Interdisciplinary studies on Coxiella burnetii: From molecular to cellular, to host, to one health research.

The Q-GAPS (Q fever GermAn interdisciplinary Program for reSearch) consortium was launched in 2017 as a German consortium of more than 20 scientists with exceptional expertise, competence, and substantial knowledge in the field of the Q fever pathogen Coxiella (C.) burnetii. C. burnetii exemplifies as a zoonotic pathogen the challenges of zoonotic disease control and prophylaxis in human, animal, and environmental settings in a One Health approach. An interdisciplinary approach to studying the pathogen is essential to address unresolved questions about the epidemiology, immunology, pathogenesis, surveillance, and control of C. burnetii. In more than five years, Q-GAPS has provided new insights into pathogenicity and interaction with host defense mechanisms. The consortium has also investigated vaccine efficacy and application in animal reservoirs and identified expanded phenotypic and genotypic characteristics of C. burnetii and their epidemiological significance. In addition, conceptual principles for controlling, surveilling, and preventing zoonotic Q fever infections were developed and prepared for specific target groups. All findings have been continuously integrated into a Web-based, interactive, freely accessible knowledge and information platform (www.q-gaps.de), which also contains Q fever guidelines to support public health institutions in controlling and preventing Q fever. In this review, we will summarize our results and show an example of how an interdisciplinary consortium provides knowledge and better tools to control a zoonotic pathogen at the national level.

Before the Flood: Impact of Coordination of Care and Direct Admissions on Emergency Department Volumes.

BACKGROUND: Transfers of pediatric patients occur to access specialty and subspecialty care, but incur risk, and consume resources. Direct admissions to medical and surgical wards may improve patient experience and mitigate resource utilization. OBJECTIVE: We sought to identify common elements for direct admissions, as well as the pattern of disposition for patients referred to our emergency department (ED). DESIGN: A retrospective qualitative analysis of patients transferred to our pediatric hospital for 12 months was performed. Different physician groups were evaluated for use of direct admissions or evaluation in the ED. Patients referred to the ED were additionally tracked to evaluate their eventual disposition. RESULTS: A total of 3982 transfers occurred during the 12-month analysis period. Of those, 3463 resulted in admission, accounting for 32.55% of all admissions. Transfers accepted by nonsurgical services accounted for 82% of the transfers, whereas 18% were facilitated by one of the surgical services. Direct admissions accounted for 1707 (44.8%) of all referrals and were used more often by nonsurgical services. Of patients referred to the ED (2101 or 55.2% of all referrals), most patients were admitted and 343 (16% of those referred to the ED) were discharged home. CONCLUSIONS: The direct admission process helped avoid ED assessments for some patients; however, some patients referred to the ED were able to be evaluated, treated, and discharged. Consistent triage of the patients being transferred as direct admissions may improve ED throughput and potentially improve the patient's experience, reduce redundant services, and expedite care.

Multifaceted quality improvement initiative to decrease pediatric asthma readmissions.

BACKGROUND: Asthma is the most common chronic disease of childhood and a leading cause of hospitalization in children. A primary goal of asthma control is prevention of hospitalizations. A hospital admission is the single strongest predictor of future hospital admissions for asthma. The 30-day asthma readmission rate at our institution was significantly higher than that of other hospitals in the Children's Hospital Association. As a result, a multifaceted quality improvement project was undertaken with the goal of reducing the 30-day inpatient asthma readmission rate by 50% within two years. METHODS: Analysis of our institution's readmission patterns, value stream mapping of asthma admission, discharge, and follow-up processes, literature review, and examination of comparable successful programs around the United States were all utilized to identify potential targets for intervention. Interventions were implemented in a stepwise manner, and included increasing inhaler availability after discharge, modifying asthma education strategies, and providing in-home post-discharge follow-up. The primary outcome was a running 12-month average 30-day inpatient readmission rate. Secondary outcomes included process measures for individual interventions. RESULTS: From a peak of 7.98% in January 2013, a steady decline to 1.65% was observed by July 2014, which represented a 79.3% reduction in 30-day readmissions. CONCLUSION: A significant decrease in hospital readmissions for pediatric asthma is possible, through comprehensive, multidisciplinary quality improvement that spans the continuum of care.

Tick-borne encephalitis in a naturally infected sheep.

