Role of Goats in the Epidemiology of Coxiella burnetii

Simple Summary This review article aims to compile the information published in the scientific literature regarding Coxiella burnetii infection in goats and their role in the epidemiology of infection, namely their association with the occurrence of Q fever in humans. Q fever presents a worldwide occurrence and the risk that it represents to humans has been recognized since its first description. The characteristics of C. burnetii justify its classification as a category B biological threat agent. International public health authorities strongly recommend global monitoring of C. burnetii, especially after large-scale Q fever epidemics occurred in The Netherlands, which originated from goat infection. An approach with the characterization of the bacterium, its strategies of infection, and clinical patterns in goats will help to understand the dynamics of infection in an epidemiological analysis and to analyze the role of goats in Q fever. Abstract Since its first description in the late 1930s, Q fever has raised many questions. Coxiella burnetii, the causative agent, is a zoonotic pathogen affecting a wide range of hosts. This airborne organism leads to an obligate, intracellular lifecycle, during which it multiplies in the mononuclear cells of the immune system and in the trophoblasts of the placenta in pregnant females. Although some issues about C. burnetii and its pathogenesis in animals remain unclear, over the years, some experimental studies on Q fever have been conducted in goats given their excretion pattern. Goats play an important role in the epidemiology and economics of C. burnetii infections, also being the focus of several epidemiological studies. Additionally, variants of the agent implicated in human long-term disease have been found circulating in goats. The purpose of this review is to summarize the latest research on C. burnetii infection and the role played by goats in the transmission of the infection to humans.


Introduction
The history of Q fever, the disease caused by Coxiella burnetii, can be traced back to 1937, when it was described by Edward Holbrooke Derrick in Australia [1]. Almost simultaneously, in the United States, an unknown agent isolated from ticks recovered from Nine Mile Creak region, Montana, was described [2]. Australian and American teams shared their findings and concluded that they were studying the same agent and the same disease [3]. The potential risk of Q fever to public health and the large gaps in the knowledge of this disease were recognized early, namely by the World Health Organization (WHO) that, in 1950, encouraged the epidemiological research. Consequently, Q fever was Table 1. Human Q fever outbreaks associated with goats.

Country (Area) Period Reference
Australia 2012-2014 [20] Bulgaria 2004 [21] 2007-2011 [22] China 2018-2019 [23] France - [24] 2007 [25] Newfoundland 1999 [26] Slovakia 1993 [27] The Netherlands 2007-2020 [28,29] United Kingdom 1987 [30] USA - [31] 2011 [32] Recently, it was observed that goats played a major role in infecting humans compared with sheep in The Netherlands Q fever outbreak, which was previously associated with small ruminants [18]. Thus, the infection patterns in goats (e.g., dynamics of infection, clinical outcomes, and shedding patterns) need clarification, and experimental studies using goats have improved our scientific knowledge on this topic. Furthermore, the effective strategy of prevention and control of Q fever, in both humans and animals, requires a One Health perspective [28,30]. This review is not to intend to give a global geographic epidemiological view of Q fever, but rather focus on the role of goats in the epidemiology of this zoonotic pathogen. First, the characteristics of the agent and its pathogenesis will be briefly described, considering its significance in understanding the clinical and excretion patterns and followed by the specific infection in goats. Then, the focus will be in the epidemiological role of goats in Q fever dissemination, finally referring to the importance of molecular tools to unravel the complex epidemiology of C. burnetii.
This review will focus on C. burnetii infection, highlighting the role of goats in the epidemiology of this zoonotic pathogen. An overview of the pathogenesis, as well as of the epidemiological characteristics influencing the dissemination of the infection, will be described referring to the importance of molecular tools to unravel the complex epidemiology of C. burnetii.
