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Coxiella burnetii: A review article with a focus on the infection process in German sheep and goat herds

Berlin and Munich veterinary weekly

Coxiella burnetii: A review focusing on infections in German sheep and goat flocks

Berlin and Munich Veterinary Weekly 133

DOI: 10.2376/0005-9366-19030

© Schlütersche Verlagsgesellschaft mbH & Co. KG. 2020

Published: 04/2020

Summary

Q fever is a worldwide zoonosis caused by Coxiella (C.) burnetii caused. This bacterium is excreted in large quantities, especially in the birth material of infected small ruminants, but also in milk, feces and urine. The main route of infection for humans and animals is the inhalation of aerosols containing pathogens. Wind and other meteorological factors play a role in the spread. Even ticks like Dermacentor spp. and Ixodes ricinus could be of importance in the transfer of C. burnetii to have. Infection can occur in goats C. burnetii especially lead to late abortions. In contrast, a Coxielle infection in sheep seems to lead to far lower abortion rates than in goats. In humans, 40% of people infected have flu-like symptoms such as fever and headache. Up to 5% of all infected people can develop chronification, which is usually associated with endocarditis. In Germany, several small-scale human Q fever epidemics with up to 331 infected people per outbreak have been detected since 2000. The trigger was mostly lambing sheep. Due to the increase in dairy goat holdings in Germany, more cases of this species are expected in the future. Diagnostic evidence of infections with C. burnetii in animals it is usually done with the ELISA and / or the PCR. Isolates can be genotyped using the Multiple Loci Variable Number of Tandem Repeat Analysis (MLVA / VNTR). The genotypic MLVA cluster A was found particularly in small ruminants, while cluster C is more associated with cattle. The antibiotic treatment of infected animals with oxytetracyclines has not proven successful. Rather, the use of a vaccine with inactivated phase I bacteria reduces the risk of excretion. Due to the high tenacity of C. burnetii many disinfectants are not effective.

Summary

Q fever is a worldwide zoonosis caused by Coxiella (C.) burnetii. This bacterium is shed in huge amounts with birth products from infected small ruminants. Shedding also occurs via milk, faeces and urine. The main route of infection for humans and animals is via inhalation of contaminated aerosols. Apart from wind, many other meteorological conditions influence pathogen dissemination. So, Dermacentor spp. other Ixodes ricinus ticks could play a role in transmission. Infections in goats can lead to abortion during the last trimester of pregnancy. In comparison to goats, C. burnetii seems to cause less abortion in sheep. In humans, 40% of infected persons show flu-like symptoms such as fever and headaches. Up to 5% of all infected people can develop chronic Q fever, which often manifests as endocarditis. Since 2000, several human Q fever epidemics occurred in Germany with up to 331 reported human infections. The source of infection was mostly associated with parturition of sheep. As the number of goats has increased in Germany over the last couple of years, Q fever cases connected to this species will probably increase in the future. For the detection of C. burnetii in animals, ELISA and / or PCR are routinely used. With Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA / VNTR) it is possible to genotype isolates. MLVA-Cluster A was detected in samples from small ruminants, Cluster C is associated with cattle. Treatment with oxytetracycline is ineffective for the control of Q fever in animals, whereas the use of an inactivated Phase I whole cell vaccine reduces shedding and therefore the risk of transmission. Many disinfectants are ineffective against C. burnetii because of its high tenacity.

introduction

With the exception of New Zealand and Antarctica, Q fever is a zoonosis that is widespread worldwide. The causative agent of Q fever is Coxiella (C.) burnetii, an obligate intracellular, immobile, gram-negative bacterium. In Australia, the pathogen first triggered an outbreak among slaughterhouse workers in 1935. Since the cause was unclear, this flu-like illness was referred to as an unclear / questionable fever or "Query (Q) fever" (Hechemy 2012). An infection of animals with C. burnetii is called coxiellosis in German-speaking countries, but in international-speaking countries the name "Q fever" is also used for animals.
In the 1990s in particular, there were repeated major Q fever epidemics in Bulgaria (Georgiev et al. 2013, Serbezov et al. 1999). In 1993 alone, over 1000 human cases were confirmed using serology (Martinov 2007). The epidemics can be traced back to goat births, the number of which has more than doubled over the same period (Serbezov et al. 1999). The world's largest human Q fever outbreak to date was registered in the Netherlands. From 2007 to 2011, more than 4,000 people there contracted Q fever (Kampschreur et al. 2013). In 2009 alone there were 2357 patients, 459 of whom had to be treated as inpatients. Previous illnesses led to death in six patients after a Coxielle infection (van der Hoek et al. 2010). This epidemic was triggered by farms with large herds of dairy goats (1000 animals on average) in the south of the Netherlands. During lambing in spring, there was a strong release of pathogens in infected farms, initially associated with abortion rates of up to 60% per farm (van den Brom and Vellema 2009). In contrast, the accumulation of human infections in Germany was mostly attributed to lambing sheep. In addition, human diseases with Q fever occur more frequently along the drive pathways (Hellenbrand et al. 2001). The German goat population has played a rather subordinate role in transmission over the past few decades. Although the prevalence in cattle herds is high in some German regions (Böttcher et al. 2011b, Hilbert et al. 2014), cattle excreted cattle are of less importance as a cause of small-scale epidemics. Cattle are more likely to be responsible for individual human diseases and these may go undiagnosed. Infections in larger groups of people, on the other hand, are usually caused by small ruminants. The increased occurrence of cases of illness in humans also favors the detection of Q fever disease. Furthermore, physiological differences between large and small ruminants could be of importance, such as the different stool consistency and excretion dynamics of C. burnetii. Dairy cows excrete the pathogen over a longer period of time, especially with their milk and less with vaginal secretions and faeces (Guatteo et al. 2007, Rodolakis et al. 2007). In small ruminants, the pathogen is mainly excreted in the feces and vaginal mucus, in goats also with the milk (Arricau-Bouvery et al. 2003, Berri et al. 2001, Rodolakis et al. 2007). Thus, the environmental contamination from infected small ruminants appears to be greater than that of cattle (Arricau-Bouvery et al. 2003). In addition, the different housing conditions could play a role, since in Germany dairy cows are mostly kept indoors and sheep are mostly kept outdoors (Ganter et al. 2011).

The germ

Originally was C. burnetii taxonomically incorporated into the Rickettsiaceae family. However, analyzes of the 16S rRNA gene and later the complete genome showed that C. burnetii phylogenetically closely related to Legionella pneumophila, the causative agent of Legionnaires' disease (Seshadri et al. 2003, Stein et al. 1993, Weisburg et al. 1989), which is why the bacterium has been incorporated into a separate family Coxiellaceae within the Legionellales class. So far was C. burnetii the only species in the Coxiellaceae family. In recent years, however, other, new or Coxiella-like bacteria have been identified, such as C.cheraxi sp. nov. in the Australian crayfish (Cherax quadricarinatus), Candidatus C. massiliensis and CandidatusC. mudrowiae in ticks and Candidatus C. avium in parrots and a toucan (Angelakis et al. 2016, Gottlieb et al. 2015, Shivaprasad et al. 2008, Tan and Owens 2000). C. burnetii itself has been detected in a large number of different species of arthropods, birds and mammals (Woldehiwet 2004).
C. burnetii exists in two different forms: the so-called small-cell-variant (SCV) and large-cell-variant (LCV) (McCaul and Williams 1981). The SCV are the metabolically dormant and non-replicative form. They have a high tenacity with resistance to dehydration, heat, UV light and numerous disinfectants (Eldin et al. 2017). The bacterium can be detected in raw milk for up to 32 months and can survive in dust and wool for up to two years at 4ºC (Angelakis and Raoult 2010, Schliesser 1991). In contrast, in a more recent study by Álvarez-Alonso et al. (2018) reproductive coxia from stable dust samples can only be determined within two months after the last lambing by means of inoculation in mice with subsequent cultivation in Vero cells. This discrepancy will require further clarification in the future. The LCV are the metabolically active form and are detected in the phagolysosome of the host cell (Coleman et al. 2004, Minnick and Raghavan 2012).
Another specialty of C. burnetii is the phase-specific antigen variation. As with enterobacteria, the lipopolysaccharide (LPS) is the dominant surface antigen on the outer membrane of C. burnetii. Bacteria that express a long-chain, smooth LPS with complete O-specific polysaccharide are present as phase I (Ph I). They are virulent and can be isolated from hosts such as humans, animals, and ticks. By frequent passages in immune incompetent hosts, e.g. B. embryonated chicken eggs, the LPS is increasingly shortened. Loss of LPS is associated with a decrease in virulence. Bacteria with a severely shortened, rough LPS are low to avirulent and are referred to as phase II (Ph II) coxies (Hotta et al. 2004).