BACKGROUND: Tick-borne encephalitis (TBE) is the most important viral tick borne zoonosis in Europe. In Germany, about 250 human cases are registered annually, with the highest incidence reported in the last years coming from the federal states Bavaria and Baden-Wuerttemberg. In veterinary medicine, only sporadic cases in wild and domestic animals have been reported; however, a high number of wild and domestic animals have tested positive for the tick-borne encephalitis virus (TBEV) antibody. CASE PRESENTATION: In May 2015, a five-month-old lamb from a farm with 15 Merino Land sheep and offspring in Nersingen/Bavaria, a TBEV risk area, showed impaired general health with pyrexia and acute neurological signs. The sheep suffered from ataxia, torticollis, tremor, nystagmus, salivation and finally somnolence with inappetence and recumbency. After euthanasia, pathological, histopathological, immunohistochemical, bacteriological, parasitological and virological analyses were performed. Additionally, blood samples from the remaining, healthy sheep in the herd were taken for detection of TBEV antibody titres. At necropsy and accompanying parasitology, the sheep showed a moderate to severe infection with Trichostrongylids, Moniezia and Eimeria species. Histopathology revealed mild to moderate necrotising, lymphohistiocytic and granulocytic meningoencephalitis with gliosis and neuronophagia. Immunohistochemistry for TBEV was negative. RNA of a TBEV strain, closely related to the Kumlinge A52 strain, was detected in the brain by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and subsequent PCR product sequencing. A phylogenetic analysis revealed a close relationship to the TBEV of central Europe. TBEV was cultured from brain tissue. Serologically, one of blood samples from the other sheep in the herd was positive for TBEV in an enzyme-linked immunosorbent assay (ELISA) and in a serum neutralisation test (SNT), and one was borderline in an ELISA. CONCLUSION: To the authors' knowledge this is the first report of a natural TBEV infection in a sheep in Europe with clinical manifestation, which describes the clinical presentation and the histopathology of TBEV infection.

The LOUVRE Laboratory: State-of-the-Art Ultrafast Ultraviolet Spectroscopies for Molecular and Materials Science.

We describe the facilities for ultraviolet studies in the femtosecond to nanosecond time domain. These facilities consist of: i) a set-up for deep-ultraviolet spectroscopy in the 260-380 nm range in both pump and probe pulses for transient absorption/reflectivity or two-dimensional spectroscopy studies; ii) a set-up for ultrafast fluorescence measurements with detection down to 300 nm. The capabilities of these set-ups are demonstrated by examples on molecular systems, biosystems, nanoparticles and solid materials.

Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions.

Exposure of small ruminants to the Schmallenberg arbovirus in Germany from 2017 to 2018 - animal-specific and flock-management-related risk factors.

The Schmallenberg virus (SBV), an emerging Orthobunyavirus of mainly ruminant hosts, caused a substantial epidemic in European ruminant populations between 2011 and 2013. The pathogen is transmitted by arthropod vectors (Culicoides spp.) and can cause reproductive disorders and severe malformations of the offspring or stillbirth. The present study aimed to assess SBV seroprevalence among German sheep and goats a few years after the first virus detection in the country (November 2011). In addition, an extensive risk factor analysis including host-specific and husbandry-related factors was implemented. Seroprevalence was determined by examining serum samples from 2759 sheep and 446 goats out of a total of 70 flocks across five German federal states. The samples were withdrawn in the period between 2017 and 2018. Using a commercial competitive ELISA, antibodies against SBV were detected in all 70 investigated flocks. A percentage of 60.1 % (1657/2759) of the sheep and 40.4 % (180/446) of the goat sera contained SBV antibodies. Generalized linear mixed modeling revealed significant effects of host species (sheep > goats), age (old > young) and sex (female > male) on SBV seroprevalence. For both species, also the farming purpose, and for goats, ectoparasite treatment and the presence of cattle on the farm played a role in terms of risk for SBV exposure. The observations from this study still emphasize a wide distribution of the pathogen in Germany. Nevertheless, the observed seroprevalence might not be sufficient to achieve effective herd immunity. Pinpointing risk factors identified susceptible populations for targeted vaccination programs to reduce potential animal losses caused by SBV.

Detection of Coxiella burnetii in the mammary gland of a dairy goat.