The internalisation of phase I SCV of C. burnetii in target cells involves the recognition of several receptors [51]. It is mediated by the leukocyte response integrin (LRI) (αvβ3) and an integrin-associated protein (IAP) [39,52]. The entry occurs through a microfilament-dependent endocytosis [44,51]. Phase I LPS induces a rearrangement of Factin cytoskeleton, leading to pronounced membrane protrusions at the site of bacterial adherence. This phenomenon, called membrane ruffling, requires contact between C. burnetii and host cells, and depends on the expression of toll-like receptor type 4 (TLR4) on the host cell surface (Figure 1) [53][54][55]. The ability to use αvβ3 integrin for invasion might be exploited by C. burnetii as a mechanism to avoid the induction of an inflammatory response, as αvβ3 integrin is typically involved in the removal of apoptotic cells via phagocytosis, being generally associated with an inhibition of inflammation [52]. Thus, C. burnetii enters the cells without alerting the immune system [56]. After internalization, bacteria localize within the nascent Coxiella-containing vacuole (CCV), which traffics through the endocytic cascade. It develops into an early phagosome acquiring the small GTPase RAB5. This GTPase stimulates the fusion with early endosomes, resulting in acidification of the lumen to approximately pH 5.4 and acquisition of the early-endosomal marker protein 1 (EEA1) [57,58]. Early phagosome is converted into late phagosome acquiring acid hydrolases, which are involved in pronounced degradative activity, which C. burnetii can resist [59]. This late phagosome lacks RAB5 and EEA1 but acquires lysosome-associated membrane protein 1, 2, and 3 (LAMP1, LAMP2, and LAMP3) and vacuolar ATPase, which pumps protons into the maturing phagosome to further decrease the luminal pH to about 5.0 [58,60,61]. C. burnetii persists and replicates, at a slow rate, within the large CCV with an acidic environment [39,62,63]. The process of phagosome maturation continues with its fusion with lysosomal compartments to acquire cathepsins and hydrolases. The vacuolar ATPase further reduces the pH to around 4.5 [58,64]. Phagosome maturation depends on the balance between proinflammatory (IFN-γ, IL-12, and IL-6) and anti-inflammatory (IL-10) cytokines [65]. C. burnetii modulates the genesis of CCV and has several strategies for adaptation to the stressful environment. It encodes a significant number of basic proteins that are probably involved in buffering the acidic environment of the CCV. Moreover, four sodium-proton exchangers and transporters for osmoprotectants are codified in its genome, allowing this bacterium to confront osmotic and oxidative stresses [35].
During its biogenesis process, CCV becomes large and contains a large number of bacteria [62]. C. burnetii does not synthesize its own CCV membrane. Multiple fusion events with autophagosomes along with endolysosomal vacuoles are essential to provide sufficient membrane to enlarge the CCV [66,67]. C. burnetii continuously directs fusion with other host cell compartments and inhibits apoptotic cell death, allowing a prolonged infectious cycle [63,[68][69][70][71].
The internalised SCV, within the CCV, suffers a differentiation into replicative and metabolically active LCV ( Figure 2). The low intra-phagosomal pH and perhaps enzyme After internalization, bacteria localize within the nascent Coxiella-containing vacuole (CCV), which traffics through the endocytic cascade. It develops into an early phagosome acquiring the small GTPase RAB5. This GTPase stimulates the fusion with early endosomes, resulting in acidification of the lumen to approximately pH 5.4 and acquisition of the early-endosomal marker protein 1 (EEA1) [57,58]. Early phagosome is converted into late phagosome acquiring acid hydrolases, which are involved in pronounced degradative activity, which C. burnetii can resist [59]. This late phagosome lacks RAB5 and EEA1 but acquires lysosome-associated membrane protein 1, 2, and 3 (LAMP1, LAMP2, and LAMP3) and vacuolar ATPase, which pumps protons into the maturing phagosome to further decrease the luminal pH to about 5.0 [58,60,61]. C. burnetii persists and replicates, at a slow rate, within the large CCV with an acidic environment [39,62,63]. The process of phagosome maturation continues with its fusion with lysosomal compartments to acquire cathepsins and hydrolases. The vacuolar ATPase further reduces the pH to around 4.5 [58,64]. Phagosome maturation depends on the balance between pro-inflammatory (IFN-γ, IL-12, and IL-6) and anti-inflammatory (IL-10) cytokines [65]. C. burnetii modulates the genesis of CCV and has several strategies for adaptation to the stressful environment. It encodes a significant number of basic proteins that are probably involved in buffering the acidic environment of the CCV. Moreover, four sodium-proton exchangers and transporters for osmoprotectants are codified in its genome, allowing this bacterium to confront osmotic and oxidative stresses [35].