Transmission and infection

Animal husbandry

C. burnetii is excreted in large quantities in the birth material (placenta, amniotic fluid) of infected sheep and goats during childbirth or an abortion. Up to 109 C. burnetiiPathogens (Babudieri, quoted from Fournier et al. 1998). C. burnetii multiplies especially in the trophoblasts of the placenta, with the multiplication rate increasing towards birth (Ben Amara et al. 2010, Roest et al. 2012). Why C. burnetii finds ideal conditions for reproduction, especially in trophoblasts, is not yet fully understood. The ability of C. burnetiireprogramming the transcription in trophoblasts seems to play an essential role (Ben Amara et al. 2010). Excretion also takes place via milk, feces and urine. Due to the high tenacity of the pathogen against environmental influences, the DNA of C. burnetii ubiquitous evidence (Kersh et al. 2010). However, larger amounts are found particularly in ruminant holdings (Carrie et al. 2019, Kersh et al. 2010). The causative agent of Q fever is highly contagious. The main route of infection for humans and animals is inhalation of aerosols containing pathogens (Brooke et al. 2013, Todkill et al. 2018). The airborne infection after spreading the pathogen with the wind has led to urban outbreaks within a limited environment (small-scale epidemics) in various European countries (Gilsdorf et al. 2008, Hawker et al. 1998, Tissot-Dupont et al. 1999). A particular risk of infection exists within a 5 to 10 km zone around the outbreak, with the greatest risk within a 2 to 4 km radius (Clark and Soares Magalhães 2018). However, transmission from a distance of up to 18 km has also been reported (Hawker et al. 1998). By contrast, another working group could C. burnetii can only be detected within a radius of 50 meters from a positive goat farm (Kersh et al. 2013). In a small-scale epidemic in Jena in 2005, people who lived within 400 meters of the outbreak events fell ill (Gilsdorf et al. 2008). Additionally can C. burnetii can also be spread through fresh sheep and goat dung from infected farms. This is particularly common through amniotic fluid and postbirths that have not been removed C. burnetii contaminated. Spreading on agricultural land, especially in windy weather, has repeatedly led to human Q fever diseases and outbreaks in the past (Arricau-Bouvery and Rodolakis 2005, King et al. 2011, Reintjes et al. 2000). Although there are indications that manure application played a subordinate role in the event of the outbreak in the Netherlands (van den Brom et al. 2015a), a consensus paper later recommended that manure be stored for three months under foil and that the manure material should only be transported on windless days ( Plummer et al. 2018). In addition to the wind, other meteorological factors also play a role. So favors z. For example, dry weather can reduce the spread of coxies (Tissot-Dupont et al. 2004), while rainfall can reduce the spread and thus the risk of human infections (Gilsdorf et al. 2008).

Raw milk

Whether the enjoyment of with C. burnetii contaminated raw milk and raw milk products lead to disease in humans is controversial (Cerf and Condron 2006, Eldin et al. 2013, Raoult et al. 2000). As early as the 1960s, a study of prison inmates showed that the consumption of with C. burnetii contaminated raw milk leads to seroconversion, but not to disease (Benson et al. 1963). Such a study cannot of course be repeated for ethical reasons. It is believed that airborne infection is more effective than oral infection due to the higher number of macrophages in the lungs (Gale et al. 2015). Nevertheless, in a few cases, regular consumption of raw milk containing pathogens appears to lead to illnesses with fever and severe headaches (Fishbein and Raoult 1992, Signs et al. 2012). Dupont et al. (1992) also suspect a connection between raw milk consumption and C. burnetii-associated hepatitis. This clinical picture is particularly prevalent in France (Dupont et al. 1992, Melenotte et al. 2018). The risk of developing Q fever through the consumption of raw milk and raw milk products is currently classified as low, but not negligible (Gale et al. 2015, Pexara et al. 2018). Especially in light of the fact that raw milk cheese can contain live coxia, milk from infected herds should always be subjected to heat treatment (Barandika et al. 2019, BfR 2010). The process of pasteurization (71.66 ° C for 15 seconds) inactivates the pathogen in the milk (EFSA 2010).

Ticks

Ticks are carriers of numerous zoonotic pathogens. The role of ticks as a reservoir and carrier of C. burnetii has not yet been clearly clarified. The pathogen has been detected in various types of ticks around the world (Duron et al. 2015). In Germany is Ixodes (I.) ricinus by far the most widespread and common type of tick (Rubel et al. 2014). Although Dermacentor (D.) marginatus, also called sheep tick, does not occur across the whole of Germany (Rubel et al. 2014, Walter et al. 2016), this is considered a vector for C. burnetii (Sting et al. 2004). However, could C. burnetii only in one of 1066 ticks of the species examined D. marginatus can be detected by means of PCR, although the ticks came from an area endemic to Q fever (Sting et al. 2004). A few years later, neither in D. marginatus still in D. reticulatus Coxielle DNA can be detected (Pluta et al. 2010). After the great Q fever epidemic in the Netherlands in 1891, DNA lysates from ticks were tested using multiplex qPCR. C. burnetii was not found in any sample (Sprong et al. 2012). However, Hildebrandt et al. (2011) C. burnetii in a Thuringian endemic area in 1.9% of the investigated I. ricinus prove. Overall, therefore, both Dermacentorspp. as well as I. ricinus epidemiologically rather a subordinate role in the transmission of C. burnetii play in Germany (Kimmig 2010). Recent studies show that about 75% of all tick species Coxiella- harbor similar bacteria, this could have contributed to false positive pathogen detection in the past (Duron 2015, Jourdain et al. 2015).
The infection with C. burnetii caused by tick bites has not yet been clearly clarified either (Duron et al. 2015, González-Barrio et al. 2016). Transmission by means of tick excrement is probably more important for the infection of hosts than the tick bite (Liebisch 1977). Coxies multiply very strongly in the intestinal cells of the ticks and are excreted with the faeces (Angelakis and Raoult 2010, Liebisch 1977, Špitalská and Kocianová 2003). The pathogen-containing tick excrement (up to 1012 Coxiella / g) sticks the wool and the coxiella it contains can remain infectious for up to 2 years (Schliesser 1991).Schulz et al. (2005) were able to detect Coxielle DNA in the surrounding air space during the shearing. Human infections through inhalation of the contaminated dust are therefore possible during the shearing (Angelakis and Raoult 2010). Therefore, appropriate precautionary measures should be taken when shearing to prevent infection.

