Quiroga JM, Nebot E. Photoreactivation and dark repair in UV-treated microorganisms: effect of temperature. Appl Environ Microbiol. 2007;73(5):1594–600. https://doi.org/10.1128/AEM.02145-06.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Omsland A, Beare P, Hill J, Cockrell D, Howe D, Hansen B, et al. Isolation from animal tissue and genetic transformation of Coxiella burnetii are facilitated by an improved axenic growth medium. Appl Environ Microbiol. 2011;77(11):3720–5. https://doi.org/10.1128/aem.02826-10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Dresler J, Klimentova J, Pajer P, Salovska B, Fucikova AM, Chmel M, et al. Quantitative proteome profiling of Coxiella burnetii reveals major metabolic and stress differences under axenic and cell culture cultivation. Front Microbiol. 2019;10:2022. https://doi.org/10.3389/fmicb.2019.02022.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Klee SR, Tyczka J, Ellerbrok H, Franz T, Linke S, Baljer G, Appel B. Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii. BMC Microbiol. 2006;6:2; doi: 1471-2180-6-2 [pii] https://doi.org/10.1186/1471-2180-6-2.

  50. Ransom SE, Huebner RJ. Studies on the resistance of Coxiella burnetii to physical and chemical agents. Am J Hygiene. 1951;53(1):110–9.

    CAS  Google Scholar 

  51. Babudieri B, Moscovici C. Research on the behavior of Coxiella burnetii in relation to various physical and chemical agents. Rend - Istituto Superiore di Sanita. 1950;13(9–10):739–48.

    CAS  Google Scholar 

  52. Kirberger E. Die Resistenz Der Coxiella burneti in vitro. Z Tropenmed Parasitol. 1951;3(1):77–86.

    CAS  PubMed  Google Scholar 

  53. Little JS, Kishimoto RA, Canonico PG. In vitro studies of interaction of rickettsia and macrophages: effect of ultraviolet light on Coxiella burnetii inactivation and macrophage enzymes. Infect Immun. 1980;27(3):837–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Dai T, Vrahas MS, Murray CK, Hamblin MR. Ultraviolet C irradiation: an alternative antimicrobial approach to localized infections? Expert review of anti-infective therapy. 2012;10(2):185–95; https://doi.org/10.1586/eri.11.166.

  55. Rudhart SA, Gunther F, Dapper L, Stuck BA, Hoch S. UV-C light-based surface disinfection: analysis of its Virucidal Efficacy using a bacteriophage model. Int J Environ Res Public Health. 2022;19(6). https://doi.org/10.3390/ijerph19063246.

  56. Mertens K, Lantsheer L, Ennis DG, Samuel JE. Constitutive SOS expression and damage-inducible AddAB-mediated recombinational repair systems for Coxiella burnetii as potential adaptations for survival within macrophages. Mol Microbiol. 2008;69(6):1411–26. https://doi.org/10.1111/j.1365-2958.2008.06373.x. MMI6373 [pii].

    Article  CAS  PubMed  Google Scholar 

  57. Oguma K, Katayama H, Mitani H, Morita S, Hirata T, Ohgaki S. Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair. Appl Environ Microbiol. 2001;67(10):4630–7. https://doi.org/10.1128/AEM.67.10.4630-4637.2001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Quek PH, Hu J. Indicators for photoreactivation and dark repair studies following ultraviolet disinfection. J Ind Microbiol Biotechnol. 2008;35(6):533–41. https://doi.org/10.1007/s10295-008-0314-0.

    Article  CAS  PubMed  Google Scholar 

  59. Hackstadt T. Estimation of the cytoplasmic pH of Coxiella burnetii and effect of substrate oxidation on proton motive force. J Bacteriol. 1983;154(2):591–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Hackstadt T, Williams JC. Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii. Proc Natl Acad Sci U S A. 1981;78(5):3240–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Hackstadt T, Williams JC. pH dependence of the Coxiella burnetii glutamate transport system. J Bacteriol. 1983;154(2):598–603.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Omsland A, Cockrell DC, Fischer ER, Heinzen RA. Sustained axenic metabolic activity by the obligate intracellular bacterium Coxiella burnetii. J Bacteriol. 2008;190(9):3203–12. https://doi.org/10.1128/JB.01911-07. doi: JB.01911-07 [pii].