During its biogenesis process, CCV becomes large and contains a large number of bacteria [62]. C. burnetii does not synthesize its own CCV membrane. Multiple fusion events with autophagosomes along with endolysosomal vacuoles are essential to provide sufficient membrane to enlarge the CCV [66,67]. C. burnetii continuously directs fusion with other host cell compartments and inhibits apoptotic cell death, allowing a prolonged infectious cycle [63,[68][69][70][71].
The internalised SCV, within the CCV, suffers a differentiation into replicative and metabolically active LCV ( Figure 2). The low intra-phagosomal pH and perhaps enzyme system and/or nutrient sources present in the vacuole seem to trigger this differentiation. Lag phase extends to approximately two days post-infection and is composed primarily of SCV to LCV morphogenesis. The exponential phase occurs over the next four days with CCV harbouring replicating LCV almost exclusively. The LCV multiplies and persists within an expanding CCV that contains lysosomal elements, including an acid pH (5.0) and degradative proteases [44,46,59,72]. system and/or nutrient sources present in the vacuole seem to trigger this differentiation. Lag phase extends to approximately two days post-infection and is composed primarily of SCV to LCV morphogenesis. The exponential phase occurs over the next four days with CCV harbouring replicating LCV almost exclusively. The LCV multiplies and persists within an expanding CCV that contains lysosomal elements, including an acid pH (5.0) and degradative proteases [44,46,59,72]. A dramatic expansion of the CCV occurs concomitantly with the appearance of replicating LCV, occupying nearly the entire cytoplasm [44,66]. These metabolically active LCVs also play an important role in cell-to-cell spread during acute infection. This process is facilitated by the display of unique LCV antigens such as a porin protein termed P1. The stationary phase begins six days post-infection, concomitantly with the re-appearance of SCV. Following the accumulation of large numbers of LCVs, C. burnetii converts back into SCVs, which are released from heavily infected cells by an undefined mechanism [44].
The resistance properties of these SCVs strongly implicate this form as responsible for long-term extracellular survival and aerosol transmission of C. burnetii [44,45].

Infection and Clinical Outcomes in Goats
It is globally recognized that C. burnetii infection occurs mainly by inhalation of contaminated aerosols and, because C. burnetii is a highly infective pathogen, low doses cause a high risk of illness [73,74]. So far, experimental studies on goats were not focused on estimating the infectious dose. However, in humans, it was estimated that the 50% infectious dose was around one bacterium [75].
Alveolar macrophages are the first-line defence that confronts C. burnetii [49,76]. The ability of these cells to rapidly respond recruiting additional immune cells is central for an effective antibacterial response in early stages of infection [65,77]. In primary infections, after entry into the organism, a bacteraemia occurs, leading to a systemic infection with the involvement of organs such as liver, spleen, lungs, and bone marrow [38]. The organism can subsequently disseminate to colonize and replicate in resident macrophages of different tissues and organs [78]. In pregnant goats, the main target cells are the trophoblasts in the allanthocorion, causing a placentitis and necrosis of placental tissues [79,80]. The amount of C. burnetii DNA detected increases until parturition and decreases drastically after parturition, probably by the disappearing of trophoblasts, the replication niche of C. burnetii during pregnancy [79,81]. This strong tropism of C. burnetii towards A dramatic expansion of the CCV occurs concomitantly with the appearance of replicating LCV, occupying nearly the entire cytoplasm [44,66]. These metabolically active LCVs also play an important role in cell-to-cell spread during acute infection. This process is facilitated by the display of unique LCV antigens such as a porin protein termed P1. The stationary phase begins six days post-infection, concomitantly with the re-appearance of SCV. Following the accumulation of large numbers of LCVs, C. burnetii converts back into SCVs, which are released from heavily infected cells by an undefined mechanism [44].
The resistance properties of these SCVs strongly implicate this form as responsible for long-term extracellular survival and aerosol transmission of C. burnetii [44,45].

Infection and Clinical Outcomes in Goats
It is globally recognized that C. burnetii infection occurs mainly by inhalation of contaminated aerosols and, because C. burnetii is a highly infective pathogen, low doses cause a high risk of illness [73,74]. So far, experimental studies on goats were not focused on estimating the infectious dose. However, in humans, it was estimated that the 50% infectious dose was around one bacterium [75].