Bacteremia and immune response

In humans and animals, the Coxielle infection occurs mainly aerogenically by means of contaminated dusts and / or droplets (Kimmig 2010). The alveolar macrophages may play a decisive role here, since the first contact with the host with the host occurs in the lungs when contaminated dusts and / or aerosols are inhaled (Khavkin and Tabibzadeh 1988). Followed by this initial uptake and multiplication of the pathogen, a hematogenous spread and specific organ manifestation (to be emphasized here are the lungs, brain, liver and reproductive organs (Kimmig 2010, Schüle 2008)) as a consequence of bacteremia. The organs affected have not yet been fully recorded with regard to human infections. During the systemic spread, alveolar macrophages and Kupffer's stellate cells in the liver are particularly affected by infections at the cellular level. In the further course, pneumonia or granulomatous hepatitis can be observed in humans (Kimmig 2010). C. burnetii is excreted through various body fluids during bacteremia and beyond (Rousset et al. 2009a, Schüle 2008). The tropism of the pathogen particularly affects epithelial cells of the cotyledons in ruminants and results in a high concentration of bacteria in the amniotic fluid, the placenta and the cochial secretion. The high pathogen load can cause premature births or abortions and increases the postnatal risk of infection. Primary host cells of C. burnetii are macrophages. Infections of trophoblasts, endothelial and epithelial cells as well as fibroblasts have been detected (Maurin and Raoult 1999, Voth and Heinzen 2007). As an intracellular pathogen, it is possible for coxies to evade antibody opsonization and elimination via the complement system (Bonkowski 2016). Macrophages take up bacteria into intracellular phagosomes via the mechanism of phagocytosis. This vacuolar cell compartment matures through fusion with endosomes and lysosomes into a degradation-active phagolysosome, in which the ingested bacteria can generally be killed (Flannagan et al. 2009). Coxials have developed mechanisms to survive and multiply in the phagolysosomes (Howe et al. 2010). In addition to macrophages, dendritic cells (DCs) are also host cells for C. burnetii (Shannon et al. 2005) (Fig. 1). With the help of toll-like receptors (TLRs) or damage-associated molecular pattern receptors (DAMP-R), DCs have important sensors of the innate immune system and modulate subordinate immune responses (Mosser and Edwards 2008). A central mechanism of innate immunity is the stimulation of pathogen pattern receptors (pattern recognition receptors, PRRs) and DAMP-Rs of the immune cells concerned. Integrins (αvβ3, for LPS phase types I and II) and complement receptor 3 (CR3, only LPS phase type II) serve as relevant uptake receptors (Capo et al. 1999). After penetrating the host cell, coxials use an autophagic pathway intended for intracellular degradation and cell-autonomous immune defense for their intracellular reproduction (Colombo et al. 2006). Autophagy is an evolutionarily conserved process of all eukaryotic cells, which normally serves to break down and / or recycle cell components that are no longer required or defective. In addition, autophagy after infection can eliminate intracellular pathogens by means of xenophagic degradation, in which autophagy is directed against pathogens. After the uptake of coxia by the host cell along the phagocytic pathway, a parasitophoric vacuole (PV) forms. In the further course, this fuses with autophagic compartments, an important prerequisite for successful intracellular adaptation to the host cell. Interestingly, escapes C. burnetii the degradation and can multiply unhindered in the autophagy compartments (Voth et al. 2007). Using different autophagy markers, the intracellular transport process of coxiae could be traced in detail (Colombo et al. 2006, Lührmann et al. 2017, Schulze-Luehrmann et al. 2016, Voth et al. 2007). With their type IVB secretion system (T4BSS), coxies are ideally adapted to an intracellular way of life and can export virulence proteins, so-called effector proteins, into the host cell (van Schaik et al. 2013) (described in more detail in the following section). Functionally, bacterial effectors (Lührmann et al. 2017) are responsible for the formation of replicative PVs (Beare et al. 2011, Carey et al. 2011), the inhibition of xenophagia (Newton et al. 2014, Winchell et al. 2014), the Modulation of the maturation of PV (Larson et al. 2013, Latomanski et al. 2016, Martinez et al. 2016), the inhibition of cell death (Beare et al. 2011, Bisle et al. 2016, Cunha et al. 2015, Lührmann et al. 2010), the remodeling of the cytoskeleton (Weber et al. 2016) and the undermining of the immune response (Clemente et al. 2018). In the case of monocytes / macrophages, a distinction is made between classic M1 and alternative M2 polarization. While antigen-presenting proinflammatory M1 monocytes / macrophages control intracellular bacterial multiplication, this is favored in anti-inflammatory M2 monocytes / macrophages (Benoit et al. 2008b). The interaction of monocytes / macrophages with C. burnetii leads to different polarization. So induce coxials in dormant monocytes in which C. burnetii survives, but does not multiply, an M1 polarization (Ghigo et al. 2004). However, in macrophages, in which C. burnetii replicated, the corresponding infection to M2 polarization with induction of anti-inflammatory cytokines (interleukin (IL) -10, transforming growth factor (TGF) -β and IL-1Ra), which support both the multiplication and the persistence of the pathogen (Benoit et al. 2008a). Furthermore, it was found that there was no induction of an antigen presentation characteristic of the M1 polarization of infected macrophages (Sobotta et al. 2016). Since the stimulation of the immune system via conservative PRRs such as extracellular TLRs (Ammerdorffer et al. 2015, Bradley et al. 2016, Ramstead et al. 2016, Zamboni et al. 2004) and intracellular NLRs (NOD-like receptors) (Ammerdorffer et al . 2015), these are the focus of current studies C. burnetii. In humans, some genetic PRR variants are associated with a higher probability of developing a chronic form of Q fever (Schoffelen et al. 2015). In this context it is interesting that the LPS of virulent Coxielle strains may act as a kind of TLR4 antagonist in infected macrophages (Zamboni et al. 2004). Although the TLR4 is used for late control of the C. burnetii-Infection seems dispensable, it controls the early events of infection, including macrophage phagocytosis, granuloma formation, and cytokine production (Honstettre et al. 2004).
When infecting DCs with C. burnetii there is neither an induction of functional DC maturation nor a corresponding cytokine production (Shannon et al. 2005). Although the responsible molecular mechanism is still unclear, it is assumed that the Coxielle LPS as a virulence factor blocks functional DC stimulation (Shannon et al. 2005). It is postulated that it may sterically impede the access of bacterial surface molecules to the corresponding TLRs of infected DCs. The lack of maturation of infected DCs presumably leads to tolerance to it C. burnetiiAntigens and can thus be responsible for the establishment and / or manifestation of persistent infections (Steinman and Nussenzweig 2002, Verhasselt et al. 2004). A successful presentation of antigens with the help of activated antigen-presenting cells (APCs) leads to the initiation of the adaptive immune response, which plays a crucial role in the defense against coxia. In the mouse system with a lack of T and B cell immunity, an unhindered one was found C. burnetiiInfection (Andoh et al. 2007, Islam et al. 2013, Kishimoto et al. 1978). T-cell-depleted test animals which only have an antibody-based immune response cannot eliminate an infection either (Read et al. 2010). The TH1-mediated cellular immunity required to fight coxia is largely based on the release of proinflammatory cytokines such as interferon-γ (IFN-γ). Limited or absent IFN-γ production by T cells is expressed in an increased mortality rate in infected test animals (Andoh et al. 2007). Although the IFN-γ activation of infected cells the C. burnetii-Replication mainly through the production of reactive oxygen (ROS) and nitrogen species (RNO) inhibits (van Schaik et al. 2013), the bacteria seem to be able to prevent ROS production via the secretion of an acid phosphatase ( Hill and Samuel 2011). It is assumed that this prevents the formation of the NADPH oxidase complex in host cells (Siemsen et al. 2009). The production of RNO requires the new synthesis of the inducible nitric oxide synthase iNOS (Vila-del Sol et al. 2007), as can be observed after TLR stimulation and subsequent induction of proinflammatory cytokines. However, due to the absence of this stimulation (Benoit et al. 2008a), the coxia infection of macrophages does not lead to a corresponding RNO synthesis. Interestingly, recently published studies on bovine and human macrophages with C. burnetii-Strains of different genotypes were infected that the upregulation of activation markers (CD40 and CD80) and the proinflammatory cytokine response of infected macrophages, by the respective genotype of the C. burnetiiStrain is determined (Sobotta et al. 2016).
Coxielle-host cell interactions therefore interfere with immunity in a variety of ways. Coxiella are able to escape the cell's own defense and use auto- / xenophagia for themselves. They also specifically manipulate the immune response directed against them through subversion. More intensive studies are needed in order to have the immunological understanding of infections C. burnetii to improve and to develop suitable immune prophylaxis and therapies.