The zoonotic bacterium Coxiella (C.) burnetii can be excreted by infected goats through birth products and milk. The detection of C. burnetii DNA in the mammary gland tissue of infected dairy goats and intermittent milk shedders has been reported, but confirmation of C. burnetii bacteria in the udder remained pending. The pathogen caused abortions in a 152-head dairy goat herd, resulting in the vaccination against C. burnetii of the entire herd with annual boosters. To monitor the C. burnetii shedding at herd level, monthly bulk tank milk (BTM) samples were analyzed using PCR (IS1111). Despite vaccination, C. burnetii DNA was detected in BTM samples within the first 16 months of the study. Therefore, individual milk samples were tested on four different occasions several months apart to identify potential intermittent milk shedders. Only one goat (#67455) tested positive three times. This goat was necropsied to investigate the presence of C. burnetii in the udder and other organs. PCR detected C. burnetii DNA solely in both mammary glands and the left teat cistern. Immunohistological examination identified C. burnetii antigen in mammary gland tissue, confirmed by the detection of C. burnetii bacteria in the mammary epithelial cells using fluorescence in situ hybridization. The removal of goat #67455 led to negative BTM samples until the end of the study. The findings demonstrate the occurrence of C. burnetii in the mammary gland of a naturally infected and vaccinated goat. The presence possibly contributed to intermittent milk shedding of goat #67455, and the mammary gland tissue may serve as a replicative niche for C. burnetii.

The immune response to a Coxiella burnetii vaccine in sheep varies according to their natural pre-exposure.

Q fever in humans is caused by Coxiella (C.) burnetii. In 2008 and 2012, cases of Q fever in humans were linked to an infected flock of approximately 650 ewes. Since 2013 gimmers (G'13, G'14, G'15 etc.) were primary vaccinated (two doses) with an inactivated C.burnetii vaccine without any revaccination. In 2013, 30 ewes were primary vaccinated (A'13). Shedding was annually monitored by qPCR-testing of vaginal and nasal swabs collected at lambing. Animals were tested for Phase I- (PhI) and PhII-antibodies (Ab) and for PhII-specific-interferon-γ (IFN-γ) before and after vaccination. The effect of a revaccination was determined in 2018 and 2023. Groups of randomly selected gimmers primary vaccinated in 2015, 2016 and 2017 and a mixed group of older animals (A'13, G'13 and G'14) were revaccinated once in 2018. The trial was repeated in 2023 on groups primary vaccinated in 2019-2023. Major shedding after the outbreak in 2012 ceased in 2014. Thereafter C.burnetii was only sporadically detected at low-level in 2018, 2021 and 2023. Sheep naturally exposed to C.burnetii during the outbreak in 2012 (A'13, G'13) mounted a strong and complete (PhI, PhII, IFN-γ) recall immune response after vaccination. A serological PhI+/PhII+ pattern dominated after vaccination. In contrast, since 2014 a weaker immune response (PhII-titre, IFN-γ) and a dominance of the PhI-/PhII+ pattern was observed in vaccinated gimmers. The number of serologically non-responding gimmers to vaccination increased to 25.0 % in G'16/G'17 and 40.4 % in G'19/G'20. But revaccination even three (G'15 in 2018) and four (G'19 in 2023) years after primary vaccination resulted in a strong and complete immune response. No difference of the immune response nor to more recently primary vaccinated animals (G'23 in 2023) nor to those animals that were present during the outbreak (A'13/G'13/G'14 in 2018) was observed.

Humoral and cellular immune responses in sheep following administration of different doses of an inactivated phase I vaccine against Coxiella burnetii.

An inactivated Coxiella burnetii Phase I (PhI) vaccine (Coxevac®) is licensed in several European countries for goats and cattle to prevent coxiellosis. The vaccine is also applied to sheep, although detailed information about the ovine immune response and vaccine dose is missing. Eighteen gimmers from a C. burnetii unsuspected flock were randomly divided into three groups of six. Group 1 (Cox1) and 2 (Cox2) were vaccinated twice with 1 ml and 2 ml Coxevac®, respectively, three weeks apart (primary vaccination). The same procedure was applied with Cox3 (2 ml sodium chloride, control group). A third injection (booster) was performed after nine months. Potential side effects were determined by measuring the rectal body temperature and skin thickness at the injection site. Blood samples were collected to detect phase-specific IgM and IgG antibodies and interferon-É£ (IFN-É£) release by immunofluorescence assay and ELISAs, respectively. Moreover, a cell infection neutralization assay determined the appearance of neutralizing sera. Body temperatures increased for one day post vaccination, and the skin swelled only slightly. Regardless of the vaccine volume, immunized sheep reacted first with an IgM and IgG PhII response. Ten weeks after the primary vaccination, IgG PhI antibodies predominated. Boosting eight months after primary vaccination resulted in a robust IgG PhI increase and strong IFN-É£ response. In the vaccinated animals, the neutralizing effect is more widespread after the administration of 1 ml than after the treatment with 2 ml. In summary, differences between 1 and 2 ml Coxevac® are minor, and a vaccine volume of 1 ml seems to be sufficient. A booster after the primary vaccination is apparently necessary to stimulate the cell-mediated immune response in naïve sheep.