Alveolar macrophages are the first-line defence that confronts C. burnetii [49,76]. The ability of these cells to rapidly respond recruiting additional immune cells is central for an effective antibacterial response in early stages of infection [65,77]. In primary infections, after entry into the organism, a bacteraemia occurs, leading to a systemic infection with the involvement of organs such as liver, spleen, lungs, and bone marrow [38]. The organism can subsequently disseminate to colonize and replicate in resident macrophages of different tissues and organs [78]. In pregnant goats, the main target cells are the trophoblasts in the allanthocorion, causing a placentitis and necrosis of placental tissues [79,80]. The amount of C. burnetii DNA detected increases until parturition and decreases drastically after parturition, probably by the disappearing of trophoblasts, the replication niche of C. burnetii during pregnancy [79,81]. This strong tropism of C. burnetii towards placenta does not seem to occur for other tissues of nonpregnant goats and kids, suggesting that pregnant females are more susceptible to C. burnetii infection [79,82].
Cell-mediated immunity probably plays a critical role in controlling C. burnetii infection [49,55]. Cells belonging to monocyte-macrophage lineage express polarized functional properties. This polarization seems to be closely related to the ability to control C. burnetii infection, explaining the bacterial persistence in chronic infections [83]. Classically, M1 polarized macrophages are induced by LPS, IFN-γ, and TNF-α, and participate in the resistance against intracellular pathogens involved in Th1 responses. In contrast, M2-polarized macrophages are induced by IL-4, IL-13, or IL-10 and promote Th2 responses. So, it is thought that the course of infection differs according to the macrophage polarization in response to C. burnetii infection [83]. If M1-associated molecules are expressed by macrophages, the bacterial replication will be controlled [62,83], while the stimulation of an M2 response will account for the persistence of C. burnetii in macrophages, which become highly permissive to C. burnetii replication [83][84][85].
Beyond cell-mediated response, an antibody-mediated immunity also seems to be important in C. burnetii infection [49]. Treatment of C. burnetii infection with immune sera makes the bacterium more susceptible to phagocytosis and destruction by macrophages [86]. Specific immunoglobulins are secreted following infection [38] and the infection of dendritic cells with antibody-opsonized bacteria results in increased expression of maturation markers and inflammatory cytokines in mice [49]. It can be concluded from field studies that C. burnetii antibodies are highly persistent, lasting for several months up to years [87,88]. Thus, both humoral and cellular immunity play a role in C. burnetii infection.
However, the immune control of C. burnetii might not lead to its eradication from the infected host [55]. It is also hypothesized that the uterus could be a site of latent infection, hence reactivation during pregnancy can occur [89,90].
In goats, as well as in other domestic ruminants, C. burnetii infection often goes unnoticed owing to the absence of symptoms, and the term Coxiellosis is usually used to refer this condition [8]. In the early stages after infection, C. burnetii can be detected in the blood, lungs, spleen, and liver. However, it is not clear if its presence in organs other than placenta affects the functions of these organs, as only mild lesions have been described [79,81,91,92]. Experimental infection of non-pregnant goats showed that, at late stages of infection, C. burnetii was present in mammary glands, emphasizing the milk as an important shedding route [82]. Infection of pregnant goats may cause a wide range of conditions including abortion, delivery of premature offspring, stillbirth, and weak offspring. Of these, one of the most important outcomes of the C. burnetii infection is the abortion, which occur at the end of pregnancy without premonitory signs. In dairy goat herds that experience abortions caused by C. burnetii, an increased incidence of metritis can be noticed. Notwithstanding, a clinically normal progeny, which may or may not be congenitally infected, may occur, as described in infection of non-pregnant goats [7,79,82]. However, it seems that apparently healthy kids born from infected mothers may develop respiratory and digestive tract disorders [7].
In the season that follows an abortion storm, the multiplication of the organism may be reactivated during pregnancy, leading to reproductive failures [93][94][95]. Even in asymptomatic infections, a latent infection may develop and a reactivation late in pregnancy can occur several days before parturition. Generally, when late-term abortions, stillbirths, or birth of stunted animals are observed in goat flocks, Q fever should be suspected. Usually, up to 90% of the reproductive females within the flock are infected. This is why it is mentioned that C. burnetii may cause epidemic herd outbreaks with significant animal losses owing to abortion waves and weak offspring during the parturition period [96,97].