Apoptosis

As an obligate intracellular bacterium can C. burnetii multiply under natural conditions only within infected cells. As already described above, the bacterium is able to avoid intracellular degradation by macrophages and develop a corresponding PV (Lührmann et al. 2017). Another possible defense strategy of infected macrophages in this situation is the induction of apoptosis, whereby infected cells are eliminated by a kind of cellular "suicide" without triggering an inflammatory reaction of the adaptive immune system. C. burnetii however, has the ability to inhibit this crucial host cell defense (Lührmann and Roy 2007, Voth et al. 2007). This is essential for the slowly replicating intracellular bacterium to maintain its intracellular replicative niche (Friedrich et al. 2017). The exact mechanisms by which C. burnetii keeps its host cell alive have not yet been fully clarified. However, it is clear that the T4BSS and the virulence factors it exports are essential for this activity (Beare et al. 2011). To date, around 150 of these bacterial effector proteins have been identified. AnkG, CaeA and CaeB inhibit apoptosis and IcaA inhibit pyroptosis, a pro-inflammatory death program (Bisle et al. 2016, Cunha et al. 2015, Eckart et al. 2014, Klingenbeck et al. 2013, Lührmann et al. 2010, Schäfer et al. 2017). However, it is assumed that there are other bacterial factors that prevent or induce cell death of infected host cells (Martinez et al. 2014) or induce (Schoenlaub et al. 2016, Zhang et al. 2012). While an early inhibition of cell death is important for the intracellular multiplication of C. burnetii the induction of cell death at a later stage of the cell infection could play a decisive role in the dissemination of the bacteria in the host organism.
The ability of C. burnetii To replicate intracellularly in macrophages depends largely on the polarization of the macrophage. So replicated C. burnetii in atypical M2 macrophages, while they are controlled or killed in M1 macrophages (Benoit et al. 2008a, Fernandes et al. 2016, Meghari et al. 2007, Mehraj et al. 2013). If C. burnetii influences the polarization of macrophages is not yet known. However, it has been shown that macrophages are activated by contact with apoptotic lymphocytes in the direction of M2 (Benoit et al. 2008c). This could also explain why patients with valvulopathies have an increased risk of developing endocarditis. These patients have more apoptotic leukocytes in the blood, which then leads to the M2 activation of macrophages and thus to the optimal multiplication of C. burnetii leads (Benoit et al. 2008c).

Clinic for goats and sheep

Infection can occur in goats C. burnetii lead to late abortions and the birth of weak lambs (Achard and Rodolakis 2017). Overall, more than 90% of the goats in a herd can be affected (Palmer et al. 1983). Mostly, however, abortion rates of 5 to 20% are observed (Palmer et al. 1983, van den Brom and Vellema 2009). Goats that are due to a C. burnetii-Abort infection, then seem to develop more likely to develop metritis (van den Brom et al. 2015c). With regard to the intensity of pathogen excretion, there is no difference between abortion and the physiological birth of healthy lambs (Roest et al. 2012, Rousset et al. 2009a). The clinical picture of coxiellosis in sheep has not been clearly established (Agerholm 2013). Late miscarriages and the birth of life-weak lambs can also occur (Berri et al. 2005, Hazlett et al. 2013, Zeman et al. 1989). Infected sheep can also give birth to healthy lambs (Böttcher et al. 2019, Hamann et al. 2009, Welsh et al. 1951). The conditions under which a late abortion or the birth of lifeless lambs occur remains unclear. Basically, it lies through a C. burnetii-Infection-causing abortion rate in sheep is lower than in goats (Agerholm 2013). For example, an average abortion rate in sheep of 5% has been reported (van den Brom and Vellema 2009). In the subsequent lambing, sheep generally no longer suffer an abortion (Berri et al. 2002). With proof of at least 104 Pathogens in the placenta or in the vaginal swab C. burnetii as the cause of the abortion (Bru et al. 2013, Hazlett et al. 2013). Such and higher amounts of pathogen are also found in normal births (Böttcher et al. 2019). The aborted fetuses look unchanged and usually look fresh; they are only occasionally autolytic (Moeller 2012, van den Brom et al. 2015c). Occasionally, macroscopic placentitis with brownish-purulent coatings and intercotyledonal thickening can be seen (Moeller 2012, van den Brom et al. 2015c). Histologically, mild mononuclear infiltration up to severe necrosis and purulent exudation can be found in the intercotyledonal trophoblasts and at the base of the chorionic villi (van den Brom et al. 2015c). In addition, mixed infections with other abortion pathogens such as Chlamydia (Chl.) Abortus and Toxoplasma gondii (Berri et al. 2001, Eibach et al. 2013, Hazlett et al. 2013). Therefore, especially in sheep, the question of the primary cause of the abortion arises, and little about the effect of a co-infection of the two intracellular pathogens C. burnetii and Chl. abortus is known in small ruminants.
Before the lamb or the abortion is C. burnetii not excreted via vaginal secretions, but excreted via faeces (Arricau-Bouvery et al. 2003, Roest et al. 2012). With the excretion intensity of C. burnetii There are differences between goats and sheep in terms of milk, faeces and vaginal mucus. While goats mainly excrete the pathogen through their milk, sheep excrete coxia more through vaginal mucus and feces (Rodolakis et al. 2007). There are different details about the elimination period. Arricau-Bouvery et al. (2003) were able to post partum in experimentally infected goats by means of PCR C. burnetii Detect up to 14 days in vaginal samples, 20 days in the feces and 52 days in the milk. In contrast, Roest et al. (2012) in goats post partum C. burnetii Detect almost 100 days in vaginal swabs and faeces, but only 38 days in milk. In sheep it was possible C. burnetii can be detected in vaginal swabs up to 71 days and in feces and milk up to eight days after birth (Berri et al. 2001). Furthermore, small ruminants can shed the pathogen for up to four months during the second lambing if no control measures are taken (Astobiza et al. 2011a, Berri et al. 2007). These differences with regard to the elimination time could also be due to the different sensitivity of the different detection methods in the respective studies. Contamination during sampling in the environment highly contaminated with coxies can also lead to false-positive results (Plummer et al. 2018, Roest et al. 2012).Intermittent shedding or even permanent shedding have been described for dairy cattle (Böttcher et al. 2013, Lucchese et al. 2015). These divorce C. burnetii with the milk for a longer period of time. Van den Brom et al. (2013) were probably able to detect chronic milk excretors in goats as well. This has not yet been described in sheep.

Epidemiological situation in Germany (2000-2018)