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Ghorbal SKB, Chatti A, Sethom MM, Maalej L, Mihoub M, Kefacha S, et al. Changes in membrane fatty acid composition of Pseudomonas aeruginosa in response to UV-C radiations. Curr Microbiol. 2013;67(1):112–7. https://doi.org/10.1007/s00284-013-0342-5.

    Article  CAS  PubMed  Google Scholar 

  64. Maalej L, Chatti A, Khefacha S, Salma K, Gottardi D, Vannini L, et al. UV-C pre-adaptation of Salmonella: effect on cell morphology and membrane fatty acids composition. World J Microbiol Biotechnol. 2014;30(3):925–30. https://doi.org/10.1007/s11274-013-1510-2.

    Article  CAS  PubMed  Google Scholar 

  65. Sana K, Abdelwaheb C, Lobna M, Davide G, Selma K, Lucia V, et al. Survival and fatty acid composition of UV-C treated Staphylococcus aureus. Ann Microbiol. 2015;65(1):235–40. https://doi.org/10.1007/s13213-014-0855-6.

    Article  CAS  Google Scholar 

  66. Santos AL, Moreirinha C, Lopes D, Esteves AC, Henriques I, Almeida A, et al. Effects of UV radiation on the lipids and proteins of bacteria studied by mid-infrared spectroscopy. Environ Sci Technol. 2013;47(12):6306–15. https://doi.org/10.1021/es400660g.

    Article  CAS  PubMed  Google Scholar 

  67. Chourabi K, Campoy S, Rodriguez JA, Kloula S, Landoulsi A, Chatti A. UV-C adaptation of Shigella: morphological, outer membrane proteins, secreted proteins, and Lipopolysaccharides effects. Curr Microbiol. 2017;74(11):1261–9. https://doi.org/10.1007/s00284-017-1311-1.

    Article  CAS  PubMed  Google Scholar 

  68. Sandoz KM, Popham DL, Beare PA, Sturdevant DE, Hansen B, Nair V, Heinzen RA. Transcriptional profiling of Coxiella burnetii reveals extensive cell Wall Remodeling in the small cell variant Developmental Form. PLoS ONE. 2016;11(2):e0149957. https://doi.org/10.1371/journal.pone.0149957.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Hoover TA, Culp DW, Vodkin MH, Williams JC, Thompson HA. Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii nine mile strain. Infect Immun. 2002;70(12):6726–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Millar JA, Beare PA, Moses AS, Martens CA, Heinzen RA, Raghavan R. Whole-genome sequence of Coxiella burnetii Nine Mile RSA439 (phase II, clone 4), a Laboratory Workhorse strain. Genome Announcements. 2017;5(23). https://doi.org/10.1128/genomeA.00471-17.

  71. Nikaido H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biology Reviews: MMBR. 2003;67(4):593–656. https://doi.org/10.1128/MMBR.67.4.593-656.2003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Wang Z, Wang J, Ren G, Li Y, Wang X. Influence of core oligosaccharide of Lipopolysaccharide to outer membrane behavior of Escherichia coli. Mar Drugs. 2015;13(6):3325–39. https://doi.org/10.3390/md13063325.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Smith HL, Howland MC, Szmodis AW, Li Q, Daemen LL, Parikh AN, Majewski J. Early stages of oxidative stress-induced membrane permeabilization: a neutron reflectometry study. J Am Chem Soc. 2009;131(10):3631–8. https://doi.org/10.1021/ja807680m.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Wang C, Lu S, Zhang Z. Inactivation of airborne bacteria using different UV sources: performance modeling, energy utilization, and endotoxin degradation. Sci Total Environ. 2019;655:787–95. https://doi.org/10.1016/j.scitotenv.2018.11.266.

    Article  CAS  PubMed  Google Scholar 

  75. Seo HS. Application of radiation technology in vaccines development. Clin Experimental Vaccine Res. 2015;4(2):145–58. https://doi.org/10.7774/cevr.2015.4.2.145.