Epidemiological Highlights
C. burnetii is a category B biological threat agent because of its impressive stability and resistance, its ability to aerosolize, and its virulence [43][44][45]98]. These characteristics allow the survival of this pathogen in the environment for long periods while keeping its infectivity [45,53]. In fact, viable microorganisms can be recovered after several years in dust, two years at -20 • C, seven to ten months on wool at environment temperature, 150 days in soil, for more than one month on fresh meat, and seven days in water or in milk at room temperature [17,38].
The transmission of C. burnetii may occur by direct, indirect, or vectorial transmission. The majority of natural C. burnetii infections occur by airborne transmission, resulting from the inhalation of aerosolized bacteria [79,93,96]. The resistance of C. burnetii allows it to be dispersed by wind far away from its original source. This may cause a long-distance transmission of infection leading to inter-herd transmission of C. burnetii or even to dispersion of bacteria to residential locations, causing human outbreaks [99][100][101]. Several studies on human outbreaks report this mid-to long-distance transmission [99,102,103]. Moreover, areas with high wind speed, open landscape, and high temperature increase the risk of infection [104].
Milk is considered a relevant route of bacterial shedding in the infected goats [105] and several studies evidence the presence of C. burnetii in goat milk (Table 2). Despite the knowledge that C. burnetii remains viable in unpasteurized milk, the assumption of the risk of infection by ingestion of C. burnetii milk is controversial [108,116,117]. In France, C. burnetii was detected in commercially available milk products, but, because its viability was not confirmed, the transmission by consumption of these products was not considered important [118]. Moreover, in a report concerning the public health risks related to raw drinking milk, C. burnetii is not mentioned as a biohazard to be transmitted via milk [119]. Thus, for risk assessment, it is assumed that multiplication of the pathogen in milk and milk products does not occur. Furthermore, there are insufficient data for a dose-response model for the oral route in humans [117]. It is known that the pasteurization procedure by ultra-high temperature treatment of milk (72 • C for 15 s) is adequate to eliminate viable C. burnetii from whole raw milk [120,121]. Notwithstanding, the risk of infection by consuming unpasteurized milk and raw milk dairy products may not be negligible.
Infected goats also shed high concentrations of C. burnetii in placental membranes, birth fluids, and/or abortion products [79,122]. This is an important excretion route in peri-partum period contributing to a high contamination of the environment. Furthermore, C. burnetii can also be excreted through faeces and vaginal mucus [79,113]. The shedding is normally very high at the first parturition after the infection, but occasionally, it occurs at subsequent pregnancies accompanied by a considerable number of bacteria excreted through placenta [79,94]. So, normal deliveries in infected females may contribute to the environmental contamination and should, therefore, be considered as a major zoonotic risk [79]. In the environment, bacteria can be easily aerosolized from desiccation of infected placenta and body fluids or from contaminated manure, reaching new hosts [96,101,103].
The risk of infection has been studied at the herd level and at the individual level in prevalence studies by detection of antibodies (Tables 3 and 4, respectively). At the herd level, the risk factors for C. burnetii infection are as follows: the proximity of an infected farm, the high animal density in a municipality, the high wind speed, an open landscape, the high temperature [123,134,135], the increased size of the herd [123,129,134], the poor hygiene and bio-security measures in the farm, the presence of ticks, the presence of dogs and cats in the farm [134,135], and the presence of swine on farms [123]. At an individual level, it was shown that the risk of positivity increases with age [129].  The number of Q fever cases varies geographically, and a seasonal variation is also described [100]. In the Northern Hemisphere, acute Q fever cases are more often reported in spring and early summer, showing a slow rise in reported cases in March and April, probably associated with the start of lambing/kidding, and the main peak occurs between May and July [27,147,148]. This occurs probably because of the "outside" lambing/kidding during spring associated with heavy environmental contamination with C. burnetii. It is known that the lambing season in October is not related to a higher incidence in humans, which might be owing to "indoor" lambing [96], which is also consistent with the study conducted in the South of France, showing that autumn is not a very windy season, which might explain the lower incidence of human Q fever at this time of the year [99].