Sheep farming in Germany varies greatly. In a state comparison, Schleswig-Holstein has (20.77 sheep / km2), Thuringia (20.66 sheep / km2) and Baden-Württemberg (17.20 sheep / km2) most sheep per km2 agricultural area, followed by Hesse (16.64 sheep / km2), Rhineland-Palatinate (11.33 sheep / km2) and North Rhine-Westphalia (11.07 sheep / km2) (Fig. 2). In contrast, most goats per km2 utilized agricultural area in Baden-Württemberg (2.10 goats / km2), Thuringia (1.61 goats / km2), Hesse (1.22 goats / km2) and Bavaria (1.21 goats / km2) held. Goat husbandry plays a subordinate role in the other federal states (Fig. 3). The number of sheep and goat holdings per km2 agricultural area is in Baden-Württemberg (0.3685 holdings / km2), Hesse (0.2834 establishments / km2) and Bavaria (0.2618 establishments / km2) the highest. The number of sheep and goat farms is lowest in the northern federal states, with North Rhine-Westphalia (0.2080 farms / km2) and Schleswig-Holstein (0.1996 holdings / km2) are the exception (Fig. 4). If one looks at the average number of small ruminants per farm, the opposite picture emerges. The federal states of Saxony-Anhalt (an average of 197 small ruminants / farm), Brandenburg (an average of 155 small ruminants / farm) and Mecklenburg-Western Pomerania (an average of 139 small ruminants / farm) have few sheep and goat farms per km2 of agricultural area, however the few farms keep a large number of animals on average. Thuringia has by far the highest average number of animals per holding (an average of 241 small ruminants / holding), while Bavaria has a large number of small sheep and goat holdings (an average of 62 small ruminants / holding; Fig. 5). Overall, Baden-Württemberg is the federal state with many sheep and goats and with the most sheep and goat farms per km2 agricultural area. These are farms with a comparatively medium number of animals (90 small ruminants / farm on average).
Regardless of the number of animals, the number of farms and the average herd size, sheep farming in Germany can also be divided into two halves according to the breeds of sheep kept and the associated reproductive behavior. In southern Germany (Baden-Wuerttemberg, Bavaria, Hesse, Rhineland-Palatinate, Saarland, Saxony, Thuringia), mainly merino breeds are kept that are out of season and therefore lamb all year round (von Korn 2001). In contrast, in the northern federal states (Berlin, Brandenburg, Bremen, Hamburg, Mecklenburg-Western Pomerania, Lower Saxony, North Rhine-Westphalia, Saxony-Anhalt, Schleswig-Holstein), meat sheep breeds are preferred that are seasonal and therefore mainly from February to April lammen (von Korn 2001).
Evidence of C. burnetii or coxiellose is notifiable according to the ordinance on notifiable animal diseases in ruminants (TKrMeldpflV 2015). The heads of the veterinary examination offices, the animal health offices or other public or private examination centers as well as veterinarians who detect notifiable diseases in the course of their profession are obliged to immediately report the occurrence of the disease or the pathogen to the authority responsible under state law, stating the date of the detection of the affected To report animal species, the affected population and the district or the urban district. A report of only antibody-positive animals is therefore not required, unless these are detected in connection with symptoms that indicate coxiellosis. The reported cases are entered into the animal disease messaging system (TSN) by the responsible veterinary authorities. In the period from 2000 to 2018, far more cases were reported in cattle (8359 records reported) than in sheep (1349 records reported) and goats (212 records reported) (Fig. 6). There could be several reasons for this. Firstly, despite the legal requirements, the sometimes different reporting practices may lead to a distortion of the data. Another cause is presumably that in this period far more cattle (4,100,863 dairy cows ≥ 2 years, DESTATIS 2018a) than sheep (1,098,700 female sheep for breeding, DESTATIS 2018b) and goats (88,451 female goats, statistical recording of all three Years since 2010, DESTATIS 2016) were held in Germany. Second is the awareness of C. burnetii as a possible cause of reproductive disorders in cattle farming has increased (Lehner et al. 2017). Furthermore, when interpreting the reporting data, it should be noted that there is no active monitoring system for ruminants in Germany that could representatively monitor the prevalence of infected animals or herds. It must therefore be assumed that the actual number of affected establishments is higher than the number of reports. In Baden-Wuerttemberg, Hesse, Lower Saxony, North Rhine-Westphalia, Rhineland-Palatinate, Saxony and Thuringia, the costs are wholly or at least partially for the C. burnetii- Vaccination taken over by the Tierseuchenkasse (Landwirtschaftskammer Nordrhein-Westfalen 2014, Lehner et al. 2017, Tierseuchenkasse Baden-Württemberg 2018, Tierseuchenkasse Hessen 2016, Tierseuchenkasse Rheinland-Pfalz 2017, Tierseuchenkasse Sachsen 2015, Tierseuchenkasse Thüringen 2019). The prerequisite is usually the molecular biological detection of the pathogen in the animal material. In addition, research on Q fever in cattle has been ongoing in Bavaria for a long time (Boettcher et al. 2017, Böttcher et al. 2011b, 2013, 2019). These two activities could have contributed to higher numbers of cattle registrations in individual federal states. In the case of small ruminants, there are mainly reports from southern Germany (Fig. 6). Overall, there are more sheep and goat farms in southern Germany than in the north. In addition, humane small-scale epidemics in Baden-Württemberg, Bavaria, Hesse and North Rhine-Westphalia, caused by lambing sheep, have increased. Therefore it is likely that awareness of C. burnetii is higher in these regions, which means that this pathogen is examined more specifically. In addition, the number of female goats increased by 17% to 88,451 between 2010 and 2016 (DESTATIS 2016). At the same time, according to our own observations, there has been a shift from small farms to intensive, large dairy goat farms with several hundred animals. In addition, smaller groups of goats in large flocks of sheep are increasingly being used for landscape maintenance. Therefore, with increased reports of C. burnetii expected for this species in the next few years. A similar structural change had taken place in the Netherlands in the 2000s. In retrospect, however, the higher number of animals per farm was not the only risk for one C. burnetiiInfection increased (Hogerwerf et al. 2013, Schimmer et al. 2011). Other factors such as the density of cattle holdings in the region, the implementation of artificial insemination for goats and the access of cats and dogs to the goat shed also played a role (Hogerwerf et al. 2013, Schimmer et al. 2011).
In the absence of a comprehensive and systematic active monitoring system for C. burnetii Active studies in German sheep herds must be used when it comes to the question of how the pathogen is spread. Only a few have been carried out on this in the last 20 years. Information on goat herds or mixed farms (sheep and goats) is particularly sparse, although goat-associated Q fever cases have been reported in northern and southern Germany in the past (Ganter et al. 2009, Sting et al. 2013). The collection of data from sheep is limited to a few federal states. In Thuringia, a herd prevalence of 28% (n = 39) was detected using ELISA (Hilbert et al. 2012). In the same study, vaginal swabs were also examined using PCR. Only 5% of the herds (n = 39) were positive. At a later point in time, 21.2% (n = 99) of the examined flocks of sheep in Thuringia were able to serologically test C. burnetiiInfection can be detected (Moog et al. 2016). In the same study, 28 flocks of goats were also tested for antibody activity against C. burnetii tested. 14.3% of the herds were positive. Overall, herds of sheep and goats with an animal number> 399 were more frequently serologically positive than smaller farms. In 2004, 95 sheep herds of various sizes in Lower Saxony were also serologically tested for antibodies C. burnetii investigated (Runge et al. 2012). At least one positive or questionably positive animal could be found in 9.5% of the herds examined. In addition, an individual animal prevalence of 2.7% (n = 1714) was determined for Lower Saxony. The high proportion of positively tested sheep per farm in three migrating sheep herds was particularly noticeable. Sting et al. (2004) reported evidence of seropositive sheep of an average of 8.7% (n = 3460) from Baden-Württemberg. For this study, sera were analyzed which were originally taken in 2001 for brucellosis monitoring. In a further study in nine municipalities in Baden-Württemberg, 7.5% of the sheep (n = 1036) were positive C. burnetii-Antibodies tested (Brockmann 2014). In 2008, a total of 1187 sheep from 48 Bavarian herds were examined using phase-specific ELISA (Böttcher et al. 2011a). 9.8% of the animals analyzed had Ph-I antibodies and 17.4% of the sheep had Ph-II antibodies. In the two following years (2009: n = 997; 2010: n = 1032) the detection rate for both Ph-I antibodies (2009: 0.5%; 2010: 0.6%) and Ph-II antibodies was Antibodies (2009: 0.8%; 2010: 2.0%) significantly lower. The comparatively high values ​​in 2008 can be traced back to a Q fever outbreak in the administrative district of Lower Franconia. Due to the different study designs and the tests used, the results of the studies carried out can only be compared to a limited extent.
The direct and indirect evidence of an acute infection with C. burnetii in humans is subject to notification by name according to the Infection Protection Act (IfSG) (IfSG 2019). The reports have been recorded since 2001 with the "SurvStat" monitoring program at the Robert Koch Institute (RKI). At the end of the 1990s there was a steady increase in the number of reported human Q fever cases, the number of which has remained at a constant level since the 2000s (Hellenbrand et al. 2001). Basically there is a difference in the number of reported human cases between northern and southern Germany. Some of the peaks can be traced back to small-scale epidemics (Fig. 7). The three largest small-scale epidemics were reported from the districts of Soest and Heidenheim an der Brenz as well as from the city of Jena. In May 2003, for example, many of the approximately 10,000 visitors were infected at a farmers' market near the city of Soest in North Rhine-Westphalia. 299 visitors contracted Q fever (Porten et al. 2006). Based on epidemiological studies using ELISA, a Texel sheep that lambed and gave birth to live twins at the exhibition could be identified as the source of the infection. This ewe came from a herd with a detection rate of 25% serologically positive sheep (n = 67). In June 2005, a small group of first lambing merino sheep that lambed near a residential area were responsible for the infection of 331 people (Gilsdorf et al. 2008). 228 visitors attended a sheep farm festival in the district of Heidenheim an der Brenz in Baden-Württemberg in June 2014 C. burnetii infected (Fischer et al. 2016). Asymptomatic infection was diagnosed in only 20% of the patients examined. A chronic infection was suspected in five people (2.2%).
Due to the often only subclinical course, a high number of unreported cases can be assumed when Q fever infections are recorded in humans. Epidemiological evaluations of the Dutch outbreak suggest that only about every tenth infection was detected (van der Hoek et al. 2012).
The epidemiological situation of Q fever infections for the period from 1947 to 1999 was described by Hellenbrand et al. (2001) evaluated. From their research, the authors concluded that C. burnetii has been endemic in cattle in many areas of Germany since the 1980s. In contrast, the occurrence in sheep is concentrated according to Hellenbrand et al. (2001) to a few federal states in the south, east and west of Germany. Since 2000, most evidence has been found in both small ruminants and humans in Baden-Württemberg, Bavaria, Hesse and North Rhine-Westphalia. Thuringia had a human small-scale epidemic in 2005 and detection rates of serologically positive goat and sheep herds between 14.3% and 28% were determined (Hilbert et al. 2012, Moog et al. 2016), but with the exception of 2005 this only happened few reports in humans and small ruminants. This is probably due to the subsidization of the vaccine for sheep and goats by the Thuringian animal disease fund and the increased vaccination of sheep and goat herds as a result (Moog et al. 2016, Tierseuchenkasse Thüringen 2019). Several factors certainly play a role in the occurrence of small-scale Q fever epidemics in humans. Frank and Höhle (2011) were able to establish a positive correlation between the sheep density per hectare of agricultural area and human Q fever. However, the authors found no connection with cattle and goats. Although no presumption of causality can yet be derived from the spatial correlation between animal density and human Q fever cases, this result can serve as a basis for further studies. It is also noticeable that the three major epidemics since 2000 began in early summer, a warm and dry season. During this time of the year, particularly non-seasonal breeds such as merino sheep lamb outdoors. Most of the out-of-season breeds are preferably kept in southern Germany. In contrast, the largest sheep density per agricultural area can be found in Schleswig-Holstein. However, in both human and veterinary medicine, there is only a small amount of evidence of Coxielle infections in this state. This could be due to the seasonal lambing of the sheep in the barn in February and March, a particularly humid and still cold season. In summary, the out-of-season lambing in the warm and dry seasons as well as a high sheep density could lead to increased Q fever infections. Hellenbrand et al. (2001) also saw the presence of D. marginatus in southern Germany as a risk factor. However, this type of tick does not occur in North Rhine-Westphalia (Rubel et al. 2014, Walter et al. 2016). If the risk factors mentioned come into contact with a human population (residential area, market visitors, etc.), this can lead to cases of illness or small-scale epidemics. These assumptions need to be checked in the future. In addition, with the rise in professional goat husbandry in Germany, this species is likely to play an increasingly important role in the Q fever epidemiology. In both Bulgaria and the Netherlands, a connection between the increase in goats and increasing human Q fever diseases was found (Mori and Roest 2018).