    Article  Google Scholar 

  76. Ferguson M, Wood DJ, Minor PD. Antigenic structure of poliovirus in inactivated vaccines. J Gen Virol. 1993;74(Pt 4):685–90. https://doi.org/10.1099/0022-1317-74-4-685.

    Article  CAS  PubMed  Google Scholar 

  77. Goldstein MA, Tauraso NM. Effect of formalin, beta-propiolactone, merthiolate, and ultraviolet light upon influenza virus infectivity chicken cell agglutination, hemagglutination, and antigenicity. Appl Microbiol. 1970;19(2):290–4. https://doi.org/10.1128/am.19.2.290-294.1970.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Fertey J, Bayer L, Kahl S, Haji RM, Burger-Kentischer A, Thoma M, et al. Low-energy Electron irradiation efficiently inactivates the Gram-negative Pathogen Rodentibacter pneumotropicus-A New Method for the generation of bacterial vaccines with increased efficacy. Vaccines (Basel). 2020;8(1). https://doi.org/10.3390/vaccines8010113.

  79. Fertey J, Bayer L, Grunwald T, Pohl A, Beckmann J, Gotzmann G, et al. Pathogens inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and induce Protective Immune responses. Viruses. 2016;8(11). https://doi.org/10.3390/v8110319.

  80. Tsunetsugu-Yokota Y. Large-scale preparation of UV-inactivated SARS coronavirus virions for vaccine antigen. Methods Mol Biol. 2008;454:119–26. https://doi.org/10.1007/978-1-59745-181-9_11.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Nadin Engelhardt for her excellent technical support especially under BSL3 conditions, Mandy Rabe, Petra Sippach, Lisa Wolf and Hannes Axt for their excellent technical support.

Funding

This research was funded by the Federal Ministry of Education and Research, Zwanzig20 Partnerschaft für Innovation, InfectControl, grant number 03ZZ0825A.

Open Access funding enabled and organized by Projekt DEAL.

Author information

Author notes

  1. Katja Mertens-Scholz and Amira A. Moawad contributed equally to this work.

Authors and Affiliations

  1. Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Jena, Germany

    Katja Mertens-Scholz, Amira A. Moawad, Jennifer Andrack & Heinrich Neubauer

  2. Institute for Infectious Diseases and Infection Control and Center for Sepsis Care and Control (CSCC), Jena University Hospital, Jena, Germany

    Katja Mertens-Scholz & Mathias W. Pletz

  3. Friedrich-Loeffler-Institut, Institute of Molecular Pathogenesis, Jena, Germany

    Elisabeth M. Liebler-Tenorio

  4. Department of In Vitro Diagnostics Development, Research Centre of Medical Technology and Biotechnology, Erfurt, Germany

    Andrea Helming

  5. Research Centre of Medical Technology and Biotechnology, Bad Langensalza, Germany

    Peter Miethe & Ina-Gabriele Richter

Authors
  1. Katja Mertens-Scholz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amira A. Moawad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elisabeth M. Liebler-Tenorio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrea Helming
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jennifer Andrack
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter Miethe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Heinrich Neubauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mathias W. Pletz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ina-Gabriele Richter
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization, IGR and KMS; methodology, ELT, IGR and KMS; software, not applicable.; validation, KMS; formal analysis, KMS; investigation, AAM, ELT, AH, IGR, KMS; resources, PM, IGR and KMS; data curation, ELT, IGR and KMS; writing—original draft preparation, AAM and KMS.; writing—review and editing, AAM, ELT, JA, HN, MWP, IGR and KMS; visualization, ELT and KMS; supervision, IGR and KMS.; project administration, IGR and KMS; funding acquisition, IGR, PM and KMS.

Corresponding author

Correspondence to Katja Mertens-Scholz.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mertens-Scholz, K., Moawad, A.A., Liebler-Tenorio, E.M. et al. Ultraviolet C inactivation of Coxiella burnetii for production of a structurally preserved whole cell vaccine antigen. BMC Microbiol 24, 118 (2024). https://doi.org/10.1186/s12866-024-03246-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12866-024-03246-z

Keywords

BMC Microbiology

ISSN: 1471-2180

Contact us