In most European countries, Q fever cases in humans and animals are reported regularly [8,148]. In humans, after the largest ever recorded outbreak in The Netherlands, the number of notified cases has suffered in general a sustained decrease in Europe and, nowadays, small outbreaks still occur, as shown on Figure 3, mainly in areas with infected livestock herds [148][149][150].
Regarding the report of infection in goats, the WAHIS Interface from the World Organization for Animal Health (WOAH) indicates the number of outbreaks reported in each country per year, which is systematized for European countries in the Figure 4. However, the analysis must be performed carefully because it is based on a notification procedure, thus it may not indicate the true prevalence as differences in the procedure of notification may differ between countries. Thus, data from epidemiological studies (Tables 3 and 4) can be more reliable within the reality in a region/country.
C. burnetii infection in humans is usually considered an occupational threat. Some professional groups are more prone to exposure with C. burnetii. For instance, the farming workforce constitutes a relevant occupational risk group because of their contact with infected livestock, namely during breeding practices [151,152]. Moreover, veterinarians, laboratory workers, and abattoir workers are also at risk of being infected [8,[153][154][155][156], as well as workers in the wool, tanneries, fur, meat, leather, and timber industries [152]. Regarding the report of infection in goats, the WAHIS Interface from the World Organization for Animal Health (WOAH) indicates the number of outbreaks reported in each country per year, which is systematized for European countries in the Figure 4. However, the analysis must be performed carefully because it is based on a notification procedure, thus it may not indicate the true prevalence as differences in the procedure of notification may differ between countries. Thus, data from epidemiological studies (Tables 3 and 4) can be more reliable within the reality in a region/country. C. burnetii infection in humans is usually considered an occupational threat. Some professional groups are more prone to exposure with C. burnetii. For instance, the farming workforce constitutes a relevant occupational risk group because of their contact with infected livestock, namely during breeding practices [151,152]. Moreover, veterinarians, laboratory workers, and abattoir workers are also at risk of being infected [8,[153][154][155][156], as well as workers in the wool, tanneries, fur, meat, leather, and timber industries [152].
However, community outbreaks have been described very often. Factors as living on rural or sub urban areas [133] and in the proximity of positive farms [157] substantially increase the risk of Q fever.
The danger of C. burnetii being used as a biological weapon is a global concern. The low infectious dose for humans and its ability to spread over large distances on the wind would cause an enormous impact on human health. Additionally, wide-scale  Regarding the report of infection in goats, the WAHIS Interface from the World Organization for Animal Health (WOAH) indicates the number of outbreaks reported in each country per year, which is systematized for European countries in the Figure 4. However, the analysis must be performed carefully because it is based on a notification procedure, thus it may not indicate the true prevalence as differences in the procedure of notification may differ between countries. Thus, data from epidemiological studies (Tables 3 and 4) can be more reliable within the reality in a region/country. C. burnetii infection in humans is usually considered an occupational threat. Some professional groups are more prone to exposure with C. burnetii. For instance, the farming workforce constitutes a relevant occupational risk group because of their contact with infected livestock, namely during breeding practices [151,152]. Moreover, veterinarians, laboratory workers, and abattoir workers are also at risk of being infected [8,[153][154][155][156], as well as workers in the wool, tanneries, fur, meat, leather, and timber industries [152].
However, community outbreaks have been described very often. Factors as living on rural or sub urban areas [133] and in the proximity of positive farms [157] substantially increase the risk of Q fever.
The danger of C. burnetii being used as a biological weapon is a global concern. The low infectious dose for humans and its ability to spread over large distances on the wind would cause an enormous impact on human health. Additionally, wide-scale However, community outbreaks have been described very often. Factors as living on rural or sub urban areas [133] and in the proximity of positive farms [157] substantially increase the risk of Q fever.
The danger of C. burnetii being used as a biological weapon is a global concern. The low infectious dose for humans and its ability to spread over large distances on the wind would cause an enormous impact on human health. Additionally, wide-scale consequences would occur because of animal infection (i.e., domesticated, and wild animals) that could represent secondary sources of infection for humans [98] and even compromise agriculture and food production.