Diagnosis

serology

For the serological detection of a C. burnetii-Infection is missing a gold standard in veterinary medicine (van den Brom et al. 2015c). In addition, veterinary Q fever serology is still a long way from being standardized (Rousset et al. 2007). In routine diagnostics, the complement fixation reaction (CFR) has long been the method of choice for the detection of C. burnetii-Antibodies used. This method basically uses a mixture of Ph-I and Ph-II antigens from the Nine Mile strain (OIE 2018). In the KBR, titers between 1:10 and 1:40 are considered latent infections in ruminants. Titers of 1:80 and above are classified as the acute phase of the infection (OIE 2018). In the meantime, the KBR has been replaced by the ELISA as the standard method (OIE 2018), since in comparative studies the KBR has a lower sensitivity than the ELISAs used (Kittelberger et al. 2009, Natale et al. 2012, Rousset et al. 2007, Sting et al. 2004). Schmeer (1985) justifies this with the partial inhibition of the complement-binding IgG1 by IgG2 and IgM, which manifests itself in the KBR as a titer reduction and incomplete inhibition of hemolysis. As a result, false negative results can be obtained with the KBR, especially in low titer ranges (Schmeer 1985). However, IgM also binds a complement in the KBR. Therefore, in contrast to IgG-based ELISAs, the early humoral immune response can also be detected using KBR (Kittelberger et al. 2009). Commercially available ELISAs are based on a mixture of Ph-I and Ph-II antigens and only detect IgG antibodies. The ELISAs currently approved in Germany in accordance with the Animal Health Act (TierGesG 2019) do not provide any reliable information about the infection process within a herd.However, due to their ease of automation, they are suitable for examining large numbers of samples (OIE 2018). The ELISAs were mostly validated on the basis of the KBR, without clearly defined negative and positive controls. The ELISAs delivered more positive reagents than the KBR, which was interpreted as a higher sensitivity. However, a positive result with the KBR or the ELISA only provides information about whether the animal has been in contact with C. burnetii and there was a humoral immune response. Both tests do not allow any conclusions to be drawn about the pathogen excretion. Animals that tested serologically negative can shed the pathogen (de Cremoux et al. 2012, Joulié et al. 2017, Natale et al. 2012, Rodolakis et al. 2007). In an infection attempt by Arricau-Bouvery et al. (2003) there were first abortions and thus pathogen excretion 12 days and 25 days after subcutaneous infection C. burnetii. The first goats were serologically tested positive by ELISA only 42 days after the experimental infection. Therefore, the serological tests alone are not suitable to confirm that individual animals and herds are free from infection (Angelakis and Raoult 2010, OIE 2018). However, they can provide information about the presence of antibody activity at the herd level. To individual animals or herds of small ruminants as free from C. burnetii currently only the PCR test, preferably of Pueperal swabs, provides reliable results (OIE 2018).
In order to gain a deeper insight into the infection process in herds, a phase-specific detection can be carried out both with the indirect immunofluorescence antibody test (IFAT) and with phase-specific ELISAs. Both test methods are currently not approved for diagnostics in Germany under the Animal Health Act (TierGesG 2019) and have so far been used for scientific purposes. So far, these tests have mainly examined serum samples from cattle and goats (Böttcher et al. 2011b, Muleme et al. 2016, Roest et al. 2013, Sting et al. 2013). In an infection test with goats it was possible to show that both IgM Ph-II and IgG Ph-II antibodies in the phase-specific ELISA simultaneously from the second week after intranasal inoculation of C. burnetii increase (Roest et al. 2013). At the same time there was also an increase in IgM Ph-I antibodies, but to a lesser extent. IgG Ph I increased slowly but steadily from the 5th week after infection. From this, Roest et al. (2013) that an increase in IgM Ph II in the absence of IgG Ph-I antibodies suggest an acute infection in goats. From the 10th week post infection, both IgG Ph-II and IgG Ph-I antibodies were at the same high level. Sting et al. Came to a similar conclusion. (2013) when investigating a Q fever outbreak in dairy goats. They defined an acute infection particularly with the presence of IgG Ph-II antibodies and only weak signals of IgG Ph-I antibodies. IgG Ph-I titers can be detected in goats for two years, while IgG Ph-II titers are usually no longer detectable at this point in time (Hatchette et al. 2003). Extensive long-term studies on the phase-specific course of IgM and IgG are lacking for both goats and sheep. Although extensive validations of the phase-specific tests are still pending, the phase-specific serology appears to be a valuable diagnostic tool for analyzing the infection dynamics in herds and for detecting new infections (Muleme et al. 2017). However, further studies over a longer period of time with targeted infections for the individual ruminant species are necessary in order to be able to make more binding statements. In addition, there is a lack of clear case definitions with regard to the infection status (free, acutely or chronically infected) in veterinary medicine. A differentiation between a vaccinated or naturally infected animal is not possible with any serological method (EFSA 2010).
In addition to serum samples, individual milk samples or tank milk samples can also be examined by ELISA. Using tank milk samples with an inner herd prevalence of at least 10–15%, an infection was also found C. burnetii can be detected in sheep and goat herds with the ELISA (Ruiz-Fons et al. 2011, van den Brom et al. 2012). When using tank milk samples, ELISA was more frequently used than PCR (Muleme et al. 2017, van den Brom et al. 2012). The cell-mediated immunity to C. burnetii can be determined by interferon-γ (IFN-γ) recall or release tests. During an infection, pathogens are taken up by antigen-presenting cells (APCs) and T lymphocytes are activated by the presentation of the pathogen antigens on the cell surface together with co-stimulating signals. Activated TH1 cells then release IFN-γ upon renewed contact with the pathogen. This is used in an IFN-γ recall test. Have TH1 cells of the animal to be examined been infected with C. burnetii-specific antigens, there is a strong production of IFN-γ. This test can be used to detect a cellular immune response. This test procedure was used in veterinary medicine to detect a C. burnetiiInfection has not yet been used very little (Boettcher et al. 2017, Janowetz et al. 2018, Roest et al. 2013). Proof of T-cell immunity to coxies has also been established for human medical diagnostics (Limonard et al. 2012). It can be used as a supplementary method for the diagnosis of chronic Q fever and its therapy control.