Co-exposure to Anaplasma spp., Coxiella burnetii and tick-borne encephalitis virus in sheep in southern Germany.

The intracellular bacteria Anaplasma spp. and Coxiella burnetii and the tick-borne encephalitis virus (TBEV) are tick-transmitted pathogens circulating in the southern German sheep population. Knowledge of interaction among Anaplasma spp., C. burnetii and TBEV in sheep is lacking, but together they might promote and reinforce disease progression. The current study aimed to identify co-exposure of sheep to Anaplasma spp., C. burnetii and TBEV. For this purpose, 1,406 serum samples from 36 sheep flocks located in both southern German federal states, Baden-Wuerttemberg and Bavaria, were analysed by ELISAs to determine the antibody levels of the three pathogens. Inconclusive and positive results from the TBEV ELISA were additionally confirmed by a serum neutralisation assay. The proportion of sheep with antibodies against Anaplasma spp. (47.2%), C. burnetii (3.7%) and TBEV (4.7%) differed significantly. Significantly more flocks with Anaplasma spp. seropositive sheep (91.7%) were detected than flocks with antibodies against TBEV (58.3%) and C. burnetii (41.7%), but there was no significant difference between the number of flocks which contained TBEV and C. burnetii seropositive sheep. Seropositivity against at least two pathogens was detected in 4.7% of sheep from 20 flocks. Most co-exposed sheep had antibodies against Anaplasma spp./TBEV (n = 36), followed by Anaplasma spp./C. burnetii (n = 27) and Anaplasma spp./C. burnetii/TBEV (n = 2). Only one sheep showed an immune response against C. burnetii and TBEV. Flocks with sheep being positive against more than one pathogen were widely distributed throughout southern Germany. The descriptive analysis revealed no association between the antibody response of the three pathogens at animal level. Taking the flocks as a cluster variable into account, the exposure to TBEV reduced the probability of identifying C. burnetii antibodies in sheep significantly (odds ratio 0.46; 95% confidence interval 0.24-0.85), but the reason for this is unknown. The presence of Anaplasma spp. antibodies did not influence the detection of antibodies against C. burnetii and TBEV. Studies under controlled conditions are necessary to evaluate any possible adverse impact of co-exposure to tick-borne pathogens on sheep health. This can help to clarify rare disease patterns. Research in this field may also support the One Health approach due to the zoonotic potential of Anaplasma spp., C. burnetii and TBEV.

Two Years after Coxiella burnetii Detection: Pathogen Shedding and Phase-Specific Antibody Response in Three Dairy Goat Herds.

The infection dynamics of Coxiella (C.) burnetii were investigated in three dairy goat herds (A, B, and C) 2 years after the first pathogen detection. A total of 28 and 29 goats from herds A and B, and 35 goats from herd C, were examined. Sera were analyzed on three sampling dates using phase-specific serology. Pathogen shedding was assessed using post-partum vaginal swabs and monthly bulk tank milk (BTM) samples. Dust samples from a barn and milking parlor were also collected monthly. These samples were analyzed with PCR (target IS1111). In herd A, individual animals tested seropositive, while vaginal swabs, BTM, and most dust samples tested negative. Herds B and C exhibited high IgG phase I activity, indicating a past infection. In herd B, approximately two-thirds of the goats shed C. burnetii with vaginal mucus, and irregular positive results were obtained from BTM. Herd C had two positive goats based on vaginal swabs, and BTM tested positive once. Dust samples from herds B and C contained C. burnetii DNA, with higher quantities typically found in samples from the milking parlor. This study highlights the different infection dynamics in three unvaccinated dairy goat herds and the potential use of dust samples as a supportive tool to detect C. burnetii at the herd level.

Host-pathogen associations revealed by genotyping of European strains of Anaplasma phagocytophilum to describe natural endemic cycles.

BACKGROUND: The zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied. METHODS: Sequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets. RESULTS: The highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals. CONCLUSIONS: In this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host-pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe.

Detection and Characterization of Alongshan Virus in Ticks and Tick Saliva from Lower Saxony, Germany with Serological Evidence for Viral Transmission to Game and Domestic Animals.