Molecular Epidemiology: An Added Value
Nowadays, molecular epidemiology is crucial in monitoring programs of C. burnetii and in investigation of Q fever outbreaks. The genetic heterogeneity of C. burnetii can be assessed by several molecular techniques. Today, the most adopted methods to define phylogeny are the multi loci variable-number tandem repeat analysis (MLVA) and the multispacer sequence typing (MST) [96,158,159]. A large number of MLVA data exist for European countries, even if the lack of consensus between scientists hampers comparison. Overall, a common pool of MLVA genotypes is present in Europe, together with novel genotypes sporadically found in specific countries [160][161][162][163]. Different MLVA genotypes could correspond to an identical MST type, indicating the MLVA method as more discriminatory than MST. Furthermore, the availability of a free-access database on the internet increased the interest in these methods to characterize C. burnetii strains circulating in a region in a normal context or in the case of outbreak [106,164,165].
A systematic genotyping provides a descriptive database, enabling to monitor the temporal and geographical evolution of strains, thus helping to trace the origins of the outbreaks and to identify interspecies transmission. These data can help to explain different scenarios of dissemination and contribute to finding efficient control measures [37,159,165,166]. For instance, in Portugal, the involvement of different genotypes in acute and in long-term infections of Q fever was found, but only one genotype found in long-term Q fever was linked to the one identified in goats from the same region [161]. Moreover, in Belgium, an emerging CbNL01-like genotype was identified in goats and this strain was isolated from half of the field samples isolated from the Dutch outbreak. However, no impact on the number of human cases was observed [106]. In Poland, examination of nine C. burnetii samples from goats revealed the presence of three MLVA genotypes (I, J, and PL1) and one sequence type (ST61). These MLVA and MST profiles were different to the strains' profiles involved in the Q fever outbreak in the Netherlands [109]. Table 5 shows the C. burnettii genotypes identified by MST in goats and in other hosts (human, ruminants, and vectors), showing the wide transmission of specific genotypes. Cattle [167] An analysis of MLVA and MST genotypes published in international databases showed that human isolates of C. burnetii frequently belong to the same genomic group of caprine isolates [168]. Furthermore, a geographical niche for C. burnetii genotypes was demonstrated. Although some isolates present a worldwide distribution, others show a geographic localization. Some genomic groups occur predominantly in Northern and Central Asia, Eastern and Central Europe, and Africa, while others are found mainly in central Europe or even in other areas [168]. For instance, MST8 reported in goats from Spain (Table 5) was the most found genotype in goat milk in the United States [169].
Thus, genotyping demonstrates that isolates in human Q fever are frequently genetically related to isolates circulating in goat populations, which reinforces the role of goats as important sources of infection for human populations.

Conclusions
In conclusion, C. burnetii presents a wide host range; however, it is mostly recognized in domestic ruminants. Q fever is not very commonly diagnosed in humans, in part because primary infection is frequently asymptomatic. The development of molecular tools has allowed to unravel the dynamic of genotypes' circulation. Genotyping of C. burnetii indicates that, often, infected goats are the source of infection in human Q fever outbreaks. The pathogenesis is complex and not entirely understood at the host level, which highlights the requirement of future research to grasp the dynamics of the infections in specific hosts, as well as to unravel strain characteristics that may determine its virulence, affect the course of the disease and the clinical outcome, or influence the affinity to specific hosts. Yet, the association of genotypes with high virulence or host specificity remains to be demonstrated. An international genotype database facilitates the identification of emerging genotypes and their epidemiological features, also relying on a standard molecular method, which allows interlaboratory comparison. Thus, a worldwide surveillance in goats based on molecular epidemiology will be an important strategy to effective control of this zoonotic and highly resistant pathogen that fits in the One Health approach that considers animals, environment, and humans. Data Availability Statement: Publicly available datasets were analyzed in this study. This data can be found here: WAHIS Interface. Available online: https://wahis.woah.org/#/home and Database on Multi Spacers Typing of Coxiella burnetii. Available online: https://ifr48.timone.univ-mrs.fr/mst/ coxiella_burnetii/.

Conflicts of Interest:
The authors declare no conflict of interest.