Molecular biology

For the direct detection of C. burnetii Nowadays a PCR is mostly used. There are currently four approved real-time PCR test kits in Germany for the examination of veterinary samples with which a transposase element (IS1111) from C. burnetii is amplified. These PCRs can be used, for example, to detect C. burnetii-specific DNA from placenta samples, swabs from vaginal mucosa and milk samples from domestic ruminants can be used. Depending on the Coxielle isolate, this repetitive element can occur between 7 and 110 times per genome per bacterial cell. In most isolates, however, the insertion sequence occurs between 10 and 30 times (Klee et al. 2006). This means that PCR diagnostics based on the detection of the IS1111 region are extremely sensitive. An exact quantification of the amount of pathogen is due to the variable number of insertion elements with different C. burnetii-Strains not possible, but of minor importance in the context of follow-up investigations into the excretion of the amount of pathogen. Exact pathogen quantifications are possible with the help of PCRs whose target sequences use single-copy genes such as com1 (Kersh et al. 2010).
The so-called Multiple Loci Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) enables the typing of entire genome sequences with high discriminatory power (Arricau-Bouvery et al. 2006). As a result, two main clusters within the ruminant species could be identified for Germany (Frangoulidis et al. 2014). Cluster A was detected in particular from isolates from small ruminants, with subcluster A1 being more associated with goats and A2 being detected mainly in sheep. Cluster C was primarily detected in isolates from cattle samples. In addition, there were also different geographical distributions of these clusters. Thus there should be different isolate groups in different regions within the ruminant species. However, no statements can yet be made about the host specificity, pathogenicity and virulence of the respective cluster.
In addition to the VNTR / MLVA method, the SNP-based multispacer sequence typing (MST) method is also used regularly (Glazunova et al. 2005). However, this method only achieves a very rough resolution with regard to regional distributions (Mendung et al. 2012).
For the monitoring of dairy goat and dairy sheep populations using tank milk samples, in addition to the use of ELISAs, especially the PCR methods have proven themselves (Ruiz-Fons et al. 2011, Sidi-Boumedine et al. 2010, van den Brom et al. 2012, 2015b.). Intra-herd seroprevalences from 15% can be detected with PCR (van den Brom et al. 2012). Samples should be taken two weeks after vaccination at the earliest C. burnetii-Antigens take place as vaccine associated C. burnetiiDNA is excreted in the milk up to 9 days after vaccination (Hermans et al. 2011). Reliable and active surveillance is lacking worldwide, especially for non-dairy sheep and goat populations. Therefore, one possible approach could be the use of environmental samples. Infected animals divorce during an abortion or normal birth C. burnetii massive and thus contaminate their stables. Dust from window sills and stable equipment can be used as evidence of C. burnetii used in animal husbandry (Carrié et al. 2019, Joulié et al. 2015). In a study by Carrié et al. (2019), especially in herds of small ruminants, Coxiae could be detected by means of PCR, sometimes with up to 108 genome equivalents. The probability that the pathogen was detected in the house dust increased with the increase in the number of dams. Stable air samples (Astobiza et al. 2011a, Joulié et al. 2015) and manure samples (Avbersek et al. 2019) were also used to detect C. burnetii used in animal husbandry. If the PCR is positive, however, no statement can be made as to whether it is an acute Q fever event or a pathogen excretion in the past. Environmental samples can remain positive over a longer period of time, even if the pathogen is no longer excreted (Astobiza et al. 2011b). Furthermore, it is not possible in routine diagnostics to make a statement about the viability of the coxia. Álvarez-Alonso et al. (2018) were unable to detect reproductive Coxiae in dust samples two months after the last lambing by means of complex inoculation in mice. Van den Brom et al. (2015a) did not succeed in cultivating Coxielle from goat manure that had already been stored for three months. The authors therefore assume that there were no longer any viable coxia in their samples. These results need to be checked in further studies in the future and the suitability of environmental samples as a sample matrix to be evaluated. In the event of repeated negative results from dust samples, it can be assumed that the animals do not excrete any coxies. In addition, surface samples could be used as markers after the cleaning and disinfection of stables to monitor the success of these measures.
Vaginal swabs can also be used to monitor shedding in herds of small ruminants. Particularly in vaccinated herds, a statement can be made about the pathogen excretion and thus the success of the vaccination. These should not be taken later than 8 days after an abortion or birth (OIE 2018). The number and time of sampling vary greatly depending on the author. For example, regardless of the herd size, it is recommended that 10 dams be sampled with vaginal swabs that have lambed within the last three months (CVUA Stuttgart 2018). In contrast, de Cremoux et al. (2018), regardless of the herd size, only sampled two to six animals using vaginal swabs that aborted within a week.
Van Leuken et al. (2016) recommend a combination of air samples inside and outside the stable with vaginal swabs and tank milk samples to monitor Q fever. The large number of recommendations for surveillance makes it clear that the standardization of active surveillance is absolutely necessary. When using molecular biological evidence, the pathogen has already been excreted. As a result, there is no active surveillance that detects positive herds at an early stage C. burnetii is eliminated.