The newly discovered group of Jingmenviruses has been shown to infect a wide range of hosts and has been associated with febrile illness in humans. During a survey for Jingmenviruses in ticks from Lower Saxony, Germany, Alongshan virus (ALSV) was identified in Ixodes spp. ticks. Additional virus screenings revealed the presence of ALSV in the bodies and saliva of ticks collected at several locations in Lower Saxony. Vector competence studies that included Ixodes ricinus and Dermacentor reticulatus validated the replication of ALSV within those tick species. In vitro feeding experiments with ALSV-injected Ixodes ricinus demonstrated effective viral transmission during blood feeding. To evaluate the potential viral transmission during a natural blood meal, sera from wild game and domestic animals were investigated. One serum sample from a red deer was found to be positive for ALSV RNA, while serological screenings in game and domestic animals revealed the presence of ALSV-specific antibodies at different locations in Lower Saxony. Overall, those results demonstrate the broad distribution of ALSV in ticks in Lower Saxony and hypothesize frequent exposure to animals based on serological investigations. Hence, its potential risk to human and animal health requires further investigation.

Exciton decay mechanism in DNA single strands: back-electron transfer and ultrafast base motions.

The photochemistry of DNA systems is characterized by the ultraviolet (UV) absorption of π-stacked nucleobases, resulting in exciton states delocalized over several bases. As their relaxation sensitively depends on local stacking conformations, disentangling the ensuing electronic and structural dynamics has remained an experimental challenge, despite their fundamental role in protecting the genome from potentially harmful UV radiation. Here we use transient absorption and transient absorption anisotropy spectroscopy with broadband femtosecond deep-UV pulses (250-360 nm) to resolve the exciton dynamics of UV-excited adenosine single strands under physiological conditions. Due to the exceptional deep-UV bandwidth and polarization sensitivity of our experimental approach, we simultaneously resolve the population dynamics, charge-transfer (CT) character and conformational changes encoded in the UV transition dipoles of the π-stacked nucleotides. Whilst UV excitation forms fully charge-separated CT excitons in less than 0.3 ps, we find that most decay back to the ground state via a back-electron transfer. Based on the anisotropy measurements, we propose that this mechanism is accompanied by a structural relaxation of the photoexcited base-stack, involving an inter-base rotation of the nucleotides. Our results finally complete the exciton relaxation mechanism for adenosine single strands and offer a direct view into the coupling of electronic and structural dynamics in aggregated photochemical systems.

Surveillance of Coxiella burnetii Shedding in Three Naturally Infected Dairy Goat Herds after Vaccination, Focusing on Bulk Tank Milk and Dust Swabs.

Q fever outbreaks on three dairy goat farms (A-C) were monitored after the animals had been vaccinated with an inactivated Coxiella burnetii phase I vaccine. The antibody response was measured before vaccination by serum samples with two C. burnetii phase-specific ELISAs to characterize the disease status. Shedding was determined by vaginal swabs during three kidding seasons and monthly bulk tank milk (BTM) samples. Dust swabs from one windowsill of each barn and from the milking parlors were collected monthly to evaluate the indoor exposure. These samples were analyzed by qPCR. The phase-specific serology revealed an acute Q fever infection in herd A, whereas herds B and C had an ongoing and past infection, respectively. In all three herds, vaginal shedders were present during three kidding seasons. In total, 50%, 69%, and 15% of all collected BTM samples were C. burnetii positive in herds A, B, and C, respectively. Barn dust contained C. burnetii DNA in 71%, 45%, and 50% of examined swabs collected from farms A, B, and C, respectively. The largest number of C. burnetii positive samples was obtained from the milking parlor (A: 91%, B: 72%, C: 73%), indicating a high risk for humans to acquire Q fever during milking activity.

Impact of Coxiella burnetii vaccination on humoral immune response, vaginal shedding, and lamb mortality in naturally pre-infected sheep.

Introduction: Sheep are considered to be one of the main reservoirs for Coxiella burnetii, a gram-negative bacterium with high zoonotic potential. Infected sheep shed tremendous amounts of the pathogen through birth products which caused human Q fever epidemics in several countries. Information about the impact of an inactivated C. burnetii Phase I vaccine on humoral immune response, vaginal shedding, and lamb mortality in naturally pre-infected sheep is scarce. Methods: Two identically managed and naturally C. burnetii-infected sheep flocks were examined for two lambing seasons (2019 and 2020). One flock (VAC) received a primary vaccination against Q fever before mating and the second flock served as control (CTR). In each flock, one cohort of 100 ewes was included in follow-up investigations. Serum samples at eight different sampling dates were analyzed by C. burnetii phase-specific ELISAs to differentiate between the IgG Phase I and II responses. Vaginal swabs were collected within three days after parturition and examined by a C. burnetii real-time PCR (IS1111). Lamb losses were recorded to calculate lamb mortality parameters. Results: After primary vaccination, almost all animals from cohort VAC showed a high IgG Phase I response up until the end of the study period. In cohort CTR, the seropositivity rate varied from 35.1% to 66.3%, and the Phase I and Phase II pattern showed an undulating trend with higher IgG Phase II activity during both lambing seasons. The number of vaginal shedders was significantly reduced in cohort VAC compared to cohort CTR during the lambing season in 2019 (p < 0.0167). There was no significant difference of vaginal shedders in 2020. The total lamb losses were low in both cohorts during the two investigated lambing seasons (VAC 2019: 6.8%, 2020: 3.2%; CTR 2019: 1.4%, 2020: 2.7%). Discussion: Neither the C. burnetii vaccine nor the C. burnetii infection seem to have an impact on lamb mortality. Taken together, the inactivated C. burnetii Phase I vaccine induced a strong IgG Phase I antibody response in naturally pre-infected sheep. It might also reduce vaginal shedding in the short term but seems to have little beneficial impact on lamb mortality.

Long-term control of Coxiellosis in sheep by annual primary vaccination of gimmers.

Coxiella (C.) burnetii, a Gram-negative intracellular bacterium, causes Q fever in humans and Coxiellosis in animals. Ruminants are a primary source of human infection with C.burnetii. In 2013, vaccination was implemented in a sheep flock with 650 ewes associated with two outbreaks of Q fever in humans in 2008 and 2012. Only gimmers (yearlings) received two doses of a commercial C.burnetii phase I whole cell vaccine three weeks apart (primary vaccination) without any revaccination. Vaginal and nasal swabs collected shortly after lambing were tested by qPCR. Additionally, a group of non-vaccinated sentinels was serologically monitored for phase I (PhI), II (PhII) antibodies and for Interferon γ (IFN-γ) after stimulation of whole blood cells with PhII-antigen with and without an IL-10-neutralizing monoclonal antibody. In 2021, 679 sera collected in 2014-2021 were retested retrospectively with three commercial ELISA kits and one batch of an in-house PhI/PhII-ELISA. A low-level shedding of C.burnetii (<103 mean C.burnetii/swab) was observed until 2014. In 2021 C.burnetii was detected in two animals (<103.1C.burnetii/swab), but vaginal swabs collected at two subsequent lambing seasons remained negative. Seroconversion of sentinels was detected until 2017. However, the retrospective analysis of sentinels in 2021 revealed additional single seropositive animals from 2018 to 2021. IFN-γ reactivity was observed during the whole study period; it peaked in 2014 and in 2018 and decreased thereafter. The sporadic detection of C.burnetii and the immune responses of sentinels suggested that a subliminal infection persisted despite vaccination. Nevertheless, vaccination of gimmers prevented the development of a major outbreak, it controlled the infection and reduced the risk of human infection.

Seroprevalence and Risk Factors of Anaplasma spp. in German Small Ruminant Flocks.

Knowledge about the distribution of Anaplasma spp. in small ruminants from Germany is limited. Therefore, serum samples were examined from 71 small ruminant flocks (2731 sheep, 447 goats) located in the five German federal states: Schleswig-Holstein (SH), Lower Saxony (LS), North Rhine-Westphalia (NRW), Baden-Wuerttemberg (BW) and Bavaria (BAV). Antibodies to Anaplasma spp. were determined by a cELISA based on the MSP5 antigen. A risk factor analysis at animal and flock level was also performed. Antibodies to Anaplasma spp. were detected in 70/71 flocks without significant difference in the intra-flock prevalence (IFP) between the federal states. The mean antibody levels from sheep were significantly lower in northern Germany (LS, SH) compared to west (NRW) and south Germany (BW, BAV). Sheep had a 2.5-fold higher risk of being seropositive than goats. Females and older animals (>2 years) were more likely to have antibodies to Anaplasma spp. in one third and one quarter of cases, respectively. Flocks used for landscape conservation had a five times higher risk of acquiring an IFP greater than 20%. Cats and dogs on the farms increased the probability for small ruminant flocks to have an IFP of above 20% 10-fold and 166-fold, respectively. Further studies are necessary to assess the impact of Anaplasma species on the health of small ruminants in Germany.