activities

Immediate action

If there is an acute infection in goat and sheep populations, various measures should be taken to minimize the risk of infection in the population as far as possible. Such steps can be found in the recommendations for hygiene measures when keeping ruminants (BMEL 2014) and in the guidelines for Q fever in Baden-Württemberg (CVUA Stuttgart 2018) as well as in textbooks (Selbitz and Ganter 2018). Lambing and clipping should only take place in closed rooms and postbirths and abortions must be kept in closed containers until they are collected by processing plants of animal by-products. External persons must not have access to the animals. Raw milk and its products should not be sold or consumed. The milk must be pasteurized. Furthermore, work clothes and work materials must be cleaned and disinfected. The frequently required use of quicklime (approx. 10 kg / m2, Hydrated lime, Ca (OH)2, BMEL 2007) for the creation of a solid manure package is used by the authors in the case of C. burnetii-Fighting not shared due to the risk of spontaneous combustion. In a Dutch study, 97 days after storage of large solid goat manure packages (at least 10 meters long, 4.5 meters wide and 3.5 meters high) under the film, it was still possible C. burnetiiDNA can be detected, but the pathogen was no longer cultivated (van den Brom et al. 2015a). Therefore, the manure from infected stocks should not be spread immediately, but stored for at least three months under foil and away from residential areas (Plummer et al. 2018). The manure must then be spread on arable land in calm weather and immediately worked into the soil. Furthermore, it is required to identify permanent excretors in a herd, for example using a vaginal swab, in order to subsequently kill them and dispose of them harmlessly (BMEL 2014). Individual with C. burnetii infected goats excrete the pathogen intermittently in the milk even after vaccination (van den Brom et al. 2013). These separators should be identified and slaughtered using individual milk samples. In these goats was C. burnetii-DNA only detected in the udder (van den Brom et al. 2013). For this reason, the udder should be removed intact and discarded during slaughter. Long-term excretors have not yet been identified in sheep (Astobiza et al. 2011b). A chronification of the C. burnetii-Infection has not yet been detected in sheep either. As part of the control measures, close cooperation and coordination between the health authorities, the veterinary authorities, the general practitioners and veterinarians as well as the animal health services and in particular the animal owners concerned should be sought (Selbitz and Ganter 2018).

Antibiotic treatment

The use of oxytetracycline to treat coxiellosis did not reduce excretion in small ruminants (Astobiza et al. 2013, de Cremoux et al. 2012). However, the use of oxytetracycline in the outbreak is associated with a simultaneous infection Chl. abortus justified (Eibach et al. 2013). In contrast to small ruminants, cattle excreted fewer coxiides after treatment with oxytetracycline (Taurel et al. 2012). Due to the very low pH in the phagolysosomes of the host cell, in which C. burnetii increased, there is inactivation of tetracyclines by epimerization at the C4 atom (Rogalski 1985). In contrast, doxycycline is considered to be the drug of choice in human medicine for the treatment of Q fever. Doxycycline not only has higher in vitro activity and better pharmacokinetic parameters (Rogalski 1985), it is also significantly more resistant to low pH values. Doxycycline-resistant strains of C. burnetiithat have been described in the literature (Rolain et al. 2005). Mechanisms that could be responsible for this resistance are not yet known (Rouli et al. 2012). There is currently no knowledge about the effectiveness of other antibiotics used in veterinary medicine.

vaccination

A vaccine approved for cattle and goats has been available in Germany since 2010 (Coxevac®, Ceva Santé Animale, Libourne, France). This is an inactivated Ph-I vaccine. This has a better effect than Ph II vaccines (Arricau-Bouvery et al. 2005). The mechanism of protective immunity of Ph-I vaccines has not yet been fully elucidated (Achard and Rodolakis 2017). The basic vaccination course every three weeks should be completed four weeks before the first covering. In this way, the excretion of C. burnetii greatly reduced in naively vaccinated goats, but not completely prevented (Arricau-Bouvery et al. 2005). In already infected goat herds, vaccination is used to eliminate C. burnetii reduced in the long term. Vaccination of young animals before the first covering reduces the pathogen excretion in positive farms to a particularly high degree (Achard and Rodolakis 2017, de Cremoux et al. 2012, Rousset et al. 2009b). This could presumably be due to the fact that C. burnetii trophoblasts are required for successful establishment of the infection (Ben Amara et al. 2010, Roest et al. 2012, Sánchez et al. 2006). Vaccination of young goats before the first covering therefore seems to be the establishment of C. burnetii to complicate. To maintain immunity, the goats must be boosted annually.In goats, vaccination can temporarily cause slight to moderate swelling of the skin at the injection site and, in the short term, fever (> 40.5 ºC) and a decline in milk yield (EMA 2014, Vellema et al. 2010). The subcutaneous swelling is already with C. burnetii infected goats were more massive than in naive animals (Vellema et al. 2010). The vaccine is not approved for use in pregnant animals, but in the past it was used in pregnant sheep (Eibach et al. 2013, Joulié et al. 2017) and goats (personal experience of the authors Ganter and Bauer). In some cases, it has been possible to reduce excretion in sheep (Eibach et al. 2013). In contrast, Astobiza et al. (2013, 2011a) found no significant difference between vaccinated and unvaccinated sheep with regard to the excretion of C. burnetii determine via the vaginal secretions. In their first study there were two C. burnetii positive dairy sheep flocks were vaccinated with a Ph-I vaccine six or three weeks before artificial insemination. A quarter of the ewes remained unvaccinated as a control group. Vaginal swabs were taken within four weeks of lambing and examined by PCR. Compared to the previous year, pathogen excretion decreased significantly in both herds, regardless of the animals' vaccination status (herd 1: from 6.5 log to 3.4 log C. burnetii per swab, foci 2: from 7.3 log to 2.9 log C. burnetii per swab). However, there was no significant difference between vaccinated and unvaccinated sub-herds (Astobiza et al. 2011a). This result was confirmed again in a similar study by the authors (Astobiza et al. 2013). Despite the lack of approval for the vaccine for sheep, it can, however, be used across species (StIKoVet 2018). A product information sheet from the manufacturer Ceva from 2008 recommends a vaccination dose of Coxevac® 1 ml for sheep. Current data are not available from the manufacturer. In practice, the vaccination dose has been used for years both nationally (Eibach et al. 2013, Hamann et al. 2009) and internationally (personal communications Renée De Cremoux, France, Ana García-Peréz, Spain and René van den Brom, Netherlands) 1 ml successfully applied to sheep. However, these are only empirical values ​​that have not yet been scientifically proven. In summary, it is so far only possible with vaccination C. burnetii-To prevent or control infections (Bontje et al. 2016). Therefore vaccination should be used as an important control tool to prevent herds C. burnetii to protect and prevent human infections (Bontje et al. 2016, EFSA 2010).

disinfection

Due to the high environmental resistance of C. burnetii many disinfectants are not sufficiently effective. Especially the different tenacity of the morphological cell forms of C. burnetii, with the vegetative LCV and the spore-like SCV, complicates the selection of suitable disinfection measures.
In general, there is a lack of precise information and recommendations regarding the selection and use of disinfectants C. burnetii decontaminate positive animal husbandry areas (Frentzel et al. 2013, Rodolakis 2009). In general, appropriate disinfectants from the list of the German Veterinary Medical Society are used for bacterial animal disease pathogens. V. (column 4a, "special disinfection", DVG 2019), which are entered as effective in the usage concentration within 2 hours (BMEL 2007). There are many specific statements about the use of disinfectants. The RKI (2012) recommends 10–20% chlorine lime solutions, a 1% Lysol solution or 5% hydrogen peroxide solutions for disinfecting stables. When using a 2% sodium hydroxide solution, the germ load was reduced by at least a factor of 10 on sandy, loam and loess soil4 can be achieved (Dörner 2011). The use of a 1% peroxygen compound led to a reduction in infectivity of over 90% after 30 minutes (Priestley 2007). After intensive cleaning and subsequent disinfection with 0.5% peracetic acid, the pathogen load of C. burnetii can be significantly reduced (Moog et al. 2018). It should also be noted that many stables for small ruminants are made of natural materials (e.g. wooden walls and gates and clay floors). These cannot be cleaned and disinfected sufficiently. Therefore, only a pathogen reduction and no pathogen freedom can be achieved. The review of potentially effective disinfectants is urgently required in order to provide evidence-based and practical recommendations for the disinfection of C. burnetii